HK40090308A - Anti ccr8 antibody therapy: biomarkers & combination therapies - Google Patents

Description

Anti-CCR8 antibody therapy: biomarkers and combination therapies

Technical Field

This invention relates to tools and methods for generating antibodies that specifically bind to chemokine receptors such as CC or CXC chemokine receptors. Isolated sulfated polypeptides and conjugates thereof are provided, which can be used, for example, as antigens or for off-target panning to promote the generation of antibodies against human, cynomolgus monkey, and/or mouse chemokine receptors, for example, to generate antibodies having fully human CDRs and/or other advantageous properties for therapeutic use.

This invention also relates to antibodies and conjugates thereof that can be obtained by applying the tools and methods described above. Antibodies that specifically bind to human, cynomolgus monkey, and/or mouse CCR8 and have advantageous properties for therapeutic use are provided, such as cross-reactive antibodies, fully human antibodies, low-internalizing (including non-internalizing) antibodies, and antibodies that effectively induce ADCC and/or ADCP in Treg cells.

The invention also provides medical uses and/or treatment methods for the antibodies or conjugates of the present invention, including administering these antibodies, alone or in combination, to patients or subjects. Finally, it provides biomarkers, stratification methods, and diagnostic methods for predicting or evaluating responses to monotherapy or combination therapy against CCR8 antibodies.

The present invention also provides tools and methods for generating the aforementioned antibodies, pharmaceutical compositions, diagnostic uses of the antibodies, and kits with instructions for use.

Technical issues

Antibodies that produce CC and CXC chemokine receptors

CXC and CC chemokine receptors are specific seven-transmembrane G protein-coupled receptors that mediate cell migration along chemokine gradients. Due to their inherent structural, biophysical, and biological properties, chemokine receptors are a challenging target class for antibody production. Several reasons contribute to the difficulty in obtaining optimal antibodies against chemokine receptors, which will be discussed in an illustrative manner, focusing on human CCR8.

Chemokine receptors are characterized by seven domains embedded in the cell membrane, making them difficult to purify in their native form. Purified native chemokine receptors are removed from their membrane environment and may therefore be conformationally compromised. These damaged structures are generally unsuitable for antibody production, as antibodies require recognition of intact antigens presented on the cell surface. While some chemokine receptors, such as CXCR4 and CCR5, possess some inherent stability that allows purification in mild detergents, this is not the case for most chemokine receptors (Hutchings, Catherine J., et al. "Opportunities for therapeutic antibodies directed at G-protein-coupled receptors." Nature Reviews Drug Discovery 16.11 (2017): 787.). This is underscored by the fact that, to date, no X-ray crystal structure for CCR8 is available in the Protein Database PDB (rcsb.org).

Therefore, for chemokine receptors, solubilization is difficult in order to obtain the required amount of protein in the native conformation and the correct orientation and folding for use as an immunogen (see cf. Klarenbeek, Alex, et al. "Targeting chemokines and chemokine receptors with antibodies." Drug Discovery Today: Technologies 9.4 (2012): e237-e244.).

A schematic diagram of the overall structure of human CCR8 is shown in Figure 2b. Of the 355 amino acids, residues 1-35 (N-terminus), 94-107 (ECL1), 172-202 (ECL2), and 264-280 (ECL3) are predicted to be extracellular domains (uniprot.org). These extracellular domains are assumed to have a tertiary structure, stabilized by disulfide bonds. Therefore, on average, less than 30% of the chemokine receptor is exposed on the cell surface. Consequently, chemokine receptors, especially CCR8, are not readily accessible for antibody binding, as described in WO200744756.

Furthermore, antibodies generated against peptides corresponding to the extracellular domains of chemokine receptors often fail to recognize the intact receptors on the cell, likely due to differences in secondary structure. Consequently, researchers in this field have a low success rate in antibody development (Tschammer, Nuska, ed. Chemokines: chemokines and their receptors in drug discovery. Vol. 14. Springer, 2015, Chapter by J.E. Pease & R. Horuk, section 6, page 12, 2nd para).

Obtaining antibodies against mouse CCR8 appears to be slightly less difficult, and inventors can replicate conventional methods, such as those described by Kremer and Márquez (D’Ambrosio, Daniele, and Francesco Sinigaglia. Cell Migration in Inflammation and Immunity. Springer, 2004., Chapter by Kremer and Márquez, pp. 243-260). The sequence identity between mouse and human CCR8 is approximately 70%, and even lower for the extracellular domains, as described in more detail in Example 2. However, Kremer and Márquez discuss that the generation of monoclonal antibodies against mouse chemokine receptors is a formidable challenge, as antibodies against mouse chemokine receptors are relatively scarce despite the passage of time since their amino acid sequences were first reported.

Therapeutic antibodies against CCR8 and their pharmaceutical applications

Despite the challenges in their production, antibodies targeting the CC chemokine receptor have been suggested as promising therapeutic tools for a variety of diseases, such as those based on mechanistic insights into diseases involving immune cells or various cancer indications characterized by aberrant expression of the chemokine receptor.

In 2004, Curiel et al. demonstrated that in 104 ovarian cancer patients, CD4+CD25+FOXP3+ regulatory T cells (Treg cells or Tregs) inhibited tumor-specific T cell immunity and promoted the growth of human tumors in vivo (Curiel, Tyler J., et al. "Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival." Nature medicine 10.9 (2004): 942-949.).

Tumor cells and tumor microenvironment macrophages produce chemokines, such as CCL22, which mediate the transport of Treg cells to the tumor. High levels of Tregs in the tumor microenvironment are not only associated with poor prognosis in many cancers (such as ovarian, breast, kidney, and pancreatic cancer), but also suppress immune responses against these cancers, for example, by inhibiting the role of immune system effector cells. Therefore, this specific recruitment of Treg cells represents a mechanism by which tumors can promote immune privilege. Consequently, it has been proposed to block Treg cell migration or function to combat human cancers.

Following Curiel's discovery, two independent teams, one led by Plitas/Rudensky and the other by De Simone/Abrignani/Pagani, identified selective CCR8 expression as a characteristic feature of tumor-infiltrating Treg cells. Indeed, the selectivity of Treg cell depletion is important for tumor-infiltrating Tregs, as systemic depletion of Treg cells can lead to severe autoimmunity (Nishikawa, Hiroyoshi, and Shimon Sakaguchi. "Regulatory T cells in tumor immunity." International Journal of Cancer 127.4 (2010): 759-767). Because Treg cells exhibit molecular patterns similar to effector lymphocytes, the search for specific Treg markers becomes complex (see Example 11.2). Peripheral Tregs and tumor-specific effector cells should not be subjected to friendly fire during intratumoral Treg depletion, as peripheral Tregs are important for avoiding autoimmunity, while tumor-specific effector cells help control the tumor.

Based on these findings, several teams have suggested using CCR8 antibodies to selectively deplete tumor-infiltrating regulatory T cells. While some data have confirmed the mechanistic concept of Treg depletion by using anti-human CCR4 or anti-mouse CCR8 antibodies to reduce tumors in tumor models, therapeutic anti-human CCR8 antibodies, for example, with superior performance in therapeutic applications, are still needed.

Existing technology

1.1 Antigens, Methods, and Antibodies

The choice of antigen in antibody production is crucial to the properties of the resulting antibody and can cause serious problems, such as with chemokine receptors like CCR8, discussed elsewhere in this paper. In some cases, whole cells have been used as antigens to obtain antibodies; these cells are engineered to overexpress chemokine receptors. These methods appear to be limited by the low amount of binding produced, at least for some chemokine receptors. Furthermore, antibodies obtained with these antigens often exhibit off-target binding to chemokine receptors and low specificity. If whole cells are used as antigens, immunodominant epitopes can mask other less antigenic epitopes, which may produce the desired selective and specific antibodies.

For CCR8, a successful “whole-cell” approach is described in WO2007044756 (ICOS). In short, anti-CCR8 monoclonal antibodies were developed by immunizing Balb/c mice with CCR8-transfected radiated cells expressing high levels of CCR8 on their cell surface. Spleen cells from these mice were fused using standard methods to generate antibody-producing hybridomas. Positive libraries were identified by FACS and cloned via limiting dilutions. Two of these antibodies, 433H and 459M, showed specific reactivity to human CCR8 in immunohistochemistry. Antibody 433H remains available and can be purchased from BD.

Biolegend distributed clone L263G8, a purified mouse IgG2a anti-human CCR8 antibody produced using human CCR8 transfectants as an immunogen.

Kremer and Márquez described the generation of monoclonal antibodies against mouse CCR8 (D'Ambrosio, Daniele, and Francesco Sinigaglia. Cell Migration in Inflammation and Immunity. Springer, 2004., Chapter by Kremer and Márquez, pp. 243-260). In short, Kremer and Márquez described the use of peptides derived from the extracellular domains of mouse CCR8 as immunogens, but did not imply tyrosine sulfation. Indeed, the inventors found that the method described by Kremer and Márquez could be successfully applied to antibodies recognizing mouse CCR8, but the success rate for antibodies recognizing human CCR8 was very low.

Schaerli et al. generated anti-human CCR8 antibodies by immunizing rabbits with human CCR8 peptide conjugates (corresponding to positions 1-34 of the N-terminal region of CCR8 conjugated with KLH or BSA), but similarly, tyrosine sulfate is not recommended (Schaerli, Patrick, et al. "A skin-selective homing mechanism for human immune surveillance T cells." The Journal of Experimental Medicine 199.9 (2004): 1265-1275.).

Haque et al. described a mouse monoclonal antibody that binds to a 26-amino acid peptide derived from the extracellular N-terminal portion of CCR8 (Haque, Nasreen S., et al. "The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned medium." Blood, The Journal of the American Society of Hematology 97.1 (2001): 39-45.).

None of these methods for antibody production use isolated sulfated peptides containing tyrosine-rich domains (TRDs) of chemokine receptors or transmembrane proteins as antigens. Using isolated sulfated peptides containing TRDs of chemokine receptors or transmembrane proteins as antigens affects the structural and functional characteristics of the resulting antibody genomes, as discussed elsewhere in this paper.

Therefore, the antibodies according to the present invention are indeed different in structure and function from the antibodies of the prior art described above, for example, at least in terms of their affinity for sulfated CCR8, their affinity for unsulfated CCR8, the manner and extent to which they modulate receptor signaling, their internalization behavior, cross-reactivity, clearance rate and pharmacokinetic behavior, and ultimately Treg depletion and/or efficacy for therapeutic applications.

Furthermore, the antigens disclosed herein can generate fully human antibodies. In contrast, the prior art antibodies against CCR8 discussed are of non-human origin and differ from some of the antibodies of this invention, at least because they do not contain human CDRs.

1.2 Medical Uses and Mechanisms of Action

Cancer immunotherapy involves using a subject's immune system to treat or prevent cancer. Immunotherapy typically leverages the fact that cancer cells often have subtly different molecules on their surface that can be detected by the immune system—cancer antigens. Therefore, immunotherapy involves stimulating the immune system to attack tumor cells through these cancer antigens. However, some cancers, such as solid tumors or blood cancers, can evade immune surveillance. For example, tumor infiltration of regulatory T cells (Treg cells or Tregs), and more specifically, a low ratio of effector T cells (Teff) to Tregs, has been considered a key factor in tumors evading the immune system (Smyth, Mark J., Shin Foong Ngiow, and Michele WL Teng. "Targeting regulatory T cells in tumor immunotherapy." Immunology and Cell Biology 92.6 (2014): 473-474.).

It is well known that regulatory T cells expressing Foxp3 are indispensable for preventing autoimmunity and can effectively suppress tumor immunity. The massive infiltration of Treg cells into tumor tissue is often associated with poor prognosis in cancer patients. Depletion of Treg cells can enhance anti-tumor immune responses but may also trigger autoimmunity. A key challenge in tailored cancer immunotherapies targeting Tregs is specifically depleting Treg cells infiltrating tumor tissue without affecting tumor-responsive effector T cells, while simultaneously suppressing autoimmunity.

As early as 2010, various teams had already studied how Treg depletion, for example by using anti-CD25 antibodies, could enhance anti-tumor immunity in mice. It has been shown that depleting Tregs before inoculation with tumor cells leads to their effective rejection, while Treg depletion occurring simultaneously with or after tumor inoculation does not lead to tumor regression. It is also believed that this is because the applied depletion antibody also removes effector T cells expressing CD25, while CD25-Foxp3+ Tregs persist (Klages, Katjana, et al. "Selective depletion of Foxp3+regulatory T cells improves effective therapeutic vaccination against established melanoma." Cancer Research 70.20 (2010): 7788-7799.).

In 2015, the fucosylated humanized anti-human CCR4 monoclonal antibody mogamulizumab (KW-0761) was evaluated in a clinical study in CCR4-positive cancer patients (Kurose, Koji, et al. "Phase I study of FoxP3+CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients." Clinical Cancer Research 21.19(2015):4327-4336.). CCR4 is expressed on regulatory T cells. Indeed, mogamulizumab effectively depletes Treg cells and an enhancement or induction of specific immune responses to cancer antigens was observed. However, mogamulizumab targets peripheral and intratumoral Tregs, as well as other effector cell populations, leading to immunological side effects such as rash or Stevens-Johnson syndrome.

Therefore, while the enormous potential of Treg depletion in cancer treatment is evident, the concept of selectively targeting Tregs and avoiding obvious autoimmunity is lacking. This changed with the work of the teams of Plitas/Rudensky and De Simone/Abrignani/Pagani in 2015 and 2016.

Plitas et al. demonstrated that CCR8 is selectively expressed by human breast cancer invasive Treg cells and immediately concluded that targeting CCR8 represents a promising immunotherapy for treating patients with breast cancer and other tumors (Plitas, G., et al. "Abstract P4-04-11: Preferential expression of the chemokine receptor 8 (CCR8) on regulatory T cells (Treg) infiltrating human breast cancers represents a novel immunotherapeutic target." (2016): P4-04.; Plitas, George, et al. "Regulatory T cells exhibit distinct features in human breast cancer." Immunity 45.5 (2016): 1122-1134.; US10087259).

Shortly after the initial work by Plitas et al., De Simone et al. published an analysis of the transcriptional landscape of tumor-infiltrating regulatory T cells, finding that tumor-infiltrating Treg cells have a high immunosuppressive effect and express CCR8 on their cell surface as a specific characteristic molecule (De Simone, Marco, et al. "Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating Tregulatory cells." Immunity 45.5 (2016): 1135-1147., WO2017198631). The authors described the association of CCR8 with poor prognosis and concluded that CCR8 may be an interesting therapeutic target that could inhibit Treg cell transport to the tumor site without interfering with the recruitment of other effector T cells that do not express CCR8.

WO2018112032 and WO2018112033 describe methods for reducing the number or activity of tumor-infiltrating regulatory T cells (TITR) in tumors, including the administration of agents that induce cytotoxicity in cells expressing gene products such as CCR8.

WO2018112033 and EP3431105 describe a molecule that can regulate the expression and/or function of at least one marker (selectively dysregulated in tumor-infiltrating regulatory T cells) for the prevention and/or treatment of the tumor.

WO2018181425 relates to an antibody against CCR8 with ADCC activity for a method of treating cancer, wherein the antibody against CCR8 is a CCR8 neutralizing antibody. WO2018181425 does not disclose a specific antibody sequence, but rather refers to the rat anti-mouse CCR8 clone SA214G2 distributed by BioLegend under catalog number 150302. This antibody is used as a tool antibody to generalize the previously described antitumor activity of mouse Treg depletion.

According to the present invention, a method is provided to promote the production of anti-CCR8 antibodies and generate antibodies with excellent properties for therapeutic use. Comparison of the antibodies according to the present invention with prior art antibodies reveals differences in structure, function, and therapeutic efficacy, as discussed elsewhere herein.

Solution to the problem

1.1 Antibody generation of CC and CXC chemokine receptors

Because existing methods for generating antibodies against the CC chemokine receptor have low success rates and the resulting antibodies typically perform poorly (see, for example, Example 3), the inventors have developed a new method for generating antibodies against the chemokine receptor, and specifically, for generating anti-CCR8 antibodies.

Using specially modified isolated peptides as antigens to select human anti-human CCR8 antibodies solves the problem of providing improved methods for antibody generation against CC and CXC chemokine receptors, for example, allowing for the first-ever complete human anti-CCR8 antibody, see Example 6. More specifically, the antigen is formed by modifying peptides containing human CCR8 tyrosine-rich domains (optionally including LID domains) by introducing sulfate modification at specific sites, see Example 4, Table 4.1. Phage display methods are optionally used in conjunction with this specially modified antigen to select complete human anti-human CCR8 antibodies. Alternatively, sulfated antigens can be used in a variety of other methods, such as in conventional immunization methods.

The obtained antibodies exhibited excellent binding characteristics to sulfated antigens and biological targets expressed under physiological conditions (see Examples 6 and 10.1.1), but showed relatively low or no affinity for unmodified antigens (see Examples 10.1.2 and 10.1.3), indicating that sulfated residues are indeed crucial for antigen-antibody binding. The antibodies of this invention also showed excellent and specific binding to cell lines engineered to express human, cynomolgus monkey, or mouse CCR8, as well as activated human Tregs (see Example 10.1.1).

Of the six CDR rings of the antibody, the H3 ring exhibits the greatest structural diversity and is located at the center of the binding site. It also acquires the most mutations through affinity maturation and has the highest average number of contacts with the antigen. Therefore, it plays a crucial role in antigen binding. Analysis of the specific structure of the antibody obtained according to the method of the present invention surprisingly revealed that the composition of HCDR3 differs structurally from the typical human HCDR3 domain (see Example 9). In particular, the HCDR3 domain of the antibody possessing the most therapeutically beneficial properties and specific binding to CCR8 is characterized by an average frequency of ~21% tyrosine residues and an average content of ~10% histidine residues, highlighting the beneficial effect of these residues on the recognition of specific sulfated antigens (see Table 9.2).

Unbound by theory, the inventors believe that these structural features translate into certain functional features, making the resulting antibodies more suitable for therapeutic applications. For example, compared to tested prior art antibodies, several antibodies according to the invention block G protein-dependent signaling (see Example 10.4.3) without affecting G protein-independent pathways (see Examples 10.4, 10.4.1, 10.4.2) and are substantially not internalized into cells with endogenous expression of target chemokine receptors (see Example 10.5). Furthermore, particularly preferred antibodies according to the invention are specific to target receptors and target cells (see Examples 10.2, 10.11) and are particularly suitable/exhibit superior properties in cancer treatment methods (see Example 12ff). Additionally, the use of these synthetic polypeptides as antigens or for off-target panning to promote or generate cross-reactive antibodies, such as antibodies specifically binding to human CCR8 and/or cynomolgus monkey CCR8 (see Example 10.1.1).

1.2 Provide therapeutic antibodies that specifically bind to chemokine receptors

1.2.1 Obtaining chemokine receptor antibodies with human CDR

While humanizing antibodies with mouse CDRs may enhance their immunogenicity, residual immunogenicity remains in the CDR regions (Harding, Fiona A., et al. "The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions." MAbs. Vol. 2. No. 3. Taylor & Francis, 2010). Therefore, antibodies containing human CDRs are considered more suitable as human therapeutic agents compared to antibodies containing CDRs from other species.

For example, antibodies with human CDRs can be produced using phage display technology or by using transgenic animals capable of producing fully human antibodies. However, obtaining fully human antibodies is not always trivial. While the diversity of the HCDR3 region is theoretically almost limitless, in practice, the generation of antibody library diversity appears to be carefully regulated by a number of mechanisms that limit the diversity and antigen-binding sites of in vivo libraries. The same is true for human phage display libraries, which are typically designed to reflect in vivo libraries. Thus, in some cases, rodent CDRs can meet the structural requirements of “complex” antigens that human CDRs cannot easily recognize. Based on the observation that tyrosine is on average about 50% less active in human HCDR3 than in mouse HCDR3, it can be assumed that finding human anti-human antibodies is clearly more challenging in cases where unique binding capabilities of tyrosine and histidine are required for target binding. In fact, the inventors are unaware of any prior art fully human anti-human CCR8 antibodies. According to the present invention, CCR8 antibodies comprising human CDRs are provided, as well as fully human antibodies, see Examples 6, 7, and 8.

1.2.2 Obtaining cross-reactive chemokine receptor antibodies

Cross-reactive anti-chemokine receptor antibodies, such as cross-reactive anti-CCR8 antibodies, are advantageous for the development of therapeutic antibodies because they can be used in non-human animal models to characterize therapeutics in terms of pharmacological data and safety before administration to humans. However, cross-reactive antibodies, such as those with similar binding behavior in two species, are difficult to generate and do not readily mature for affinity, for example because the extracellular portions of chemokine receptors such as CCR8 have low homology across species (see Example 2). According to the invention, cross-reactive antibodies against CCR8 can be generated, in particular, by using small sulfated tyrosine residues containing motifs that are more conserved across species, such that cross-reactive antibodies binding to chemokine receptors such as CCR8 from two or more species can be obtained, for example, in a simple and convenient manner with the same order of magnitude of affinity (see Examples 6, 7, 10.1.1).

1.2.3 Obtaining chemokine receptor antibodies for therapeutic use

To induce the killing of cells expressing target chemokine receptors or CCR8, several modes of action can be envisioned. One mode of action involves conjugating an antibody targeting the chemokine receptor or CCR8 with a drug to form an antibody-drug conjugate (ADC). Other possible modes of action include inducing antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). For ADCC, CDC, and ADCP, a two-step mechanism is involved: on the one hand, the antibody or fragment needs to effectively bind to the target cell, for example, via the Tregs of CCR8; on the other hand, the FC portion of the antibody (or an alternative binding portion that can be conjugated with the antibody or fragment, as described elsewhere in this document) must bind to the effector cell, thus mediating the killing of the target cell. For ADCP, the binding of effector cells to macrophages typically occurs through the interaction of the antibody's FC portion with FcγRIIa (CD32a) expressed by macrophages. Conversely, ADCC is mediated through the interaction of the antibody or fragment with FcγRIIIa. In humans, FcγRIII exists in two distinct forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). FcγRIIIa is expressed as a transmembrane receptor on mast cells, macrophages, and natural killer cells, while FcγRIIIb is expressed only on neutrophils. These receptors bind to the Fc moiety of IgG antibodies, thereby activating antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

1.2.4 Obtaining low-internalized (including non-internalized) chemokine receptor antibodies

When the inventors analyzed known prior art anti-human CCR8 antibodies, they found that these antibodies readily internalized into cells expressing endogenous targets but did not remain on the cell surface for extended periods. The internalization behavior of antibodies not only affects their clearance and pharmacological behavior but also their suitability for specific modes of action for therapeutic purposes.

While high internalization is desirable for the formation of certain antibody-drug conjugates, it is undesirable for ADCC-induced depletion of tumor cells or Treg cells. More specifically, antibody-drug conjugates are required to deliver the drug into the cells to achieve effective and specific depletion of the target cells. Conversely, the ADCC mode of action requires the antibody and its Fc domain to be exposed externally to the target cell, where immune effector cells can bind the Fc domain to lyse the target cell. Therefore, low or even no internalization increases the duration of action of ADCC/ADCP-induced antibodies.

Upon characterizing the antibodies obtained using the novel method, the inventors were surprised to find that several antibodies according to the invention exhibited particularly low or even nonexistent internalization characteristics in cells with endogenous CCR8 expression levels (see Example 10.5), while prior art antibodies recognizing the same target showed higher internalization rates. For example, prior art antibodies 433H and L263G8 readily internalize into target cells and do not remain on the cell surface for extended periods. Due to their low internalization properties, some antibodies according to the invention are particularly suitable for ADCC, ADCP, CDC, or mixed methods, such as combined ADCC/ADCP methods.

Typically, target selection is a crucial factor strongly influencing antibody internalization into cells. According to Islam SA et al., CCL1 ligand binding induces Ca2+ flux and rapid receptor internalization of CCR8 (Islam, Sabina A., et al. "Identification of human CCR8 as a CCL18 receptor. CCR8 is a CCL18 receptor." The Journal of Experimental Medicine 210.10 (2013): 1889-1898.). The inventors were surprised to find that antibodies themselves could influence internalization properties to such a great extent. However, when they characterized the ability of the antibodies of this invention to modulate the G protein-independent signaling pathway of their target chemokine receptors, they found that all prior art antibodies tested not only blocked G protein-dependent signaling but also modulated G protein-independent signaling, see Example 10.4 ff. G protein-independent signaling has previously been associated with internalization behavior; see Fox, James M., et al. "Structure/function relationships of CCR8 agonists and antagonists: Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist." Journal of Biological Chemistry 281.48 (2006): 36652-36661.

1.2.5 Obtaining chemokine receptor antibodies that regulate target receptor signaling

Antibodies regulate chemokine receptors in several ways. For example, antibodies can...

a) Block G protein-independent signaling.

b) Block G protein-dependent signaling,

c) Block both G protein-dependent and G protein-independent signal transduction.

d) Increase G protein-independent signaling,

e) Increase G protein-dependent signaling,

f) Increase both G protein-dependent and G protein-independent signaling.

Unbound by theory, the inventors hypothesize that the sulfated TRD of the corresponding chemokine receptor may be associated with ligand-induced chemokine receptor signaling. Interestingly, most of the antibodies of this invention effectively block ligand-induced G protein-dependent signaling but do not affect G protein-independent signaling, see Example 10.4ff. Conversely, all prior art antibodies similarly block ligand-induced G protein-dependent signaling but activate G protein-independent signaling. Unbound by theory, these differences may lead to differences in internalization behavior.

Avoiding therapeutic antibody-induced G protein-independent signaling may also be advantageous, as nonspecific signaling can lead to uncontrollable downstream effects, such as increased cell proliferation; see Hutchings, Catherine J., et al. (2017); Webb, David R., et al. "Opportunities for functional selectivity in GPCR antibodies." Biochemical pharmacology 85.2 (2013): 147-152.; Fox, James M., et al. (2006).

1.2.6 Obtaining ADCC/ADCP-induced chemokine receptor antibodies

The antibodies according to the invention are characterized by excellent induction of ADCC and ADCP, as shown, for example, in Examples 10.3.3 and 10.3.4. In some preferred embodiments, the antibody is engineered to obtain a high ADCC rate by eliminating fucose at N297 (fucosylation-free), see Example 10.3.1. Interestingly, many of the anti-CCR8 antibodies of the present invention appear to induce target cell exhaustion through a combined mode of action of both mechanisms (i.e., ADCC and ADCP).

1.3 Treatment Methods

While several research groups have suggested the use of CCR8-binding antibodies in therapeutics based on mechanistic insights, methods for providing antibodies with optimal therapeutic properties have been lacking. By providing an antibody production method according to the invention, chemokine receptor antibodies with excellent therapeutic properties can now be readily obtained. According to the invention, antibodies with various sequence definitions possessing advantageous properties are disclosed that can be used in therapeutic methods, for example, in ADCC-based methods, ADCP-based methods, CDC-based methods, or in mixed ADCC/ADCP-based methods, as part of a conjugate. Example 12ff shows that the alternative antibody produced using the method according to the invention exhibits significant efficacy in terms of Treg depletion, overall response rate, and tumor-to-control ratio.

1.4 Combined Treatment

While significant response rates were achieved with monotherapy using the anti-CCR8 antibody of the present invention, it was surprisingly found that the therapeutic benefit could be further enhanced by combining the antibody with immune checkpoint inhibitors, further targeted therapies, or even nonspecific chemotherapy or radiotherapy. These combination therapies were particularly beneficial in challenging tumor models. In particular, certain combination therapy regimens were found to be effective in which a second therapeutic agent or therapy was administered only after the anti-CCR8 antibody had induced substantial Treg depletion (see Example 12.6ff.). Furthermore, significant therapeutic effects were observed even with monotherapy using the anti-CCR8 antibody (see Example 12.4.2).

1.5 Stratification Scheme and Diagnostic Methods

In evaluating the responsiveness of different syngeneic mouse models to anti-CCR8 antibody therapy, the inventors were able to identify several mechanisms, biomarkers, and combinations of biomarkers that could predict treatment efficacy and overall response. For example, surprisingly, the degree of response to immune checkpoint inhibitors (such as PD-1, PD-L1, or CTLA4 antibodies) was also found to predict response to anti-CCR8 antibody therapy. Since PD-L1 expression is sometimes used as a surrogate biomarker for predicting responsiveness to immune checkpoint inhibitors, the inventors evaluated whether PD-L1 could also be used to predict responsiveness to anti-CCR8 antibody therapy. This hypothesis was ultimately confirmed with relevant data, see Example 12.7.1. In further attempts to identify those subjects most likely to benefit from anti-CCR8 therapy, further biomarker genes, such as immune cell and Treg biomarkers, were evaluated as biomarkers (see Examples 12.1.2, 12.7.2, 12.8).

Attached Figure Description

Figure 1: Comparison of human CC chemokine receptor and CXC chemokine receptor.

Figure 2: Human CCR8, cynomolgus monkey CCR8, and mouse CCR8 sequences retrieved from Uniprot and aligned using Clustal Omega. b. Schematic diagram of the CCR8 receptor structure, which includes 7 transmembrane domains (black boxes), three extracellular loops (ECL), multiple disulfide bonds (SS), an intracellular C-terminal portion, and an extracellular N-terminal domain containing the "LID" domain and a tyrosine-rich domain (TRD).

Figure 3: Evaluation of three existing-technology antibodies by FACS staining in CCR8-positive target cells. None of the existing-technology antibodies showed changes in FACS staining compared to the isotype control. hAP-0068 and MAB1429-100 were tested on 293T cells stably transfected with human CCR8, while MAB8324 was tested on BW5147.3 cells expressing mouse CCR8.

Figure 4: Selection strategies for finding lead compounds: Two main sulfated peptide selection strategies are described. In all strategies, a step of removing relevant or irrelevant biotinylated proteins is included before each round of selection.

Figure 5: Selection strategy for obtaining anti-mouse CCR8 antibodies: The main selection strategy based on a mixture of sulfated/non-sulfated peptides is described. A detinction step of the relevant biotinylated protein is performed before each round of selection.

Figure 6: FACS data of the antibody of the present invention in combination with CHO cells expressing human CCR8.

Figure 7: FACS data of the antibody of the present invention in combination with CHO cells expressing cynomolgus monkey CCR8.

Figure 8: FACS data of candidates binding with activated human Tregs (donor 1). Right: Percentage of CCR8 expression determined using BioLegend clone L263G8.

Figure 9: FACS data of the binding of the antibodies TPP-21181 and TPP-23411 of the present invention to CHO cells expressing human CCR8.

Figure 10: FACS data of the binding of the antibodies TPP-21181 and TPP-23411 of the present invention to CHO cells expressing cynomolgus monkey CCR8.

Figure 11: Binding of TPP-21360, L263G8 and 433H to CHO cells expressing human CCR8.

Figure 12: Binding of TPP-21360, L263G8, and 433H to CHO cells expressing cynomolgus monkey CCR8. The prior art antibodies showed only very low overall binding (low saturation) to cynomolgus monkey CCR8. However, the EC50 value for each antibody could be determined.

Figure 13: Staining of human Tregs activated with the antibody TPP-23411 of this invention, using a gating strategy. CD4+ and CD25+ sorted cells from peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 beads (Treg amplification kit, Miltenyi). Middle cell: CD4+CD25+CD127low and Foxp3+ cells. Bottom cell: CCR8 expression on Tregs after staining with TPP-23411 or L263G8.

Figure 14: Binding of TPP-21360 to CHO cells with mimic transfectants. No nonspecific binding was observed.

Figure 15: Off-target binding in HEK cells transiently transfected with human CCR1. Most candidates of this invention show only low off-target binding to CCR1.

Figure 16: Off-target binding in HEK cells transiently transfected with human CCR4. Most candidates of this invention exhibit only low off-target binding to CCR4.

Figure 17: Different immune cell populations from healthy donor PBMCs (CD4+ T cells, CD8+ T cells, B cells marked by CD19+ expression, bone marrow cells marked by CD11b+ expression, CD4 T cells activated with anti-CD3 and anti-CD28 beads for 4 days, and CD8+ T cells activated with anti-CD3 and anti-CD28 beads for 4 days).

Figure 18: CCR8 is upregulated in human Tregs after activation. Following activation, the percentage of CCR8+ cells in CD4+CD25+ purified cells significantly increased with increasing MFI, while the percentage of CCR8+ cells in the PBMC population increased slightly. Anti-CCR8 antibodies from BioLegend were used in these experiments. N = 4 donors.

Figure 19: ADCC induced by wild-type or unfucosylated antibodies of the present invention, TPP-19546, TPP-21360, or TPP-23411, in HEK cells expressing human CCR8 as target cells. Isotype control: TPP-9808.

Figure 20: Left panel: Percentage of CCR8+ expression in activated human Tregs as target cells on day 3. Right panel: ADCC induced by the present invention's antibodies TPP-21360 and TPP-23411 (wild-type or unfucosylated) in activated human Tregs as target cells. Isotype control: TPP-9808. Results were reproducible in different donors (data not shown).

Figure 21: Protocol for ADCP determination.

Figure 22: ADCP assays of the wild-type and non-fucosylated forms of the antibodies of this invention show phagocytosis induced in HEK cells expressing human CCR8 as target cells and in in vitro differentiated M2c macrophages as effector cells. The wild-type and non-fucosylated forms of the antibodies of this invention, TPP-19546, TPP-21360, and TPP-23411, induced ADCP. TPP-9808 was an isotype control. Mogamulizumab is a marketed anti-CCR4 antibody.

Figure 23: ADCP assays of the wild-type and non-fucosylated forms of the antibodies TPP-21360 and TPP-23411 of the present invention show phagocytosis induced in activated human Tregs from two different donors as target cells (top or bottom) and in M2c macrophages as effector cells.

Figure 24: ADCP assays of wild-type and non-fucosylated forms of the antibodies TPP-21360 and TPP-23411 of the present invention show phagocytosis induced in activated human Treg cells as target cells and M1 macrophages as effector cells.

Figure 25: Activation of β-arrestin signaling, measured using DiscoverX assays against CCL1, TPP-23411, L263G8, or 433H, in CHO cells co-expressing human CCR8 and EA labeled with ProLink. As expected, the CCR8 ligand CCL1 (human) induces β-arrestin signaling. Additional time points were analyzed against TPP-23411 to ensure that β-arrestin activation did not occur at earlier time points. None of the analyzed antibodies induced β-arrestin signaling activation.

Figure 26: Evaluation of the blocking effect of TP-23411, L263G8, or 433H on CCL1-induced β-arrestin signaling using a DiscoverX assay. CCL1 was used at its EC80 (20 ng/ml). Prior art antibodies L263G8 and 433H both blocked CCL1-induced β-arrestin signaling at low antibody concentrations, while the present invention antibody TPP-23411 showed no effect at these concentrations. β-arrestin signaling has been suggested to play a role in internalization.

Figure 27: Phospho Erk1/2 ELISA assay. CHO cells expressing human CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at their respective time points.

Figure 28: Phospho Erk1/2 ELISA assay. Activated human Treg cells expressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at their respective time points. Prior art antibodies Biolegend L263G8 and BD antibody 433H both induce a significant increase in phosphorylated Erk1/2 levels, for example, after 15 minutes in activated human Tregs, which is not the case with the antibodies of the present invention.

Figure 29: Phosphorylated AKT ELISA assay. CHO cells expressing human CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at their respective time points.

Figure 30: Phosphorylated AKT ELISA assay. Activated human Treg cells expressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, and cell lysates were collected at their respective time points. Existing antibodies Biolegend L263G8 and BD 433H both induced a significant increase in phosphorylated AKT levels in activated human Tregs, for example, after 15 minutes, while this was not the case with the invented antibody.

Figure 31: Internalization studies. a. FACS analysis of commercial anti-human CCR8 antibody Biolegend L263G8 or BD antibody 433H with the endogenous CCR8-expressing cell line HuT78. b. Internalization studies of commercial anti-human CCR8 antibody and the endogenous CCR8-expressing cell line HuT78 based on cytoplasmic strength points. TPP-5657: Isotype control.

Figure 32: Internalization studies. a. FACS analysis of commercial anti-mouse CCR8 antibody SA214G2 and mouse endogenous CCR8-expressing cell line BW5147.3. b. Internalization study of commercial anti-mouse CCR8 antibody SA214G2 and mouse endogenous CCR8-expressing cell line BW5147.3. The existing anti-mouse CCR8 antibody SA214G2 induced internalization.

Figure 33: Internalization studies of the anti-human CCR8 antibodies TPP-21360 and TPP-23411 of the present invention with the endogenously CCR8-expressing cell lines TALL-1(a) and HuT78(b). Both antibodies showed the same internalization behavior, comparable to the isotype control TPP-5657.

Figure 34: FACS analysis of the anti-human CCR8 antibodies TPP-21360 and TPP-23411 of the present invention with endogenous CCR8-expressing cell lines TALL-1(a) and HuT78(b). Both antibodies showed the same binding potency to the cell lines.

Figure 35: CCR8 mRNA expression in 11,642 samples representing a comprehensive set of human tissues and cell types, as measured by the affymetrix probe 208059at. All samples were common normalized using the refRMA method (Katz, Simon, et al. "A summary approach for Affymetrix GeneChip data using a reference training set from a large, biologically diverse database." BMCbioinformatics 7.1 (2006): 1–11). Dark gray boxes indicate median samples in the corresponding groups with probe signal intensities significantly higher than background noise (estimated by the affymetrix MAS5 algorithm, see Pepper, Stuart D., et al. "The utility of MAS5 expression summary and detection call algorithms." BMCbioinformatics 8.1 (2007): 1–12). Median CCR8 expression significantly higher than background noise was observed only in activated regulatory T cells and tumor-infiltrating lymphocytes. Groups were ranked based on decreasing mean expression.

Figure 36: CCR8 mRNA expression measured by RNA-seq in 50 different TCGA (https://www.cancer.gov/tcga) tumor indications (dark gray boxes), corresponding normal tissues (white boxes), and the entire CCLE tumor cell line map, also grouped by tumor indication (light gray boxes) (Barretina, Jordi, et al. "The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity." Nature 483.7391(2012):603-607). Groups were ranked according to median expression. The indications with the highest CCR8 expression were breast cancer, lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), head and neck malignancies, and esophageal tumors. In all indications except pancreatic adenocarcinoma and melanoma, expression in tumors was higher than in the corresponding normal tissues. The observation of little or no expression in corresponding epithelial-derived tumor cell lines indicates that CCR8 is not expressed by tumor cells, but only by tumor-infiltrating T cells. In principle, every tumor indication with CCR8+ Treg cell infiltration should meet the criteria for anti-CCR8 antibody therapy.

Figure 37: Immunohistochemical staining of Treg markers FOXP3 and CCR8 (clone 433H) in human non-small cell lung cancer (NSCLC) or human melanoma tissues.

Figure 38: Staining of T cell populations sorted by FACS based on CCR4, CCR8, OX40, GITR, and CD25. Only CCR8 is specific for activated Tregs. OX40, GITR, and CD25 are significantly expressed in stimulated CD8+ Teff cells and CD4+ T cells (CD4+CD25+Foxp3-). Dark gray: isotype control; light gray: target staining.

Figure 39: CCR8 expression in different T cell subsets extracted from different tumor entities, measured by single-cell RNA-seq. For these tumors, CCR8 mRNA expression was largely confined to regulatory T cells in tumor tissue (light gray box), and virtually absent in regulatory T cells in normal tissue and CD4 helper T and CD8 cytotoxic T cells (medium gray and dark gray boxes, respectively). Top panel: Colorectal tumor tissue (tumor) or adjacent normal tissue (normal). Middle panel: Liver cancer tissue (tumor) or adjacent normal tissue (normal). Bottom panel: Lung cancer tissue (tumor) or adjacent normal lung tissue and peripheral blood (normal). Sample size N represents the number of cells in each category. Cells were designated as regulatory T cells, helper T cells, or cytotoxic T cells based on the expression of the marker genes FOXP3, CD4 (but without FOXP3), and CD8A/B, respectively.

Figure 40: Tregs, macrophages, T cells, or tumor cell populations from NSCLC, CRC (colorectal cancer), or RCC (renal cancer) stained with anti-CCR8 antibody or allotype controls and analyzed by flow cytometry. X-axis: logarithmic shift up to ^10⁵ ; Y-axis: normalized to pattern intensity. Light gray: allotype control. Dark gray: CCR8. Specific CCR8 expression on human Tregs within the tumor is indicated by arrows.

Figure 41: CT26 tumor growth after treatment with different doses of anti-CCR8 antibody or anti-PDL1 antibody.

Figure 42: Spider web diagram of mice carrying CT26 tumors after treatment with different doses of anti-CCR8 antibody, anti-PDL1 antibody or isotype control.

Figure 43: FACS analysis of intratumoral immune cells 24 hours after the second antibody treatment. Analysis of intratumoral Tregs. For 10 mg/kg, 1 mg/kg, or 0.1 mg/kg, anti-CCR8 antibody TPP15285 resulted in a mean reduction of intratumoral Tregs to 13%, 33%, or 47% of the Tregs in the isotype control, respectively. For 10 mg/kg, 1 mg/kg, or 0.1 mg/kg, anti-CCR8 antibody TPP15286 resulted in a mean reduction of intratumoral Tregs to 10%, 9%, or 14% of the Tregs in the isotype control, respectively. See Table 12.2.1 for the analysis of intratumoral CD8+ T cells. Table 12.2.1 shows the mean percentage increase in the ratio of CD8+ T cells to Treg cells in the isotype control. Anti-CCR8 antibody TPP15286 resulted in a mean increase in the ratio of CD8+ cells to Treg cells of 44 (10 mg/kg), 87 (1 mg/kg), or 68 (0.1 mg/kg). The anti-CCR8 antibody TPP15285 resulted in an average increase in the ratio of CD8+ cells to Treg cells of 64 (10 mg/kg), 16 (1 mg/kg), or 10 (0.1 mg/kg). The ratio of CD4+ conv T cells to Treg cells was analyzed.

Figure 44: Growth of CT26 tumors treated with glycosylated (TPP-14095, TPP-14099, TPP-15285, TPP-15286) or non-glycosylated (TPP-18208, TPP-18209) anti-CCR8 antibodies. Glycosylation largely eliminates the anti-tumor effect.

Figure 45: Spider web diagram of CT26 tumor-bearing mice treated with glycosylated (TPP-14095, TPP-14099, TPP-15285, TPP-15286) or non-glycosylated (TPP-18208, TPP-18209) anti-CCR8 antibodies. Glycosylation largely eliminates the antitumor effect, indicating an ADCC/ADCP-dependent mechanism of antitumor efficacy.

Figure 46: FACS analysis of immune cells 24 hours after the second antibody treatment. Absolute number of CD45+ cells in the tumor. Absolute number of CD45+CD8+ T cells in the tumor. Absolute number of CD8+ T cells in the tumor. Absolute number of CD4+conv cells in the tumor. Analysis of Treg depletion in the tumor by flow cytometry, showing the absolute cell count. The mean percentage of residual Tregs relative to the corresponding isotype control after antibody administration was 15.6% for TPP-14095, 28.2% for TPP-14099, 8.7% for TPP-15285, and 36.5% for TPP-15286. No reduction in Tregs was observed for non-glycosylated antibodies. Percentage of CD8+ T cells among CD45+ cells in the tumor. CD8+ T cell:Treg ratio. CD4+conv:Treg ratio. Percentage of Treg cells among CD4+ T cells. Absolute number of 4-1BB+ Treg cells in the tumor. The absolute number of 4-1BB+CD4+conv cells within the tumor.

Figure 47: EMT6 tumor growth after treatment with different doses of anti-CCR8 antibody TPP-15285. Significance was determined by 1-way ANOVA plus a log-transformed Sidak post-test.

Figure 48: EMT6 tumor growth on day 19 after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody.

Figure 49: Spider web diagram of EMT6 tumor-bearing mice treated with different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody.

Figure 50: FACS analysis of immune cells from EMT6 tumor-bearing mice after treatment with different doses of CCR8 antibody TPP-15285 or anti-CTLA4 antibody, 24 hours after the second treatment, or at the end of the study. Absolute number of Treg cells within the tumor. Significant differences were observed, for example, when using 10 mg/kg anti-CCR8 antibody at both time points. Ratio of CD8-positive cells to Tregs. Ratio of CD4+ conv cells to Tregs. Percentage of Treg cells among CD4+ T cells.

Figure 51: FACS analysis of immune cells from EMT6 tumor-bearing mice after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody, 24 hours after the second treatment, or at the end of the study. Absolute number of CD45+ cells within the tumor. Absolute number of CD4+ conv cells within the tumor. Absolute number of CD4+ T cells within the tumor. Absolute number of activated CD8+ T cells within the tumor. Absolute number of NK cells within the tumor. Absolute number of CD8+ T cells within the tumor. Differences were observed, for example, when 10 mg/kg of anti-CCR8 antibody was used at the end of the study.

Figure 52: F9 tumor growth after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-PDL1 antibody.

Figure 53: F9 tumor volume on day 16 after starting treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-PDL1 antibody. Significant improvement was observed at least for antibody doses of 10 mg/kg.

Figure 54: Spider diagram of F9 tumor-bearing mice after treatment with different doses of anti-CCR8 antibody or anti-PDL1 antibody.

Figure 55: Effects of anti-CCR8 or anti-PDL1 antibodies on immune cell populations and their ratios in F9 tumors, analyzed by FACS 24 hours after the second antibody treatment. Compared with matched isotype controls, anti-CCR8 antibody increased the absolute numbers of intratumoral CD45+ T cells, intratumoral CD4+ T cells, intratumoral CD8+ T cells, intratumoral CD4+ convT cells, and intratumoral NK cells. 10 mg/kg of anti-CCR8 antibody decreased the absolute number of intratumoral Treg cells (CD4+, CD25+, FoxP3+).

Figure 56: Effects of anti-CCR8 or anti-PDL1 antibodies on immune cell populations and their ratios in F9 tumors, analyzed by FACS 24 hours after the second treatment. The plots are shown as the ratio of CD8+ T cells to Tregs, the frequency of Tregs to CD4+ T cells, the frequency of 4-1BB+ cells in CD8+ T cells, the frequency of 4-1BB+ cells in Tregs, and the frequency of 4-1BB+ cells in conventional CD4+ T cells. At 10 mg/kg, anti-CCR8 antibody increased the ratio of CD8+ cells to Tregs to approximately 54 or higher (see Table 12.5.1).

Figure 57: Efficacy of combination therapy in C38 tumor-bearing mice. a. C38 tumor growth after treatment with anti-CCR8 replacement antibody TPP-14099 or TPP-15285 or anti-PD-L1 antibody, or in combination with 3 mg/kg anti-PDL1 antibody and 10 mg/kg TPP-15285. Significance was determined by 1-Way ANOVA plus a log-transformed Sidak post-test. b. Survival of C38 tumor-bearing mice after treatment with anti-mouse CCR8 antibody TPP-15285 (10 mg/kg), anti-mouse PD-L1 antibody (PDL1, 3 mg/kg), or a combination of TPP-1528 (10 mg/kg) and anti-mouse PD-1 antibody (3 mg/kg).

Figure 58: Analysis of tumor Treg exhaustion in C38 tumors by flow cytometry (sampled 24 hours after secondary antibody treatment). C38 tumor-bearing mice received single-agent treatment with anti-CCR8 alternative antibody TPP-14099 or TPP-15285 or anti-PD-L1 antibody, or combination therapy with TPP-15285 and anti-PD-L1 antibody. Absolute Treg exhaustion. CD8+ T cell/Treg cell ratio.

Figure 59: CD11+F4/80+ macrophages in C38 tumors analyzed by flow cytometry (sampled at the end of the study).

Figure 60: B16F10-OVA tumor growth after treatment with TPP-15285, anti-CTLA4 antibody, or both.

Figure 61: EMT-6 tumor growth after treatment with anti-mouse CCR8 antibody TPP-15285 (1 mg/kg), anti-mouse PD-1 antibody (CDRs: atezolizumab, 10 mg/kg), or a combination of TPP-15285 (1 mg/kg) and anti-mouse PD-1 antibody (10 mg/kg) (twice weekly i.p.). Mean plus standard deviation.

Figure 62: FACS analysis of immune cell populations in EMT-6 tumors by flow cytometry, sampled 24 hours after the second antibody treatment. Ratios of CD4+ T cells, Tregs, CD8+ T cells, NK cells, and CD8+ T cells to Tregs. Boxes and whiskers, minimum to maximum and median values (GraphPad).

Figure 63: Analysis of C38 tumor-bearing mice treated with anti-mouse CCR8 antibody TPP-15285 (5 mg/kg), anti-mouse PD-1 antibody (CDR: atezolizumab, 5 mg/kg), or a combination of TPP-15285 (5 mg/kg) and anti-mouse PD-1 antibody (5 mg/kg), (twice weekly i.p.). a. Tumor volume. Mean plus standard deviation. b. Survival plot.

Figure 64: Analysis of the ratio of tumor CD4+ T cells, Tregs, CD8+ T cells, NK cells, or CD8+ T cells to Tregs in C38 tumors by flow cytometry (sampled 24 hours after the second antibody treatment).

Figure 65: MB49 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs: atezolizumab, 10 mg/kg) alone, or TPP-1528 (10 mg/kg) and anti-PD-1 monoclonal antibody (10 mg/kg) in sequence (all twice weekly i.p.).

Figure 66: Analysis of CD45+ cells, Tregs, CD8+ T cells, NK cells, and the ratio of CD8 to Treg in MB49 tumors by flow cytometry (samples taken at the end of the study).

Figure 67: EMT-6 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (5 mg/kg, q3/4d i.p.), oxaliplatin (5 mg/kg, q4d i.p.), doxorubicin (6 mg/kg, i.v. SD), docetaxel (10 mg/kg, q2dx5, i.v.), or TPP-15285 in combination with oxaliplatin, doxorubicin, or docetaxel.

Figure 68: MB49 tumor growth after treatment with anti-CCR8 antibody TPP-15285, gemcitabine, or a combination of TPP-15285 and gemcitabine.

Figure 69: Lewis lung tumor growth after treatment with anti-CCR8 antibody TPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs: atezolizumab, 10 mg/kg) alone, anti-PD-L1 antibody (10 mg/kg) alone, anti-CTLA4 antibody alone, combination of TPP-15285 (10 mg/kg) and anti-mouse PD-1 antibody (10 mg/kg) alone, or combination of TPP-15285 (10 mg/kg) and anti-PD-L1 antibody alone.

Figure 70: EMT-6 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (3 mg/kg) or radiotherapy (RT, 3 x 2 Gy) alone or in combination. Significance was determined by 1-way ANOVA plus a log-transformed post-test of Sidak.

Figure 71: mRNA expression levels of different immune cell markers in different syngeneic tumor models treated with allotype control (TPP-9809) or anti-CCR8 antibody (TPP-14099). Median expression in the allotype-treated control group was set to zero. Gene expression was estimated as part of transcript (TPM) using RSEM algorithm. Treg marker Foxp3. Significantly higher Foxp3 levels were observed in the CT26, H22, Hepa1-6, and RM1 models treated with TPP-14099, indicating increased Treg infiltration after at least three doses of TPP-1409.

Figure 72: Inflammatory marker lfng. Significantly higher lfng levels were observed in the H22, CT26, and RM1 models treated with TPP-14099, indicating that TPP-1409 induces strong pro-inflammatory activity.

Figure 73: Macrophage marker Ms4a7. Significantly higher Ms4a8 levels were observed in the CT26, H22, and Hepa1-6 models treated with TPP-14099, indicating that TPP-1409 leads to increased macrophage infiltration.

Figure 74: Cytotoxic T cell markers Cd8a and Cd8b1. Significantly higher levels of cytotoxic T lymphocytes were observed in the CT26, H22, RM1, and Hepa1-6 models treated with TPP-14099, indicating that TPP-1409 induces cytotoxic T cell infiltration and/or proliferation.

Figure 75: Natural killer (NK) cell marker Ncrl. Significantly higher NK cell levels were observed in the CT26, H22, and Hepa1-6 models treated with TPP-14099.

Figure 76: Pan T cell marker Cd3e/d/g. Significantly higher T cell levels were observed in the CT26, H22, and Hepa1-6 models treated with TPP-14099.

Figure 77: Activated Treg markers and antibody target Ccr8. Significantly higher Ccr8 levels were observed in the CT26, H22, and Hepa1-6 models treated with TPP-14099.

Figure 78: Acod1 (a), a highly specific pro-inflammatory M1 macrophage marker, and Mrc1 (b), a highly specific anti-inflammatory M2 macrophage marker. Significantly higher M1 macrophage levels were observed in the CT26, 4T1, H22, Hepa1-6, and RM1 models treated with TPP-14099, while no significantly higher M2 macrophage levels were observed in any of the models treated with TPP-14099.

Figure 79: Ratio of highly specific pro-inflammatory M1 macrophage marker Acod1 to highly specific anti-inflammatory M2 macrophage marker Mrc1. In summary, multiple doses of the anti-CCR8 antibody TPP-14099 increased the M1/M2 macrophage ratio in these tumor models.

Figure 80: B-cell markers Cd19 and Cd22. Significantly higher B-cell levels were observed in the CT26, H22, RM1, and Hepa1-6 models treated with TPP-14099. The anti-CCR8 antibody appears to recruit B cells to the tumor. Unbound by theory, B-cell recruitment may influence the anti-tumor response induced by TPP-14099.

Figure 81: Expression levels of LTta/b and Cxcr5 and its ligand Cxcl13. These markers are crucial for the development of lymph nodes and tertiary lymphoid structures (Cyster, Jason G. "Blown away: the unexpected role of lymphotoxin in lymphoid organ development." The Journal of Immunology 192.5 (2014): 2007-2009, and Cupedo, Tom, et al. "Induction of secondary and tertiary lymphoid tissue"). Tertiary lymphoid structures in the skin (Immunity 21.5 (2004): 655-667) are key drivers of antitumor responses in humans (see Dieu-Nosjean, Marie-Caroline, et al. "Tertiary lymphoid structures, drivers of the antitumor responses in human cancers." Immunological reviews 271.1 (2016): 260-275). The full contents of all three published articles are incorporated herein by reference.

Figure 82: Spider plot of tumor growth (in mm3) over time for the TPP-14099 treatment group (shown as triangles) and the isotype control group (shown as circles) in different tumor models. T cell infiltration levels at the end of the study, determined by Cd8a mRNA levels in a large number of tumor samples, are represented by gray shading (black and light gray correspond to the highest and lowest Cd8a levels, respectively). In H22 and CT26, tumor size showed a very strong inverse correlation with Cd8a levels.

Figure 83: Correlation between tumor invasion and tumor size of different immune cell populations as determined by mRNA levels of different immune cell markers. Tumors treated with TPP-14099 are shown as triangles, and tumors treated with the same type are shown as circles. Tumor size is measured in mm3. Cytotoxic T cell infiltration (determined by Cd8a+Cd8b1 mRNA levels) was strongly negatively correlated with CT26 tumor size. Compared with the control group, TPP-14099-treated tumors were smaller and showed higher levels of Cd8a+Cd8b1 T cell infiltration, indicating that the increased T cell infiltration after TPP-14099 treatment led to reduced tumor growth. In TPP-14099-treated tumors, there was a strong negative correlation between T cell infiltration (measured by Cd3 mRNA levels) and CT26 tumor size. TPP-14099-treated tumors were smaller and had higher levels of T cell infiltration, i.e., higher Cd3 mRNA levels were associated with smaller tumors. NK cell infiltration (assessed by the expression of the NK marker Ncr1) was negatively correlated with CT26 tumor size. TPP-14099-treated tumors were smaller and showed higher NK cell infiltration. Tumor size was strongly negatively correlated with NK infiltration; higher NK levels were associated with smaller tumors. In TPP-14099-treated tumors, the correlation between the hypothetical induced tertiary lymphoid structures and CT26 tumor size was assessed based on Lta/Ltb/Cxcr5 and Cxcl13 mRNA levels. For each of the four markers, TPP-14099 treatment significantly increased their levels, and tumor size was strongly negatively correlated with expression levels, indicating that increased tertiary lymphoid structure formation after TPP-14099 treatment led to reduced tumor growth.

Figure 84: Correlation between Cd8a infiltration level (as determined by Cd8a mRNA expression) and tumor size in different tumor models from TPP-14099-treated tumors (shown as triangles) and isotype-treated control groups (shown as circles). TPP-1409-treated tumors were smaller and had higher Cd8a infiltration levels. Tumor size was strongly negatively correlated with Cd8a infiltration; higher Cd8a levels were associated with smaller tumors.

Figure 85: Efficacy of CCR8 antibody TPP-15285 in EMT6 tumor mice treated with four different regimens: single treatment, two BIW treatments, three BIW treatments, or four BIW treatments. Each group was treated with either a load-controlled regimen or TPP-15285 at doses of 0.1 mg/kg, 1 mg/kg, or 5 mg/kg.

Figure 86: As shown in Table 12.6.9.1, the therapeutic effects of the present invention's anti-CCR8 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, or paclitaxel, tested alone or in combination, in MBT2 syngeneic tumor-bearing mice.

Figure 87: The therapeutic efficacy of the anti-CCR8 antibody of the present invention after quantitative depletion of CD8a+ expression in diphtheria toxin-treated DTR mice. Depletion of Cd8+ T cells eliminates the anti-tumor growth effect of TPP15825 (see group 02b v. 01b).

Figure 88: The therapeutic efficacy of the anti-CCR8 antibody of the present invention after quantitative depletion of CD19+ expression in diphtheria toxin-treated DTR mice. Depletion of Cd19+ B cells enhances the anti-tumor growth effect of TPP15825 (see group 02b v. 01b).

Figure 89: Internalization curves of various anti-CCR8 antibodies of the present invention in HUVEC cells. Anti-CD71 antibodies with known internalization characteristics were used as positive controls. Corresponding data are shown in Table 10.5.2. The antibodies obtained using the method according to the present invention did not show significant internalization, making them particularly useful for ADCC/ADCP-based methods.

Brief description of SEQ ID

The full contents of the sequence list provided with the application via electronic archiving are included herein. SEQ ID NO: 1 to SEQ ID NO: 108 and SEQ ID NO: 157 to SEQ ID NO: 168 relate to isolated polypeptides that can be used as antigens or for off-target screening. SEQ ID NO: 109 to SEQ ID NO: 156 relate to chemokine receptor proteins from different species. SEQ ID NO: 201 to SEQ ID NO: 965 relate to antibodies of the present invention. The sulfation column is provided for convenience only and is not intended to limit the respective sequences in any way.

definition

Unless otherwise defined, all scientific and technical terms used in the specification, drawings, and claims have their ordinary meanings as commonly understood by one of ordinary skill in the art. The entire contents of all publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference. In case of conflict, this specification (including definitions) shall prevail. If two or more documents incorporated by reference contain conflicting and/or inconsistent disclosures, the document with the later effective date shall prevail. If databases are referenced, the valid data should be the version number applicable to May 26, 2021, unless otherwise stated. Materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise stated, the following terms used in this document, including those in the specification and claims, have the following definitions.

The expressions “about” or “~” used herein refer to a value within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, according to practice in the art, “about” can mean within one or more standard deviations. The term “about” is also used to indicate that the quantity or value in question can be a specified value or other values that are approximately the same. The phrase is intended to convey that similar values promote equivalent results or effects as described herein. In this case, “about” can refer to a range higher and/or lower than by up to 10%. Whenever the term “about” is specified for a particular measurement or implementation method, this definition applies to the specific context.

The terms “comprising,” “including,” “containing,” “having,” etc., should be read in an expanded or open-ended manner. When used in a specification, the term “comprising” includes “consisting of.”

Singular forms such as "a/an" or "the" include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "monoclonal antibody" includes a single monoclonal antibody as well as multiple identical or different monoclonal antibodies. Similarly, "cell" includes a single cell as well as multiple cell types.

Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. The terms "at least one" and "at least one of" include, for example, one, two, three, four, five, or more elements.

It should also be understood that slight variations above and below the specified range can be used to achieve substantially the same results as values within that range. Furthermore, unless otherwise indicated, the range is intended to be a continuous range including every value between the minimum and maximum values.

The term "amino acid" or "amino acid residue" as used in this article generally refers to naturally occurring amino acids. One-letter codes are used to denote the corresponding amino acids. As used herein, "charged amino acids" are amino acids that are either negatively or positively charged. "Negatively charged amino acids" are aspartic acid (D) and glutamic acid (E). "Positively charged amino acids" are arginine (R), lysine (K), and histidine (H). "Polar amino acids" are all amino acids that form hydrogen bonds as donors or acceptors. These are charged amino acids and asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine (C). "Polarly uncharged amino acids" are asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine (C). "Dibasic amino acids" are tryptophan (W), tyrosine (Y), and methionine (M). "Aromatic amino acids" are phenylalanine (F), tyrosine (Y), and tryptophan (W). The "hydrophobic amino acids" are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and cysteine. The "small amino acids" are glycine (G), alanine (A), serine (S), proline (P), threonine (T), aspartic acid (D), and asparagine (N).

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and long chains, which are commonly referred to in the art as proteins and come in many types. “Polypeptide” includes, for example, bioactive fragments, basic homologous peptides, oligopeptides, homodimers, heterodimers, peptide variants, modified peptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

When a gene or protein of a species (such as a mouse) is generally referenced, it should also refer to a human analogue unless otherwise stated or clearly incompatible. This is especially true for biomarkers.

When applied to nucleic acids, peptides, proteins, or antibodies, the term "isolated" means that the nucleic acid, peptide, protein, or antibody is substantially free of other cellular components associated with it in their natural state. It is preferably in a homogeneous state. It can be a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The protein, peptide, or antibody, as the main substance present in the formulation, is substantially purified. In particular, isolated genes are separated from open reading frames flanking the gene and encoding proteins other than the gene of interest. However, isolated peptides can be immobilized on beads or particles, for example, using suitable adapters.

The term "purification" means that nucleic acids or proteins essentially produce a single band in an electrophoresis gel. Specifically, this means that the nucleic acids or proteins are at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

As used herein, the term "synthesis," such as synthetic nucleic acid molecules, synthetic genes, or synthetic peptides, refers to nucleic acid or polypeptide molecules produced through recombinant and/or chemical synthesis methods. As used herein, recombinant production using recombinant DNA methods means expressing proteins encoded by cloned DNA using well-known molecular biology methods.

Post-translational modifications (PTMs) refer to covalent modifications of peptides or proteins that are introduced after protein biosynthesis under natural conditions. This term includes, but is not limited to, glycosylation, phosphorylation, acylation, adenineization, farnesylation, ubiquitination, and sulfation. Post-translational modifications can affect the activity of peptides or proteins. In 2004, Gutiérrez et al. described the sulfation and glycosylation status of the mouse CCR8 chemokine receptor and how these post-translational modifications affect CCR8 activity. They suggested that tyrosine residues at positions 14 and 15 of mouse CCR8 are sulfated amino acid residues, while asparagine 8 and threonine residues at positions 10 and 12 are glycosylated. Furthermore, they showed that sulfation is important for the activity of CCR8 (Gutiarrez, Julio, et al. "Analysis of post-translational CCR8 modifications and their influence on receptor activity." Journal of Biological Chemistry 279.15 (2004): 14726-14733).

"Sulfation" is a post-translational modification in which a sulfate group is added to a tyrosine residue of an amino acid, such as a polypeptide or protein. Tyrosine sulfation occurs in all multicellular organisms. Under physiological conditions, it is catalyzed by tyrosine protein sulfur transferases (TPSTs) 1 and 2, which are Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphate sulfate) to the protein substrate. Synthetic sulfation of tyrosine can be performed using techniques known in the art, such as those described in Bunschoten, Anton, et al. "A general sequence-dependent solid phase method for the site-specific synthesis of multiple sulfated-tyrosine containing peptides." Chemical Communications 21 (2009): 2999-3001. A sulfated polypeptide is a polypeptide containing at least one sulfate group. A non-sulfated polypeptide is a polypeptide that does not contain a sulfate group.

The tyrosine-rich domain (TRD) is a conserved domain that characterizes seven-transmembrane proteins, such as CXC and CC chemokine receptors. The TRD is typically located at the N-terminus of the chemokine receptor and is usually linked to the LID domain via a cysteine residue (see Figure 2b). Therefore, as used herein, the term TRD refers to the amino acid or protein sequence of the CXC or CC chemokine located at the N-terminus of the first cysteine residue counted from the N-terminus. The TRD may or may not include a signal peptide. The TRD may be modified or left unmodified. In addition to tyrosine residues, the TRD typically contains negatively charged amino acid residues, such as aspartic acid. The TRD is considered an important structure for the interaction of chemokine receptors with their endogenous ligands. Under physiological conditions, the tyrosine residues in the TRD can be sulfated, unsulfated, or partially sulfated. Specific sequences of the corresponding chemokine receptor TRDs are provided in Table 4.1. However, it is clear that mutations can be introduced into the TRD sequence without altering the overall charge and interaction patterns. Preferably, according to Table 4.1, the TRD has at least 90%, 95%, or 99% sequence identity or sequence similarity with at least one TRD sequence.

As used herein, the term "N-terminus/N-terminus" for chemokine receptors refers to the N-terminal amino acid of a chemokine receptor that contains at least the TRD domain. When the peptide or protein contains a signal peptide, the N-terminus may also refer to the N-terminal sequence following the native cleavage site of the peptide or protein. According to some preferred embodiments, the N-terminus contains both the LID and TRD domains of the chemokine receptor, but excluding the native cysteine residue between these two domains. Instead, the cysteine residue may be removed or substituted with a different amino acid.

The "LID" domain of the chemokine receptor used in this article refers to the amino acid sequence located at the C-terminus of the chemokine receptor TRD. The TRD and LID domains are usually separated by a single cysteine residue.

"Sequence identity" or "percentage identity" is a number describing the similarity between a query sequence and a target sequence; more precisely, it's how many characters in each sequence are identical after alignment. The most popular tool for calculating sequence identity is BLAST (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/), which performs comparisons between sequence pairs, searching for locally similar regions. Suitable alignment methods are known in the art, such as the Needleman-Wunsch algorithm for global alignment, which uses a BLOSUM62 matrix with a gap opening penalty of 11 and a gap expansion penalty of 1. It counts identical residue pairs in the alignment and then divides by the total length of the alignment (including internal and external gaps) to obtain the identity percentage value.

For “percentage similarity” or “sequence similarity” values, the same method as for percentage identity values can be used, except that the aligned residue pairs are calculated with non-negative BLOSUM62 values, instead of identical residue pairs (i.e., ≥0).

The "seven-transmembrane receptor" (7-TM receptor) is a complete membrane protein containing seven transmembrane helices. As used in this article, the 7-TM receptor is a G protein-coupled receptor.

"Chemokine receptors" are seven-transmembrane receptors. The chemokine receptor family in humans contains 24 members, which can be further divided into four subfamilies based on the types of chemokines they bind: CX3CR, CXCR, CCR, and XCR. They all activate G proteins. ACKR contains six atypical receptors that do not activate G proteins upon ligand binding.

CXC chemokine receptors (CXCRs) are intact membrane proteins that specifically bind to and respond to CXC chemokine family cytokines. They represent a subfamily of chemokine receptors because they cross the cell membrane seven times, forming a larger family of G protein-linked receptors known as seven-transmembrane (7-TM) proteins. Currently, seven CXC chemokine receptors are known in mammals, named CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, and CXCR6. CXCR6 is structurally more closely related to CC chemokine receptors than to other CXC chemokine receptors.

"CC chemokine receptors" (CCRs, also known as β-chemokine receptors) are intact membrane proteins that specifically bind to and respond to cytokines of the CC chemokine family. They represent a subfamily of chemokine receptors because they are a large family of G protein receptors that cross the cell membrane seven times, and are called seven-transmembrane (7-TM) proteins. The CC chemokine receptor subfamily includes CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10.

The term "CCR8" refers to C-C chemokine receptor type 8. The CCR8 protein is encoded by the gene CCR8 (NCBI gene ID 1237). Synonyms for CCR8 include CC-CKR-8, CCR-8, CDw198, CKRL1, CMKBR8, CMKBRL2, GPRCY6, CY6, and TER1. CCR8 proteins include human, mouse, rat, rhesus monkey, and other mammalian and non-mammal homologs. The human CCR8 sequence is accessible via the UniProt identifier P51685 (CCR8_human), for example, human isotypes P51685-1 or P51685-2 (UniProt, November 29, 2019). The mouse CCR8 sequence is accessible via the UniProt identifier P56484 (CCR8_MOUSE). The rhesus monkey CCR8 sequence is accessible via the UniProt identifier O97665 (CCR8_MACMU). Different species may have different isotypes and variants, all of which are composed of the term CCR8. This also includes pre- and post-maturation CCR8 molecules, i.e., cleavage independent of one or more pre-domains. Furthermore, synthetic variants of the CCR8 protein can be generated and are included in the term CCR8. In addition, the CCR8 protein can undergo various modifications, such as synthetic or naturally occurring modifications, including post-translational modifications. Recombinant human CCR8 is commercially available or can be manufactured as is known in the art. CCR8 is a receptor for the chemokine CCL1/SCJA1/I-309. Barington et al. reported the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8 (Barington, Line, et al. "Role of conserved disulfide bridges and aromatic residues in extracellular loop 2 of chemokine receptor CCR8 for chemokine and small molecule binding." Journal of Biological Chemistry 291.31(2016): 16208-16220). Furthermore, they found that two distinct aromatic residues in ECL-2, Tyr184 (Cys+1) and Tyr187 (Cys+4), are crucial for the binding of CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but are not important for the binding of small molecules.

Programmed death-1 (PD-1) refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes, but is not limited to, human PD-1 (hPD-1), variants, isotypes, and species homologs of hPD-1, as well as analogs sharing at least one common epitope with hPD-1. The complete hPD-1 sequence is available in GenBank accession number U64863 (November 29, 2019).

Programmed death-ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes, but is not limited to, human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1, as well as analogs sharing at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank accession number Q9NZQ7 (November 29, 2019).

Tumor proportion score (TPS) is the percentage of live tumor cells that show partial or complete membrane staining at any intensity. For example, if TPS ≥ 1%, the sample should be considered to have PD-L1 expression; if TPS ≥ 50%, the sample should be considered to have high PD-L1 expression. For example, PD-L1 protein expression in NSCLC is often determined using tumor proportion score (TPS).

The "combined positive score" (CPS) is calculated by dividing the number of stained cells (tumor cells, lymphocytes, macrophages) by the total number of viable tumor cells, multiplied by 100. For example, if CPS ≥ 1, the sample should be considered to have PD-L1 expression; if CPS ≥ 10, the sample should be considered to have high PD-L1 expression. The FDA has approved the use of the PD-L1 IHC 22C3pharmDx assay to determine the suitability of the therapeutic antibody pembrolizumab in patients.

FOXP3 is a 50-55 kDa transcription factor, also known as the Forkhead Box protein P3, Scurfin, JM2, or IPEX. Compared to most cell surface markers, it is considered a master regulatory gene and a more specific marker for regulatory T cells. Transduced expression of FOXP3 in CD4+/CD25- cells has been shown to induce GITR, CD103, and CTLA4, conferring a regulatory T cell phenotype. Biolegend antibody clones 206D and 259D recognize human FOXP3 epitopes in the amino acid 105-235 region. Poly6238 recognizes human and mouse FOXP3 and targets the N-terminal portion of FOXP3.

The term "regulation" refers to any alteration of an existing process or behavior, such as blocking (antagonism) and induction (excitation). For example, regulation of G protein-independent signaling refers to any significant change in G protein-independent signaling.

The term "internalization" for antibodies, fragments, or conjugates refers to the uptake of antibodies, fragments, and conjugates into cells. Preferably, internalization is determined in cell lines with endogenous target expression (e.g., human or mouse CCR8 as described elsewhere herein). Preferably, internalization is determined by measuring the total internalization fluorescence intensity per cell and quantifying it relative to an isotype control, for example as described in Example 10.5. In short, antibodies, fragments, or conjugates are labeled with a dye and a matched isotype control is used, and the internalization fluorescence of the antibody, fragment, or conjugate is measured and quantified relative to the isotype control. A "non-internalizing antibody" is defined as an antibody that exhibits substantially the same internalization as the corresponding isotype control. A "low-internalizing antibody" is defined as showing internalization equal to or less than 10-fold of the isotype control internalization, preferably 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.4, 1.3, 1.2, or 1.1-fold less than the isotype control internalization. "Intermediate internalization antibody" is defined as an antibody that shows internalization equal to or less than 21 times that of the isotype control and more than 10 times that of the isotype control. "High internalization antibody" is defined as an antibody that shows internalization more than 21 times that of the isotype control.

In an alternative approach, internalization can also be quantified based on t(1/2), i.e., the time until half of the antibody, fragment, or conjugate is internalized. Preferably, the antibody according to the invention is characterized by a time until half of the antibody, fragment, or conjugate is internalized, which is >2 hours, preferably >4, >5, >6, >7, >8, >9, >10, >11, >12, >13, >14, >15, >16, >17, >18, >19, >20, >21, >22, >23, >24, >26, >28, >30, or >48 hours. Most preferably, the antibody according to the invention is not internalized at all, i.e., the time before half of the antibody, fragment, or conjugate is internalized cannot be defined.

"Isotype control" is an antibody or fragment that does not bind to the target but has the same class and type as a reference antibody or fragment that recognizes the target.

If an antibody or fragment binds to antigens from two or more different species, for example, with a KD value of 10⁻⁷ M or less, more preferably less than 10⁻⁸ M, or even more preferably in the range of 10⁻⁹ M to 10⁻¹¹ M, then the antibody or fragment is referred to as “cross-reactive” or “cross-reactive”.

As used herein, the term "specific binding" refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample: antibodies characterized by substantial nonspecific binding would lack therapeutic applicability, and therefore such implementations are excluded. However, as is known in the art, specific binding of an antibody or conjugate does not necessarily preclude binding to other antigens/target molecules. An antibody that specifically binds to an antigen from one species may also bind to antigens from one or more other species. This cross-species reactivity itself does not alter the antibody's specific classification.

In some contexts, the terms "specific binding" or "specifically binding" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than binding to the protein in general. If an antibody is specific for epitope "A," then in a reaction involving epitope "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

In cases of doubt, the specific binding of an antibody or conjugate preferably describes the binding of the antibody, antibody fragment, or conjugate to its antigen/target with an affinity of at least 10⁻⁷ M (as a KD value; i.e., preferably a KD value less than 10⁻⁷ M), wherein the antibody or conjugate has at least twice the lower affinity for a nonspecific antigen that is not a predetermined antigen/target molecule or a closely related antigen/target molecule.

"Multispecificity," also known as "multireactivity" or "nonspecific binding," refers to the ability of a conjugate or antibody to bind to a defined set of unrelated antigens. Nonspecific binding is substantial if the (therapeutic) suitability of the antibody is compromised. Multispecificity of non-protein structures, including but not limited to target-negative cell lines or tissues, baculovirus particles (BVP), insulin, or DNA, can be assessed as known in the art and described herein. For example, nonspecific binding to target-negative human cell lines can be determined, for instance, by using FACS analysis of simulated transfected CHO or HEK cells. In a second embodiment, nonspecific binding to different tissues can be analyzed by FACS analysis of cell lines or cell line groups derived from the respective tissues. In a third example, nonspecific binding to immune cell populations can be analyzed by FACS after classification of immune cell populations as known in the art. In the fourth example, ELISA can be used to analyze the non-specific binding of BVP, insulin, or DNA, as in Isidro, et al. "A strategy for risk mitigation of antibodies with fast clearance." MAbs. Vol. 4. No. 6. Taylor & Francis, 2012.; Avery, Lindsay B., et al. "Establishing in vivo correlations to screen monoclonal antibodies for physiological prophylaxis." The entire contents of the following are incorporated herein by reference, particularly regarding the technical details required for the analysis and quantification of nonspecific binding: "Properties related to favorable human pharmacokinetics." MAbs. Vol. 10. No. 2. Taylor & Francis, 2018., and Jain, Tushar, et al. "Biophysical properties of the clinical-stage antibody landscape." Proceedings of the National Academy of Sciences 114.5 (2017): 944-949. Antibodies that do not exhibit substantial nonspecific binding are preferably characterized by nonspecific binding lower than that of the reference antibody Gantenerumab (Roche) and most preferably lower than that of the reference antibody Remicade (Janssen Biotech).

The term "off-target binding" refers to the ability of an antibody to bind to a single protein that is different from its intended target, such as a protein from a target protein family. Off-target binding can be assessed using commercially available assays known in the art, such as the Retrogenix Off-Target Assay. In short, the antibody is tested on a microarray containing HEK293 cells that individually express thousands of human membrane and secretory proteins. Binding of the antibody to a potential off-target must be confirmed using FACS on cells that overexpress the potential off-target.

The term "affinity" is a term used in the art and describes the binding strength between a conjugate, antibody, or antibody fragment and a target. The "affinity" of antibodies and their fragments to a target can be determined using techniques known in the art or described herein, such as by ELISA, isothermal titration (ITC), surface plasmon resonance (SPR), flow cytometry, or fluorescence polarization assay. Preferably, affinity is provided as a dissociation constant KD.

The "dissociation constant" (KD) has molar units (M) and corresponds to the concentration of the conjugate/antibody when half of the target protein is in equilibrium. The smaller the dissociation constant, the higher the affinity between the conjugate or antibody and its target.

According to the present invention, the antibody preferably has a target affinity of at least 10⁻⁷ M (as a KD value), more preferably at least 10⁻⁸ M, and even more preferably in the range of 10⁻⁹ M to 10⁻¹¹ M. The KD value can preferably be determined by surface plasmon resonance spectroscopy, as described elsewhere herein. If it is found that the assay conditions affect the measured KD, the assay settings with the smallest standard deviation should be used.

The "half-maximum effective concentration" (EC50) refers to the concentration of a drug, antibody, fragment, conjugate, or molecule that induces half of the response between baseline and maximum after a specified incubation time. In the case of antibody binding, EC50 thus reflects the antibody concentration required for half of the maximum binding. EC50 can be determined if the inflection point can be determined by mathematical modeling (e.g., nonlinear regression) of the dose-response curve describing the relationship between the concentration of the applied drug, antibody, fragment, conjugate, or molecule and the signal. For example, if the dose-response curve follows an sigmoid curve, EC50 can be determined. When the response is inhibitory, EC50 is called the half-maximum inhibitory concentration (IC50). EC80 can be determined with necessary modifications.

The term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to an antigen or responds to an immune response, such as, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgE, IgA1, IgA2; mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, IgD, IgE, or IgM; rat IgG1, IgG2a, IgG2b, IgG2c, IgA, IgD, IgE, or IgM; rabbit IgA1, IgA2, IgA3, IgE, IgG, IgM; goat IgA, IgE, IgG1, IgG2, IgE, IgM; or chicken IgY. Antibodies or antibody fragments contain complementarity-determining regions (CDRs), also known as hypervariable regions, within variable domains of the light and heavy chains. Highly conserved portions within the variable domains are called frames (FRs). As is known in the art, the amino acid positions/boundaries depicting the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. As used herein, the numbering of immunoglobulin amino acid residues follows the immunoglobulin nucleic acid residue numbering system of Kabat et al. The variable domains of both the natural heavy and light chains each contain four FR regions. The three CDRs in each chain are tightly linked together by the FR regions and, together with CDRs from the other chain, contribute to the formation of antibody-antigen binding sites; see Kabat, E.A., et al. "Sequences of Proteins of Immunological Interest" (Natl. Inst. Health, Bethesda, MD), GPO Publ. "No. 165-462 (1987). The term antibody as used herein also refers to antibody fragments unless otherwise explicitly stated. Depending on the context, the term antibody may also refer to any protein-binding molecule with immunoglobulin-like function.

The term "CDR" refers to the complementarity-determining region (CDR) of an antibody. As is known in the art, a complementarity-determining region (CDR) is part of a variable chain in antibodies and T-cell receptors. A set of CDRs constitutes a paraepitope. CDRs are crucial for the diversity of antigen specificity. There are three CDRs (CDR1, CDR2, and CDR3), which are arranged discontinuously on the amino acid sequence of the variable domain of an antigen receptor. Since antigen receptors typically contain two variable domains (on two different polypeptide chains, a heavy chain and a light chain), each antigen receptor typically has six CDRs that can co-contact with the antigen. The CDRs of the light chain are LCDR1, LCDR2, and LCDR3. The CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. HCDR3 is the most variable complementarity-determining region (see, for example, Chothia, Cyrus, and Arthur M. Lesk. "Canonical structures for the hypervariable regions of immunoglobulins." Journal of Molecular Biology 196.4 (1987): 901-917.; Kabat, E.A., et al. "Sequences of proteins of immunological interest. Bethesda, MD: USDa department of health and human services." Public Health Service, National Institutes of Health (1991): 103-511).

The "constant region" refers to the portion of an antibody molecule that confers the function of an effector. The heavy chain constant region can be selected from any of five isotypes: alpha (α), delta (δ), epsilon (ε), gamma (g), or mu (μ).

As used herein, the terms “Fc domain,” “Fc region,” or “Fc portion” refer to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. This terminology includes native sequence Fc regions and variant Fc regions. For example, the human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.

The antibody or binding fragment according to the invention can be modified to alter the biological effector function mediated by at least one constant region. For example, in some embodiments, the antibody can be modified to reduce or enhance the biological effector function mediated by at least one constant region relative to an unmodified antibody, such as reducing or improving binding to the Fc receptor (FcγR). FcγR binding may be reduced, for example, by mutating a specific region in the immunoglobulin constant region fragment of the antibody that is used for Fcγ receptor interaction (see, for example, Canfield, Stephen M., and Sherie L. Morrison. "The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated b y the hinge region. "The Journal of Experimental Medicine 173.6 (1991): 1483-1491; and Lund, John, et al. "Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG." The Journal of Immunology 147.8 (1991): 2657-2662). FcγR binding may be enhanced, for example, through afucosylation. Reduced FcγR binding may also reduce other effector functions dependent on Fcγ receptor interactions, such as opsonization, phagocytosis, and antigen-dependent cytotoxicity (“ADCC”).

Furthermore, resolving the interaction between Fc and FcRn allows for the regulation of the antibody's half-life in vivo. Eliminating this interaction, for example, by introducing a mutant H435A, results in an extremely short half-life because the antibody is no longer protected against lysosomal degradation by the FcRn cycle. In some preferred embodiments according to all aspects, the antibody according to the invention comprises a mutant H435A or is otherwise engineered to reduce its half-life.

Conversely, antibodies containing “YTE” mutations (M252Y/S254T/T256E) and/or equivalent mutations (such as “LS” mutations (M428L/N434S)) have been shown to significantly prolong half-life through more efficient recovery from preclinical species and human endosomes (Dall’Acqua, William F., et al. "Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences." The Journal of Immunology 169.9 (2002): 5171-5180.; Zalevsky, Jonathan, et al. "Enhanced antibody half-life improves in vivo activity." Nature Biotechnology 28.2 (2010): 157-159). In some preferred embodiments according to all aspects, the antibody according to the invention comprises a YTE mutation (M252Y/S254T/T256E) and/or an equivalent mutation, such as LS (M428L/N434S), or is otherwise engineered to improve half-life. Haraya, Kenta, Tatsuhiko Tachibana, and Tomoyuki Igawa. "Improvement of pharmacokinetic properties of therapeutic antibodies by antibody engineering." Drug Metabolism and Pharmacokinetics 34.1 (2019): 25-41., and/or Lee, Chang-Han, et al. "An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence." Nature Communications 10.1 (2019): 1-11. Suitable Fc engineering methods for extending half-life can be found, all of which are incorporated herein by reference.

"Unfucosylated" antibodies are engineered antibodies so that the oligosaccharide in the Fc region of the antibody does not contain any fucose units. Glycosylation of an antibody can alter its function. For example, if the glycosylation at N297 in the CH2 domain of IgG is completely removed, binding to FcγRs is lost. However, modulation of the specific carbohydrate composition at N297 can have the opposite effect and enhance the antibody's ADCC activity. In short, the antibody's affinity for activating FcγRs depends on the composition of the N297 N-linked oligosaccharide. There are 32 possible combinations of oligosaccharides that can appear at this position. Naturally occurring human IgG and human IgG produced by hybridomas or other common expression systems typically contain N-acetylglucosamine (GlcNAc) and three mannose residues forming the core carbohydrate. This core is linked to two additional GlcNAc groups to form a bibranch. The addition of galactose to each branch and the terminal addition of sialic acid to these galactose molecules can both occur. Fucose is typically part of the core GlcNAc. This fucose sterically hinders the interaction between the antibody and FcγRIIIA. Therefore, eliminating this fucose molecule while maintaining other forms of glycosylation at this site increases the binding of the antibody to activated FcγRs, enhancing its ability to induce ADCC and/or ADCP (Almagro, Juan C., et al. "Progress and challenges in the design and clinical development of antibodies for cancer therapy." Frontiers in Immunology 8 (2018): 1751). A method for preparing fucose-free antibodies involves growing the cells in rat myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels of FUT8 mRNA encoding α-1,6-fucosyltransferase (an enzyme essential for peptide fucosylation). Non-fucosylated antibodies are preferred for this invention.

Antibody-dependent cytotoxicity (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is a cell-mediated immune defense mechanism in which immune cells actively lyse target cells whose membrane surface antigens have been bound by specific antibodies. ADCC is mediated through the interaction of antibodies or fragments with FcγRIIIa. In humans, FcγRIII exists in two distinct forms: FcγRIIIa (CD16a) and FcγRIIb (CD16b). While FcγRIIIa is expressed as a transmembrane receptor on monocytes, neutrophils, mast cells, macrophages, and natural killer cells, FcγRIIIb is expressed only on neutrophils. These receptors bind to the Fc portion of IgG antibodies, thereby activating antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

Different assay systems for determining ADCC induction in human subjects have been described in the literature and are applicable to the characterization of the subject matter disclosed herein. For example, Yao Te Hsieh et al. investigated different ADCC assay systems based on (i) natural killer cells (FcγRIIIA+ primary NK) from human donors, (ii) FcγRIIIA-engineered NK-92 cells, and (iii) FcβRIIIA/NFAT-RE/luc2-engineered Jurkat T cells (Hsieh, Yao-Te, et al. "Characterization of FcγRIIIA effector cells used in in vitro ADCC bioassay: comparison of primary NK cells with engineered NK-92 and Jurkat T cells." Journal of Immunological Methods 441 (2017): 56-66, the entire contents of which are incorporated herein by reference; in particular, the methodologies described for these assays are described). In summary, all three effector cell systems differentially express FcγRIIIA and provide dose-dependent ADCC pathway activity, but only primary NK cells and engineered NK-92 cells are able to induce ADCC-mediated cell lysis. For functional assessment of ADCC activity, primary NK or NK-92 (V-158) cells therefore better reflect the physiologically relevant mechanisms of ADCC action. As an engineered cell line, NK-92 cells may exhibit more reproducibility than primary NK cells and are therefore the preferred assay system for determining ADCC responses in human subjects, for example, in cases of suspicion.

The antibody or antigen-binding fragment that induces ADCC is an antibody that can induce massive lysis of target cells in the presence of NK effector cells. Preferably, ADCC induction results in at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lysis of target cells.

Antibody-dependent phagocytosis (ADCP) refers to the mechanism by which antibody-mediated target cells activate FcγRs on the surface of macrophages to induce phagocytosis, leading to the internalization and degradation of target cells. For ADCP, binding to macrophages, which are effector cells, usually occurs through the interaction of the antibody FC portion with FCγRIIa (CD32a) expressed by macrophages.

The antibody or antigen-binding fragment that induces ADCP is an antibody that can trigger a large-scale phagocytosis of target cells in the presence of macrophages. Preferably, ADCP induction results in phagocytosis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the target cells.

Complement-dependent cytotoxicity (“CDC”) is the effector function of IgG and IgM antibodies. When they bind to surface antigens on target cells (e.g., cells infected by bacteria or viruses), the classical complement pathway is triggered by the binding of protein C1q to these antibodies, leading to the formation of the membrane attack complex (MAC) and target cell lysis. The complement system is effectively activated by human IgG1, IgG3, and IgM antibodies, weakly activated by IgG2 antibodies, and not activated by IgG4 antibodies. This is one mechanism by which therapeutic antibodies (and specific embodiments of antibodies according to the invention) can achieve antitumor effects. Several laboratory methods exist to determine the potency of CDC and are known in the art.

The antibody or antigen-binding fragment that induces CDC is an antibody that can trigger the formation of a large number of membrane attack complexes and target cell lysis. Preferably, CDC induction results in at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of target cells lysed.

Antibodies containing the Fc region may or may not contain modifications that promote the binding of the first and second subunits of the Fc domain.

"Modifications that promote the binding of the first and second subunits of the Fc domain" refer to manipulation of the peptide backbone or post-translational modifications of the Fc subunits that reduce or prevent the binding of a peptide containing an Fc subunit to the same peptide to form a homodimer. Antibodies containing the Fc region may or may not contain modifications that promote the binding of the first and second subunits of the Fc domain. Modifications promoting binding as used herein specifically include individual modifications to each of the two Fc domain subunits to which binding is desired (i.e., the first and second subunits of the Fc structure), wherein the modifications are complementary to each other to promote the binding of the two Fc domain subunits. For example, modification-promoted binding can alter the structure or charge of one or both Fc domain subunits, thereby making their binding spatially or electrostatically favorable. Thus, (hetero)dimerization occurs between peptides containing a first Fc domain subunit and peptides containing a second Fc subunit, which may be dissimilar, for example, in the sense that other components fused to each subunit (e.g., antigen-binding moieties) are different. In some embodiments, modification-promoted binding includes amino acid mutations in the Fc domain, particularly amino acid substitutions. In one particular implementation, the modification promotes binding in each of the two subunits of the Fc domain, including individual amino acid mutations, particularly amino acid substitutions.

The “fragments” of antibodies used in this article need to retain the required affinity of the full-length antibody. Therefore, a suitable fragment of an anti-human CCR8 antibody will retain the ability to bind to target chemokine receptors, such as the human CCR8 receptor. An antibody fragment comprises a portion of the full-length antibody, typically its antigen-binding region or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain antibody molecules, biantibodies, and domain antibodies; see Holt, Lucy J., et al. “Domain antibodies: proteins for therapy.” Trends in biotechnology 21.11 (2003): 484-490.

The “Fab fragment” contains the constant domain of the light chain and the first constant domain (CH2) of the heavy chain.

The “Fab′ fragment” differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH2 domain, including one or more cysteine residues from the antibody hinge region.

The “F(ab′) fragment” is produced by the cleavage of the disulfide bond at the hinge cysteine residue of the F(ab′)2 pepsin digestion product. Other chemical conjugations of antibody fragments are known to those skilled in the art. The Fab and F(ab′)2 fragments lack the Fc fragment of the intact antibody, are cleared from the animal circulation more rapidly, and may have less nonspecific tissue binding than the intact antibody, see, for example, Wahl, Richard L., Charles W. Parker, and Gordon W. Philpott. “Improved radioimaging and tumor localization with monoclonal F(ab′)2.” Journal of nuclear medicine: official publication, Society of Nuclear Medicine 24.4 (1983): 316-325.

The “Fv fragment” is the smallest fragment of an antibody containing both complete target recognition and binding sites. This region consists of a dimer of a heavy chain and a light chain variable domain bound together in a tight, non-covalent manner (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Typically, six CDRs confer antigen-binding specificity to the antibody. However, in some cases, even a single variable domain (or half of the Fv fragment, which contains only three target-specific CDRs) may have the ability to recognize and bind antigens, although its affinity is lower than that of the entire binding site.

A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody within a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

A “single-domain antibody” contains a single VH or VL domain that exhibits sufficient affinity for the target. In certain embodiments, a single-domain antibody is a camelified antibody, see, for example, Riechmann, Lutz, and Serge Muyldermans. “Singledomain antibodies: comparison of camel VH and camelised human VH domains.” Journal of Immunological Methods 231.1-2 (1999): 25-38.

A "bispecific antibody" is a monoclonal antibody that has binding specificity to at least two different epitopes on the same or different antigens. In this disclosure, one binding specificity may target a chemokine receptor such as CCR8, and the other may target any other antigen, such as, but not limited to, cell surface proteins, receptors, receptor subunits, tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins. The bispecific antibody constructs according to the invention also include multispecific antibody constructs comprising multiple binding domains/binding sites, such as trispecific antibody constructs, wherein the construct comprises three binding domains.

“Derived antibodies” are typically modified via glycosylation, acetylation, polyethylene glycolation, phosphorylation, sulfation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or linking to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, the derivative may contain one or more non-natural amino acids, for example, using Wolfson, Wendy. “Amber codon flashing ambrx augmentations proteins with unnatural amino acids.” Chemistry & Biology 13.10 (2006): 1011-1012. Antibodies according to the invention can be derivatized, for example, by glycosylation or sulfation.

"Monoclonal antibodies" are a basic homogeneous group of antibodies that bind to a specific antigen. Monoclonal immunoglobulins can be obtained by methods well known to those skilled in the art (e.g., see Georges, and Cesar Milstein. "Continuous cultures of fused cells secreting antibody of predefined specificity." Nature 256.5517 (1975): 495-497, and U.S. Patent No. 4,376,110). Immunoglobulins or immunoglobulin fragments with specific binding affinity can be isolated, enriched, or purified from prokaryotes or eukaryotes. Conventional methods known to those skilled in the art can produce immunoglobulins or immunoglobulin fragments and protein-binding molecules with immunoglobulin-like functions in prokaryotes and eukaryotes. The antibodies according to the invention are preferably monoclonal.

"Humanized antibodies" contain CDR regions derived from non-human species (e.g., mice), which, along with any necessary framework inverse mutations, are inserted into a human sequence-derived V region. Therefore, in most cases, humanized antibodies are created by replacing residues of the hypervariable region of the receptor for a human immunoglobulin (receptor antibody) with residues of the hypervariable region from a non-human species (donor antibody) (e.g., mouse, rat, or non-human primate) that possess the desired specificity, affinity, and ability. See, for example, U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; and 5,859,205, each incorporated herein by reference. In some cases, framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the receptor antibody or donor antibody. These modifications are made to further improve antibody performance (e.g., to achieve the desired affinity). Typically, humanized antibodies will contain at least one, usually two, variable domains, wherein all or substantially all hypervariable regions correspond to those of non-human immunoglobulins, and all or substantially all frame regions are those of human immunoglobulin sequences. Humanized antibodies optionally contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins. For further details, see Jones, Peter T., et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321.6069 (1986): 522-525.; Riechmann, Lutz, et al. "Reshaping human antibodies for therapy." Nature 332.6162 (1988): 323-327.; and Presta, Leonard G. "Antibody engineering." Current Opinion in Structural Biology 2.4 (1992): 593-596, each incorporated herein by reference.

Fully human antibodies (human antibodies) include human-derived CDRs, i.e., human-derived CDRs. Preferably, the fully human antibody according to the invention is an antibody having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the closest human VH germline gene (e.g., a sequence extracted from a recommended list and analyzed in IMGT/Domain gap align).

As accepted by commonly used nomenclature systems such as the INN species subsystem, which was valid before 2017, whole-human antibodies may contain a small amount of phylogenetic bias compared to the closest human germline reference determined based on the IMGT database (http://www.imgt.org, November 29, 2019). For example, whole-human antibodies according to the present invention may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15 phylogenetic biases in their CDRs compared to the closest human germline reference. Completely human antibodies can be developed from human B cells using cloning techniques combined with cell enrichment or immortalization steps. However, most fully human antibodies used clinically are either isolated from transgenic mice targeting the human IgG locus or from complex combinatorial libraries via phage display (Brüggemann, Marianne, et al. "Human antibody production in transgenic animals." Archivum immunologiae et therapiae experimentalis 63.2 (2015): 101-108.; Carter, Paul J. "Potent antibody therapy by design." Nature reviews immunology 6.5 (2006): 343-357.; Frenzel, A. ndré, Thomas Schirrmann, and Michael Hust. "Phage display-derived humanantibodies in clinical development and therapy." MAbs.Vol.8.No.7.Taylor&Francis, 2016.; Nelson , Aaron L., Eugen Dhimolea, and Janice M. Reichert. "Development trends for human monoclonal antibody therapeutics." Nature reviews drug discovery 9.10 (2010): 767-774).

Several techniques exist for generating fully human antibodies or antibodies containing human CDRs (see WO2008112640). Cambridge Antibody Technologies (CAT) and Dyax have obtained antibody cDNA sequences from peripheral B cells isolated from immunized individuals and designed phage display libraries to identify human variable region sequences with specific specificity. In short, the antibody variable region sequence is fused to the structure of gene III or gene VIII of M13 phage. These antibody variable region sequences are expressed at the ends of phages carrying the corresponding sequences in either a Fab or single-stranded Fv (scFv) structure. Through a multi-round screening process using different levels of antigen-binding conditions (strictness), phages expressing Fab or scFv structures specific to the antigen of interest can be selected and isolated. The antibody variable region cDNA sequence of the selected phages can then be elucidated using standard sequencing procedures. These sequences can then be used to reconstruct a fully human antibody with the desired isotype using established antibody engineering techniques. Antibodies constructed according to this method are considered fully human antibodies (including CDRs). To enhance the immunoreactivity (antigen-binding affinity and specificity) of selected antibodies, in vitro maturation processes can be introduced, including combined binding of different heavy and light chains, deletion/addition/mutation at CDR3 of heavy chains (to mimic V-J and V-D-J recombination), and random mutation (to mimic somatic hypermutation). An example of a “fully human” antibody produced by this approach is the anti-tumor necrosis factor α antibody Humira (adalimumab).

The term "polynucleotide" refers to recombinant or synthetically produced polymeric deoxyribonucleotides or their analogues, or modified polynucleotides. This term includes double-stranded and single-stranded DNA or RNA. Polynucleotides can be integrated into, for example, small loops, plasmids, visceral particles, small chromosomes, or artificial chromosomes. Polynucleotides can also be isolated from or integrated into another nucleic acid molecule, such as in expression vectors or chromosomes of eukaryotic host cells.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating a nucleic acid molecule linked thereto. The term also includes plasmids (non-viral) and viral vectors. Certain vectors are capable of directing the expression of the nucleic acids or polynucleotides to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Expression vectors for eukaryotic use can be constructed by inserting a polynucleotide sequence encoding at least one protein of interest (POI) into a suitable vector backbone. The vector backbone may include necessary elements to ensure the maintenance of the vector and, if desired, to provide amplification within the host. For viral vectors, such as lentiviral or retroviral vectors, further virus-specific elements, such as structural elements or other elements well known in the art, may be required. These elements may be provided, for example, in cis (on the same plasmid) or trans (on separate plasmids). Viral vectors may require auxiliary viruses or packaging lines for large-scale transfection. Vectors may contain other elements, such as enhancer elements (e.g., viruses, eukaryotic cells), introns, and viral origins for plasmid replication in mammalian cells. According to the invention, expression vectors typically have a promoter sequence that drives POI expression. Expression of POIs and/or selective marker proteins can be constitutive or regulatory (e.g., induced by the addition or removal of small molecule inducers). Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of POI expression in mammalian cells, such as regulatory elements, promoters, and/or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter Ad-LP), or polyomas. For further descriptions of viral regulatory elements and their sequences, see, for example, U.S. 5,168,062, U.S. 4,510,245, and U.S. 4,968,615.

As used herein, the term "connector" or "spacer" refers to any molecule capable of achieving a direct topological connection between two parts. Parts can be, in particular, peptides, proteins, antibodies, antibody fragments, cytotoxic parts, binding parts, detection parts such as fluorophores, immobilization or recovery parts such as beads or magnetic beads, reactive parts, or any other molecule. The two parts can be of the same type or different. The connector may be part of a conjugate and may even contribute to its function. For example, in a conjugate containing a peptide and biotin, a spacer of about 5 atoms between the carboxyl group of biotin and the first bulky amino acid of the peptide allows biotin to reach (strept) the avidin binding bag. Various connectors are known in the art and can be selected based on the parts to be connected. Connector lengths are typically between 4 atoms and more than 200 atoms. Connectors longer than 60 atoms typically comprise groups of compounds with an average length.

"Peptide linkers" can be linked via amide linkages or any other functional residues. Peptide linkers can connect to the N-terminus or C-terminus of the peptide, or via reactive functional groups or amino acid side chains. Peptides can be linked to, for example, biotin, proteins such as human serum albumin (HSA), carrier proteins such as keyhole limpet hemocyanin (KLH), ovalbumin (OVA), or bovine serum albumin (BSA), fluorescent dyes, short amino acid sequences such as Flag tags, HA tags, Myc tags, or His tags, reactive tags such as maleimide, iodoacetamide, alkyl halides, 3-mercaptopropyl or 4-azidobutyric acid, or various other suitable moieties. Non-limiting examples of suitable linkers, such as those used for peptide coupling, include β-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), PEG2 spacers (8-amino-3,6-dioxanoic acid), PEG3 spacers (12-amino-4,7,10-trioxadodecanoic acid), PEG4 spacers (15-amino-4,8,10,13-tetraoxapentadecanoic acid), and Ttds (trioxadecanosuccinic acid). In some cases, the linker may be derived from a reactive moiety, such as maleimide, iodoacetamide, alkyl halides, 3-mercaptopropyl, or 4-azidobutyric acid. In some cases, the linker may comprise polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of polyethylene glycol or polypropylene glycol.

"Antibody adapters" refer to adapters that establish covalent links between different antibody moieties, including peptide adapters and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of polyethylene glycol and polypropylene alcohol.

"Treating" a subject's disease or "treating" a subject with a disease means administering medication to the subject, such as administering a drug to reduce or prevent the worsening of at least one symptom of the disease.

The term “preventing” refers to reducing the probability of a subject having a disease, disorder, or symptom, even if the subject does not have a disease, disorder, or symptom but is at risk or susceptible to such a disease, disorder, or symptom.

The terms “effective dose” or “therapeutic effective dose” are used interchangeably herein and refer to an amount sufficient to achieve a specific biological outcome or to modulate or improve symptoms or symptom onset time in a subject, typically at least about 10%; typically at least about 20%, preferably at least about 30%, or more preferably at least about 50%. The effectiveness of antibody use in cancer treatment can be assessed based on changes in tumor burden. Tumor shrinkage (objective response) and time to disease progression are important endpoints in cancer clinical trials. The standardized response criteria, RECIST (Responsiveness Evaluation Criteria for Solid Tumors), were published in 2000. An update (RECIST 1.1) was published in 2009. RECIST criteria are commonly used in clinical trials where objective response is the primary endpoint, as well as in trials assessing stable disease, tumor progression, or time to progression, because these outcome measurements are based on an assessment of anatomical tumor burden and its changes during the trial. The effective dose for a specific subject may vary depending on factors such as treatment conditions, the subject's overall health, method of administration, route and dose, and severity of side effects. When combined, the effective dose is proportional to the combination of components, and the effect is not limited to the individual components.

Unless otherwise defined, “complete remission” (CR) is defined as the disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must be reduced to <10 mm on the short axis. For “partial remission” (PR), a reduction of at least 30% of the sum of the diameters of the target lesions must be achieved, with reference to the baseline total diameter. For “progressive disease” (PD), the sum of the diameters of the target lesions must increase by at least 20% (including the baseline sum if the smallest sum is observed in the study) with reference to the smallest sum in the study. In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. In “stable disease” (SD), no sufficient contraction is observed to meet the requirements for PR, nor is a sufficient increase observed to meet the requirements for PD, with reference to the smallest sum diameter during the study period.

Secondary outcome measures that may be used to determine the therapeutic benefit of the antibodies of the present invention described herein include the following: "Objective Response Rate" (ORR) is defined as the proportion of subjects achieving a complete response (CR) or partial response (PR). "Progression-Free Survival" (PFS) is defined as the time from the date of first administration of the antibody to disease progression or death, whichever occurs first. "Overall Survival" (OS) is the length of time a patient diagnosed with the disease remains alive from the date of diagnosis or the start of treatment. "Duration of Overall Response" (DOR) is defined as the time from a participant's initial CR or PR to disease progression. "Depth of Response" (DpR) is defined as the percentage of tumor shrinkage observed at the point of maximum response compared to baseline tumor burden. The clinical endpoints of ORR and PFS can be determined according to the RECIST 1.1 criteria described above.

In the case of analyzing non-human subjects, the parameters described above used to determine therapeutic effects and benefits must be adjusted as discussed elsewhere in this document, see Example 12ff.

Typical "subjects" according to the present invention include human and non-human subjects. Subjects can be mammals, such as mice, rats, cats, dogs, primates, and/or humans.

A “pharmaceutical composition” (also known as a “therapeutic formulation”) of an antibody, fragment, or conjugate can be prepared by mixing an antibody of desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer, for example, according to Remington’s Pharmaceutical Sciences (18th ed.; Mack Pub. Co.: Eaton, Pa., 1990), in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to the receptor at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzalkonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of parabens, such as methyl or propyl paraben sulfonate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); and low molecular weight (less than about 10 residues). Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

A “host cell” is a cell used to receive, maintain, reproduce, and amplify a vector. Host cells can also be used to express polypeptides, such as antibodies or fragments thereof encoded by the vector. When a host cell divides, the nucleic acids contained in the vector are replicated, thereby amplifying the nucleic acids. Preferred host cells are mammalian cells, such as CHO cells or HEK cells. Further preferred host cells are rat myeloma YB2/0 cells.

"Cells with endogenous target expression" refers to cells that express target proteins at levels comparable to physiological or disease conditions. Typically, engineered cells that have been overexpressed express target proteins at much higher levels.

In the context of cells, structures, proteins, antibodies, or markers, the terms “intratumoral,” “within tumor,” “tumor-infiltrating,” or “tumor-related” refer to their location within tumor tissue.

Cells that are “positive” or “+” for a certain marker or protein are characterized by the high expression of that marker or protein. Marker or protein expression can be determined and quantified as is known in the art, for example, by defining different cell populations. For the characterization of (immune) cell populations, marker expression can be determined by FACS or using any other techniques described herein.

"White blood cells" are immune cells that express CD45. In this article, "CD45+ cells" refers to all white blood cells. CD45 can serve as a marker to distinguish between immune cells and non-immune cells.

The term "lymphocyte" refers to all immature, mature, undifferentiated, and differentiated white lymphocyte populations, including tissue-specific and specialty varieties. As a non-limiting example, it includes B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages, including pre-B cells, progenitor B cells, early pre-B cells, late pre-B cells, large pre-B cells, and small pre-B cells, as well as immature B cells, mature B cells, plasma B cells, memory B cells, B-1 cells, B-2 cells, and apoptotic AN1/T3 cell populations.

"T cells" are immune cells that express TCRαβ, CD3, and CD8 or CD4. As used herein, the term includes naive T cells, CD4+ T cells, CD8+ T cells, regulatory T cells, memory T cells, activated T cells, apotent T cells, tolerant T cells, chimeric B cells, and antigen-specific T cells, as well as other T cell populations known in the art. The presence of T cell receptors (TCRs) on the cell surface distinguishes T cells from other lymphocytes.

CD8+ T cells (also known as cytotoxic T cells, TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T lymphocytes, or killer T cells) are T cells that express CD3, CD45, and CD8. CD8+ T cells can kill cancer cells, infected cells (especially those infected by viruses), or other damaged cells.

CD4+ T cells (also known as helper T cells or Th cells) are immune cells that express CD3, CD4, and CD45. Helper T cells have several subsets, including, but not limited to, Th1, Th2, and Th17. CD4+ T lymphocytes help suppress or regulate immune responses. They are crucial in B cell antibody class switching, the activation and growth of cytotoxic T cells, and maximizing the bactericidal activity of phagocytes such as macrophages.

As used herein, the term "Treg cell" (also known as "Tregs," "regulatory T cells," "T regulatory cells," or "suppressive T cells") refers to immune cells that express CD3, CD4, CD45, and FoxP3, in addition to high levels of CD25 and low levels of CD127. Identification of Treg cells can be performed as described elsewhere in this document. Treg cells also typically express high levels of CTLA-4, GITR, and LAG-3. In the literature, Tregs have also been classified based on the memory marker CD45RO.

Under physiological conditions, Treg cells maintain immune tolerance. During the immune response, Treg cells cease T cell-mediated immunity and suppress autoreactive T cells in the thymus that escape negative selection. Treg cells can also suppress other types of immune cells, such as NK cells and B cells. Adaptive Treg cells (called Th3 or Tr1 cells) are thought to be generated during the immune response.

Treg cells also play a crucial role in immune escape by suppressing anti-tumor immunity, thereby providing an environment of immune tolerance. T cells that recognize cancer cells are typically abundant in tumors, but their cytotoxic function is suppressed by nearby immunosuppressive cells. Tregs are abundant in many different cancers, highly enriched in the tumor microenvironment, and are well-known for their role in tumor progression.

Activated Treg cells express CD4, CD45, FoxP3, CD69, and CCR8. In addition, CD25 is highly expressed, while CD127 is poorly expressed. CD69 is a marker of T cell activation.

“CCR8-positive regulatory T cells” or “CCR8+ regulatory T lymphocytes” are Tregs that express CCR8.

"CD4conv cells" are traditional CD4+, CD25- T cells.

"γδT cells" are T cells that express the unique T cell receptor TCRγδ on their surface. γδT cells also express CD3.

B cells are immune cells that express CD19, while mature B cells express CD20 and CD22. Upon activation by CD40, B cells differentiate, undergoing somatic hypermutation and enhanced immunoglobulin conversion, forming mature B lymphocytes or plasma cells (capable of secreting antibodies). B cells participate in humoral immunity within the adaptive immune system and are antigen-presenting cells.

Macrophages are immune cells that express low levels of CD14 and high levels of CD16, CD11b, CD68, CD163, and CD206. Macrophages engulf and digest cellular debris, foreign substances, microorganisms, or cancer cells through phagocytosis. In addition to phagocytosis, macrophages play a crucial role in innate immunity and help initiate adaptive immunity by recruiting other immune cells. For example, macrophages are important as antigen presenters for T cells. Macrophages that promote inflammation are called M1 macrophages, while those that reduce inflammation and promote tissue repair are called M2 macrophages.

As used in this article, "M1 macrophages" are a subset of macrophages that express ACOD1. M1 macrophages have pro-inflammatory, bactericidal, and phagocytic functions.

As used in this article, “M2 macrophages” are a subset of macrophages that express MRC1 (CD206). M2 macrophages secrete anti-inflammatory interleukins, play a role in wound healing, and are required for revascularization and epithelial cell redifferentiation. Tumor-associated macrophages predominantly possess the M2 phenotype and appear to actively promote tumor growth.

Dendritic cells (DCs) are bone marrow-derived leukocytes and are the most efficient antigen-presenting cells. Dendritic cells are specialized for capturing and processing antigens, converting proteins into peptides on the major histocompatibility complex (MHC) molecule recognized by T cells. As defined herein, DCs are characterized by the expression of CD1c, CD14, CD16, CD141, CD11c, and CD123. Different dendritic cell subsets exist. In humans, DC1 is immunogenic, while DC2 cells are tolerant. Mature DCs express CD83, while plasmacytoid DCs express CD123.

NK cells (also known as natural killer cells) are immune cells that express CD45, CD16, CD56, and NKG2D but are CD3 negative. NK cells do not require activation to kill cells lacking the MHC1 class of "self" markers. NCR1 (also known as CD335 or NKp46) is expressed on a subset of NK cells and NKT cells.

Natural killer T (NKT) cells are a heterogeneous group of T cells that possess characteristics of both T cells and natural killer cells.

"iNKT cells" (also known as "invariant natural killer T cells") express invariant αβTCR (Vα24-Jα18, CD241o), CD44hi, NK1.1 (mouse), and NKG2D. The invariant TCR recognizes glyco-like protein antigens presented by the non-polymorphic MHCI class molecule CD1d. These cells can influence immune responses by rapidly producing large amounts of cytokines (i.e., IFNg).

As is known in the art, an "effect cell" is an immune cell that actively supports an immune response upon stimulation. As used herein, an effector cell refers to an immune cell that expresses the Fcγ receptor and is therefore capable of mediating ADCC or ADCP. Non-limiting examples of effector cells include monocytes, neutrophils, mast cells, preferably macrophages and natural killer cells.

"Tertiary lymphoid structures" are intratumoral structures characterized by increased expression of LTta, LTtb, Cxcr5, and Cxcl13.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T-cell surface receptor designed to be expressed on immune effector cells and specifically bind to antigens. CARs can be used as a therapeutic approach for adoptive cell transfer. Monocytes are taken from a patient (blood, tumor, or ascites) and modified to express a receptor for a specific form of antigen. In some embodiments, the CAR is already specifically expressed for tumor-associated antigens. CARs may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain containing a tumor-associated antigen-binding region. In some aspects, CARs include a single-chain variable fragment (scFv) fused to both the CD3ζ transmembrane and intracellular domains. The specificity of the CAR design may be derived from a ligand of the receptor (e.g., a peptide). In some embodiments, CARs can target cancer by redirecting monocytes/macrophages expressing tumor-associated antigen-specific CARs.

Dosing regimens are abbreviated as known in the art, such as daily (QD), every 2 days (Q2D), or every 3 days (Q3D).

Implementation

Antigens and antibodies that bind to chemokine receptors

Aspect 1 - Antigen

According to a first aspect, an isolated sulfated polypeptide is provided comprising a tyrosine-rich domain (TRD) of a seven-transmembrane receptor. The tyrosine-rich domain is a conserved N-terminal domain characterized by a seven-transmembrane receptor, such as CXC and CC chemokine receptors, see Example 1. As used herein, the term TRD refers to the amino acid or protein sequence of the CXC or CC chemokine receptor located at the N-terminus of the first cysteine residue counted from the N-terminus. In addition to tyrosine, the TRD typically includes negatively charged amino acid residues, such as aspartic acid. The TRDs of all CC and CXC chemokine receptors are listed in Table 4.1 of Example 4, applicable to mice, monkeys, and humans.

Tyrosine sulfation is a ubiquitous post-translational protein modification that occurs in all multicellular organisms. It is catalyzed by tyrosine protein sulfotransferases (TPSTs) 1 and 2, two Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphate sulfate) to an environment-dependent tyrosine residue in the protein substrate. Currently, only a small fraction of sulfated proteins are known, and the understanding of biological sulfation mechanisms and specific modification sites is ongoing; see Example 4. While post-translational modifications such as glycosylation, phosphorylation, acylation, adenineization, farnesylation, ubiquitination, and sulfation are common in proteins of living systems, antibody production is typically based on unmodified target sequences.

The inventors have developed an unusual method for antibody generation and surprisingly discovered that the synthesis of sulfated peptides according to the first aspect can improve the success rate of antibody generation for chemokine receptors themselves, as well as the success rate of chemokine antibodies with excellent functional properties for therapeutic use, as discussed elsewhere herein. The increased success rate of highly specific chemokine receptor antibodies is particularly surprising because research antibodies designed for independent detection of sulfated tyrosine residues as post-translational modifications are rare.

Seven-transmembrane receptors can come from any species that express chemokine receptors characterized by TRD, such as humans, monkeys, macaques (cynomolgus monkeys), rhesus monkeys, rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels.

According to some first embodiments of the first aspect, an isolated polypeptide is provided, wherein the isolated polypeptide comprises a tyrosine-rich domain (TRD) of a seven-transmembrane receptor, further characterized in that at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated.

For example, in highly successful antibody campaigns, the TRD of human or cynomolgus monkey CCR8 was sulfated at positions Y3, Y15, and Y17, while Y16 was omitted, meaning 75% of the tyrosine residues in the TRD were sulfated (Table 6.1). In another example, the TRD of human CCR4 was sulfated at positions 19 and 22 for off-target binding, meaning 50% of the tyrosine residues in the TRD were sulfated (Table 8.1). In yet another approach, the TRD of mouse CCR4 was sulfated at position 22 for off-target binding, meaning 25% of the tyrosine residues in the TRD were sulfated (Table 6.1).

Table 4.1 shows a list of sulfated peptides, including preferred positions of tyrosine sulfate. Without being bound by theory, the inventors believe that the introduction of the additional charge in the form of tyrosine sulfate enables the antibody to recognize specific negatively charged patterns. This recognition appears to require at least an increase in the percentage of tyrosine and positively charged amino acids in the antibody's HCDR3; that is, the use of the isolated sulfated peptides according to the invention also affects the structural composition of the antibody, particularly the amino acid composition of HCDR3, see Example 9.

According to some second embodiments of the first aspect, which may be the same as or different from the first embodiment of the first aspect, a separate polypeptide is provided, wherein the seven-transmembrane receptor is human, cynomolgus monkey or mouse.

In some of these second embodiments, the seven-transmembrane receptor is mouse. In some preferred embodiments of these second embodiments, the seven-transmembrane receptor is human and/or cynomolgus monkey. In some preferred embodiments of these second embodiments, the seven-transmembrane receptor is human. In some of these second embodiments, the seven-transmembrane receptor is cynomolgus monkey.

According to some third embodiments of the first aspect, which may be the same as or different from the first and/or second embodiments of the first aspect, a separated polypeptide is provided, wherein the seven-transmembrane receptor is a chemokine receptor, preferably.

a) CCR chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10.

b) CXC chemokine receptors, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6, or

c) CX3CR1 or CXCR1.

In some of these third embodiments, the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor. In some embodiments of these third embodiments, the seven-transmembrane receptor is a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10. In some preferred embodiments of these third embodiments, the seven-transmembrane receptor is CCR8 or CCR4. In some of these third embodiments, the seven-transmembrane receptor is a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6. In some of these third embodiments, the seven-transmembrane receptor is CX3CR1 or CXCR1.

In some preferred embodiments of the first aspect, an isolated polypeptide is provided comprising a human or cynomolgus monkey seven-transmembrane receptor TRD, characterized in that at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated, wherein the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor, and preferably, wherein the seven-transmembrane receptor is CCR8 or CCR4.

According to some embodiments A of the third embodiment of the first aspect, the isolated sulfated polypeptide comprises the following sequence or a sequence having at least 90%, 95%, or 98% sequence identity with the following sequence.

a) SEQ ID NO: 1 (CCR1_HUMAN_TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 7 (CCR2_HUMAN_TRD), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 13 (CCR3_HUMAN_TRD), preferably wherein Y16 and/or Y17 have been sulfated, or

d) SEQ ID NO: 19 (CCR4_HUMAN_TRD), preferably wherein at least Y22 has been sulfated, and preferably Y16, Y19 and/or Y20 have been sulfated; or

e) SEQ ID NO: 25 (CCR5_HUMAN_TRD), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

f) SEQ ID NO: 31 (CCR6_HUMAN_TRD), preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated, or

g) SEQ ID NO: 37 (CCR7_HUMAN_TRD), preferably wherein one or both of Y8 and Y17 have been sulfated, or

h) SEQ ID NO: 43 (CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

i) SEQ ID NO: 61 (CCR9_HUMAN_TRD), wherein at least Y28 and preferably Y17 and/or Y37 have been sulfated, or

j) SEQ ID NO: 67 (CCR10_HUMAN_TRD), preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

k)SEQ ID NO: 73(CXCR1_HUMAN_TRD), preferably wherein Y27 has been sulfated, or

1) SEQ ID NO: 79 (CXCR2_HUMAN_TRD), preferably wherein Y23 and/or Y25 have been sulfated, or

m)SEQ ID NO: 85(CXCR3_HUMAN_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 91 (CXCR4_HUMAN_TRD), preferably wherein at least Y12 and/or Y21 has been sulfated, or

o)SEQ ID NO: 97(CXCR5_HUMAN_TRD), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p)SEQ ID NO: 103 (CXCR6_HUMAN_TRD), preferably wherein at least one or both of Y6 and Y10 have been sulfated, or

q)SEQ ID NO: 157(CX3CR1_HUMAN_TRD), preferably wherein at least Y14 has been sulfated, or

r)SEQ ID NO: 163(CXCR1_HUMAN_TRD), preferably wherein at least Y27 has been sulfated.

According to some embodiments B of the third embodiment of the first aspect, the isolated sulfated polypeptide comprises the following sequence or a sequence having at least 90%, 95%, or 98% sequence identity with the following sequence.

a) SEQ ID NO: 3 (CCR1_MOUSE_TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 9 (CCR2_MOUSE_TRD), preferably wherein at least Y37 and/or Y39 has been sulfated, or

c) SEQ ID NO: 15 (CCR3_MOUSE_TRD), preferably wherein Y20 and/or Y22 have been sulfated, or

d) SEQ ID NO: 21 (CCR4_MOUSE_TRD), preferably wherein at least Y22 has been sulfated, and preferably in addition, Y16, Y19 and/or Y20 have been sulfated; or

e) SEQ ID NO: 27 (CCR5_MOUSE_TRD), preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

f) SEQ ID NO: 33 (CCR6_MOUSE_TRD), preferably wherein two or three of Y13, Y18 and Y19 have been sulfated, or

g) SEQ ID NO: 39 (CCR7_MOUSE_TRD), preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

h) SEQ ID NO: 45 (CCR8_MOUSE_TRD), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated, or

i) SEQ ID NO: 63 (CCR9_MOUSE_TRD), preferably wherein at least Y28 has been sulfated, and preferably Y19 has also been sulfated; or

j) SEQ ID NO: 69 (CCR1 0_MOUSE_TRD), preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

k)SEQ ID NO: 75(CXCR1_MOUSE_TRD), preferably wherein at least Y6 has been sulfated, or

1) SEQ ID NO: 81 (CXCR2_MOUSE_TRD), preferably wherein Y24 has been sulfated, or

m)SEQ ID NO: 87 (CXCR3_MOUSE_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 93 (CXCR4_MOUSE_TRD), preferably wherein at least Y23, Y13 and/or Y14 have been sulfated, or

o)SEQ ID NO: 99(CXCR5_MOUSE_TRD), preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, or

p)SEQ ID NO: 105(CXCR6_MOUSE_TRD), preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

q)SEQ ID NO: 159 (CX3CR1_MOUSE_TRD), preferably wherein at least Y15 has been sulfated, or

r)SEQ ID NO: 165(CXCR1_MOUSE_TRD), preferably wherein at least Y6 has been sulfated.

According to some embodiments C of the third embodiment of the first aspect, the isolated sulfated polypeptide comprises the following sequence or a sequence having at least 90%, 95%, or 98% sequence identity with the following sequence.

a) SEQ ID NO: 2 (CCR1_MACFA_TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 8 (CCR2_MACMU_TRD), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 14 (CCR3_MACFA_TRD), preferably wherein Y16 has been sulfated, or

d) SEQ ID NO: 20 (CCR4_MACFA_TRD), preferably wherein at least Y22 has been sulfated, and preferably Y16, Y19 and/or Y20 have been sulfated, or

e) SEQ ID NO: 26 (CCR5_MACMU_TRD), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

f) SEQ ID NO: 32 (CCR6_MACFA_TRD), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

g) SEQ ID NO: 38 (CCR7_MACFA_TRD), preferably wherein one or both of Y8 and Y17 have been sulfated, or

h) SEQ ID NO: 44 (CCR8_MACFA_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

i) SEQ ID NO: 62 (CCR9_MACFA_TRD), preferably wherein at least Y28, preferably Y17 and/or Y37 have also been sulfated, or

j) SEQ ID NO: 68 (CCR10_MACFA_TRD), preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

k)SEQ ID NO: 74(CXCR1_MACFA_TRD), preferably wherein at least one of Y14 and Y28 has been sulfated, or

1) SEQ ID NO: 80 (CXCR2_MACFA_TRD), preferably wherein Y20 and/or Y22 have been sulfated, or

m)SEQ ID NO: 86(CXCR3_MACFA_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 92 (CXCR4_MACFA_TRD), preferably wherein at least Y12 and/or Y21 has been sulfated, or

o)SEQ ID NO: 98(CXCR5_MACFA_TRD), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p)SEQ ID NO: 104(CXCR6_MACFA_TRD), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

q)SEQ ID NO: 158 (CX3CR1_MACFA_TRD), preferably wherein at least Y20 has been sulfated, or

r)SEQ ID NO: 164(CXCR1_MACMU_TRD), preferably wherein at least Y14 has been sulfated.

In some fourth embodiments of the first aspect, which may be the same as or different from the first, second and/or third embodiments of the first aspect, the isolated polypeptide comprises the N-terminus of a seven-transmembrane receptor containing TRD and LID domains, preferably wherein at least the cysteine between the TRD and LID domains has been removed or has been changed to a different amino acid.

The extracellular domain of chemokine receptors can be divided into four regions:

(i) The N-terminal structural domain can be further subdivided into

(a) The distal membrane domain rich in tyrosine (TRD),

(b) Cysteine, and

(c) LID structural domain,

(iii) Extracellular domain 1 (ECL1),

(iii) Extracellular domain 2 (ECL2), and

(iv) Extracellular domain 3 (ECL3).

Some peptides according to the present invention are difficult to process, for example, due to a high tendency to aggregate. Without being bound by theory, the inventors believe that the high “stickiness” is due to an increase in the number of charged amino acid and charged sulfate residues. For peptides containing the N-terminus of a chemokine receptor, aggregation properties can be improved by removing cysteine residues or amino acid exchanges between the TRD and LID domains. Optimal results were obtained by changing cysteine to serine (Example 5, Table 4.1).

In some fourth embodiments according to the first aspect, cysteine may be omitted, and TRD and LID are directly linked. In some different fourth embodiments according to the first aspect, cysteine may be replaced by different polar, uncharged amino acids. In some preferred fourth embodiments according to the first aspect, cysteine is replaced by serine (see Table 4.1). In some fourth embodiments according to the first aspect, cysteine may be replaced by at least one different amino acid.

According to some embodiments A of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises the following sequence or a sequence having at least 90%, 95%, or 98% sequence identity with the following sequence.

a) SEQ ID NO: 4 (CCR1_HUMAN_N term), wherein at least Y10 and/or Y18 has been sulfated.

b) SEQ ID NO: 10 (CCR2_HUMAN_N term), wherein at least Y26 has been sulfated.

c) SEQ ID NO: 16 (CCR3_HUMAN_N term), wherein Y16 and/or Y17 have been sulfated.

d) SEQ ID NO: 22 (CCR4_HUMAN_N term), wherein at least Y22 has been sulfated and preferably Y16, Y19 and/or Y20 have also been sulfated.

e) SEQ ID NO: 28 (CCR5_HUMAN_N term), wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated.

f) SEQ ID NO: 34 (CCR6_HUMAN_N term), wherein at least two or three of Y18, Y26 and Y27 have been sulfated.

g) SEQ ID NO: 40 (CCR7_HUMAN_N term), wherein one or both of Y8 and Y17 have been sulfated.

h) SEQ ID NO: 46 (CCR8_HUMAN_N term), wherein at least two or all of Y3, Y15 and Y17 have been sulfated.

i) SEQ ID NO: 64 (CCR9_HUMAN_N term), wherein at least Y28, and preferably Y17 and/or Y37, has been sulfated.

j) SEQ ID NO: 70 (CCR10_HUMAN_N term), wherein at least one or both of Y14 and Y22 have been sulfated.

k)SEQ ID NO: 76 (CXCR1_HUMAN_N term), where Y27 has been sulfated.

1) SEQ ID NO: 82 (CXCR2_HUMAN_N term), wherein Y23 and/or Y25 have been sulfated.

m)SEQ ID NO: 88 (CXCR3_HUMAN_N term), wherein at least one or both of Y27 and Y29 have been sulfated.

n)SEQ ID NO: 94 (CXCR4_HUMAN_N term), wherein at least Y12 and/or Y21 have been sulfated.

o)SEQ ID NO: 100(CXCR5_HUMAN_N term), wherein at least one of Y3 and Y27 has been sulfated, or

p)SEQ ID NO: 106 (CXCR6_HUMAN_N term), wherein at least one or both of Y6 and Y10 have been sulfated, or

q)SEQ ID NO: 160 (CX3CR1_HUMAN_N term), preferably wherein at least Y14 has been sulfated, or

r)SEQ ID NO: 166 (CXCR1_HUMAN_N term), preferably wherein at least Y27 has been sulfated.

According to some embodiments B of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises the following sequence or a sequence having at least 90%, 95%, or 98% sequence identity with the following sequence.

a) SEQ ID NO: 6 (CCR1_MOUSE_N term), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 12 (CCR2_MOUSE_N term), preferably wherein at least Y37 and/or Y39 has been sulfated, or

c) SEQ ID NO: 18 (CCR3_MOUSE_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

d) SEQ ID NO: 24 (CCR4_MOUSE_N term), preferably wherein at least Y22 has been sulfated and preferably in addition, Y16, Y19 and/or Y20 have been sulfated, or

e) SEQ ID NO: 30 (CCR5_MOUSE_N term), preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

f) SEQ ID NO: 36 (CCR6_MOUSE_N term), preferably wherein at least two or three of Y13, Y18 and Y19 have been sulfated.

g) SEQ ID NO: 42 (CCR7_MOUSE_N term), preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

h) SEQ ID NO: 48 (CCR8_MOUSE_N term, C = X or S), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated, or

i) SEQ ID NO: 66 (CCR9_MOUSE_N term), preferably wherein at least Y28 has been sulfated, and preferably Y19 has also been sulfated, or

j) SEQ ID NO: 72 (CCR10_MOUSE_N term), preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

k)SEQ ID NO: 78 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated, or

1) SEQ ID NO: 84 (CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, or

m)SEQ ID NO: 90 (CXCR3_MOUSE_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 96 (CXCR4_MOUSE_N term), preferably wherein at least Y23 and/or Y14 has been sulfated, or

o)SEQ ID NO: 102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, or

p)SEQ ID NO: 108 (CXCR6_MOUSE_N term), preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

q)SEQ ID NO: 162 (CX3CR1_MOUSE_N term), preferably wherein at least Y15 has been sulfated, or

r)SEQ ID NO: 168 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated.

According to some embodiments C of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises the following sequence or a sequence having at least 90%, 95%, or 98% sequence identity with the following sequence.

a) SEQ ID NO: 5 (CCR1_MACFA_N term), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 11 (CCR2_MACMU_N term), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 17 (CCR3_MACFA_N term), preferably wherein Y16 has been sulfated, or

d) SEQ ID NO: 23 (CCR4_MACFA_N term), preferably wherein at least Y22 has been sulfated and preferably in addition, Y16, Y19 and/or Y20 have been sulfated, or

e) SEQ ID NO: 29 (CCR5_MACMU_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

f) SEQ ID NO: 35 (CCR6_MACFA_N term), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

g) SEQ ID NO: 41 (CCR7_MACFA_N term), preferably wherein one or both of Y8 and Y17 have been sulfated, or

h) SEQ ID NO: 47 (CCR8_MACFA_N term), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

i) SEQ ID No: 65 (CCR9_MACFA_N term), preferably wherein at least Y28, and preferably Y17 and/or Y37 have been sulfated, or

j) SEQ ID NO: 71 (CCR10_MACFA_N term), preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

k)SEQ ID NO: 77 (CXCR1_MACFA_N term), preferably wherein at least one of Y14 and Y28 has been sulfated, or

l) SEQ ID NO: 83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

m)SEQ ID NO: 89 (CXCR3_MACFA_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 95 (CXCR4_MACFA_N term), preferably wherein at least Y12 and/or Y21 has been sulfated, or

o)SEQ ID NO: 101 (CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p)SEQ ID NO: 107 (CXCR6_MACFA_N term), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

q)SEQ ID NO: 161 (CX3CR1_MACFA_N term), preferably wherein at least Y20 or Y22 has been sulfated, or

r)SEQ ID NO: 167 (CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 has been sulfated.

In some fourth embodiments of the first aspect, the isolated sulfated polypeptide comprises the following sequence

a) SEQ ID NO: 4 (CCR1_HUMAN_N term), SEQ ID NO: 5 (CCR1_MACFA_N term), or SEQ ID NO: 6 (CCD1_MOUSE_N term), preferably wherein at least Y10 and/or Y18 have been sulfated, or

b) SEQ ID NO: 10 (CCR2_HUMAN_N term) or SEQ ID NO: 11 (CCR2_MACMU_N term), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 12 (CCR2_MOUSE_N term), preferably wherein at least Y37 and/or Y39 has been sulfated, or

d) SEQ ID NO: 16 (CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17 have been sulfated, or

e) SEQ ID NO: 17 (CCR3_MACFA_N term), preferably wherein Y16 has been sulfated, or

f) SEQ ID NO: 18 (CCR3_MOUSE_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

g) SEQ ID NO: 22 (CCR4_HUMAN_N term), SEQ ID NO: 23 (CCR4_MACFA_N term), or SEQ ID NO: 24 (CCR4_MOUSE_N term), preferably wherein at least Y22 has been sulfated and preferably in addition Y16, Y19 and/or Y20 has been sulfated, or

h) SEQ ID NO: 28 (CCR5_HUMAN_N term) or SEQ ID NO: 29 (CCR5_MACMU_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

i) SEQ ID NO: 30 (CCR5_MOUSE_N term), preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

j) SEQ ID NO: 34 (CCR6_HUMAN_N term), preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated, or

k)SEQ ID NO: 35 (CCR6_MACFA_N term), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

l) SEQ ID NO: 36 (CCR6_MOUSE_N term), preferably wherein at least two or three of Y13, Y18 and Y19 have been sulfated, or

m) SEQ ID NO: 40 (CCR7_HUMAN_N term) or SEQ ID NO: 41 (CCR7_MACFA_N term), preferably wherein one or both of Y8 and Y17 have been sulfated, or

n)SEQ ID NO: 42 (CCR7_MOUSE_N term), preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

o) SEQ ID NO: 46 (CCR8_HUMAN_N term, C=X or S), or SEQ ID NO: 47 (CCR8_MACFA_N term, C=X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

p)SEQ ID NO: 48 (CCR8_MOUSE_N term, C = X or S), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated, or

q) SEQ ID NO: 64 (CCR9_HUMAN_N term) or SEQ ID NO: 65 (CCR9_MACFA_N term), preferably at least Y28, and even more preferably Y17 and/or Y37 have been sulfated, or

r)SEQ ID NO: 66 (CCR9_MOUSE_N term), preferably wherein at least Y28 has been sulfated, and even more preferably Y19 has been sulfated; or

s) SEQ ID NO: 70 (CCR10_HUMAN_N term), or SEQ ID NO: 71 (CCR10_MACFA_N term), preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

t)SEQ ID NO: 72 (CCR10_MOUSE_N term), preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

u)SEQ ID NO: 76 (CXCR1_HUMAN_N term), preferably wherein Y27 has been sulfated, or

v)SEQ ID NO: 77 (CXCR1_MACFA_N term), preferably wherein at least one of Y14 and Y28 has been sulfated, or

w)SEQ ID NO: 78 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated, or

x)SEQ ID NO: 82 (CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have been sulfated, or

y)SEQ ID NO: 83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

z)SEQ ID NO: 84 (CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, or

aa) SEQ ID NO: 88 (CXCR3_HUMAN_N term), SEQ ID NO: 89 (CXCR3_MACFA_N term), or SEQ ID NO: 90 (CXCR3_MOUSE_N term), preferably at least one or both of Y27 and Y29 have been sulfated, or

bb)SEQ ID NO: 94 (CXCR4_HUMAN_N term), or SEQ ID NO: 95 (CXCR4_MACFA_N term), preferably wherein at least Y12 and/or Y21 have been sulfated, or

cc)SEQ ID NO: 96 (CXCR4_MOUSE_N term), preferably wherein at least Y23 and/or Y14 has been sulfated, or

dd)SEQ ID NO: 100 (CXCR5_HUMAN_N term), or SEQ ID NO: 101 (CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27 has been sulfated, or

ee)SEQ ID NO: 102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, or

ff)SEQ ID NO: 106 (CXCR6_HUMAN_N term), preferably wherein at least one or both of Y6 and Y10 have been sulfated, or

gg)SEQ ID NO: 107(CXCR6_MACFA_N term), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

hh)SEQ ID NO: 108 (CXCR6_MOUSE_N term), preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

ii) SEQ ID NO: 160 (CX3CR1_HUMAN_N term), preferably wherein at least Y14 has been sulfated, or

jj)SEQ ID NO: 161 (CX3CR1_MACFA_N term), preferably wherein at least Y20 or Y22 has been sulfated, or

kk)SEQ ID NO: 162 (CX3CR1_MOUSE_N term), preferably wherein at least Y15 has been sulfated, or

ll)SEQ ID NO: 166 (CXCR1_HUMAN_N term), preferably wherein at least Y27 has been sulfated, or

mm)SEQ ID NO: 167 (CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 has been sulfated, or

nn)SEQ ID NO: 168(CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated.

In some first, second, third, or fourth embodiments of the first aspect, the isolated sulfated polypeptide comprises the following sequence or a sequence having at least 90% sequence identity with the following sequence.

a) SEQ ID NO: 1 (CCR1_HUMAN_TRD), SEQ ID NO: 4 (CCR1_HUMAN_N term), SEQ ID NO: 2 (CCR1_MACFA_TRD), SEQ ID NO: 5 (CCR1_MACFA_N term), SEQ ID NO: 3 (CCR1_MOUSE_TRD) or SEQ ID NO: 6 (CCR1_MOUSE_N term), preferably wherein at least Y10 and/or Y18 have been sulfated, or

b) SEQ ID NO: 7 (CCR2_HUMAN_TRD), SEQ ID NO: 10 (CCR2_HUMAN_N term), SEQ ID NO: 8 (CCR2_MACMU_TRD) or SEQ ID NO: 11 (CCR2_MACMU_N term), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 9 (CCR2_MOUSE_TRD) or SEQ ID NO: 12 (CCR2_MOUSE_N term), preferably wherein at least Y37 and/or Y39 has been sulfated, or

d) SEQ ID NO: 13 (CCR3_HUMAN_TRD) or SEQ ID NO: 16 (CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17 have been sulfated, or

e) SEQ ID NO: 14 (CCR3_MACFA_TRD) or SEQ ID NO: 17 (CCR3_MACFA_N term), preferably wherein Y16 has been sulfated, or

f) SEQ ID NO: 15 (CCR3_MOUSE_TRD) or SEQ ID NO: 18 (CCR3_MOUSE_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

g) SEQ ID NO: 19 (CCR4_HUMAN_TRD), SEQ ID NO: 22 (CCR4_HUMAN_N term), SEQ ID NO: 20 (CCR4_MACFA_TRD), SEQ ID NO: 23 (CCR4_MACFA_N term), SEQ ID NO: 21 (CCR4_MOUSE_TRD) or SEQ ID NO: 24 (CCR4_MOUSE_N term), preferably wherein at least Y22 has been sulfated and preferably additionally Y16, Y19 and/or Y20 has been sulfated, or

h) SEQ ID NO: 25 (CCR5_HUMAN_TRD), SEQ ID NO: 28 (CCR5_HUMAN_N term), SEQ ID NO: 26 (CCR5_MACMU_TRD) or SEQ ID NO: 29 (CCR5_MACMU_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

i) SEQ ID NO: 27 (CCR5_MOUSE_TRD) or SEQ ID NO: 30 (CCR5_MOUSE_N term), preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

j) SEQ ID NO: 31 (CCR6_HUMAN_TRD) or SEQ ID NO: 34 (CCR6_HUMAN_N term), preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated, or

k) SEQ ID NO: 32 (CCR6_MACFA_TRD) or SEQ ID NO: 35 (CCR6_MACFA_N term), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

l) SEQ ID NO: 33 (CCR6_MOUSE_TRD) or SEQ ID NO: 36 (CCR6_MOUSE_N term), preferably wherein at least two or three of Y13, Y18 and Y19 have been sulfated, or

m) SEQ ID NO: 37 (CCR7_HUMAN_TRD), SEQ ID NO: 40 (CCR7_HUMAN_N term), SEQ ID NO: 38 (CCR7_MACFA_TRD) or SEQ ID NO: 41 (CCR7_MACFA_N term), preferably one or both of Y8 and Y17 are sulfated, or

n) SEQ ID NO: 39 (CCR7_MOUSE_TRD) or SEQ ID NO: 42 (CCR7_MOUSE_N term), preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

o) SEQ ID NO: 43 (CCR8_HUMAN_TRD), SEQ ID NO: 44 (CCR8_MACFA_TRD), SEQ ID NO: 46 (CCR8_HUMAN_N term, C=X or S), or SEQ ID NO: 47 (CCR8_MACFA_N term, C=X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

p) SEQ ID NO: 45 (CCR8_MOUSE_TRD) or SEQ ID NO: 48 (CCR8_MOUSE_N term, C=X or S), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated, or

q) SEQ ID NO: 61 (CCR9_HUMAN_TRD), SEQ ID NO: 64 (CCR9_HUMAN_N term), SEQ ID NO: 62 (CCR9_MACFA_TRD) or SEQ ID NO: 65 (CCR9_MACFA_N term), preferably at least Y28, and even more preferably Y17 and/or Y37 have been sulfated, or

r) SEQ ID NO: 63 (CCR9_MOUSE_TRD) or SEQ ID NO: 66 (CCR9_MOUSE_N term), preferably wherein at least Y28 has been sulfated, and preferably Y19 has also been sulfated, or

s) SEQ ID NO: 67 (CCR10_HUMAN_TRD), SEQ ID NO: 70 (CCR 10_HUMAN_N term), SEQ ID NO: 68 (CCR 10_MACFA_TRD) or SEQ ID NO: 71 (CCR10_MACFA_N term), preferably at least one or both of Y14 and Y22 have been sulfated, or

t) SEQ ID NO: 69 (CCR10_MOUSE_TRD) or SEQ ID NO: 72 (CCR10_MOUSE_N term), preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

u) SEQ ID NO: 73 (CXCR1_HUMAN_TRD) or SEQ ID NO: 76 (CXCR1_HUMAN_N term), preferably wherein Y27 has been sulfated, or

v) SEQ ID NO: 74 (CXCR1_MACFA_TRD) or SEQ ID NO: 77 (CXCR1_MACFA_N term), preferably wherein at least one of Y14 and Y28 has been sulfated, or

w) SEQ ID NO: 75 (CXCR1_MOUSE_TRD) or SEQ ID NO: 78 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated, or

x) SEQ ID NO: 79 (CXCR2_HUMAN_TRD) or SEQ ID NO: 82 (CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have been sulfated, or

y) SEQ ID NO: 80 (CXCR2_MACFA_TRD) or SEQ ID NO: 83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

z) SEQ ID NO: 81 (CXCR2_MOUSE_TRD) or SEQ ID NO: 84 (CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, or

aa) SEQ ID NO: 85 (CXCR3_HUMAN_TRD), SEQ ID NO: 88 (CXCR3_HUMAN_N term), SEQ ID NO: 86 (CXCR3_MACFA_TRD), SEQ ID NO: 89 (CXCR3_MACFA_N term), SEQ ID NO: 87 (CXCR3_MOUSE_TRD) or SEQ ID NO: 90 (CXCR3_MOUSE_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

bb)SEQ ID NO: 91 (CXCR4_HUMAN_TRD), SEQ ID NO: 94 (CXCR4_HUMAN_N term), SEQ ID NO: 92 (CXCR4_MACFA_TRD) or SEQ ID NO: 95 (CXCR4_MACFA_N term), preferably wherein at least Y12 and/or Y21 have been sulfated, or

cc) SEQ ID NO: 93 (CXCR4_MOUSE_TRD) or SEQ ID NO: 96 (CXCR4_MOUSE_N term), preferably wherein at least Y23 and/or Y14 has been sulfated, or

dd) SEQ ID NO: 97 (CXCR5_HUMAN_TRD), SEQ ID NO: 100 (CXCR5_HUMAN_N term), SEQ ID NO: 98 (CXCR5_MACFA_TRD) or SEQ ID NO: 101 (CXCR5_MACFA_N term), preferably at least one of Y3 and Y27 has been sulfated, or

ee) SEQ ID NO: 99 (CXCR5_MOUSE_TRD) or SEQ ID NO: 102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, or

ff)SEQ ID NO: 103 (CXCR6_HUMAN_TRD) or SEQ ID NO: 106 (CXCR6_HUMAN_N term), preferably wherein at least one or both of Y6 and Y10 have been sulfated, or

gg)SEQ ID NO: 104 (CXCR6_MACFA_TRD) or SEQ ID NO: 107 (CXCR6_MACFA_N term), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

hh)SEQ ID NO: 105 (CXCR6_MOUSE_TRD) or SEQ ID NO: 108 (CXCR6_MOUSE_N term), preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

ii) SEQ ID NO: 157 (CX3CR1_HUMAN_TRD) or SEQ ID NO: 160 (CX3CR1_HUMAN_Nterm), preferably wherein at least Y14 has been sulfated, or

jj)SEQ ID NO: 158(CX3CR1_MACFA_TRD), preferably wherein at least Y20 has been sulfated, or

kk)SEQ ID NO: 161 (CX3CR1_MACFA_N term), preferably wherein at least Y20 or Y22 has been sulfated, or

ll) SEQ ID NO: 159 (CX3CR1_MOUSE_TRD) or SEQ ID NO: 162 (CX3CR1_MOUSE_Nterm), preferably wherein at least Y15 has been sulfated, or

mm) SEQ ID NO: 163 (CXCR1_HUMAN_TRD) or SEQ ID NO: 166 (CXCR1_HUMAN_N term), preferably wherein at least Y27 has been sulfated, or

nn)SEQ ID NO: 164(CXCR1_MACMU_TRD), preferably wherein at least Y14 has been sulfated, or

oo)SEQ ID NO: 167 (CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 has been sulfated, or

pp) SEQ ID NO: 165 (CXCR1_MOUSE_TRD) or SEQ ID NO: 168 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated.

Preferably, the isolated peptides, according to the present aspect, are immobilized, for example, via a linker. Immobilization can occur, without limitation, suitable beads, particles, proteins, or solid supports.

Aspect 2 - Contains conjugates of isolated peptides

According to a second aspect of the invention, a conjugate comprising the sulfated polypeptide isolated according to the first aspect is provided.

For example, the conjugate may contain a polypeptide according to a first embodiment of the first aspect. For example, the conjugate may contain a polypeptide according to a second embodiment of the first aspect. For example, the conjugate may contain a polypeptide according to a third embodiment of the first aspect. For example, the conjugate may contain polypeptides of some embodiments A, B, and C of the third embodiment of the first aspect. For example, the conjugate may contain a polypeptide according to a fourth embodiment of the first aspect. For example, the conjugate may contain polypeptides of some embodiments A, B, and C of the fourth embodiment of the first aspect.

For example, isolated sulfated polypeptides can be attached to tags or adapters for immobilization or recovery. Suitable tags are selected from those known in the art, such as small organic molecules like biotin (which binds strongly and non-covalently to streptavidin), their derivatives, or short peptide sequences such as Flag tags, HA tags, Myc tags, or His tags (see Table 4.1 or Example 10.1.2). For some applications, the tag can be a protein, such as human serum albumin (see Table 4.1 or Example 10.1.2), or a carrier protein, such as KLH, OVA, or BSA, or a larger structure, such as beads or magnetic particles. Preferably, the tag can be attached via the C-terminus or N-terminus of the TRD or the N-terminus of the chemokine receptor, but it can also be attached via reactive residues or amino acid side chains, such as lysine residues within the TRD. In some embodiments, the tag is attached via an adapter, which can be any adapter known in the art. Suitable linkers include trioxane-tetrasmosuccinic acid (Ttds) linkers (see Table 4.1), β-alanine, GABA, AEA, Ava, Ahx, PEG2 spacers, PEG3 spacers, PEG4 spacers, O1Pen, O2Oc, or O1Pen-O1.

Aspect 3 - Antigen Production Methods

Sulfated peptides may be difficult to synthesize, for example because sulfates are unstable under acidic conditions (Houben-Weyl, Methods of Organic Chemistry Vol. E 22b, Synthesis of Peptides and Peptidomimetics, 4th Edition, section 6.6.1.2 Synthesis of Sulfated Tyrosine Peptides with Tyrosine O-Sulfate Synthons, p. 440 ff.in: Felix, Arthur et al.: 2004).

According to a third aspect, a method is provided for producing a sulfated polypeptide isolated according to a first aspect or a conjugate according to a second aspect, wherein the method comprises synthesizing the isolated polypeptide and sulfated the corresponding tyrosine residues.

The synthesis of the isolated peptides according to the first aspect and the sulfation of the corresponding tyrosine residues can be carried out as described in Example 5, or according to any other method known in the art. In Chapter 6.6.1, “Methods of Organic Chemistry Vol. E 22b, Synthesis of Peptides and Peptidomimetics,” Houben-Weyl describes various chemical methods for synthesizing sulfated tyrosine peptides. This chapter, and in particular these methods, are incorporated herein by reference in their entirety.

For example, the sequence-independent solid-phase method previously described by Bunschoten et al. (Bunschoten, Anton, et al. "A general sequence-independent solid-phase method for the site-specific synthesis of multiple sulfated-tyrosine containing peptides." Chemical communications 21 (2009): 2999-3001), the entire contents of which are incorporated herein by reference. In short, the peptide is synthesized according to the Fmoc tBu strategy, and then the tyrosine residues to be sulfated are selectively deprotected and a protected sulfate group is introduced. After the synthesis of the sulfated peptide is complete, it is cleaved from the resin by acid hydrolysis to remove the protecting groups other than the sulfate protecting groups, thereby preventing undesirable acid-induced removal of sulfate groups during this step. Finally, the sulfate protecting groups can be removed in a micro-acid reduction step, leaving the sulfate groups unaffected.

Chen et al. reported a short and efficient one-step route for obtaining sY-containing peptides, in which Fmoc-protected fluorosulfated tyrosine (Y(OSO2F)) is incorporated into the peptide of interest via a Fmoc-based solid-phase synthesis strategy (see Chen, Wentao, et al. "Synthesis of Sulfotyrosine-Containing Peptides by Incorporating Fluorosulfated Tyrosine Using an Fmoc-Based Solid-Phase Strategy." Angewandte Chemie 128.5 (2016): 1867-1870), the entire contents of which are incorporated herein by reference. Standard simultaneous peptide resin cleavage and removal of acid-labile side-chain protecting groups yield crude peptides containing fluorosulfated tyrosine. Basic ethylene glycol is used as both solvent and reactant to convert the fluorosulfated tyrosine peptides to sulfotyrosine peptides in high yield.

Global sulfation of the peptide is also possible, for example, using pyridine trioxide. According to the invention, this route can be used if all tyrosines must be sulfated, or if a "crude" mixture of partially sulfated peptides is used for further steps. Furthermore, sulfation can occur enzymatically, for example in biotransformation reactions using natural sulfatases or engineered versions thereof. Preferably, the sulfation of the corresponding tyrosines can be carried out chemically or enzymatically. Preferably, the isolated peptide is synthesized using the Fmoc-tBu strategy.

According to some embodiments of the third aspect, the method includes purifying the obtained peptide. Purification can be performed, for example, by HPLC, using a C18 column. According to some embodiments of the third aspect, the method includes analytical characterization of the peptide. Analytical characterization can be performed using spectroscopic or mass spectrometric methods without limitation.

Aspect 4 - Uses/Methods of Antigens, including the uses of antigens.

According to the fourth aspect, the sulfated polypeptide isolated according to the first aspect or the conjugate according to the second aspect is provided for use in antibody production, as an antigen, or for off-target screening and/or for antibody characterization. For example, the sulfated polypeptide isolated according to the first aspect or the conjugate according to the second aspect can be used to generate fully human antibodies or fragments thereof. For example, the sulfated polypeptide isolated according to the first aspect or the conjugate according to the second aspect can be used to generate cross-reactive antibodies.

According to some first embodiments of the fourth aspect, the use of the sulfated polypeptide isolated according to the first aspect or the conjugate according to the second aspect for antibody production is provided. In particular, as described elsewhere herein, the sulfated polypeptide isolated according to the first aspect can be used to promote the production of antibodies that specifically recognize chemokine receptors.

According to some second embodiments of the fourth aspect, which may be the same as or different from the first embodiment of the fourth aspect, the isolated sulfated polypeptide of the first aspect or the conjugate of the second aspect may be used as an antigen, for example, an antibody, antibody fragment or molecule that specifically binds to a chemokine receptor, see Examples 6 and 8.

According to some third embodiments of the fourth aspect, which may be the same as or different from the first and/or second embodiments of the fourth aspect, the isolated sulfated peptides according to the first aspect or the conjugates according to the second aspect are used for off-target screening to select antibodies that do not bind to specific seven-transmembrane receptors, such as chemokine receptors (off-target receptors), see Examples 6 and 8.

According to some fourth embodiments, the method according to the fourth aspect includes characterizing the antibody using a sulfated polypeptide isolated according to the first aspect or a conjugate according to the second aspect. Preferably, the antibody is an antibody according to the invention. For example, antibody characterization may include using ELISA, surface plasmon resonance, mass spectrometry, competitive assay, staining, IHC, FACS, or various other assays known in the art.

Aspect 5 - Antibody Production Methods

According to a fifth aspect, a method for obtaining an antibody or conjugate is provided, the method comprising using a sulfated polypeptide isolated according to a first aspect or a conjugate according to a second aspect.

According to some first embodiments, the method according to the fifth aspect includes using a sulfated polypeptide isolated according to the first aspect or a conjugate according to the second aspect as an antigen.

According to some preferred first embodiments of the fifth aspect, the method includes using at least one additionally isolated polypeptide or a conjugate thereof, wherein the at least one additionally isolated polypeptide comprises the following TRD

a) Seven-transmembrane receptors that are different from the first seven-transmembrane receptor, or

b) Among the first seven transmembrane receptors derived from different species

Preferably, the at least one additionally separated polypeptide is a polypeptide separated according to the first aspect.

According to some embodiments A of the first embodiment of the fifth aspect, the method includes using at least one additionally isolated polypeptide or conjugate, preferably as an antigen or for off-target selection, according to the first or second aspect. In these embodiments, the first isolated sulfated polypeptide comprises a TRD of a first seven-transmembrane receptor (e.g., a chemokine receptor), and the additionally isolated polypeptide or conjugate comprises a TRD of a seven-transmembrane receptor different from the first seven-transmembrane receptor. When the additionally isolated polypeptide or conjugate is used as an antigen, the method is a method for generating antibodies or conjugates that recognize at least two different seven-transmembrane receptors, such as two different members of the chemokine receptor family.

When additionally isolated peptides or conjugates are used for off-target screening, this method is used to generate antibodies or conjugates that recognize only specific seven-transmembrane receptors, for example, to avoid off-target binding with other members of the chemokine receptor family.

According to some embodiments B of the first embodiment of the fifth aspect, the method includes using at least one additionally isolated polypeptide or conjugate, preferably according to the first or second aspect, as an antigen or for off-target selection. In these embodiments, the first isolated polypeptide comprises a TRD of a first seven-transmembrane receptor of a first species (e.g., a human chemokine receptor), and the additionally isolated polypeptide comprises a TRD of the same seven-transmembrane receptor derived from a different species (e.g., a cynomolgus monkey chemokine receptor). For example, the first polypeptide may comprise a TRD of a human chemokine receptor, and the second polypeptide may comprise a TRD of a cynomolgus monkey chemokine receptor.

These implementations B have particular advantages because the generation of cross-reactive chemokine antibodies or conjugates against both humans and suitable model species is difficult for chemokine receptors, particularly CCR8. While the overall concordance between transmembrane domains is relatively high, the concordance of extracellular domains of chemokine receptors is low between different chemokine receptor family members and between species of specific chemokine receptors (Figs. 1, 2a). However, according to the invention, it is now found that small sulfated tyrosine residues containing motifs within the TRD are sufficiently conserved for specific chemokine receptors to yield a large number of cross-reactive antibodies. Example 6 describes the generation of cross-reactive antibodies against humans and cynomolgus monkeys, and Example 10.1.1 demonstrates the excellent affinity of various antibodies according to the invention in both species. Cynomolgus monkeys are the preferred model system compared to rodents and mice because mouse models fail to predict immune side effects.

The method according to the fifth aspect can be any method known in the art for generating antibodies. For example, the method can be a conventional immunization method, such as in which the peptide according to the first aspect is conjugated with KLH and administered to a suitable animal for immunization.

For example, following immunization, spleen cells from an immunized animal can be used to generate hybridoma cells that produce animal antibodies. A second step involves screening for antibodies that specifically bind to the antigen using methods known in the art, such as ELISA, and for binding off-target (if applicable). Alternatively, spleen cells can be directly screened to produce antibodies that bind to the antigen in a droplet-based microfluidic system or cell culture assay. Only the selected spleen cells are then directly used for sequencing to obtain the antibody or hybridoma-generated sequence. Another approach may include panning the antigen with an antibody library, which could be, for example, a phage display library, as described elsewhere, or a mammalian library. After screening the library on the antigen, enriched antibodies can be screened using methods known in the art (e.g., ELISA or SPR) to specifically bind to the antigen, as described in more detail herein.

According to some second embodiments of the fifth aspect, which may be the same as or different from the first embodiment of the fifth aspect, the method for obtaining an antibody according to the fifth aspect is a method comprising using a phage display library, a transgenic animal, or any other technique available in the art for producing an antibody containing a human CDR.

In some embodiments A of the second embodiment of the fifth aspect, the method includes using a human phage display library, see Examples 6 and 8. For example, the phage display library may be a fully human antibody phage display library (e.g., BioInvent n-CoDeR Fab library). In some preferred embodiments, the phage display library is rich in tyrosine and/or histidine. In the first step, the phage display library containing phages displaying antibodies or antibody fragments externally may be bound to an immobilized isolated peptide according to the first aspect or a conjugate according to the second aspect to allow binding to the isolated peptide or conjugate.

In an optional second depletion step, which may be performed before or after the first step, a phage display library containing a phage displaying an antibody or antibody fragment on its exterior may be combined with an immobilized isolated polypeptide or conjugate according to the first aspect, different from the isolated polypeptide of the first step, to deplete the off-target conjugate.

In an optional third step, which may be performed before or after the first step or before or after the second step, a phage display library containing an antibody or antibody fragment externally may be combined with an immobilized separation peptide according to the first aspect or a conjugate according to the second aspect, different from the separation peptide of the first step and optionally the separation peptide of the second step, to obtain a cross-reactive conjugate.

Each step can be repeated multiple times, for example, one, two, three, four, five, six, seven, eight, nine, ten, or more times. The bound antibody or fragment can be recovered for amplification by infecting a suitable bacterial host, and the DNA can be sequenced as known in the art to obtain the sequence of the seven-transmembrane receptor antibody or fragment.

In some embodiments B of the second embodiment of the fifth aspect, the method for obtaining antibodies according to the fifth aspect is a method comprising using a transgenic animal described by Lonberg (Lonberg, Nils. "Human antibodies from transgenic animals." Nature biotechnology 23.9 (2005): 1117-1125), the entire contents of which are incorporated herein by reference. For example, the transgenic animal may be a XenoMouse (Abgenix Inc., Fremont, CA, e.g. U.S. Patent No. 5,939,598), a HuMAb mouse (GenPharm-Medarex, San Jose, CA), a RenMab mouse (Biocytogen), or any other animal known in the art for producing fully human antibodies.

In some embodiments C of the second embodiment of aspect 5, the method of obtaining the antibody according to aspect 5 includes the use of in vitro activated B cells (U.S. Pat. Nos. 5,567,610 and 5,229,275, the entire contents of which are incorporated herein by reference).

According to the third embodiment of the fifth aspect, a method for obtaining an antibody or antibody fragment or conjugate that specifically binds to human and/or cynomolgus monkey and/or mouse CC or CXC chemokine receptors is provided, the method comprising:

a) Synthetically sulfated peptides containing tyrosine-rich domains (TRDs) and

b) Select antibodies, antibody fragments, or conjugates that recognize sulfated peptides, and

c) Optionally generate antibodies, antibody fragments, or conjugates.

In some embodiments, the use/method of obtaining an antibody having the advantageous properties as described elsewhere is according to the fourth aspect or the fifth aspect, preferably wherein the antibody

a) Includes human-derived CDRs, and/or

b) is a human, rat, or mouse IgG antibody, preferably a human IgG1 antibody or a mouse IgG2a antibody, and/or

c) exhibits cross-reactivity to two different seven-transmembrane receptors, and/or

d) Cross-reactivity with seven-transmembrane receptors in humans and cynomolgus monkeys, and/or

e) Characterized in that the HCDR3 region contains 10% to 34% tyrosine and/or 2% to 20% histidine, preferably 7% to 20% histidine and/or

f) G protein-independent signaling that does not regulate chemokine receptors, and/or

g) is a non-internalizing antibody, or characterized by internalization into cells expressing an endogenous target at a rate of 1.5, 2, 3, 4, 5, 6, 7 or 10 times lower than that of the isotype control.

The methods described above can be used to obtain a large number of antibodies that bind to chemokine receptors. Interestingly, many of these antibodies are characterized by properties that differ from those of known antibodies obtained using conventional methods described elsewhere.

Aspect 6 - Antigen Definition of Antibodies

According to the present invention, an isolated antibody or an antigen-binding fragment thereof, or a conjugate obtained by means or use according to the prior aspects, is provided.

As understood by those skilled in the art, antibodies and/or binding fragments are inherently "modules." Throughout this disclosure, various specific aspects and embodiments of various "modules" comprising antibodies and/or binding fragments are described. Various specific embodiments of VH CDRs, VH chains, VL CDRs, and VL chains, or functional features, are described as specific, non-limiting examples. It is intended that all specific embodiments can be combined with each other as if each specific combination were individually and explicitly described. Various specific functional embodiments are described as specific, non-limiting examples. It is intended that all specific embodiments can be combined with each other as if each specific combination were individually and explicitly described.

According to a sixth aspect, a separated antibody or antigen-binding fragment thereof is provided, which binds (specifically) to a first separated sulfated polypeptide comprising a tyrosine-rich domain (TRD) of a seven-transmembrane receptor, wherein at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated. Preferably, the first separated sulfated polypeptide further comprises a LID domain of the seven-transmembrane receptor. Preferably, the cysteine residue between the TRD and LID domains has been removed or exchanged for a different amino acid.

Preferably, the first isolated sulfated polypeptide comprises the N-terminus of a seven-transmembrane receptor, which includes its tyrosine-rich domain (TRD) and optionally includes its LID domain; even more preferably, at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated.

In a preferred embodiment, the isolated antibody or its antigen-binding fragment binds to its target with the following KD values: 5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1.5E-9M, <1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, or <9E-11M. For example, the antibodies of the present invention can bind to their targets with KD values between <8E-9M and >4E-10M. When the isolated antibody or its antigen-binding fragment binds to more than one target, it is most preferably bound to its targets with the same order of magnitude of affinity.

According to some preferred embodiments, the isolated antibody or its antigen-binding fragment...

a) Including human CDRs, and/or

b) Cross-reactivity with humans and cynomolgus monkeys, and/or

c) Characterized by the HCDR3 region comprising 10 to 34% tyrosine and/or 7 to 20% histidine, and/or

d) G protein-independent signaling that does not regulate seven-transmembrane receptors, and/or

e) is a non-internalizing antibody or is characterized by internalization into cells expressing an endogenous target at a rate of 1.5, 2, 3, 4, 5, 6, 7, or 10 times lower than that of the isotype control, and/or

f) Inducing ADCC and/or ADCP and/or

g) is a human, rat, or mouse IgG antibody, preferably a human IgG1 antibody or a mouse IgG2a antibody, and/or

h) is a fragment of scFv, Fab, Fab’ or F(ab’)2.

According to some first embodiments of the sixth aspect, the seven-transmembrane receptor is a chemokine receptor. In some of these first embodiments, the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor. In some of these first embodiments, the seven-transmembrane receptor is a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10. In some preferred embodiments of these first embodiments, the seven-transmembrane receptor is CCR8 or CCR4. In some most preferred embodiments of these first embodiments, the seven-transmembrane receptor is CCR8. In some first embodiments, the seven-transmembrane receptor is a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6. In some first embodiments, the seven-transmembrane receptor is CX3CR1 or CXCR1.

According to some second embodiments of the sixth aspect, which may be the same as or different from the first embodiment of the sixth aspect, the seven-transmembrane receptor may be derived from any species expressing a chemokine receptor characterized by TRD, such as humans, monkeys, macaques (cynomolgus monkeys), rhesus monkeys, rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels. In some of these second embodiments of the sixth aspect, the seven-transmembrane receptor is mouse. In some of the most preferred embodiments of these second embodiments of the sixth aspect, the seven-transmembrane receptor is human. In some of these second embodiments of the sixth aspect, the seven-transmembrane receptor is cynomolgus monkey. In some preferred embodiments of these embodiments, the seven-transmembrane receptor is human, cynomolgus monkey, or mouse. In some of these preferred second embodiments of the sixth aspect, the seven-transmembrane receptor is human or cynomolgus monkey.

In some preferred embodiments, the seven-transmembrane receptor is a seven-transmembrane receptor from humans, cynomolgus monkeys, or mice, and the seven-transmembrane receptor is...

a) CCR chemokine receptors, preferably CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10.

b) CXC chemokine receptors, preferably CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6, or

c) CX3CR1 or CXCR1.

According to some third embodiments of the sixth aspect, which may be the same as or different from the first and/or second embodiments of the sixth aspect, (first) the isolated sulfated polypeptide comprises or consists of the following sequences:

a) SEQ ID NO: 1 (CCR1_HUMAN_TRD), SEQ ID NO: 4 (CCR1_HUMAN_N term), SEQ ID NO: 2 (CCR1_MACFA_TRD), SEQ ID NO: 5 (CCR1_MACFA_N term), SEQ ID NO: 3 (CCR1_MOUSE_TRD) or SEQ ID NO: 6 (CCR1_MOUSE_N term), preferably wherein at least Y10 and/or Y18 have been sulfated, or

b) SEQ ID NO: 7 (CCR2_HUMAN_TRD), SEQ ID NO: 10 (CCR2_HUMAN_N term), SEQ ID NO: 8 (CCR2_MACMU_TRD) or SEQ ID NO: 11 (CCR2_MACMU_N term), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 9 (CCR2_MOUSE_TRD) or SEQ ID NO: 12 (CCR2_MOUSE_N term), preferably wherein at least Y37 and/or Y39 has been sulfated, or

d) SEQ ID NO: 13 (CCR3_HUMAN_TRD) or SEQ ID NO: 16 (CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17 have been sulfated, or

e) SEQ ID NO: 14 (CCR3_MACFA_TRD) or SEQ ID NO: 17 (CCR3_MACFA_N term), preferably wherein Y16 has been sulfated, or

f) SEQ ID NO: 15 (CCR3_MOUSE_TRD) or SEQ ID NO: 18 (CCR3_MOUSE_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

g) SEQ ID NO: 19 (CCR4_HUMAN_TRD), SEQ ID NO: 22 (CCR4_HUMAN_N term), SEQ ID NO: 20 (CCR4_MACFA_TRD), SEQ ID NO: 23 (CCR4_MACFA_N term), SEQ ID NO: 21 (CCR4_MOUSE_TRD) or SEQ ID NO: 24 (CCR4_MOUSE_N term), preferably wherein at least Y22 has been sulfated and preferably additionally Y16, Y19 and/or Y20 has been sulfated, or

h) SEQ ID NO: 25 (CCR5_HUMAN_TRD), SEQ ID NO: 28 (CCR5_HUMAN_N term), SEQ ID NO: 26 (CCR5_MACMU_TRD) or SEQ ID NO: 29 (CCR5_MACMU_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

i) SEQ ID NO: 27 (CCR5_MOUSE_TRD) or SEQ ID NO: 30 (CCR5_MOUSE_N term), preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

j) SEQ ID NO: 31 (CCR6_HUMAN_TRD) or SEQ ID NO: 34 (CCR6_HUMAN_N term), preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated, or

k) SEQ ID NO: 32 (CCR6_MACFA_TRD) or SEQ ID NO: 35 (CCR6_MACFA_N term), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

1) SEQ ID NO: 33 (CCR6_MOUSE_TRD) or SEQ ID NO: 36 (CCR6_MOUSE_N term), preferably wherein at least two or three of Y13, Y18 and Y19 have been sulfated, or

m) SEQ ID NO: 37 (CCR7_HUMAN_TRD), SEQ ID NO: 40 (CCR7_HUMAN_N term), SEQ ID NO: 38 (CCR7_MACFA_TRD) or SEQ ID NO: 41 (CCR7_MACFA_N term), preferably one or both of Y8 and Y17 are sulfated, or

n) SEQ ID NO: 39 (CCR7_MOUSE_TRD) or SEQ ID NO: 42 (CCR7_MOUSE_N term), preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

o) SEQ ID NO: 43 (CCR8_HUMAN_TRD), SEQ ID NO: 44 (CCR8_MACFA_TRD), SEQ ID NO: 46 (CCR8_HUMAN_N term, C=X or S), or SEQ ID NO: 47 (CCR8_MACFA_N term, C=X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

p) SEQ ID NO: 45 (CCR8_MOUSE_TRD) or SEQ ID NO: 48 (CCR8_MOUSE_N term, C=X or S), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated, or

q) SEQ ID NO: 61 (CCR9_HUMAN_TRD), SEQ ID NO: 64 (CCR9_HUMAN_N term), SEQ ID NO: 62 (CCR9_MACFA_TRD) or SEQ ID NO: 65 (CCR9_MACFA_N term), preferably at least Y28, and even more preferably Y17 and/or Y37 have been sulfated, or

r) SEQ ID NO: 63 (CCR9_MOUSE_TRD) or SEQ ID NO: 66 (CCR9_MOUSE_N term), preferably wherein at least Y28 has been sulfated, and preferably Y19 has also been sulfated, or

s) SEQ ID NO: 67 (CCR10_HUMAN_TRD), SEQ ID NO: 70 (CCR10_HUMAN_N term), SEQ ID NO: 68 (CCR10_MACFA_TRD) or SEQ ID NO: 71 (CCR10_MACFA_N term), preferably at least one or both of Y14 and Y22 have been sulfated, or

t) SEQ ID NO: 69 (CCR10_MOUSE_TRD) or SEQ ID NO: 72 (CCR10_MOUSE_N term), preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

u) SEQ ID NO: 73 (CXCR1_HUMAN_TRD) or SEQ ID NO: 76 (CXCR1_HUMAN_N term), preferably wherein Y27 has been sulfated, or

v) SEQ ID NO: 74 (CXCR1_MACFA_TRD) or SEQ ID NO: 77 (CXCR1_MACFA_N term), preferably wherein at least one of Y14 and Y28 has been sulfated, or

w) SEQ ID NO: 75 (CXCR1_MOUSE_TRD) or SEQ ID NO: 78 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated, or

x) SEQ ID NO: 79 (CXCR2_HUMAN_TRD) or SEQ ID NO: 82 (CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have been sulfated, or

y) SEQ ID NO: 80 (CXCR2_MACFA_TRD) or SEQ ID NO: 83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

z) SEQ ID NO: 81 (CXCR2_MOUSE_TRD) or SEQ ID NO: 84 (CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, or

aa) SEQ ID NO: 85 (CXCR3_HUMAN_TRD), SEQ ID NO: 88 (CXCR3_HUMAN_N term), SEQ ID NO: 86 (CXCR3_MACFA_TRD), SEQ ID NO: 89 (CXCR3_MACFA_N term), SEQ ID NO: 87 (CXCR3_MOUSE_TRD) or SEQ ID NO: 90 (CXCR3_MOUSE_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

bb)SEQ ID NO: 91 (CXCR4_HUMAN_TRD), SEQ ID NO: 94 (CXCR4_HUMAN_N term), SEQ ID NO: 92 (CXCR4_MACFA_TRD) or SEQ ID NO: 95 (CXCR4_MACFA_N term), preferably wherein at least Y12 and/or Y21 have been sulfated, or

cc) SEQ ID NO: 93 (CXCR4_MOUSE_TRD) or SEQ ID NO: 96 (CXCR4_MOUSE_N term), preferably wherein at least Y23 and/or Y14 has been sulfated, or

dd) SEQ ID NO: 97 (CXCR5_HUMAN_TRD), SEQ ID NO: 100 (CXCR5_HUMAN_N term), SEQ ID NO: 98 (CXCR5_MACFA_TRD) or SEQ ID NO: 101 (CXCR5_MACFA_N term), preferably at least one of Y3 and Y27 has been sulfated, or

ee) SEQ ID NO: 99 (CXCR5_MOUSE_TRD) or SEQ ID NO: 102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, or

ff)SEQ ID NO: 103 (CXCR6_HUMAN_TRD) or SEQ ID NO: 106 (CXCR6_HUMAN_N term), preferably wherein at least one or both of Y6 and Y10 have been sulfated, or

gg)SEQ ID No: 104 (CXCR6_MACFA_TRD) or SEQ ID No: 107 (CXCR6_MACFA_N term), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

hh)SEQ ID NO: 105 (CXCR6_MOUSE_TRD) or SEQ ID NO: 108 (CXCR6_MOUSE_N term), preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

ii) SEQ ID NO: 157 (CX3CR1_HUMAN_TRD) or SEQ ID NO: 160 (CX3CR1_HUMAN_Nterm), preferably wherein at least Y14 has been sulfated, or

jj)SEQ ID NO: 158(CX3CR1_MACFA_TRD), preferably wherein at least Y20 has been sulfated, or

kk)SEQ ID NO: 161 (CX3CR1_MACFA_N term), preferably wherein at least Y20 or Y22 has been sulfated, or

1l) SEQ ID NO: 159 (CX3CR1_MOUSE_TRD) or SEQ ID NO: 162 (CX3CR1_MOUSE_Nterm), preferably wherein at least Y15 has been sulfated, or

mm) SEQ ID NO: 163 (CXCR1_HUMAN_TRD) or SEQ ID NO: 166 (CXCR1_HUMAN_N term), preferably wherein at least Y27 has been sulfated, or

nn)SEQ ID NO: 164(CXCR1_MACMU_TRD), preferably wherein at least Y14 has been sulfated, or

oo)SEQ ID NO: 167 (CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 has been sulfated, or

pp) SEQ ID NO: 165 (CXCR1_MOUSE_TRD) or SEQ ID NO: 168 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated.

According to some embodiments A of the third embodiment of the sixth aspect, (first) the isolated sulfated polypeptide comprises or consists of the following sequences:

a. SEQ ID NO: 1 (CCR1_HUMAN_TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b. SEQ ID NO: 7 (CCR2_HUMAN_TRD), preferably wherein at least Y26 has been sulfated, or

c. SEQ ID NO: 13 (CCR3_HUMAN_TRD), preferably wherein Y16 and/or Y17 have been sulfated, or

d. SEQ ID NO: 19 (CCR4_HUMAN_TRD), preferably wherein at least Y22 has been sulfated, and preferably Y16, Y19 and/or Y20 have been sulfated; or

e.SEQ ID NO: 25 (CCR5_HUMAN_TRD), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

f. SEQ ID NO: 31 (CCR6_HUMAN_TRD), preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated, or

g. SEQ ID NO: 37 (CCR7_HUMAN_TRD), preferably wherein one or both of Y8 and Y17 have been sulfated, or

h.SEQ ID NO: 43 (CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

i. SEQ ID NO: 61 (CCR9_HUMAN_TRD), preferably Y17 and/or Y37 have also been sulfated, or

j.SEQ ID NO: 67 (CCR10_HUMAN_TRD), preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

k.SEQ ID NO: 73 (CXCR1_HUMAN_TRD), preferably wherein Y27 has been sulfated, or

l.SEQ ID NO: 79 (CXCR2_HUMAN_TRD), preferably wherein Y23 and/or Y25 have been sulfated, or

m.SEQ ID NO: 85(CXCR3_HUMAN_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n.SEQ ID NO: 91(CXCR4_HUMAN_TRD), preferably wherein at least Y12 and/or Y21 has been sulfated, or

o.SEQ ID NO: 97 (CXCR5_HUMAN_TRD), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p.SEQ ID NO: 103 (CXCR6_HUMAN_TRD), preferably wherein at least one or both of Y6 and Y10 have been sulfated, or

q.SEQ ID NO: 157(CX3CR1_HUMAN_TRD), preferably wherein at least Y14 has been sulfated, or

r.SEQ ID NO: 163(CXCR1_HUMAN_TRD), preferably wherein at least Y27 has been sulfated.

According to some embodiments B of the third embodiment of the sixth aspect, (first) the isolated sulfated polypeptide comprises or is composed of the following sequences:

a) SEQ ID NO: 3 (CCR1_MOUSE_TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 9 (CCR2_MOUSE_TRD), preferably wherein at least Y37 and/or Y39 has been sulfated, or

c) SEQ ID NO: 15 (CCR3_MOUSE_TRD), preferably wherein Y20 and/or Y22 have been sulfated, or

d) SEQ ID NO: 21 (CCR4_MOUSE_TRD), preferably wherein at least Y22 has been sulfated, and preferably in addition, Y16, Y19 and/or Y20 have been sulfated; or

e) SEQ ID NO: 27 (CCR5_MOUSE_TRD), preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

f) SEQ ID NO: 33 (CCR6_MOUSE_TRD), preferably wherein two or three of Y13, Y18 and Y19 have been sulfated, or

g) SEQ ID NO: 39 (CCR7_MOUSE_TRD), preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

h) SEQ ID NO: 45 (CCR8_MOUSE_TRD), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated, or

i) SEQ ID NO: 63 (CCR9_MOUSE_TRD), preferably wherein at least Y28 has been sulfated, and preferably Y19 has also been sulfated; or

j) SEQ ID NO: 69 (CCR1 0_MOUSE_TRD), preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

k)SEQ ID NO: 75(CXCR1_MOUSE_TRD), preferably wherein at least Y6 has been sulfated, or

1) SEQ ID NO: 81 (CXCR2_MOUSE_TRD), preferably wherein Y24 has been sulfated, or

m)SEQ ID NO: 87(CXCR3_MOUSE_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 93 (CXCR4_MOUSE_TRD), preferably wherein at least Y23 and/or Y14 has been sulfated, or

o)SEQ ID NO: 99(CXCR5_MOUSE_TRD), preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, or

p)SEQ ID NO: 105(CXCR6_MOUSE_TRD), preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

q)SEQ ID NO: 159 (CX3CR1_MOUSE_TRD), preferably wherein at least Y15 has been sulfated, or

r)SEQ ID NO: 165(CXCR1_MOUSE_TRD), preferably wherein at least Y6 has been sulfated.

According to some embodiments C of the third embodiment of the sixth aspect, (first) the isolated sulfated polypeptide comprises or is composed of the following sequences:

a) SEQ ID NO: 2 (CCR1_MACFA_TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 8 (CCR2_MACMU_TRD), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 14 (CCR3_MACFA_TRD), preferably wherein Y16 has been sulfated, or

d) SEQ ID NO: 20 (CCR4_MACFA_TRD), preferably wherein at least Y22 has been sulfated, and preferably Y16, Y19 and/or Y20 have been sulfated, or

e) SEQ ID NO: 26 (CCR5_MACMU_TRD), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

f) SEQ ID NO: 32 (CCR6_MACFA_TRD), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

g) SEQ ID NO: 38 (CCR7_MACFA_TRD), preferably wherein one or both of Y8 and Y17 have been sulfated, or

h) SEQ ID NO: 44 (CCR8_MACFA_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

i) SEQ ID NO: 62 (CCR9_MACFA_TRD), preferably wherein at least Y28, preferably Y17 and/or Y37 have also been sulfated, or

j) SEQ ID NO: 68 (CCR10_MACFA_TRD), preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

k)SEQ ID NO: 74(CXCR1_MACFA_TRD), preferably wherein at least one of Y14 and Y28 has been sulfated, or

l) SEQ ID NO: 80 (CXCR2_MACFA_TRD), preferably wherein Y20 and/or Y22 have been sulfated, or

m)SEQ ID NO: 86(CXCR3_MACFA_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 92 (CXCR4_MACFA_TRD), preferably wherein at least Y12 and/or Y21 has been sulfated, or

o)SEQ ID NO: 98(CXCR5_MACFA_TRD), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p)SEQ ID NO: 104(CXCR6_MACFA_TRD), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

q)SEQ ID NO: 158 (CX3C R1_MACFA_TRD), preferably wherein at least Y20 has been sulfated, or

r)SEQ ID NO: 164(CXCR1_MACMU_TRD), preferably wherein at least Y14 has been sulfated.

According to some fourth embodiments of the sixth aspect, which may be the same as or different from the first, second and/or third embodiments of the sixth aspect, the first isolated sulfated polypeptide comprises an N-terminus of a seven-transmembrane receptor containing a tyrosine-rich domain (TRD) and preferably a LID domain, and at least 25%, at least 50% or at least 75% of the tyrosine residues of the TRD are sulfated, preferably at least one cysteine located between the TRD and LID domains is removed or exchanged for a different amino acid.

According to some embodiments A of the fourth embodiment of the sixth aspect, (first) the isolated sulfated polypeptide comprises or is composed of the following sequences:

a) SEQ ID NO: 4 (CCR1_HUMAN_N term), preferably wherein at least Y10 and/or Y18 has been sulfated.

b) SEQ ID NO: 10 (CCR2_HUMAN_N term), preferably wherein at least Y26 has been sulfated.

c) SEQ ID NO: 16 (CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17 have been sulfated.

d) SEQ ID NO: 22 (CCR4_HUMAN_N term), preferably wherein at least Y22 has been sulfated and preferably in addition, Y16, Y19 and/or Y20 have been sulfated.

e) SEQ ID NO: 28 (CCR5_HUMAN_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated.

f) SEQ ID NO: 34 (CCR6_HUMAN_N term), preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated.

g) SEQ ID NO: 40 (CCR7_HUMAN_N term), preferably wherein one or both of Y8 and Y17 have been sulfated.

h) SEQ ID NO: 46 (CCR8_HUMAN_N term), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated.

i) SEQ ID NO: 64 (CCR9_HUMAN_N term), preferably wherein at least Y28, and preferably Y17 and/or Y37, has been sulfated.

j) SEQ ID NO: 70 (CCR10_HUMAN_N term), preferably wherein at least one or both of Y14 and Y22 have been sulfated.

k)SEQ ID NO: 76 (CXCR1_HUMAN_N term), preferably wherein Y27 has been sulfated.

l) SEQ ID NO: 82 (CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have been sulfated.

m)SEQ ID NO: 88 (CXCR3_HUMAN_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated.

n)SEQ ID NO: 94 (CXCR4_HUMAN_N term), preferably wherein at least Y12 and/or Y21 has been sulfated,

o)SEQ ID NO: 100 (CXCR5_HUMAN_N term), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p)SEQ ID NO: 106 (CXCR6_HUMAN_N term), preferably wherein at least one or both of Y6 and Y10 have been sulfated, or

q)SEQ ID NO: 160 (CX3CR1_HUMAN_N term), preferably wherein at least Y14 has been sulfated, or

r)SEQ ID NO: 166 (CXCR1_HUMAN_N term), preferably wherein at least Y27 has been sulfated.

According to some embodiments B of the fourth embodiment of the sixth aspect, (first) the isolated sulfated polypeptide comprises or is composed of the following sequences:

a) SEQ ID NO: 6 (CCR1_MOUSE_N term), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 12 (CCR2_MOUSE_N term), preferably wherein at least Y37 and/or Y39 has been sulfated, or

c) SEQ ID NO: 18 (CCR3_MOUSE_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

d) SEQ ID NO: 24 (CCR4_MOUSE_N term), preferably wherein at least Y22 has been sulfated and preferably in addition, Y16, Y19 and/or Y20 have been sulfated, or

e) SEQ ID NO: 30 (CCR5_MOUSE_N term), preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

f) SEQ ID NO: 36 (CCR6_MOUSE_N term), preferably wherein at least two or three of Y13, Y18 and Y19 have been sulfated.

g) SEQ ID NO: 42 (CCR7_MOUSE_N term), preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

h) SEQ ID NO: 48 (CCR8_MOUSE_N term, C = X or S), preferably

Of which at least two or all of Y3, Y14 and Y15 have been sulfated, or

i) SEQ ID NO: 66 (CCR9_MOUSE_N term), preferably wherein at least Y28 has been sulfated, and preferably Y19 has also been sulfated, or

j) SEQ ID NO: 72 (CCR10_MOUSE_N term), preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

k)SEQ ID NO: 78 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated, or

l) SEQ ID NO: 84 (CXCR2_MOUSE_N term), preferably wherein Y24 has been sulfated, or

m)SEQ ID NO: 90 (CXCR3_MOUSE_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 96 (CXCR4_MOUSE_N term), preferably wherein at least Y13 and/or Y14 has been sulfated, or

o)SEQ ID NO: 102 (CXCR5_MOUSE_N term), preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, or

p)SEQ ID NO: 108 (CXCR6_MOUSE_N term), preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

q)SEQ ID NO: 162 (CX3CR1_MOUSE_N term), preferably wherein at least Y15 has been sulfated, or

r)SEQ ID NO: 168 (CXCR1_MOUSE_N term), preferably wherein at least Y6 has been sulfated.

According to some embodiments C of the fourth embodiment of the sixth aspect, (first) the isolated sulfated polypeptide comprises or is composed of the following sequences:

a) SEQ ID NO: 5 (CCR1_MACFA_N term), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) SEQ ID NO: 11 (CCR2_MACMU_N term), preferably wherein at least Y26 has been sulfated, or

c) SEQ ID NO: 17 (CCR3_MACFA_N term), preferably wherein Y1 6 has been sulfated, or

d) SEQ ID NO: 23 (CCR4_MACFA_N term), preferably wherein at least Y22 has been sulfated and preferably in addition, Y16, Y19 and/or Y20 have been sulfated, or

e) SEQ ID NO: 29 (CCR5_MACMU_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, or

f) SEQ ID NO: 35 (CCR6_MACFA_N term), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

g) SEQ ID NO: 41 (CCR7_MACFA_N term), preferably wherein one or both of Y8 and Y17 have been sulfated, or

h) SEQ ID NO: 47 (CCR8_MACFA_N term), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

i) SEQ ID NO: 65 (CCR9_MACFA_N term), preferably wherein at least Y28, and preferably Y17 and/or Y37 have been sulfated, or

j) SEQ ID NO: 71 (CCR10_MACFA_N term), preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

k)SEQ ID NO: 77 (CXCR1_MACFA_N term), preferably wherein at least one of Y14 and Y28 has been sulfated, or

1) SEQ ID NO: 83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have been sulfated, or

m)SEQ ID NO: 89 (CXCR3_MACFA_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

n)SEQ ID NO: 95 (CXCR4_MACFA_N term), preferably wherein at least Y12 and/or Y21 has been sulfated, or

o)SEQ ID NO: 101 (CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p)SEQ ID NO: 107 (CXCR6_MACFA_N term), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

q)SEQ ID NO: 161 (CX3CR1_MACFA_N term), preferably wherein at least Y20 or Y22 has been sulfated, or

r)SEQ ID NO: 167 (CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 has been sulfated.

According to some fifth embodiments of the sixth aspect, which may be the same as or different from the first, second, third and/or fourth embodiments of the sixth aspect, the isolated antibody or its antigen-binding fragment (specifically) binds to a second isolated sulfated polypeptide containing a tyrosine-rich domain (TRD) of a seven-transmembrane receptor.

Preferably, the second isolated sulfated polypeptide contains a seven-transmembrane receptor for TRD.

a) A seven-transmembrane receptor for TRD, unlike the first isolated sulfated polypeptide.

b) The first isolated sulfated polypeptide contained the corresponding seven transmembrane receptors for TRD, but from different species.

According to some preferred embodiments, a second isolated sulfated polypeptide comprising a tyrosine-rich domain (TRD) of a seven-transmembrane receptor differs from a first isolated sulfated polypeptide comprising a tyrosine-rich domain (TRD) of a seven-transmembrane receptor, and is a first isolated polypeptide having a sequence of any one of embodiments A, B, or C according to the third and/or fourth embodiments of the sixth aspect. For example, the first isolated sulfated polypeptide may comprise a TRD of a seven-transmembrane receptor of a first species, and the second isolated sulfated polypeptide may comprise a TRD of a seven-transmembrane receptor of a second species, preferably wherein said species is human and cynomolgus monkey, or human and mouse, or human and rat.

According to some embodiments A of the fifth embodiment of the sixth aspect, antibody or fragment specific binding

a) A first isolated sulfated polypeptide comprising SEQ ID NO: 1 (CCR1_HUMAN_TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID Nv: 2 (CCR1_MACFA TRD), preferably wherein at least Y10 and/or Y18 has been sulfated, or

b) A first isolated sulfated polypeptide comprising SEQ ID NO: 7 (CCR2_HUMAN_TRD), preferably wherein at least Y26 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 8 (CCL2_MACMU_TRD), preferably wherein at least Y26 has been sulfated, or

c) A first isolated sulfated polypeptide comprising SEQ ID NO: 13 (CCR3_HUMAN_TRD), preferably wherein Y16 and/or Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 14 (CCR3_MACFA_TRD), preferably wherein Y16 has been sulfated; or

d) A first isolated sulfated polypeptide comprising SEQ ID NO: 19 (CCR4_HUMAN_TRD), preferably wherein at least Y22 has been sulfated, and preferably Y16, Y19 and/or Y20 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 20 (CCR4_MACFA_TRD), preferably wherein at least Y22 has been sulfated, and preferably further preferably Y16, Y19 and/or Y20 has been sulfated, or

e) A first isolated sulfated polypeptide comprising SEQ ID NO: 25 (CCR5_HUMAN_TRD), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 26 (CCR5_MACMU_TRD), preferably wherein two, three, or all of Y3, Y20, Y15, and Y15 have been sulfated; or

f) A first isolated sulfated polypeptide comprising SEQ ID NO: 31 (CCR6_HUMAN_TRD), preferably wherein at least two or three of Y18, Y26, and Y27 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 32 (CCR6_MACFA_TRD), preferably wherein at least two or three of Y23, Y31, and Y32 have been sulfated, or

g) A first isolated sulfated polypeptide comprising SEQ ID NO: 37 (CCR7_HUMAN_TRD), preferably wherein one or both of Y8 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 38 (CCR7_MACFA_TRD), preferably wherein one or both of Y18 and Y17 have been sulfated, or

h) A first isolated sulfated polypeptide comprising SEQ ID NO: 43 (CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 44 (CCR8_MACFA_TRD), preferably wherein at least two or all of Y13, Y17 and Y15 have been sulfated, or

i) a first isolated sulfated polypeptide comprising SEQ ID NO: 61 (CCR9_HUMAN_TRD), preferably Y17 and/or Y37 having been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 62 (CCR9_MACFA_TRD), preferably wherein at least Y28, and preferably Y17 and/or Y37 having also been sulfated, or

j) A first isolated sulfated polypeptide comprising SEQ ID NO: 67 (CCR10_HUMAN_TRD), preferably wherein at least one or both of Y14 and Y22 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 68 (CCR10_MACFA_TRD), preferably wherein at least one or both of Y14 and Y12 have been sulfated; or

k) A first isolated sulfated polypeptide comprising SEQ ID NO: 73 (CXCR1_HUMAN_TRD), preferably wherein Y27 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 74 (CXCR1_MACFA_TRD), preferably wherein at least one of Y14 and Y28 has been sulfated, or

1) A first isolated sulfated polypeptide comprising SEQ ID NO: 79 (CXCR2_HUMAN_TRD), preferably wherein Y23 and/or Y25 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 80 (CXCR2_MACFA_TRD), preferably wherein Y20 and/or Y22 have been sulfated, or

m) a first isolated sulfated polypeptide comprising SEQ ID NO: 85 (CXCR3_HUMAN_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 86 (CXCR3_MACFA_TRD), preferably wherein at least one or both of Y2 and Y29 have been sulfated, or

n) A first isolated sulfated polypeptide comprising SEQ ID NO: 91 (CXCR4_HUMAN_TRD), preferably wherein at least Y12 and/or Y21 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 92 (CXCR4_MACFA_TRD), preferably wherein at least Y22 and/or Y21 has been sulfated, or

o) a first isolated sulfated polypeptide comprising SEQ ID NO: 97 (CXCR5_HUMAN_TRD), preferably wherein at least one of Y3 and Y27 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 98 (CXCR5_MACFA_TRD), preferably wherein at least one of Y23 and Y2 has been sulfated, or

p) A first isolated sulfated polypeptide comprising SEQ ID NO: 103 (CXCR6_HUMAN_TRD), preferably wherein at least one or both of Y6 and Y10 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 104 (CXCR6_MACFA_TRD), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated.

q) A first isolated sulfated polypeptide comprising SEQ ID NO: 157 (CX3CR1_HUMAN_TRD), preferably wherein at least Y14 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 158 (CX3CR1_MACFA_TRD), preferably wherein at least Y20 has been sulfated, or

r) a first isolated sulfated polypeptide comprising SEQ ID NO: 163 (CXCR1_HUMAN_TRD), preferably wherein at least Y27 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 164 (CXCR1_MACMU_TRD), preferably wherein at least Y14 has been sulfated.

According to some embodiments B of the fifth embodiment of the sixth aspect, antibody or fragment specific binding

a) A first isolated sulfated polypeptide comprising SEQ ID NO: 4 (CCR1_HUMAN_N term), preferably wherein at least Y10 and/or Y18 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 5 (CCR1_MACFA_N term), preferably wherein at least Y10 and/or Y18 has been sulfated.

b) A first isolated sulfated polypeptide comprising SEQ ID NO: 10 (CCR2_HUMAN_N term), preferably wherein at least Y26 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 11 (CCR2_MACMU_N term), preferably wherein at least Y26 has been sulfated.

c) A first isolated sulfated polypeptide comprising SEQ ID NO: 16 (CCR3_HUMAN_N term), preferably wherein Y16 and/or Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 17 (CCR3_MACFA_N term), preferably wherein Y16 has been sulfated.

d) A first isolated sulfated polypeptide comprising SEQ ID NO: 22 (CCR4_HUMAN_N term), preferably wherein at least Y22 has been sulfated and preferably additionally Y16, Y19 and/or Y20 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 23 (CCR4_MACFA_N term), preferably wherein at least Y22 has been sulfated and preferably additionally Y16, Y19 and/or Y20 has been sulfated.

e) a first isolated sulfated polypeptide comprising SEQ ID NO: 28 (CCR5_HUMAN_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 29 (CCR5_MACMU_N term), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 have been sulfated.

f) A first isolated sulfated polypeptide comprising SEQ ID NO: 34 (CCR6_HUMAN_N term), preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 35 (CCR6_MACFA_N term), preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated.

g) A first isolated sulfated polypeptide comprising SEQ ID NO: 40 (CCR7_HUMAN_N term), preferably wherein one or both of Y8 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 41 (CCR7_MACFA_N term), preferably wherein one or both of Y8 and Y17 have been sulfated.

h) A first isolated sulfated polypeptide comprising SEQ ID NO: 46 (CCR8_HUMAN_N term, C=X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 47 (CCR8_MACFA_N term, C=X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated.

i) a first isolated sulfated polypeptide comprising SEQ ID NO: 64 (CCR9_HUMAN_N term), preferably wherein at least Y28, and even more preferably Y17 and/or Y37, has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 65 (CCR9_MACFA_N term), preferably wherein at least Y28, and even more preferably Y17 and/or Y37, has been sulfated.

j) A first isolated sulfated polypeptide comprising SEQ ID NO: 70 (CCR10_HUMAN_N term), preferably wherein at least one or both of Y14 and Y22 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 71 (CCR10_MACFA_N term), preferably wherein at least one or both of Y14 and Y22 have been sulfated.

k) A first isolated sulfated polypeptide comprising SEQ ID NO: 76 (CXCR1_HUMAN_N term), preferably wherein Y27 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 77 (CXCR1_MACFA_N term), preferably wherein Y14 and Y28 have been sulfated.

1) A first isolated sulfated polypeptide comprising SEQ ID NO: 82 (CXCR2_HUMAN_N term), preferably wherein Y23 and/or Y25 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have been sulfated.

m) a first isolated sulfated polypeptide comprising SEQ ID NO: 88 (CXCR3_HUMAN_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 89 (CXCR3_MACFA_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated.

n) a first isolated sulfated polypeptide comprising SEQ ID NO: 94 (CXCR4_HUMAN_N term), preferably wherein at least Y12 and/or Y21 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 95 (CXCR4_MACFA_N term), preferably wherein at least Y12 and/or Y21 has been sulfated.

o) a first isolated sulfated polypeptide comprising SEQ ID NO: 100 (CXCR5_HUMAN_N term), preferably wherein at least one of Y3 and Y27 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 101 (CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27 has been sulfated, or

p) A first isolated sulfated polypeptide comprising SEQ ID NO: 106 (CXCR6_HUMAN_N term), preferably wherein at least one or both of Y6 and Y10 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 107 (CXCR6_MACFA_N term), preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

q) A first isolated sulfated polypeptide comprising SEQ ID NO: 160 (CX3CR1_HUMAN_N term), preferably wherein at least Y14 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 161 (CX3CR1_MACFA_N term), preferably wherein at least Y20 or Y22 has been sulfated, or

r) a first isolated sulfated polypeptide comprising SEQ ID NO: 166 (CXCR1_HUMAN_N term), preferably wherein at least Y27 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 167 (CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 has been sulfated.

According to some embodiments C1 of the fifth embodiment of the sixth aspect, the antibody or fragment specifically binds to a first isolated sulfated polypeptide comprising a sequence of some embodiments A of the third embodiment of the sixth aspect and a second isolated sulfated polypeptide comprising a sequence of some embodiments B or C of the third embodiment of the sixth aspect, wherein preferably, the first and second polypeptides comprise the same receptor but TRDs from different species.

According to some embodiments C2 of the fifth embodiment of the sixth aspect, the antibody or fragment specifically binds to a first isolated sulfated polypeptide comprising a sequence of some embodiments A of the fourth embodiment of the sixth aspect and a second isolated sulfated polypeptide comprising a sequence of some embodiments B or C of the fourth embodiment of the sixth aspect, wherein preferably, the first and second polypeptides comprise the same receptor but TRDs from different species.

According to some sixth embodiments of the sixth aspect, which may be the same as or different from the first, second, third, fourth and/or fifth embodiments of the sixth aspect, the dissociation constant or EC50 for binding the first isolated sulfated polypeptide and/or for binding the antibody or antigen-binding fragment of the seven-transmembrane receptor is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

According to some embodiments A of the sixth embodiment of the sixth aspect, the dissociation constant or EC50 of the antibody or antigen-binding fragment used to bind the first isolated sulfated polypeptide and/or the antibody or antigen-binding fragment used to bind the seven-transmembrane receptor is less than 200 nM, 199 nM, 198 nM, 197 nM, 196 nM, 195 nM, 194 nM, 193 nM, 192 nM, 191 nM, 190 nM, 189 nM, 188 nM, 187 nM, etc. nM, 186nM, 185nM, 184nM, 183nM, 182nM, 181nM, 180nM, 179nM, 178nM, 177nM, 176nM, 175nM, 17 4nM, 173nM, 172nM, 171nM, 170nM, 169nM, 168nM, 167nM, 166nM, 165nM, 164nM, 163nM, 162nM, 1 61nM, 160nM, 159nM, 158nM, 157nM, 156nM, 155nM, 154nM, 153nM, 152nM, 151nM, 150nM, 149nM, 148nM, 147nM, 146nM, 145nM, 144nM, 143nM, 142nM, 141nM, 140nM, 139nM, 138nM, 137nM, 136nM , 135nM, 134nM, 133nM, 132nM, 131nM, 130nM, 129nM, 128nM, 127nM, 126nM, 125nM, 124nM, 123n M, 122nM, 121nM, 120nM, 119nM, 118nM, 117nM, 116nM, 115nM, 114nM, 113nM, 112nM, 111nM, 110n M, 109nM, 108nM, 107nM, 106nM, 105nM, 104nM, 103nM, 102nM, 101nM, 100nM, 99nM, 98nM, 97nM, 96nM, 95nM, 94nM, 93nM, 92nM, 91nM, 90nM, 89nM, 88nM, 87nM, 86nM, 85nM, 84nM, 83nM, 82nM, 81 nM, 80nM, 79nM, 78nM, 77nM, 76nM, 75nM, 74nM, 73nM, 72nM, 71nM, 70nM, 69nM, 68nM, 67nM, 66nM , 65nM, 64nM, 63nM, 62nM, 61nM, 60nM, 59nM, 58nM, 57nM, 56nM, 55nM, 54nM, 53nM, 52nM, 51nM, 5 0nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42nM, 41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 35n M, 34nM, 33nM, 32nM, 31nM, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.25nM, 0.2nM, 0.15nM, or 0.1nM.

Preferably, the dissociation constant or EC50 of the antibody or antigen-binding fragment used to bind the first isolated sulfated polypeptide and/or the seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, 0.25 nM, 0.2 nM, 0.15 nM or 0.1 nM.

Preferably, EC50 can be measured in CHO cells overexpressing the target. As disclosed, for example, in Example 10.1.1, antibodies obtained using the methods disclosed herein have excellent affinity for their respective targets. For example, TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bind to human CCR8 in CHO cells engineered to express CCR8 at EC50 levels of 4.8 nM, 1.7 nM, 0.8 nM, 0.6 nM, ~0.9 nM, or 1.7 nM. In addition, TPP-21281, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bound to cynomolgus monkey CCR8 at EC50 values of 1.8 nM, 1 nM, 0.5 nM, 0.7 nM, ~0.55 nM, or 0.9 nM. Furthermore, TPP-17578, TPP-19546, TPP-18206, and TPP-21360 bound to human regulatory T cells at EC50 values of 25 nM, 15 nM, 23 nM, or 10 nM. Additionally, the anti-mouse CCR8 antibody TPP-14099 bound to CHO cells expressing mouse CCR8 at an EC50 value of 3 nM and to mouse iTregs at an EC50 value of 13.2 nM (see Table 10.1.1.5).

According to some embodiments B of the sixth embodiment of the sixth aspect, which may be the same as or different from embodiment A of the sixth embodiment of the sixth aspect, the dissociation constant or EC50 of the antibody or antigen-binding fragment used to bind the second isolated sulfated polypeptide and/or the antibody or antigen-binding fragment used to bind the second seventh transmembrane receptor is less than 200 nM, 199 nM, 198 nM, 197 nM, 196 nM, 195 nM, 194 nM, 193 nM, 192 nM, 191 nM, etc. M, 190nM, 189nM, 188nM, 187nM, 186nM, 185nM, 184nM, 183nM, 182nM, 181nM, 180nM, 179nM, 178nM , 177nM, 176nM, 175nM, 174nM, 173nM, 172nM, 171nM, 170nM, 169nM, 168nM, 167nM, 166nM, 165nM, 164nM, 163nM, 162nM, 161nM, 160nM, 159nM, 158nM, 157nM, 156nM, 155nM, 154nM, 153nM, 152nM, 151nM, 150nM, 149nM, 148nM, 147nM, 146nM, 145nM, 144nM, 143nM, 142nM, 141nM, 140nM, 139nM, 1 38nM, 137nM, 136nM, 135nM, 134nM, 133nM, 132nM, 131nM, 130nM, 129nM, 128nM, 127nM, 126nM, 12 5nM, 124nM, 123nM, 122nM, 121nM, 120nM, 119nM, 118nM, 117nM, 116nM, 115nM, 114nM, 113nM, 112 nM, 111nM, 110nM, 109nM, 108nM, 107nM, 106nM, 105nM, 104nM, 103nM, 102nM, 101nM, 100nM, 99n M, 98nM, 97nM, 96nM, 95nM, 94nM, 93nM, 92nM, 91nM, 90nM, 89nM, 88nM, 87nM, 86nM, 85nM, 84nM, 83 nM, 82nM, 81nM, 80nM, 79nM, 78nM, 77nM, 76nM, 75nM, 74nM, 73nM, 72nM, 71nM, 70nM, 69nM, 68nM, 6 7nM, 66nM, 65nM, 64nM, 63nM, 62nM, 61nM, 60nM, 59nM, 58nM, 57nM, 56nM, 55nM, 54nM, 53nM, 52nM, 51nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42nM, 41nM, 40nM, 39nM, 38nM, 37nM, 36n M, 35nM, 34nM, 33nM, 32nM, 31nM, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20 nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3n M, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.25nM, 0.2nM, 0.15nM, or 0.1nM.

Preferably, the dissociation constant (KD) or EC50 of the sulfated polypeptide used to bind the second isolated peptide and/or the antibody or antigen-binding fragment used to bind the second seventh transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM.

According to some embodiments AB of the sixth embodiment of the sixth aspect, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment used to bind the first isolated sulfated polypeptide and/or the first seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment used to bind the second isolated sulfated polypeptide and/or the second seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

According to some seventh embodiments of the sixth aspect, which may be the same as or different from the first, second, third, fourth, fifth and/or sixth embodiments of the sixth aspect, the dissociation constant (KD) of the antibody for binding the first isolated sulfated polypeptide is lower than the dissociation constant (KD) of the antibody for binding the first isolated non-sulfated polypeptide having the same sequence as the first isolated sulfated polypeptide. Preferably, the antibody substantially does not bind the first isolated non-sulfated polypeptide having the same sequence as the first isolated sulfated polypeptide.

According to some embodiments A of the seventh embodiment of the sixth aspect, the dissociation constant or EC50 of the antibody used to bind the first isolated non-sulfated polypeptide is higher than 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM, or 3 μM, or is undetectable. Preferably, the dissociation constant or EC50 of the antibody used to bind the first isolated non-sulfated polypeptide is higher than 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or is undetectable.

According to some embodiments B of the seventh embodiment of the sixth aspect, which may be the same as embodiment A of the seventh embodiment of the sixth aspect, the dissociation constant or EC50 of the antibody or fragment used to bind the first isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the dissociation constant or EC50 of the antibody or fragment used to bind the first isolated non-sulfated polypeptide is greater than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM or 500 nM, or is undetectable.

The antibody according to aspect 6 may comprise a CDR derived from humans, monkeys, macaques (cynomolgus monkeys), rhesus monkeys, rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels. Preferably, the antibody according to aspect 6 comprises a CDR derived from humans, rats, or mice.

According to some eighth embodiments of the sixth aspect, which may and are suggested to be combined with the first, second, third, fourth, fifth, sixth and/or seventh embodiments of the sixth aspect, the isolated antibody or antigen-binding fragment comprises a human CDR. According to some highly preferred embodiments, the deviation of the human CDR from the nearest human germline is no more than or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The nearest human germline can be determined as is known in the art, for example using IgBLAST (Ye, Jian, et al. "IgBLAST: an immunoglobulin variable domain sequence analysis tool." Nucleic acids research 41. W1 (2013): W34-W40.) with data retrieved from the IMGT human germline database. The antibodies according to these eighth embodiments can be obtained as described, for example, in Examples 6 or 8 or elsewhere herein.

According to some ninth embodiments of aspect 6, which can and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, or eighth embodiments of aspect 6, the isolated antibody or antigen-binding fragment is cross-reactive to humans and cynomolgus monkeys, see Example 10.1.1. Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment used to bind to the human chemokine receptor is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM, for example less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment used to bind to the cynomolgus monkey chemokine receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM.

According to some tenth embodiments of aspect 6, which may and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth and/or ninth embodiments of aspect 6, the isolated antibody or antigen-binding fragment is characterized in that the composition of the HCDR3 region deviates from the average HCDR3 region. Preferably, the isolated antibody or antigen-binding fragment is characterized in that the HCDR3 region contains 10 to 34% tyrosine and/or at least one histidine, preferably 2 to 20% histidine, and most preferably 7 to 20% histidine.

In some embodiments A of the tenth embodiment of aspect 6, the isolated antibody or antigen-binding fragment comprises HCDR3, which contains

a) >0 and <35%, >8 and <34%, ≥10 and ≤34%, or >15 and <11% tyrosine (Y) residues, and/or

b) >0 and ≤16%, ≥2 and ≤20% or ≥7 and ≤20% histidine (H) residues, and

c) Preferably greater than 0 ≤ 18%, ≥ 7 and ≤ 10% or ≥ 0 and ≤ 7% arginine (R) residues, and/or

d) Preferably greater than 0 ≤ 25%, ≥ 7 and ≤ 16% or ≥ 7 and ≤ 13% aspartic acid (D) residues, and/or

e) Preferably free of lysine (K) residues and/or

f) Preferably, it does not contain glutamic acid (E) residues.

In some embodiments B of the tenth embodiment of aspect 6, which may be the same as or different from embodiment A, the isolated antibody or antigen-binding fragment contains HCDR3, which contains

a) >0 and <47%, >22 and <50%, >10 and <34%, or >15 and <47% charged amino acids, and/or

b) Positively charged amino acids >0 and <32%, >8 and <30%, or >10 and <37%, and/or

c) >0 and <26%, ≥7 and <16%, or >7 and <14% of negatively charged amino acids.

In some embodiments C of the tenth embodiment of aspect 6, which may be the same as or different from embodiments A and/or B, the isolated antibody or antigen-binding fragment comprises HCDR3, which contains a total of >0 and ≤42%, ≥10 and ≤42%, or ≥36 and ≤43% histidine and tyrosine residues.

This document discloses each embodiment and description of CCR8 according to aspect 10, such as a general chemokine receptor antibody with necessary modifications. The inventors believe that the sulfated TRD motif, rather than a specific CCR8 sequence, promotes an increase in the frequency of tyrosine and/or histidine residues.

According to some eleventh embodiments of the sixth aspect, it can and is recommended to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth embodiments of the sixth aspect, wherein the isolated antibody or antigen-binding fragment at least partially modulates CCR8 signaling.

For example, the antibody according to the present invention can

a) Blocking G protein-independent signal transduction, and/or

b) Blocking G protein-dependent signaling, and/or

c) Blocking G protein-dependent and G protein-independent signal transduction, and/or

d) Increase G protein-independent signaling, and/or

e) Increase G protein-dependent signaling, and/or

f) Increase both G protein-dependent and G protein-independent signaling.

In a specific context, regulation refers to both blocking ligand-induced G protein-independent signal transduction and inducing G protein-independent signal transduction.

According to some preferred embodiments, the antibody or fragment does not regulate G protein-independent signaling of the seven-transmembrane receptor. According to some preferred embodiments, the antibody or fragment does not block ligand-induced G protein-independent signaling of the target protein. According to some preferred embodiments, the antibody or fragment does not induce G protein-independent signaling.

According to some preferred embodiments, antibodies or fragments block G protein-dependent signaling. G protein-dependent signaling can be analyzed by chemotaxis assays or, preferably, by calcium flux assays, as described elsewhere herein.

According to some preferred embodiments, antibodies or fragments block G protein-dependent signaling of chemokine receptors, but do not block G protein-independent signaling of chemokine receptors, see Example 10.4. G protein-independent signaling of chemokine receptors is any signaling activity that is not G protein-dependent.

According to some twelfth embodiments of the sixth aspect, which may and are recommended to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh embodiments of the sixth aspect, the isolated antibody or antigen-binding fragment is a low-internalized or non-internalized antibody or antibody-binding fragment.

Because overexpression can affect internalization behavior and is not well-suited for simulating internalization in a therapeutic setting, it is preferable to use model cell lines with endogenous expression of the target chemokine receptor to determine internalization, see Example 10.5. For example, internalization can be determined over a time span or at specific time points. Preferably, internalization can be measured in cells with endogenous target expression at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, or 48 hours.

According to some embodiments A of the 12th embodiment of aspect 6, the antibody or antigen-binding fragment has an internalization rate of the same order of magnitude as that of the isotype control.

According to some embodiments B of the 12th embodiment of aspect 6, the antibody or antigen-binding fragment is characterized by internalization into cells with endogenous target expression at a rate of 1.5, 2, 3, 4, 5, 6, 7, or 10 times lower than that of the isotype control internalized. The isotype control of the antibody can be selected as is known in the art to match the antibody isotype as closely as possible, but without binding to the target.

According to some embodiments C of the 12th embodiment of aspect 6, the antibody or antigen-binding fragment is characterized by entering cells with endogenous target expression at an internalization rate of 150%, 175%, 200%, 300%, 400%, or 500% lower than that of the isotype control, for example, after 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, or 48 hours.

According to some thirteenth embodiments of the sixth aspect, it can and is suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and/or twelfth embodiments of the sixth aspect, wherein isolated antibodies or antigen-binding fragments induce ADCC and/or ADCP in cells expressing antibody target receptors.

In some preferred embodiments of the 13th embodiment of aspect 6, the antibody or antigen-binding fragment is non-glycosylated.

In some embodiments A1 of the 13th embodiment of aspect 6, the antibody or antigen-binding fragment binds to the human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) of less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM, or 200 nM. Preferably, the KD can be determined using surface plasmon resonance.

In some embodiments B1 of the 13th embodiment of aspect 6, which may be the same as or different from embodiment A1, an antibody or antigen-binding fragment induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing the target receptor via human effector cells (e.g., human NK cells). ADCC induction can be analyzed using assays known in the art, such as those described in Example 10.3.3ff or elsewhere herein. Preferably, the assay is performed using Treg cells, wherein at least 80% or 85% of the Treg cells express CCR8.

In some embodiments C1 of the thirteenth embodiment of the sixth aspect, which may be the same as or different from embodiments A1 or B1, the maximum exhaustion induced by ADCC in cells expressing the target receptor is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%.

In some embodiments D1 of the thirteenth embodiment of the sixth aspect, which may be the same as or different from embodiments A1, B1 or C1, the depleted EC50 induced by ADCC of the target expression cells is less than 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 12.5 pM, 10 pM, 5 pM or 2.5 pM.

In some embodiments A2 of the 13th embodiment of aspect 6, which may be the same as or different from embodiments A1, B1, C1 and/or D1, the antibody or antigen-binding fragment binds to human FcγRIIA (CD32a) with a dissociation constant (KD) of less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

In some embodiments B2 of the thirteenth embodiment of the sixth aspect, which may be identical to embodiments A1, B1, C1 and/or D1, and may be identical to embodiment A2, the antibody or antigen-binding fragment induces antibody-dependent cell-mediated phagocytosis (ADCP) in cells expressing the target receptor via human effector cells (e.g., human macrophages). For example, the human macrophages may be M2 or M1 macrophages.

In some embodiments C2 of the thirteenth embodiment of the sixth aspect, which may be the same as embodiments A1, B1, C1 and/or D1, and may be the same as embodiments A2 and/or B2, the maximum depletion of ADCP-induced cells expressing the target receptor is at least 5, 10, 15, 20, 25, 30, 40 or 50%.

In some embodiments D2 of the thirteenth embodiment of the sixth aspect, which may be the same as embodiments A1, B1, C1 and/or D1, and may be the same as embodiments A2, B2 and/or C2, the depletion EC50 of ADCP-induced activated human regulatory T cells is less than 1500 pM, 1000 pM, 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM or 10 pM.

In some preferred embodiments of the 13th embodiment of aspect 6, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided, wherein said antibody or antigen-binding fragment

a) Binding to human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) below 530 nM, 500 nM, 450 nM, 400 nM, 300 nM, or 200 nM, and/or

b) Binds to human FcγRIIA (CD32a) with dissociation constants (KD) below 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

In some preferred embodiments of the 13th embodiment of aspect 6, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided, wherein said antibody or antigen-binding fragment

a) Inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human chemokine receptors using human effector cells (e.g., human NK cells), and/or

b) Inducing antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human chemokine receptors via human effector cells (e.g., human macrophages).

In some preferred embodiments of the 13th embodiment of aspect 6, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided, wherein

a) The maximum exhaustion induced by ADCC in target cells expressing human chemokine receptors is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, and/or

b) The maximum ADCP-induced exhaustion in target cells expressing human chemokine receptors is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%, and/or

c) The maximum depletion of target cells expressing human chemokine receptors is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%.

In some preferred embodiments of the 13th embodiment of aspect 6, an isolated antibody or antigen-binding fragment thereof that specifically binds to a chemokine receptor is provided, wherein

a) ADCC-induced depletion of EC50 in target cells expressing human chemokine receptors below 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM, or 5 pM and/or

b) ADCP-induced depletion of EC50 in target cells expressing human chemokine receptors below 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, or 25 pM.

According to some of the 14th embodiments of aspect 6, which can and are recommended to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and/or thirteenth embodiments of aspect 6, the isolated antibody or antigen-binding fragment comprises human, rat or mouse IgG antibody, preferably human IgG1 antibody or mouse IgG2a antibody, see Examples 6 and 8.

According to some of the 15th embodiments of aspect 6, which may and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and/or fourteenth embodiments of aspect 6, the isolated antibody or antigen-binding fragment is scFv, Fab, Fab′ or F(ab′)2 fragment, see Example 6.

Antibodies according to the present invention may be conjugated, as discussed elsewhere herein. Antibodies according to the present invention may be used to treat tumors or diseases characterized by the expression of seven-transmembrane receptors, as described elsewhere herein. Antibodies according to the present invention may be used as diagnostic agents in vivo or in vitro, as described elsewhere herein. Furthermore, a kit is provided comprising an antibody according to the present invention and instructions for use.

According to the preferred combination in the sixth aspect

The following embodiments disclose preferred combinations of the sixth aspect, thereby emphasizing the modular nature of the invention. According to preferred embodiment I, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to a first isolated sulfated polypeptide comprising a tyrosine-rich domain (TRD) of a seven-transmembrane receptor and optionally its LID domain, wherein at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated. According to preferred embodiment II, an isolated antibody or antigen-binding fragment according to preferred embodiment I is provided, wherein the cysteine residue between the TRD and LID domains has been removed or exchanged for a different amino acid. According to preferred embodiment III, an isolated antibody or antigen-binding fragment according to preferred embodiment I or II is provided, wherein the seven-transmembrane receptor is a human, cynomolgus monkey, or mouse seven-transmembrane receptor. According to preferred embodiment IV, an isolated antibody or antigen-binding fragment according to any one of preferred embodiments I, II, or III is provided, wherein the seven-transmembrane receptor is a) a C_C chemokine receptor, preferably CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10; b) a CX_C chemokine receptor, preferably CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6; or c) CX₃CR1 or CXCR1. According to preferred embodiment V, an isolated antibody or antigen-binding fragment according to any one of preferred embodiments I, II, III, or IV is provided, wherein the first isolated sulfated polypeptide comprises or is composed of the following sequences:

a. SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 3 or SEQ ID NO: 6, preferably wherein at least Y10 and/or Y18 has been sulfated, or

b. SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 8 or SEQ ID NO: 11, preferably wherein at least Y26 has been sulfated, or

c. SEQ ID NO: 9 or SEQ ID NO: 12, preferably wherein at least Y37 and/or Y39 has been sulfated, or

d. SEQ ID NO: 13 or SEQ ID NO: 16, preferably wherein Y16 and/or Y17 have been sulfated, or

e. SEQ ID NO: 14 or SEQ ID NO: 17, preferably wherein Y16 has been sulfated, or

f. SEQ ID NO: 15 or SEQ ID NO: 18, preferably wherein Y20 and/or Y22 have been sulfated, or

g. SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 21 or SEQ ID NO: 24, preferably wherein at least Y22 has been sulfated, and preferably in addition, Y16, Y19 and/or Y20 have been sulfated; or

h. SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 26 or SEQ ID NO: 29, preferably wherein two, three or all of Y3, Y10, Y14 and Y15 have been sulfated, or

i. SEQ ID NO: 27 or SEQ ID NO: 30, preferably wherein two or three of Y10, Y12 and Y16 have been sulfated, or

j. SEQ ID NO: 31 or SEQ ID NO: 34, preferably wherein at least two or three of Y18, Y26 and Y27 have been sulfated, or

k. SEQ ID NO: 32 or SEQ ID NO: 35, preferably wherein at least two or three of Y23, Y31 and Y32 have been sulfated, or

1. SEQ ID NO: 33 or SEQ ID NO: 36, preferably wherein at least two or three of Y13, Y18 and Y19 have been sulfated, or

m. SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 38 or SEQ ID NO: 41, preferably wherein one or both of Y8 and Y17 have been sulfated, or

n. SEQ ID NO: 39 or SEQ ID NO: 42, preferably wherein one or both of Y8 and Y17 and optionally Y20 have been sulfated, or

o. SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46 or SEQ ID NO: 47, preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or

p. SEQ ID NO: 45 or SEQ ID NO: 48, preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated, or

q. SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 62 or SEQ ID NO: 65, preferably wherein at least Y28, preferably Y17 and/or Y37 have also been sulfated, or

r. SEQ ID NO: 63 or SEQ ID NO: 66, preferably wherein at least Y28 has been sulfated, and preferably Y19 has also been sulfated; or

s. SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 68 or SEQ ID NO: 71, preferably wherein at least one or both of Y14 and Y22 have been sulfated, or

t. SEQ ID NO: 69 or SEQ ID NO: 72, preferably wherein at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or

u. SEQ ID NO: 73 or SEQ ID NO: 76, preferably wherein Y27 has been sulfated, or

v. SEQ ID NO: 74 or SEQ ID NO: 77, preferably wherein at least one of Y14 and Y28 has been sulfated, or

w. SEQ ID NO: 75 or SEQ ID NO: 78, preferably wherein at least Y6 has been sulfated, or

x. SEQ ID NO: 79 or SEQ ID NO: 82, preferably wherein Y23 and/or Y25 have been sulfated, or

y. SEQ ID NO: 80 or SEQ ID NO: 83, preferably wherein Y20 and/or Y22 have been sulfated, or

z. SEQ ID NO: 81 or SEQ ID NO: 84, preferably wherein Y24 has been sulfated, or

aa. SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 87 or SEQ ID NO: 90, preferably wherein at least one or both of Y27 and Y29 have been sulfated, or

bb. SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 92 or SEQ ID NO: 95, preferably wherein at least Y12 and/or Y21 has been sulfated, or

cc. SEQ ID NO: 93 or SEQ ID NO: 96, preferably wherein at least Y23 and/or Y14 has been sulfated, or

dd. SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 98 or SEQ ID NO: 101, preferably wherein at least one of Y3 and Y27 has been sulfated, or

ee.SEQ ID NO: 99 or SEQ ID NO: 102, preferably wherein at least Y3 and/or Y14 and/or Y20 and/or Y6 have been sulfated, or

ff. SEQ ID NO: 103 or SEQ ID NO: 106, preferably wherein at least one or both of Y6 and Y10 have been sulfated, or

gg. SEQ ID NO: 104 or SEQ ID NO: 107, preferably wherein at least two or all of Y4, Y7 and Y39 have been sulfated, or

hh.SEQ ID NO: 105 or SEQ ID NO: 108, preferably wherein at least one or both of Y11 and Y15 have been sulfated, or

ii. SEQ ID NO: 157 or SEQ ID NO: 160, preferably wherein at least Y14 has been sulfated, or

jj.SEQ ID NO: 158, preferably wherein at least Y20 has been sulfated, or

kk.SEQ ID NO: 161, preferably wherein at least Y20 or Y22 has been sulfated, or

11. SEQ ID NO: 159 or SEQ ID NO: 162, preferably wherein at least Y15 has been sulfated, or

mm. SEQ ID NO: 163 or SEQ ID NO: 166, preferably wherein at least Y27 has been sulfated, or

nn.SEQ ID NO: 164, preferably wherein at least Y14 has been sulfated or

oo.SEQ ID NO: 167, preferably wherein at least Y14 or Y28 has been sulfated, or

pp. SEQ ID NO: 165 or SEQ ID NO: 168, preferably wherein at least Y6 has been sulfated.

According to preferred embodiment VI, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I, II, III, IV, or V is provided, wherein the dissociation constant or EC50 of the antibody for binding the first separated sulfated polypeptide and/or the seven-transmembrane receptor is less than 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. According to preferred embodiment VII, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to VI is provided, wherein the separated antibody or antigen-binding fragment specifically binds a second separated sulfated polypeptide containing a seven-transmembrane receptor TRD, preferably wherein the second separated sulfated polypeptide contains a seven-transmembrane receptor of the TRD.

a. Unlike the seven-transmembrane receptor of TRD contained in the first isolated sulfated polypeptide, or

b. is the first isolated sulfated polypeptide containing the corresponding seven-transmembrane receptor of TRD, but from a different species.

According to preferred embodiment VIII, a separated antibody or antigen-binding fragment according to preferred embodiment VII is provided, wherein the dissociation constant or EC50 of the antibody for binding the second separated sulfated polypeptide and/or the antibody for binding the second seventh transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. According to preferred embodiment IX, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to VIII is provided, wherein the dissociation constant (KD) of the antibody for binding the first separated sulfated polypeptide is less than the dissociation constant (KD) of the antibody for binding a first separated non-sulfated peptide having the same sequence as the first separated sulfated polypeptide. According to preferred embodiment X, a separated antibody or antigen-binding fragment according to preferred embodiment IX is provided, wherein the dissociation constant and/or EC50 of the antibody for binding the first separated non-sulfated polypeptide is greater than 150 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or is undetectable. According to preferred embodiment XI, a separated antibody or antigen-binding fragment according to any preferred embodiment IX or X is provided, wherein the dissociation constant or EC50 of the antibody or fragment for binding the first separated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM, and wherein the dissociation constant of the antibody or fragment for binding the first separated non-sulfated polypeptide is greater than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, or 500 nM, or is undetectable. According to preferred embodiment XII, a separated antibody or antigen-binding fragment according to any preferred embodiment I to XI is provided, wherein the antibody comprises a human, rat, or mouse-derived CDR. According to preferred embodiment XIII, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to XII is provided, wherein the antibody...

a. Including human-derived CDRs, and/or

b. Cross-reactivity with humans and cynomolgus monkeys, and/or

c. Characterized by the HCDR3 region comprising 10 to 34% tyrosine and/or 2 to 20% histidine, and/or

d. G protein-independent signaling that does not regulate seven-transmembrane receptors, and/or

e. It is a non-internalizing antibody or is characterized by internalization into cells containing endogenous target expression at a rate of 1.5, 2, 3, 4, 5, 6, 7, or 10 times lower than that of the isotype control, and/or

f. Inducing ADCC and/or ADCP

g, is a human, rat, or mouse IgG antibody, preferably a human IgG1 antibody or a mouse IgG2a antibody, and/or

h is a fragment of scFv, Fab, Fab’, or F(ab’)2.

According to preferred embodiment XIV, a conjugate comprising an antibody or antigen-binding fragment according to any one of preferred embodiments I to XIII is provided, preferably wherein the conjugate comprises

a. Radioactive elements

b. Cytotoxic agents, such as auristatin, maytansinoid, kinin spindle protein inhibitors, nicotinamide phosphoribosyltransferase inhibitors, or pyrrolobenzodiazepine derivatives.

c. Another antibody or antigen-binding fragment, or

d. Chimeric antigen receptor.

According to preferred embodiment XV, an antibody or antigen-binding fragment according to any one of preferred embodiments I to XIII, or a conjugate according to preferred embodiment XIV, is provided for treating tumors or diseases characterized by cells expressing a seven-transmembrane protein, optionally in combination with an antibody targeting a checkpoint inhibitor. According to preferred embodiment XVI, an antibody or antigen-binding fragment according to any one of preferred embodiments I to XIII, or a conjugate according to preferred embodiment XIV, is provided for use as an in vivo or in vitro diagnostic agent. According to preferred embodiment XVII, a kit is provided comprising an antibody or antigen-binding fragment according to any one of preferred embodiments I to XIII, or a conjugate according to preferred embodiment XIV, and instructions for use.

Antibodies that bind to CCR8

The following aspects 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 relate to antibodies that specifically bind to CCR8. While each aspect is described individually, different structural or functional aspects may be combined and are suggested to be combined with each other, unless obviously incompatible. Furthermore, except in cases of obvious incompatibility, each embodiment in each aspect may be combined with embodiments in the same or different aspects.

Unless otherwise specified, CCR8 can be derived from any species, such as human, monkey, macaque (cynomolgus monkey), rhesus monkey, rodent, mouse, rat, horse, cattle, pig, dog, cat, and camel CCR8. Antibodies conjugating CCR8 from at least two species (one of which is human) are highly preferred and are recommended for combination with each embodiment disclosed for these aspects. Antibodies having human-derived CDRs are highly preferred and are recommended for combination with each embodiment disclosed for these aspects. According to the present aspects, antibodies having an HCDR3 domain characterized by a frequency of at least 21% tyrosine residues and/or at least 2%, 7%, or 10% histidine residues are highly preferred and are recommended for combination with each embodiment for the following aspects. According to the present aspects, antibodies that simultaneously induce ADCC and ADCP, such as non-fucosylated antibodies, are highly preferred and are recommended for combination with each embodiment disclosed for these aspects. According to the present aspects, low-internalization or non-internalization antibodies or fragments are highly preferred and are recommended for combination with each embodiment disclosed for these aspects. Some highly preferred features applicable to each of the described aspects are described in the section “Preferred combinations according to ‘all aspects’”.

7-CCR8 antibody binding to sulfated TRD

The extracellular domain of human CCR8 can be divided into four regions:

(i) The N-terminal structural domain can be further subdivided into

a) The distal end of the membrane formed by amino acids 1 to 24 is rich in tyrosine domains (TRD) (SEQ ID NO: 43)

b) Cysteine at amino acid position 25, and

c) The LID domain, formed by amino acids 26 to 35 (SEQ ID NO: 49)

(iii) Based on extracellular domain 1 (ECL1) of SEQ ID NO: 52,

(iii) Based on the extracellular domain 2 (ECL2) of SEQ ID NO: 55, and

(iv) Extracellular domain 3 (ECL3) according to SEQ ID NO: 58.

According to the seventh aspect, which may be the same as or different from the sixth aspect, a separate antibody or its antigen-binding fragment specifically binds to the sulfated tyrosine enrichment domain of CCR8.

For example, an antibody or antigen-binding fragment binds to a first isolated sulfated polypeptide, said polypeptide comprising...

a) The tyrosine-rich domain (TRD) of CCR8 or

b) The N-terminus of CCR8 containing TRD,

Optionally, at least the cysteine residue between the TRD and LID domains has been removed or replaced with a different amino acid. A sulfated polypeptide according to the present aspect is a polypeptide in which preferably at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated. When the polypeptide contains the N-terminus of a CCR8 containing the TRD, preferably the cysteine residue between the TRD and LID domains has been replaced with a serine residue or has been removed.

Without being bound by theory, the specific recognition of the provided CCR8 sulfation pattern appears to influence whether the antibody competes with CCL1 (the natural ligand of CCR8) and whether and how the antibody stimulates or antagonizes CCR8 signaling, as described in aspect 11.

In some preferred embodiments according to the present aspect, the antibody of the present invention binds to sulfated TRD of human and/or cynomolgus monkey CCR8 with a KD value of <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1.5E-9M, <1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, or <9E-11M. For example, the antibodies of the present invention bind to sulfated TRDs of human and/or cynomolgus monkey CCR8 with KD values between <8E-9M and >4E-10M. For example, the antibodies of the present invention can bind to sulfated TRDs of human and/or cynomolgus monkey CCR8 with KD values between <8E-9M and >5.5E-10M. In some further preferred embodiments of these embodiments, the antibodies of the present invention bind to the N-terminus of human and/or cynomolgus monkey CCR8 (i.e., containing the aforementioned sulfated TRD) with substantially the same KD value. In some most preferred embodiments of these embodiments, the antibodies of the present invention substantially do not bind to non-sulfated TRDs of human and/or cynomolgus monkey CCR8.

In some preferred embodiments according to the present aspect, the antibody of the present invention binds to the sulfated N-terminus of human and/or cynomolgus monkey CCR8 with a KD value of <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, < 1.5E-9M, <1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, <9E-11M, <8E-11M, <7E-11M, <6E-11M, or <5E-11M. For example, the antibody of the present invention can bind to the sulfated N-terminus of human and/or cynomolgus monkey CCR8 at KD values between <8E-9M and >8E-11M. In some further preferred embodiments of these embodiments, the antibody of the present invention binds to the sulfated TRD (i.e., the subsequence of the sulfated N-terminus described above) of human and/or cynomolgus monkey CCR8 at substantially the same KD value or within the same order of magnitude. In some of the most preferred embodiments of these implementations, the antibody of the present invention is substantially non-sulfated TRD of human and/or cynomolgus monkey CCR8.

According to some first embodiments of aspect 7, the first isolated sulfated polypeptide includes

a) SEQ ID NO: 43 (CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated.

b) SEQ ID NO: 44 (CCR8_MACFA_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

c) SEQ ID NO: 45 (CCR8_MOUSE_TRD), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

According to some second embodiments of aspect 7, the first isolated sulfated polypeptide includes

a) SEQ ID NO: 46 (CCR8_HUMAN_N term, C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated.

b) SEQ ID NO: 47 (CCR8_MACFA_N term, C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

c) SEQ ID NO: 48 (CCR8_MOUSE_N term, c = X or S), preferably wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

According to some third embodiments of the seventh aspect, which may be the same as or different from the first and/or second embodiments, the antibody or antigen-binding fragment binds to the first isolated sulfated polypeptide with a dissociation constant or EC50 of <15nM, <10nM, <5nM, <1nM or <0.6nM.

According to some preferred embodiments of these embodiments, the isolated antibody or its antigen-binding fragment binds specifically with a dissociation constant or EC50 of <15 nM, <10 nM, <5 nM, <1 nM, or <0.6 nM.

a) Human CCR8 or the isolated polypeptide according to SEQ ID NO: 46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated.

b) Cynomolgus monkey CCR8 or the polypeptide isolated according to SEQ ID NO: 47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

c) Mouse CCR8 or a polypeptide isolated according to SEQ ID NO: 48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

According to some fourth embodiments of the seventh aspect, it may and is suggested to be combined with the first, second and/or third embodiments of the seventh aspect, wherein the dissociation constant (KD) of the antibody for binding the first isolated sulfated polypeptide is lower than the dissociation constant (KD) of the antibody for binding the isolated non-sulfated polypeptide having the same sequence as the first isolated sulfated polypeptide.

According to some embodiments A of these fourth embodiments, the dissociation constant or EC50 of the antibody used to bind the isolated non-sulfated peptide is higher than 1 pM, 10 nM, 25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM, or 3 μM, or is undetectable. Preferably, the dissociation constant or EC50 of the antibody used to bind the isolated non-sulfated peptide is higher than 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or is undetectable.

According to some embodiments B of these fourth embodiments, which may be the same as embodiments A of these second embodiments, the dissociation constant or EC50 of the antibody used to bind the first isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the dissociation constant or EC50 of the antibody used to bind the isolated non-sulfated polypeptide is greater than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM or 500 nM, or is undetectable.

Aspect 8 - CCR8 antibody containing human CDR

According to aspect 8, which may be the same as or different from aspect 6 or 7, an antibody or antigen-binding fragment thereof that binds to CCR8 isolates is provided (particularly) wherein the antibody comprises a human CDR. Most preferably, the antibody or antigen-binding fragment thereof binds (particularly) a sulfated TRD of CCR8. According to some preferred embodiments, the deviation of a single CDR from the nearest human germline is less than 1, 2, 3, 4, 5, or 6. According to some highly preferred embodiments, the deviation of the human CDR from the nearest human germline is no more than or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The nearest human germline can be determined as is known in the art, for example using IgBLAST with data retrieved from the IMGT human germline database, as discussed elsewhere herein.

Although humanization of antibodies with non-human CDRs can enhance immunogenicity, residual immunogenicity remains in the CDR regions (Harding, Fiona A., et al. "The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions." MAbs. Vol. 2. No. 3. Taylor & Francis, 2010). Therefore, antibodies containing human CDRs and minimal germline bias are considered to have superior suitability as therapeutic agents according to current understanding.

Antibodies containing human CDRs can be obtained as described herein, for example, by using human phage display libraries as described in Examples 4 and 6. Alternatively, antibodies containing human CDRs can also be produced using transgenic mice that do not express functional endogenous immunoglobulins but do express human immunoglobulin genes. Furthermore, antibodies containing human CDRs can also be generated using in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275, the entire contents of which are incorporated herein by reference).

The provided isolated antibody or antigen-binding fragment containing a human CDR is preferably an antibody or combination thereof according to any one of aspects 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

Aspect 9 - Cross-reactive CCR8 antibody

According to the ninth aspect, which may be the same as or different from the sixth, seventh, and/or eighth aspects, a separated antibody or antigen-binding fragment thereof specifically binding to CCR8 is provided, wherein the antibody or antigen-binding fragment is cross-reactive to CCR8 from at least two species, preferably selected from humans, monkeys, macaques (cynomolgus monkeys), rhesus monkeys, rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels, and even more preferably selected from humans, cynomolgus monkeys, and mice. According to some of the most preferred embodiments, the antibody or antigen-binding fragment is cross-reactive to human and cynomolgus monkey CCR8.

In a preferred embodiment, the antibody or antigen-binding fragment binds to CCR8 from a first species with a first dissociation constant KD and to CCR8 from a second species with a second dissociation constant KD, wherein the first and second dissociation constants are on the same order of magnitude. As those skilled in the art will understand, the order of magnitude difference between the two values is 10-fold.

Cross-reactive anti-CCR8 antibodies are advantageous for the development of therapeutic antibodies because they can be used in non-human animal models to characterize therapeutic agents in terms of pharmacological data and safety before administration to humans. However, cross-reactive antibodies with similar binding behavior in two species are difficult to generate because the portions of CCR8 available for antibody recognition have low homology between species (see Example 2). According to the invention, cross-reactive antibodies against CCR8 can be generated by using small sulfated tyrosine residues containing motifs that are more conserved between species, making it easy and convenient to obtain cross-reactive antibodies that bind to CCR8 from two or more species with the same order of magnitude affinity. More specifically, antibodies specifically bind sulfated TRD motifs, and cross-reactive antibodies can be obtained because these sulfated TRD motifs are conserved between species.

In some preferred embodiments according to the present aspect, the antibody of the present invention binds to the sulfated TRD of human and cynomolgus monkey CCR8 with a KD value of <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1.5E-9M, <1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, or <9E-11M. For example, the antibody of the present invention can bind to the sulfated TRD of human and cynomolgus monkey CCR8 with a KD value between <8E-9M and >4E-10M. For example, the antibody of the present invention can bind to the sulfated TRD of human and cynomolgus monkey CCR8 with a KD value between <8E-9M and >5.5E-10M. In some further preferred embodiments of these embodiments, the antibody of the present invention binds to the sulfated N-terminus of human and cynomolgus monkey CCD8 (i.e., containing the aforementioned sulfated TRD) with substantially the same KD value or an order of magnitude higher. In the most preferred embodiment of these embodiments, the antibody of the present invention substantially does not bind to the non-sulfated TRD of human and cynomolgus monkey CCR8.

In some preferred embodiments according to the present aspect, the antibody of the present invention binds to the sulfated N-terminus of human and cynomolgus monkey CCR8 with a KD value of <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1 <5E-9M, <1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, <9E-11M, <8E-11M, <7E-11M, <6E-11M, or <5E-11M. For example, the antibody of the present invention can bind to the sulfated N-terminus of human and cynomolgus monkey CCR8 at KD values between <8E-9M and >8E-11M. In some further preferred embodiments of these embodiments, the antibody of the present invention binds to the sulfated TRD (i.e., the subsequence of the sulfated N-terminus described above) of human and cynomolgus monkey CCR8 at substantially the same KD value or the same order of magnitude. In some of the most preferred embodiments of these implementations, the antibody of the present invention is substantially non-sulfated TRD of human and cynomolgus monkey CCR8.

According to some first embodiments of aspect 9, antibody-specific binding

a) A first isolated sulfated polypeptide comprising SEQ ID NO: 46 (CCR8_HUMAN_N term, C=X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated; and a second isolated sulfated polypeptide comprising SEQ ID NO: 47 (CCR8_MACFA_N term, C=X or S), preferably wherein at least two or all of Y3, Y5 and Y17 have been sulfated; or

b) A first isolated sulfated polypeptide comprising SEQ ID NO: 43 (CCR8_HUMAN_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 44 (CCR8_MACFA_TRD), preferably wherein at least two or all of Y3, Y5 and Y17 have been sulfated.

Preferably, the EC50 of the antibody or antigen-binding fragment used to bind human CCR8 or to bind the first isolated sulfated polypeptide is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM, for example less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. Preferably, the EC50 of the antibody or antigen-binding fragment used to bind cynomolgus monkey CCR8 or to bind the second isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. In some preferred embodiments, the antibodies bind the first isolated sulfated polypeptide and the second isolated sulfated polypeptide with substantially the same affinity.

According to some second embodiments of aspect 9, antibody-specific binding

a) A first isolated sulfated polypeptide comprising SEQ ID NO: 46 (CCR8_HUMAN_N term, C=X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 48 (CCR8_MOUSE_N term, C=X and S), preferably wherein at least two or all of Y3, Y4 and Y15 have been sulfated; or

b) A first isolated sulfated polypeptide comprising SEQ ID NO: 43 (CCR8_UMAN_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO: 45 (CCR8_MOUSE_TRD), preferably wherein at least two or all of Y13, Y14 and Y15 have been sulfated.

Preferably, the EC50 of the antibody or antigen-binding fragment used to bind human CCR8 or to bind the first isolated sulfated polypeptide is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM, for example less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. Preferably, the EC50 of the antibody or antigen-binding fragment used to bind mouse CCR8 or to bind the second isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM. In some preferred embodiments, the antibodies bind the first isolated sulfated polypeptide and the second isolated sulfated polypeptide with substantially the same affinity.

The provided isolated antibody or antigen-binding fragment is preferably also an antibody or combination thereof according to any one of aspects 7, 8, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

Aspect 10 - CCR8 antibody defined by HCDR3 structure

According to aspect ten, which may be identical or different from aspects six, seven, eight, and/or nine, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein said antibody or antigen-binding fragment is characterized by an HCDR3 region having increased histidine and/or tyrosine frequencies. As will be understood by those skilled in the art, although the frequencies of each amino acid are discussed individually, the frequencies are interrelated and are suggested to be combined unless their incompatibility is immediately apparent to those skilled in the art. For the determination of the CDR region, the Kabat immunoglobulin amino acid residue numbering system is used herein, and should be definitive in case of doubt.

Surprisingly, the CCR8 antibody according to the invention is characterized by a significantly higher frequency of tyrosine residues in HCDR3 than that in random whole-human HCDR3 of matched length (approximately 10%, see Zemlin, Michael, et al. "Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures." Journal of molecular biology 334.4 (2003): 733-749). On average, the initial set of antibodies of the invention contains approximately 20.6% tyrosine residues, while the optimized set of specific human CCR8 binding antibodies contains an average of 21% tyrosine residues in HCDR3.

Furthermore, the number of positively charged amino acids, particularly histidine residues, was higher than expected, especially in the optimized antibody group with improved therapeutic characteristics (see Example 9). On average, when summarized, histidine and tyrosine residues accounted for 31% of all amino acids in HCDR3 of the CCR8-binding antibody in the optimized group.

Interestingly, the antigens used containing sulfated TRDs are characterized by tyrosine residues and a particularly high number of negative charges, which are further increased by sulfation. Without being bound by theory, the inventors believe that the preference for high tyrosine and histidine content in the CDRH3 of the antibodies of the present invention is caused by specific sulfated motifs of the antigen, and that the high tyrosine/histidine content in HCDR3 promotes specific antigen recognition. Without being bound by theory, the inventors further believe that this specific recognition affects the properties of the obtained antibodies as regulators of CCR8, as discussed, for example, in aspect 11.

In some highly preferred embodiments of the tenth aspect, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or fragment is characterized in the HCDR3 region, which contains 10% to 34% tyrosine and/or 2% to 20% histidine.

The provided isolated antibody or antigen-binding fragment is preferably also an antibody or combination thereof according to any one of aspects 7, 8, 9, 11, 12, 13, 14, 15, 16, 17 or 18.

In some first embodiments of the tenth aspect, the isolated antibody or antigen-binding fragment contains HCDR3 as follows.

a) >0 and <35%, >8 and <34%, ≥10 and ≤34%, or >15 and <31% of tyrosine (Y) residues, and/or

b) >0 and ≤16%, ≥2 and ≤20%, or ≥7 and ≤20% of histidine (H) residues, and

c) Preferably ≥0 and ≤18%, ≥7 and ≤10%, or ≥0 and ≤7% arginine (R) residues, and/or

d) Preferably, ≥0 and ≤25%, ≥7 and ≤16%, or ≥7 and ≤13% of aspartic acid (D) residues, and/or

e) Preferably, there are no lysine (K) residues, and/or

f) Preferably, there are no glutamic acid (E) residues.

In some second embodiments of the tenth aspect, which may be the same as or different from the first embodiment, the isolated antibody or antigen-binding fragment contains HCDR3 having the following...

a) >0 and <47%, >22 and <50%, >10 and <34%, or >15 and <47% charged amino acids, and/or

b) >0 and <32%, >8 and <30%, or >10 and <37% of positively charged amino acids, and/or

c) >0 and <26%, ≥7 and <16%, or >7 and <14% of negatively charged amino acids.

In certain third embodiments of the tenth aspect, which may be the same as or different from the first and/or second embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having a total histidine and tyrosine residues of >0 and ≤42%, ≥10 and ≤42%, or ≥36 and ≤43%.

Tyrosine (Y)

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 33%, 31%, 25%, 23%, 21%, 20%, 18%, 15%, 10%, 9%, 8%, or 0% tyrosine residues. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having (a) >0 and <35%, (b) >8 and <34%, (c) >10 and <34%, or (d) >15 and <31% tyrosine residues.

In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having >8, >9, >10, >11, >12, >13, >14, >15, >16, >17, >18, >19, >20, >21, >22, >23, >24, >25, >26, >27, >28, >29, >30, >31, >32, >33% of tyrosine residues. In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having <35, <34, <33, <32, <31, <30, <29, <28, <27, <26, <25, <24, <23, <22, <21, <20, <19, <18, <17, <16, <15, <14, <13, <12, <11, <10, <9, or <8% of tyrosine residues.

In some embodiments, the isolated antibody or antigen-binding fragment comprises fragments having the following characteristics: >0 and <34%, >1 and <34%, >2 and <34%, >3 and <34%, >4 and <34%, >5 and <34%, >6 and <34%, >7 and <34%, >8 and <34%, >9 and <34%, >10 and <34%, >11 and <34%, >12 and <34%, >13 and <34%, >14 and <34%, >15... HCDR3 with tyrosine residues <34%, >16 and <34%, >17 and <34%, >18 and <34%, >19 and <34%, >20 and <34%, >21 and <34%, >22 and <34%, >23 and <34%, >24 and <34%, >25 and <34%, >26 and <34%, >27 and <34%, >28 and <34%, >29 and <34% or >30 and <34%.

In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having >0 and <36%, >0 and <35%, >0 and <34%, >0 and <33%, >0 and <32%, >0 and <31%, >0 and <30%, >0 and <29%, >0 and <28%, >0 and <27%, >0 and <26%, >0 and <25%, >0 and <24%, >0 and <23%, >0 and <22%, >0 and <21%, >0 and <20%, >0 and <19%, >0 and <18%, >0 and <17%, >0 and <16%, or >0 and <15%, >0 and <14%, >0 and <13%, >0 and <12%, >0 and <11%, >0 and <10%, >0 and <9%, >0 and <8%, >0 and <7%, >0 and <6%, >0 and <5% tyrosine residues.

Charged aa

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 47%, 39%, 31%, 28%, 27%, 25%, 23%, 20%, 18%, 17%, 16%, 15%, 9%, or 0% charged amino acids. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having (a) >0 and <47%, (b) >22 and <50%, (c) >10 and <34%, or (d) >15 and <47% charged amino acids.

positive charge aa

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 31%, 23%, 18%, 15%, 10%, 8%, 7%, or 0% of positively charged amino acids. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having (a) >0 and <32%, (b) >8 and <30%, or (c) >10 and <37% of positively charged amino acids.

negative charge aa

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 25%, 17%, 15%, 11%, 10%, 9%, 8%, 7%, or 0% of negatively charged amino acids. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having (a) >0 and <26%, (b) ≥7 and <16%, or (c) >7 and <14% of negatively charged amino acids.

Histidine (H)

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 0%, 3%, 7%, 8%, 10%, or 15% histidine residues. Preferably, the isolated antibody or antigen-binding fragment comprises HCDR3 having at least one histidine residue. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having (a) >0 and ≤16%, (b) ≥2 and ≤20%, or (c) ≥7 and ≤20% histidine residues.

In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having ≥0, >0, >1, >2, >3, >4, >5, >6, >7, >8, >9, >10, >11, >12, >13, >14, >15, >16, >17, >18, >19, >20% histidine residues. In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having <20, <19, <18, <17, <16, <15, <14<13, <12, <11, <10, <9, <8, <7, <6, <5, <4, <3, <2, or <1% histidine residues.

In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having >0 and <24%, >1 and <24%, >2 and <24%, >3 and <24%, >4 and <24%, >5 and <24%, >6 and <24%, >7 and <24%, >8 and <24%, >9 and <24%, >10 and <24%, >11 and <24%, >12 and <24%, >13 and <24%, >14 and <24%, >15 and <24%, >16 and <24%, >17 and <24%, >18 and <24%, >19 and <24%, >20 and <24%, >21 and <24%, >22 and <24% or >23 and <24% histidine residues.

In some embodiments, the isolated antibody or antigen-binding fragment contains HCDR3 having >0 and <24%, >0 and <23%, >0 and <22%, >0 and <21%, >0 and <20%, >0 and <19%, >0 and <18%, >0 and <17%, >0 and <16%, or >0 and <15%, >0 and <14%, >0 and <13%, >0 and <12%, >0 and <11%, >0 and <10%, >0 and <9%, >0 and <8%, >0 and <7%, >0 and <6%, >0 and <5%, >0 and <4%, >0 and <3%, >0 and <2% or >0 and <1% histidine residues.

Histidine + Tyrosine

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having a total histidine and tyrosine residue content of 42%, 38%, 33%, 31%, 30%, 24%, 23%, 18%, 15%, 10%, 9%, or 0%. Preferably, the isolated antibody or antigen-binding fragment comprises HCDR3 having at least one histidine residue. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having a total histidine and tyrosine residue content of (a) > 42%, (b) ≥ 10% and ≤ 42%, or (c) ≥ 36% and ≤ 43%.

Arginine (R)

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 0%, 7%, 8%, 10%, 15%, or 18% arginine residues. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having (a) >0 and ≤18%, (b) ≥7 and ≤10%, or (c) ≥0 and ≤7% arginine residues.

Lysine (K)

In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 0% to 8% lysine residues. In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 without lysine residues.

Aspartic acid (D)

In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 0%, 7%, 8%, 9%, 10%, 11%, 15%, 16%, 17%, or 25% aspartic acid residues. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having (a) >0 and ≤25%, (b) ≥7 and ≤16%, or (c) ≥7 and ≤13% aspartic acid residues.

Glutamic acid (E)

In some embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 having 0%, 8%, or 9% glutamate residues. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises HCDR3 without glutamate residues.

length

In some embodiments, the isolated antibody or antigen-binding fragment comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Preferably, the isolated antibody or antigen-binding fragment comprises HCDR3 having 10, 11, 12, or 13 amino acid residues or a length of 8 to 13 amino acid residues.

Aspect 11 - Blocking/Neutral CCR8 Antibody

Antibodies regulate CCR8 signaling in several ways. For example, antibodies can...

a) Block G protein-independent signal transduction.

b) Block G protein-dependent signal transduction,

c) Block both G protein-dependent and G protein-independent signal transduction.

d) Increase G protein-independent signaling.

e) Increase G protein-dependent signaling,

f) Increase both G protein-dependent and G protein-independent signaling.

According to the eleventh aspect, which may be the same as or different from the sixth, seventh, eighth, ninth and/or tenth aspects, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment at least partially modulates CCR8 signaling.

For CCR8, the G protein-independent signaling pathways are β-arrestin signaling (Example 10.4.1), Erk1/2 phosphate signaling (phosphorylation of Erk1/2), and Akt phosphate signaling (phosphorylation of AKT) (Example 10.4.2). Other subtypes of antagonists or agonists can be defined based on their effects on these three G protein-independent signaling pathways.

According to some first embodiments of aspect 11, an antibody or antigen-binding fragment

a) Does not block CCL1-induced β-arrestin signaling and/or

b) Does not induce ERK1/2 phosphorylation and/or

c) Does not induce AKT phosphorylation.

Example 10.4.1 shows that prior art antibodies 433H and L268G8 effectively block CCL1-induced β-arrestin signaling, for example, with IC50 values below 20 nM (see Table 10.4.1.1), while the IC50 value of the antibodies of the present invention, such as TPP-23411, cannot be determined.

According to some embodiments A of the first embodiment of the eleventh aspect, the antibody or antigen-binding fragment does not block CCL1-induced β-arrestin signaling. In doubt, if the IC50 is less than 100 nM, the antibody blocks CCL1-induced β-arrestin signaling. In doubt, if the IC50 is ≥ 100 nM, or if the IC50 cannot be determined using the assay system, the antibody does not block CCL1-induced β-arrestin signaling. As described in the 12th aspect, CCL1-induced β-arrestin signaling is related to receptor internalization.

Examples 10.4.2 and Figures 27 and 28 show that prior art antibodies induce phosphorylation of Erk1/2 in CHO cells expressing human CCR8 or in human activated Tregs, for example, by at least 1.5-fold after 15 minutes. In contrast, the antibody TPP-23411 of the present invention does not induce significant phosphorylation of Erk1/2.

According to some embodiments B of the first embodiment of the eleventh aspect, the antibody or antigen-binding fragment does not induce ERK1/2 phosphorylation. In doubt, if—using the assay described herein—an increase in Erk1/2 phosphorylation level of at least 1.5 times compared to the control can be detected, then the antibody induces Erk1/2 phosphorylation. In doubt, if no significant increase in Erk1/2 phosphorylation level is detected, or if the increase is less than 1.5 times that of the control, then the antibody does not induce Erk1/2 phosphorylation.

Example 10.4.2, Figure 30 shows that prior art antibodies induce phosphorylation of AKT, for example, by at least 1.5-fold after 15 minutes. In contrast, the antibody TPP-23411 of the present invention does not induce significant phosphorylation of AKT.

According to some embodiments C of the first embodiment of aspect 11, the antibody or antigen-binding fragment does not induce AKT phosphorylation. In doubt, if an increase in AKT phosphorylation level of at least 1.5 times compared to the control can be detected using the assay described herein, then the antibody induces AKT phosphorylation. In doubt, if no significant increase in AKT phosphorylation level is detected or the increase is less than 1.5 times that of the control, then the antibody does not induce AKT phosphorylation.

Antibodies or fragments that do not induce G protein-independent signaling pathways (such as AKT or ERK1/2 phosphorylation) are considered to have an advantage in treatment because inducing G protein-dependent signaling pathways may lead to unwanted effects and side effects.

To analyze G protein-dependent signaling, known and described Ca flux assays in the art can be used as reads. Most of the antibodies tested in this invention have been found to be fully effective antagonists of G protein-dependent Ca signaling; however, differences in IC50 can be identified compared to prior art antibodies.

Example 10.4.3 shows that several tested anti-CCR8 antibodies blocked CCL1-induced G protein-dependent Ca signaling, for example, with IC50 values in the low nM or even sub-nM range.

According to some second embodiments of the eleventh aspect, which may be the same as or different from the first embodiment, the antibody or antigen-binding fragment blocks CCL1-induced calcium signaling. In some preferred embodiments of these embodiments, the antibody or antigen-binding fragment blocks CCL1-induced calcium signaling, for example, IC50 < 1 nM, < 0.5 nM, or < 0.01 nM.

According to some third embodiments of the eleventh aspect, which may be the same as or different from the first embodiment, the antibody or antigen-binding fragment does not block CCL1-induced G protein-dependent calcium signaling.

Aspect 12 - No and/or low internalization of CCR8 antibodies

Anti-CCR8 antibodies can be non-internalized, low-internalized, moderately internalized, or highly internalized antibodies or antigen-binding fragments.

According to the twelfth aspect, which may be the same as or different from the sixth, seventh, eighth, ninth, tenth and/or eleventh aspects, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment is a non-internalized or low-internalized antibody or antigen-binding fragment.

Depending on the specific mode of action of the antibody, fragment, or conjugate, internalization into the cell may be necessary or necessary to avoid, as discussed elsewhere in this document. Antibodies in aspect 12 are particularly suitable for ADCC/ADCP methods, or any other mode of action that relies on the recruitment of effector cells by antibody or antigen-binding fragments.

Because overexpression can affect internalization behavior and is not well-suited for simulating internalization in a therapeutic setting, internalization is preferably determined using model cell lines with endogenous expression of the target. When the target is human CCR8, HuT78 cells are preferred for endogenous target expression. When the target is mouse CCR8, mouse BW5147.3 cells are preferred. Both HuT78 and mBW5147.3 are available from ATCC.

While all prior art antibodies readily internalize into cells expressing endogenous targets, as shown in Example 10.5, the tested antibodies TPP-21360, TPP-21047 (data not shown), and TPP-23411, as well as various other antibodies according to the invention, did not internalize significantly.

For example, internalization can be determined over a time span or at a specific time point. Preferably, internalization can be determined in cells that endogenously express the target at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, or 48 hours.

According to some first embodiments of the twelfth aspect, the antibody or antigen-binding fragment is characterized by being internalized into cells expressing an endogenous target at a rate of 1.5, 2, 3, 4, 5, 6, 7, or 10 times lower than that internalized in an isotype control. An isotype control of the antibody can be selected, as is known in the art, to match the antibody isotype as closely as possible, but without binding to the target.

According to some of these embodiments, the antibody or antigen-binding fragment is characterized by being internalized into cells expressing the endogenous target at a rate of 150%, 175%, 200%, 300%, 400%, or 500% lower than that of the isotype control, for example, after 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, or 48 hours, preferably, wherein the cells expressing the endogenous target are HuT78 lymphoma cells.

According to some preferred embodiments of these implementations, the antibody or antigen-binding fragment has an internalization rate that is on the same order of magnitude as that of the isotype control.

According to some second embodiments of the twelfth aspect, which may be the same as or different from the first embodiment, the antibody or antigen-binding fragment specifically binds to human CCR8 and is characterized in that, in cells endogenously expressing the target, the time until half of the amount of antibody, fragment, or conjugate has been internalized is >2 hours, preferably >4, >5, >6, >7, >8, >9, >10, >11, >12, >13, >14, >15, >16, >17, >18, >19, >20, >21, >22, >23, >24, >26, >28, >30, or >48 hours, or such a time cannot be completely determined. Preferably, the cells endogenously expressing the target are HuT78 lymphoma cells. For example, the internalization rate of anti-CCR8 antibodies is lower than that of antibodies 433H and L263G8 (BioLegendCat.No.360602).

According to some third embodiments of the twelfth aspect, which may be the same as or different from the first or second embodiments, the antibody or antigen-binding fragment specifically binds to mouse CCR8 and has a lower internalization rate than antibody SA214G2 in cells that endogenously express the target, preferably wherein the cells that endogenously express the target are mouse lymphoma cell line BW5147.3.

The provided isolated antibody or antigen-binding fragment is preferably an antibody according to any one or a combination of aspects 7, 8, 9, 10, 11, 13, 14, 15, 16, 17 or 18.

Aspect 13-ADCC/ADCP induces CCR8 antibody

To induce the killing of CCR8-expressing cells (such as activated Tregs), several modes of action can be envisioned. One mode of action involves conjugating an antibody targeting CCR8 with a drug to form an antibody-drug conjugate (ADC). Other possible modes of action are ADCC, CDC, and ADCP. For ADCC, CDC, and ADCP, a two-step mechanism is involved: on the one hand, the antibody or fragment needs to effectively bind to the target cell, for example, via Tregs of CCR8; on the other hand, the FC portion of the antibody (or an alternative binding portion that can be conjugated with the antibody or fragment, as described elsewhere in this document) must bind to the effector cell, thus mediating the killing of the target cell. For ADCP, binding to macrophages as effector cells typically occurs through the interaction of the antibody's FC portion with FcγRIIa (CD32a) expressed by macrophages. Conversely, ADCC is mediated through the interaction of an antibody or fragment with FcγRIIIa. In humans, FcγRIII exists in two distinct forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). FcγRIIIa, as a transmembrane receptor, is expressed on mast cells, macrophages, and natural killer cells, while FcγRIIIb is expressed only on neutrophils. These receptors bind to the Fc moiety of IgG antibodies, thereby activating antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

If in doubt, when analyzing ADCC and/or ADCP induction, at least 80%, preferably at least 85%, of the target cells should show CCR8 expression.

According to aspect 13, which may be the same as or different from aspects 6, 7, 8, 9, 10, 11 and/or 12, a separate antibody or antigen-binding fragment thereof specifically binds to CCR8, wherein the antibody or antigen-binding fragment induces ADCC and/or ADCP.

For example, isolated antibodies or antigen-binding fragments thereof that specifically bind to CCR8 are provided, wherein the antibody or antigen-binding fragment is non-fucosylated and induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells such as human NK cells, and b) induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells such as human macrophages. Preferably, the maximum ADCC and ADCP induce in vitro depletion of target cells expressing human CCR8 at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%. In case of doubt, target cells with at least 85% CCR8 expression should be used to determine ADCC and ADCP.

In some preferred embodiments of aspect thirteen, the antibody or antigen-binding fragment is non-fucosylated. The non-fucosylated antibody is an engineered antibody such that the oligosaccharide in the Fc region of the antibody does not contain any fucosylate units. Non-fucosylated antibodies can be obtained as is known in the art, for example as described in Example 10.3. Example 10.3.2 demonstrates improved binding to FcγRIIIa in the non-fucosylated form of the antibody of the present invention. Binding to FcγRIIa is also slightly improved. Non-fucosylation also increases binding to cynomolgus monkey FcγRIII, see Table 10.3.2.1.

ADCC

In some first embodiments A of aspect 13, the antibody or antigen-binding fragment binds to human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) of less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM.

In some first embodiments B of aspect 13, which may be the same as or different from first embodiment A, an antibody or antigen-binding fragment induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells, such as human NK cells. ADCC induction can be analyzed using assays known in the art, for example, as described in Example 10.3.3ff.

In some first embodiments C of aspect 13, which may be the same as or different from first embodiments A or B, the maximum exhaustion of ADCC-induced activated human regulatory T cells is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, preferably wherein at least 85% of the activated human regulatory T cells express CCR8, see Tables 10.3.3.1.2 and 10.3.3.1.3.

In some first embodiments D of aspect 13, which may be the same as or different from first embodiments A, B, or C, the EC50 of ADCC-induced activated human regulatory T cells is below 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 12.5 pM, 10 pM, 5 pM, or 2.5 pM. Preferably, at least 85% of the activated human regulatory T cells express CCR8.

ADCP

In some second embodiments A of aspect 13, which may be the same as or different from first embodiments A, B, C and/or D, the antibody or antigen-binding fragment binds to human FcγRIIA with a dissociation constant (KD) of less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

In some second embodiments B of aspect 13, which may be identical to first embodiments A, B, C and/or D, and may be identical to second embodiment A, the antibody or antigen-binding fragment induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells, such as human macrophages. For example, the human macrophages may be M2c or M1 macrophages.

In some second embodiments C of aspect 13, which may be the same as first embodiments A, B, C and/or D, and may be the same as second embodiments A and/or B, the maximum exhaustion of ADCP-induced activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%.

In some second embodiments D of aspect 13, which may be the same as first embodiments A, B, C and/or D, and may be the same as second embodiments A, B and/or C, the ADCP-induced activation of human regulatory T cells exhaustion is below 1500 pM, 1000 pM, 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM or 10 pM.

In some preferred embodiments, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment

a) Binding to human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) below 530 nM, 500 nM, 450 nM, 400 nM, 300 nM, or 200 nM, and/or

b) Bind human FcγRIIA (CD32a) with a dissociation constant (KD) of less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

In some preferred embodiments, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment

a) Inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells (e.g., human NK cells), and/or

b) Inducing antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 using human effector cells (such as human macrophages).

In some preferred embodiments, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein

a) The maximum exhaustion of ADCC-induced activated human regulatory T cells is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, and/or

b) The maximum exhaustion of ADCP-induced activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%, and/or

c) The maximum depletion of regulatory T cells within the tumor, either in vitro or in the subject, is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

In some preferred embodiments, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein

a) ADCC-induced depletion of activated human regulatory T cells with EC50 values below 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM, or 5 pM and/or

b) ADCP-induced activated human regulatory T cells with depleted EC50 below 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, or 25 pM.

The provided isolated antibody or antigen-binding fragment is preferably an antibody according to any one of aspects 14, 15, 16, 17 or 18 or a combination thereof.

Aspect 14-TREG regulatory CCR8 antibody

The in vivo therapeutic effects of the antibody according to the invention are shown in Example 12ff. The ability of the antibody to regulate the absolute and relative numbers of several immune cell populations is structurally achieved through a combination of target-binding properties and FC receptor interactions, for example as described according to aspects 7, 10, 12 and/or 13 or combinations thereof.

According to aspect fourteen, which may be the same as or different from aspects six, seven, eight, nine, ten, eleven, twelfth, and/or thirteen, a specific antibody or antigen-binding fragment thereof that binds to CCR8 is provided, wherein the antibody or antigen-binding fragment depletes activated regulatory T cells, preferably intratumoral Tregs. Example 12.1.1 shows high levels of Treg depletion obtained with the antibody of the present invention in various mouse models, wherein the alternative antibody is characterized by functional features equivalent to the provided anti-human CCR8 antibody. Interestingly, better treatment responses were observed in those examples where Treg depletion was at least 50%. Treg depletion can be measured in vitro or in vivo. As will be understood by those skilled in the art, Treg depletion can be temporary. For example, Treg depletion can be analyzed at 24, 48, or 72 hours after treatment.

According to some first embodiments of aspect 14, an effective dose of antibody or antigen-binding fragment is characterized by a maximum depletion of at least 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99% of the maximum depletion of activated or intratumoral regulatory T cells in vitro or in a subject.

In vitro or in vivo assays can be performed as known in the art. In vivo assays can be performed as described in Example 12ff. Suitable subjects include, for example, humans and non-humans, such as mice (e.g., CT26 or EMT-6 models), rodents, or cynomolgus monkeys.

According to some second embodiments of aspect 14, which may be the same as or different from the first embodiment, an effective dose of the antibody or antigen-binding fragment reduces the number of activated or intratumoral regulatory T cells to less than 55%, 50%, 40%, 30%, 25%, 20%, 10%, 5%, or 1% in vitro or in a subject. The reduction may be temporary. For example, without being bound by theory, the intratumoral Treg pool may subsequently be replenished.

According to some third embodiments of aspect 14, which may be the same as or different from the first and/or second embodiments, an effective dose of antibody or antigen-binding fragment increases the ratio of intratumoral CD8+ T cells to intratumoral Tregs to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or higher in vitro or in a subject. The increase may be temporary.

According to some fourth embodiments of the fourteenth aspect, which may be the same as or different from the first, second, and/or third embodiments, an effective dose of the antibody or antigen-binding fragment reduces the percentage of regulatory T cells in intratumoral CD4+ T cells to <30%, <20%, <10%, or <5% in vitro or in subjects. The reduction may be temporary.

In a preferred embodiment, an effective dose of antibody or antigen-binding fragment.

a) Reducing the number of activated or intratumoral regulatory T cells to less than 30%, 25%, 20%, 10%, 5%, or 1% in vitro or in subjects and/or

b) In vitro or in subjects, increasing the ratio of intratumoral CD8+ T cells to intratumoral Tregs to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or higher and/or

c) Reduce the percentage of regulatory T cells among CD4+ T cells in tumors to <30%, <20%, <10%, or <5%, either in vitro or in subjects.

The antibodies according to the present aspect are superior in terms of therapeutic efficacy, see Example 12ff. The provided isolated antibody or antigen-binding fragment is also preferably an antibody according to any one or a combination of aspects 15, 16, 17 or 18.

Aspect 15 - Immune cell regulation CCR8 antibody

According to aspect fifteen, which may be the same as or different from aspects six, seven, eight, nine, ten, eleven, twelfth, thirteenth and/or fourteenth, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein an effective dose of the antibody or antigen-binding fragment increases the absolute number of immune cells in a defined tumor volume, for example, by at least 1.5, 2, 2.5, 3, 3.5 or 4 times.

Example 12.8 showed a significant increase in the mRNA expression levels of various global or specific immune cell markers in reactive tumors from different subjects at the end of the study. Increases were observed, for example, for macrophages (especially M1 macrophages), CD8+ T cells, NK cells, CD3+ T cells, B cells, and interestingly, activated Tregs, indicating an increased infiltration of these immune cell populations into the tumor after long-term treatment. The increase in absolute immune cell numbers was also confirmed by FACS, see Examples 12.3 (CT26), 12.4 (EMT6), and 12.5 (F9). Suitable subjects include, for example, human and non-human subjects, such as mice, rodents, or cynomolgus monkeys. For example, the increase in immune cells can be measured after one, two, three, or four effective doses of the antibody, see Examples 12.3, 12.4.2, and after chronic treatment, such as after the final treatment. For example, the tumor can be a tumor characterized by tumor-infiltrating lymphocytes, such as lymphocytes; a tumor characterized by tumor-infiltrating T cells; or a tumor characterized by the expression of pro-inflammatory cytokines.

According to some first embodiments of the fifteenth aspect, the immune cells are selected from at least one, two, three, four, five, six, seven, eight, or nine of the following:

a) CD45+ cells (within the tumor)

b) CD8+ T cells (within the tumor)

c) CD4+ T cells (within the tumor)

d) Intratumoral macrophages, such as M1 or M2 macrophages.

e)(tumor-intratumor) NK cells,

f)(tumor-bearing) B cells,

g)(tumor-intratumor) dendritic cells,

h)(tumor-bearing) γδ T cells (unconventional T cells), and

i) iNKT cells.

The respective cell types and populations are defined as those known in the art, and in particular as defined elsewhere in this document.

According to some highly preferred embodiments of these first embodiments, three or more effective doses of antibody or antigen-binding fragments are increased in the tumor.

a) The number of CD8+ T cells within the tumor is at least 150%, 200%, 250%, 300%, or 350%.

b) The ratio of intratumoral CD8+ T cells to intratumoral regulatory T cells is at least 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or higher.

c) The number of macrophages within the tumor is at least 2, 3, 4, or 5 times the normal value.

d) The number of ACOD1+ macrophages (M1 macrophages) within the tumor is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher.

e) The ratio of ACOD1+ macrophages (M1 macrophages) to MRC1+ macrophages (M2 macrophages) is at least 1.5, 2, 3, 4, 5, 10 or higher.

f) The number of NK cells within the tumor must reach at least 140% or 200%.

g) The number of CD3+ T cells within the tumor must be at least 150%, 200%, 300%, or 400%.

h) The number of B cells within the tumor is at least 2, 5, 10, 20, 30, or 40 times greater.

i) The number of CD45+ T cells within the tumor is at least 150%, 200%, or 300%, and/or

j) The number of CD4+ T cells within the tumor is at least 150%, 200%, 300%, or 400%.

The tumor is preferably a tumor characterized by tumor-infiltrating lymphocytes. As understood by those skilled in the art, the tumor is characterized by cells expressing the target CCR8 of the antibody or fragment of the present invention, such as intratumoral Tregs. Preferably, the tumor is a tumor derived from a syngeneic tumor model of the BALB/c strain. For example, as shown in Example 12.1.1, the tumor may be derived from a CT26 model, EMT-6 model, F9 model, C38, H22, or B16F10-OVA model, wherein the antibody specifically binds to mouse CCR8. For example, when an antibody specifically binds to human CCR8, the tumor may be selected from adrenal carcinoma (e.g., adrenocortical carcinoma or pheochromocytoma), bladder cancer (e.g., transitional cell carcinoma, transitional cell carcinoma-papillary), brain cancer (e.g., glioma-astrocytoma, glioma-astrocytoma-glioblastoma, glioma-oligodendroglioma, glioma-oligodendroglioma), breast cancer (e.g., ADC, ADC-ductal, ADC-ductal-TNBC, ADC-ductal-TPBC, ADC-lobular), colorectal cancer (e.g., ADC), esophageal cancer (e.g., ADC), esophageal cancer (e.g., SCC), gastric cancer (e.g., ADC, ADC-diffuse, ADC-intestinal type, ADC-intestinal-tubular), head and neck cancer (e.g., laryngeal cancer-SCC, SCC, oral cancer-SCC), and kidney cancer (e.g., ccRCC, Chromophobe, papillary, papillary type I). Papillary type II), liver cancer (e.g., HCC), lung cancer (e.g., NSCLC-ADC, NSCLC-ADC-mixed, NSCLC-SCC, SCLC), mesothelioma (e.g., epithelioid), ovarian cancer (e.g., ADC-cystadenocarcinoma-serous papillary carcinoma), pancreatic cancer (e.g., ADC-ductal), prostate cancer (e.g., ADC-acinoid), sarcoma (e.g., leiomyosarcoma, dedifferentiated liposarcoma, malignant fibrous histiocytoma), skin cancer (e.g., melanoma), testicular cancer (e.g., germ cell tumor-seminomatous tumor), thymoma, thyroid cancer (e.g., follicular carcinoma, papillary carcinoma-classic variant), uterine cancer (e.g., cervical-SCC, cervical-SCC-keratotic, cervical-SCC-nonkeratotic, endometrial-ADC-endometrioid, endometrial-ADC-papillary serous, endometrial-carcinosarcoma-malignant Müllerian mixed tumor), see Table 11.1.2.

In some of these implementations, immune cells are

a) CD8+ T cells, CD4+ T cells, and macrophages

b) CD8+ T cells, CD4+ T cells, and NK cells

c) CD8+ T cells, CD4+ T cells, CD45+ cells

d) CD8+ T cells, CD4+ T cells, NK cells, and macrophages

e) CD8+ T cells, CD4+ T cells, NK cells, and macrophages, such as ACOD1+ macrophages.

f) CD8+ T cells, CD4+ T cells, NK cells, CD45+ cells,

g) CD8+ T cells, CD4+ T cells, NK cells, CD45+ cells, and macrophages, such as ACOD1+ macrophages.

h) CD8+ T cells, ACOD1+ macrophages (M1 macrophages), and B cells,

i) CD8+ T cells, CD4+ T cells, and dendritic cells, wherein dendritic cells are characterized by the expression of CD1c, CD14, CD16, CD141, CD11c, and CD123, or

j) CD8+ T cells and Acod1+ macrophages (M1 macrophages).

In some preferred embodiments of these implementations, immune cells are

a) CD8+ T cells, CD4+ T cells, and macrophages, or

b) CD8+ T cells, CD4+ T cells, NK cells, and macrophages, or

c) CD8+ T cells, ACOD1+ macrophages (M1 macrophages) and B cells.

According to a further embodiment, the immune cells are CD45+ cells and CD8+ T cells, CD45+ cells and CD4+ T cells, CD45+ cells and macrophages (such as M1 macrophages or M2 macrophages), CD45+ cells and NK cells, CD45+ cells and B cells, CD45+ cells and dendritic cells, CD45+ cells and γδ T cells, CD45+ cells and iNKT cells, CD8+ T cells and CD4+ T cells, CD8+ T cells and macrophages (such as M1 macrophages or M2 macrophages), CD8+ T cells and NK cells, CD8+ T cells and B cells, CD8+ T cells and dendritic cells, CD8+ T cells and γδ T cells, CD8+ T cells and iNKT cells, CD4+ T cells and macrophages (such as M1 macrophages). The following are possible combinations of macrophages: M1 macrophages and M2 macrophages, CD4+ T cells and NK cells, CD4+ T cells and B cells, CD4+ T cells and dendritic cells, CD4+ T cells and γδ T cells, CD4+ T cells and iNKT cells, macrophages (such as M1 macrophages or M2 macrophages) and NK cells, macrophages (such as M1 macrophages or M2 macrophages) and B cells, macrophages (such as M1 macrophages or M2 macrophages) and dendritic cells, macrophages (such as M1 macrophages or M2 macrophages) and γδ T cells, macrophages (such as M1 macrophages or M2 macrophages) and iNKT cells, B cells and dendritic cells, B cells and γδ T cells, B cells and iNKT cells, dendritic cells and γδ T cells, dendritic cells and iNKT cells, or γδ T cells and iNKT cells.

The provided isolated antibody or antigen-binding fragment is preferably an antibody according to any one of aspects 16, 17 or 18 or a combination thereof.

Regarding the formation of tertiary lymphoid structures by 16-CCR8 antibodies

According to the sixteenth aspect, which may be the same as or different from the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, and/or fifteenth aspects, a method is provided that specifically binds to an antibody or antigen fragment isolated from CCR8, wherein the antibody or antigen-binding fragment induces the formation of tertiary lymphoid structures. Tertiary lymphoid structures are intratumoral structures characterized by increased expression of LTta, LTtb, Cxcr5, and their Cxcl13 ligand. Tertiary lymphoid structures have been described as key drivers of antitumor activity in humans. Without being bound by theory, the inventors believe that the formation of these substructures may contribute to altering immune cell patterns and the antitumor activity of the antibodies of the present invention, for example, in humans. As discussed in Example 12.8, long-term treatment with the antibodies of the present invention consistently increased the expression levels of LTta, LTtb, and Cxcr5 and its ligand Cxcl13.

Sequence-determined antibodies

The antibodies according to aspects 17 and 18 are obtained using the method according to aspect 3 or 4. The antibodies produced according to aspects 17 and 18 specifically bind to polypeptides containing sulfated TRDs of human chemokine receptors, for example, in which at least 50% of tyrosine residues have been sulfated. As previously described, this method affects specific structural features of the HCDR3 of the obtained antibodies and may also affect functional features, such as the regulation of G protein-independent signaling or internalization behavior.

With the CDR, variable heavy chain, variable light chain, heavy chain, or light chain disclosed, the modular nature of antibodies generally allows for their combination, as well as their combination with a variety of functional features, as described elsewhere in this document.

Aspect 17 - Anti-human CCR8 antibody (sequence definition)

According to aspect seventeen, which may be the same as or different from aspects six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fourteenth, fifteenth and/or sixteenth, an isolated anti-CCR8 antibody or its antigen-binding fragment is provided.

Preferably, the antibody according to the present aspect binds to (a) the polypeptide isolated according to SEQ ID NO: 43 and/or SEQ ID NO: 46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and (b) the polypeptide isolated according to SEQ ID NO: 44 and/or SEQ ID NO: 47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and the antibody is cross-reactive to human and cynomolgus monkey CCR8. Furthermore, the antibodies are preferably characterized by properties that make them particularly suitable for treatment, for example, antibodies according to at least one or more of aspects 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains at least one, two, preferably three, four, five, or six CDR sequences, which have at least 90%, 95%, 98%, or 100% sequence identity with any of the following sequences.

a) SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 263 or SEQ ID NO: 264 (TPP-16966),

b) SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 281 or SEQ ID NO: 282 (TPP-17575),

c) SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 299 or SEQ ID NO: 300 (TPP-17576),

d) SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 317 or SEQ ID NO: 318 (TPP-17577),

e) SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 334, SEQ ID NO: 335 or SEQ ID NO: 336 (TPP-17578),

f) SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 352, SEQ ID NO: 353 or SEQ ID NO: 354 (TPP-17579),

g) SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 370, SEQ ID NO: 371 or SEQ ID NO: 372 (TPP-17580),

h) SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 388, SEQ ID NO: 389 or SEQ ID NO: 390 (TPP-17581),

i) SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 406, SEQ ID NO: 407 or SEQ ID NO: 408 (TPP-18205),

j) SEQ ID NO: 420, SEQ ID NO: 421, SEQ ID NO: 422, SEQ ID NO: 424, SEQ ID NO: 425 or SEQ ID NO: 426 (TPP-18206),

k) SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 442, SEQ ID NO: 443 or SEQ ID NO: 444 (TPP-18207),

l) SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 460, SEQ ID NO: 461 or SEQ ID NO: 462 (TPP-19546),

m) SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 478, SEQ ID NO: 479 or SEQ ID NO: 480 (TPP-20950),

n) SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 494, SEQ ID NO: 496, SEQ ID NO: 497 or SEQ ID NO: 498 (TPP-20955),

o) SEQ ID NO: 510, SEQ ID NO: 511, SEQ ID NO: 512, SEQ ID NO: 514, SEQ ID NO: 515, or SEQ ID NO: 516 (TPP-20965),

p) SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 532, SEQ ID NO: 533 or SEQ ID NO: 534 (TPP-21045),

q) SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548, SEQ ID NO: 550, SEQ ID NO: 551 or SEQ ID NO: 552 (TPP-21047),

r) SEQ ID NO: 564, SEQ ID NO: 565, SEQ ID NO: 566, SEQ ID NO: 568, SEQ ID NO: 569 or SEQ ID NO: 570 (TPP-21181),

s)SEQ ID NO: 582, SEQ ID NO: 583, SEQ ID NO: 584, SEQ ID NO: 586, SEQ ID NO: 587 or SEQ ID NO: 588 (TPP-21183),

t) SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 604, SEQ ID NO: 605 or SEQ ID NO: 606 (TPP-21360),

u)SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 623 or SEQ ID NO: 624 (TPP-23411),

v) SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 665, SEQ ID NO: 666 or SEQ ID NO: 667 (TPP-29596),

w) SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 685, SEQ ID NO: 686 or SEQ ID NO: 687 (TPP-29597),

x) SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 708 or SEQ ID NO: 709 (TPP-18429),

y) SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 727, SEQ ID NO: 728 or SEQ ID NO: 729 (TPP-18430),

z) SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 748 or SEQ ID NO: 749 (TPP-18432),

aa) SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769 (TPP-18433),

bb)SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785, SEQ ID NO:787, SEQ ID NO:788or SEQ ID NO:789(TPP-18436),

cc) SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 805, SEQ ID NO: 807, SEQ ID NO: 808 or SEQ ID NO: 809 (TPP-19571),

dd)SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ ID NO: 831, SEQ ID NO: 832 or SEQ ID NO: 833 (TPP-27477),

ee) SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 851, SEQ ID NO: 852 or SEQ ID NO: 853 (TPP-27478),

ff) SEQ ID NO: 867, SEQ ID NO: 868, SEQ ID NO: 869, SEQ ID NO: 871, SEQ ID NO: 872 or SEQ ID NO: 873 (TPP-27479),

gg)SEQ ID NO: 887, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 891, SEQ ID NO: 892 or SEQ ID NO: 893 (TPP-27480),

hh) SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 911, SEQ ID NO: 912 or SEQ ID NO: 913 (TPP-29367),

ii) SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 931, SEQ ID NO: 932 or SEQ ID NO: 933 (TPP-29368),

and/or

jj) SEQ ID NO: 947, SEQ ID NO: 948, SEQ ID NO: 949, SEQ ID NO: 951, SEQ ID NO: 952 or SEQ ID NO: 953 (TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment comprises SEQ ID NO: 260 (TPP-16966), SEQ ID NO: 278 (TPP-17575), SEQ ID NO: 296 (TPP-17576), SEQ ID NO: 314 (TPP-17577), SEQ ID NO: 332 (TPP-17578), SEQ ID NO: 350 (TPP-17579), SEQ ID NO: 368 (TPP-17580), SEQ ID NO: 386 (TPP-17581), SEQ ID NO: 404 (TPP-18205), SEQ ID NO: 422 (TPP-18206), SEQ ID NO: 440 (TPP-18207), SEQ ID NO: 458 (TPP-19546), SEQ ID NO: 476 (TPP-20950), SEQ ID NO: 494 (TPP-20955), SEQ ID NO: 512 (TPP-20965), SEQ ID NO: 530 (TPP-21045), SEQ ID NO: 548 (TPP-21047), SEQ ID NO: 566 (TPP-21181), SEQ ID NO: 584 (TPP-21183), SEQ ID NO: 602 (21360), or SEQ ID NO: 620 (TPP-23411), SEQ ID NO: 663 (TPP-29596), SEQ ID NO: 683 (TPP-29597), S EQ ID NO: 705 (TPP-18429), SEQ ID NO: 725 (TPP-18430), SEQ ID NO: 745 (TPP-18432), SEQ ID NO: 765 (TPP-18433), SEQ ID NO: 785 (TPP-184 36), any HCDR3 sequence having at least 90%, 95%, 98% or 100% sequence identity of SEQ ID NO: 805 (TPP-19571), SEQ ID NO: 829 (TPP-27477), SEQ ID NO: 849 (TPP-27478), SEQ ID NO: 869 (TPP-27479), SEQ ID NO: 889 (TPP-27480), SEQ ID NO: 909 (TPP-29367), SEQ ID NO: 929 (TPP-29368), or SEQ ID NO: 949 (TPP_29369).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains at least one, two, preferably three, four, five, or six CDR sequences.

a) SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 263 and SEQ ID NO: 264 (TPP-16966),

b) SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 281 and SEQ ID NO: 282 (TPP-17575),

c) SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 299 and SEQ ID NO: 300 (TPP-17576),

d) SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 317 and SEQ ID NO: 318 (TPP-17577),

e) SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 334, SEQ ID NO: 335 and SEQ ID NO: 336 (TPP-17578),

f) SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 352, SEQ ID NO: 353 and SEQ ID NO: 354 (TPP-17579),

g) SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 370, SEQ ID NO: 371 and SEQ ID NO: 372 (TPP-17580),

h) SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 388, SEQ ID NO: 389 and SEQ ID NO: 390 (TPP-17581),

i) SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 406, SEQ ID NO: 407 and SEQ ID NO: 408 (TPP-18205),

j) SEQ ID NO: 420, SEQ ID NO: 421, SEQ ID NO: 422, SEQ ID NO: 424, SEQ ID NO: 425 and SEQ ID NO: 426 (TPP-18206),

k) SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 442, SEQ ID NO: 443 and SEQ ID NO: 444 (TPP-18207),

l) SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 460, SEQ ID NO: 461 and SEQ ID NO: 462 (TPP-19546),

m) SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 478, SEQ ID NO: 479 and SEQ ID NO: 480 (TPP-20950),

n) SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 494, SEQ ID NO: 496, SEQ ID NO: 497 and SEQ ID NO: 498 (TPP-20955),

o) SEQ ID NO: 510, SEQ ID NO: 511, SEQ ID NO: 512, SEQ ID NO: 514, SEQ ID NO: 515, and SEQ ID NO: 516 (TPP-20965),

p) SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 532, SEQ ID NO: 533 and SEQ ID NO: 534 (TPP-21045),

q) SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548, SEQ ID NO: 550, SEQ ID NO: 551 and SEQ ID NO: 552 (TPP-21047),

r)SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:568, SEQ ID NO:569and SEQ ID NO:570 (TPP-21181),

s)SEQ ID NO:582, SEQ ID NO:583, SEQ ID NO:584, SEQ ID NO:586, SEQ ID NO:587and SEQ ID NO:588 (TPP-21183),

t) SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 604, SEQ ID NO: 605 or SEQ ID NO: 606 (TPP-21360),

u)SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 623 or SEQ ID NO: 624 (TPP-23411),

V) SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 665, SEQ ID NO: 666 or SEQ ID NO: 667 (TPP-29596),

w) SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 685, SEQ ID NO: 686 or SEQ ID NO: 687 (TPP-29597),

x) SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 708 or SEQ ID NO: 709 (TPP-18429),

y) SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 727, SEQ ID NO: 728 or SEQ ID NO: 729 (TPP-18430),

z) SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 748 or SEQ ID NO: 749 (TPP-18432),

aa) SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 768 or SEQ ID NO: 769 (TPP-18433),

bb)SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785, SEQ ID NO:787, SEQ ID NO:788or SEQ ID NO:789(TPP-18436),

cc) SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 805, SEQ ID NO: 807, SEQ ID NO: 808 or SEQ ID NO: 809 (TPP-19571),

dd)SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ ID NO: 831, SEQ ID NO: 832 or SEQ ID NO: 833 (TPP-27477),

ee) SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 851, SEQ ID NO: 852 or SEQ ID NO: 853 (TPP-27478),

ff) SEQ ID NO: 867, SEQ ID NO: 868, SEQ ID NO: 869, SEQ ID NO: 871, SEQ ID NO: 872 or SEQ ID NO: 873 (TPP-27479),

gg)SEQ ID NO: 887, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 891, SEQ ID NO: 892 or SEQ ID NO: 893 (TPP-27480),

hh) SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 911, SEQ ID NO: 912 or SEQ ID NO: 913 (TPP-29367),

ii) SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 931, SEQ ID NO: 932 or SEQ ID NO: 933 (TPP-29368),

and/or

jj) SEQ ID NO: 947, SEQ ID NO: 948, SEQ ID NO: 949, SEQ ID NO: 951, SEQ ID NO: 952 or SEQ ID NO: 953 (TPP-29369),

Optionally, at most one, two, three, four, or five mutations have been introduced into at least one CDR. Preferably, HCDR3 comprises or has been engineered to comprise at least one or more histidine residues as described elsewhere.

For example, tyrosine can exchange with positively charged amino acids such as histidine, and vice versa. Similarly, positively charged amino acids can exchange with different positively charged amino acids, negatively charged amino acids can exchange with different negatively charged amino acids, polar amino acids can exchange with different polar amino acids, polar uncharged amino acids can exchange with different polar uncharged amino acids, small amino acids can exchange with different small amino acids, amphoteric amino acids can exchange with different amphoteric amino acids, and aromatic amino acids can exchange with different aromatic amino acids. As understood by those skilled in the art, specific amino acid exchanges that do not alter the specific interaction between the antibody and the sulfated TRD of CCR8 are possible.

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment comprises a variable heavy chain sequence and/or a variable light chain sequence having at least 90%, 95%, 98%, or 100% sequence identity with the following sequences.

a) According to the variable heavy chain sequence of SEQ ID NO: 257 and/or according to the variable light chain sequence (TPP-16966) of SEQ ID NO: 261,

b) According to the variable heavy chain sequence of SEQ ID NO: 275 and/or according to the variable light chain sequence (TPP-17575) of SEQ ID NO: 279,

c) The variable heavy chain sequence according to SEQ ID NO: 293 and/or the variable light chain sequence (TPP-17576) according to SEQ ID NO: 297.

d) According to the variable heavy chain sequence of SEQ ID NO: 311 and/or according to the variable light chain sequence (TPP-17577) of SEQ ID NO: 315,

e) According to the variable heavy chain sequence of SEQ ID NO: 329 and/or according to the variable light chain sequence (TPP-17578) of SEQ ID NO: 333,

f) The variable heavy chain sequence according to SEQ ID NO: 347 and/or the variable light chain sequence (TPP-17579) according to SEQ ID NO: 351,

g) According to the variable heavy chain sequence of SEQ ID NO: 365 and/or according to the variable light chain sequence (TPP-17580) of SEQ ID NO: 369,

h) According to the variable heavy chain sequence of SEQ ID NO: 383 and/or according to the variable light chain sequence (TPP-17581) of SEQ ID NO: 387,

i) The variable heavy chain sequence according to SEQ ID NO: 401 and/or the variable light chain sequence (TPP-18205) according to SEQ ID NO: 405,

j) The variable heavy chain sequence according to SEQ ID NO: 419 and/or the variable light chain sequence (TPP-18206) according to SEQ ID NO: 423,

k) The variable heavy chain sequence according to SEQ ID NO: 437 and/or the variable light chain sequence (TPP-18207) according to SEQ ID NO: 441,

1) According to the variable heavy chain sequence of SEQ ID NO: 455 and/or according to the variable light chain sequence (TPP-19546) of SEQ ID NO: 459,

m) The variable heavy chain sequence according to SEQ ID NO: 473 and/or the variable light chain sequence (TPP-20950) according to SEQ ID NO: 477,

n) According to the variable heavy chain sequence of SEQ ID NO: 491 and/or according to the variable light chain sequence (TPP-20955) of SEQ ID NO: 495,

o) According to the variable heavy chain sequence of SEQ ID NO: 509 and/or according to the variable light chain sequence (TPP-20965) of SEQ ID NO: 513,

p) According to the variable heavy chain sequence of SEQ ID NO: 527 and/or according to the variable light chain sequence (TPP-21045) of SEQ ID NO: 531,

q) The variable heavy chain sequence according to SEQ ID NO: 545 and/or the variable light chain sequence (TPP-21047) according to SEQ ID NO: 549,

r) According to the variable heavy chain sequence of SEQ ID NO: 563 and/or according to the variable light chain sequence (TPP-21181) of SEQ ID NO: 567,

s) According to the variable heavy chain sequence of SEQ ID NO: 581 and/or according to the variable light chain sequence (TPP-21183) of SEQ ID NO: 585,

t) According to the variable heavy chain sequence of SEQ ID NO: 599 and/or according to the variable light chain sequence (TPP-21360) of SEQ ID NO: 603,

u) The variable heavy chain sequence according to SEQ ID NO: 617 and/or the variable light chain sequence (TPP-23411) according to SEQ ID NO: 621,

v) The variable heavy chain sequence according to SEQ ID NO: 660 and/or the variable light chain sequence (TPP-29596) according to SEQ ID NO: 664,

w) According to the variable heavy chain sequence of SEQ ID NO: 680 and/or according to the variable light chain sequence (TPP-29597) of SEQ ID NO: 684,

x) The variable heavy chain sequence according to SEQ ID NO: 702 and/or the variable light chain sequence (TPP-18429) according to SEQ ID NO: 706,

y) According to the variable heavy chain sequence of SEQ ID NO: 722 and/or according to the variable light chain sequence (TPP-18430) of SEQ ID NO: 726,

z) The variable heavy chain sequence according to SEQ ID NO: 742 and/or the variable light chain sequence (TPP-18432) according to SEQ ID NO: 746.

aa) The variable heavy chain sequence according to SEQ ID NO: 762 and/or the variable light chain sequence (TPP-18433) according to SEQ ID NO: 766.

bb) According to the variable heavy chain sequence of SEQ ID NO: 782 and/or according to the variable light chain sequence (TPP-18436) of SEQ ID NO: 786,

cc) based on the variable heavy chain sequence of SEQ ID NO: 802 and/or based on the variable light chain sequence (TPP-19571) of SEQ ID NO: 806,

(dd) According to the variable heavy chain sequence of SEQ ID NO: 826 and/or according to the variable light chain sequence (TPP-27477) of SEQ ID NO: 830,

(ee) According to the variable heavy chain sequence of SEQ ID NO: 846 and/or according to the variable light chain sequence (TPP-27478) of SEQ ID NO: 850,

ff) According to the variable heavy chain sequence of SEQ ID NO: 866 and/or according to the variable light chain sequence (TPP-27479) of SEQ ID NO: 870,

(gg) According to the variable heavy chain sequence of SEQ ID NO: 886 and/or according to the variable light chain sequence (TPP-27480) of SEQ ID NO: 890,

hh) Based on the variable heavy chain sequence of SEQ ID NO: 906 and/or based on the variable light chain sequence (TPP-29367) of SEQ ID NO: 910,

ii) The variable heavy chain sequence according to SEQ ID NO: 926 and/or the variable light chain sequence (TPP-29368) according to SEQ ID NO: 930, or

jj) The variable heavy chain sequence according to SEQ ID NO: 946 and/or the variable light chain sequence according to SEQ ID NO: 950 (TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains...

a) According to the variable heavy chain sequence of SEQ ID NO: 257 and/or according to the variable light chain sequence (TPP-16966) of SEQ ID NO: 261,

b) According to the variable heavy chain sequence of SEQ ID NO: 275 and/or according to the variable light chain sequence (TPP-17575) of SEQ ID NO: 279,

c) The variable heavy chain sequence according to SEQ ID NO: 293 and/or the variable light chain sequence (TPP-17576) according to SEQ ID NO: 297.

d) According to the variable heavy chain sequence of SEQ ID NO: 311 and/or according to the variable light chain sequence (TPP-17577) of SEQ ID NO: 315,

e) According to the variable heavy chain sequence of SEQ ID NO: 329 and/or according to the variable light chain sequence (TPP-17578) of SEQ ID NO: 333,

f) The variable heavy chain sequence according to SEQ ID NO: 347 and/or the variable light chain sequence (TPP-17579) according to SEQ ID NO: 351,

g) According to the variable heavy chain sequence of SEQ ID NO: 365 and/or according to the variable light chain sequence (TPP-17580) of SEQ ID NO: 369,

h) According to the variable heavy chain sequence of SEQ ID NO: 383 and/or according to the variable light chain sequence (TPP-17581) of SEQ ID NO: 387,

i) The variable heavy chain sequence according to SEQ ID NO: 401 and/or the variable light chain sequence (TPP-18205) according to SEQ ID NO: 405,

j) The variable heavy chain sequence according to SEQ ID NO: 419 and/or the variable light chain sequence (TPP-18206) according to SEQ ID NO: 423,

k) The variable heavy chain sequence according to SEQ ID NO: 437 and/or the variable light chain sequence (TPP-18207) according to SEQ ID NO: 441,

1) According to the variable heavy chain sequence of SEQ ID NO: 455 and/or according to the variable light chain sequence (TPP-19546) of SEQ ID NO: 459,

m) The variable heavy chain sequence according to SEQ ID NO: 473 and/or the variable light chain sequence (TPP-20950) according to SEQ ID NO: 477,

n) According to the variable heavy chain sequence of SEQ ID NO: 491 and/or according to the variable light chain sequence (TPP-20955) of SEQ ID NO: 495,

o) According to the variable heavy chain sequence of SEQ ID NO: 509 and/or according to the variable light chain sequence (TPP-20965) of SEQ ID NO: 513,

p) According to the variable heavy chain sequence of SEQ ID NO: 527 and/or according to the variable light chain sequence (TPP-21045) of SEQ ID NO: 531,

q) The variable heavy chain sequence according to SEQ ID NO: 545 and/or the variable light chain sequence (TPP-21047) according to SEQ ID NO: 549,

r) According to the variable heavy chain sequence of SEQ ID NO: 563 and/or according to the variable light chain sequence (TPP-21181) of SEQ ID NO: 567,

s) According to the variable heavy chain sequence of SEQ ID NO: 581 and/or according to the variable light chain sequence (TPP-21183) of SEQ ID NO: 585,

t) According to the variable heavy chain sequence of SEQ ID NO: 599 and/or according to the variable light chain sequence (TPP-21360) of SEQ ID NO: 603,

u) The variable heavy chain sequence according to SEQ ID NO: 617 and/or the variable light chain sequence (TPP-23411) according to SEQ ID NO: 621,

v) The variable heavy chain sequence according to SEQ ID NO: 660 and/or the variable light chain sequence (TPP-29596) according to SEQ ID NO: 664,

w) According to the variable heavy chain sequence of SEQ ID NO: 680 and/or according to the variable light chain sequence (TPP-29597) of SEQ ID NO: 684,

x) The variable heavy chain sequence according to SEQ ID NO: 702 and/or the variable light chain sequence (TPP-18429) according to SEQ ID NO: 706,

y) According to the variable heavy chain sequence of SEQ ID NO: 722 and/or according to the variable light chain sequence (TPP-18430) of SEQ ID NO: 726,

z) The variable heavy chain sequence according to SEQ ID NO: 742 and/or the variable light chain sequence (TPP-18432) according to SEQ ID NO: 746.

aa) The variable heavy chain sequence according to SEQ ID NO: 762 and/or the variable light chain sequence (TPP-18433) according to SEQ ID NO: 766.

bb) According to the variable heavy chain sequence of SEQ ID NO: 782 and/or according to the variable light chain sequence (TPP-18436) of SEQ ID NO: 786,

cc) According to the variable heavy chain sequence of SEQ ID NO: 802 and/or according to the variable light chain sequence (TPP-19571) of SEQ ID NO: 806,

(dd) According to the variable heavy chain sequence of SEQ ID NO: 826 and/or according to the variable light chain sequence (TPP-27477) of SEQ ID NO: 830,

(ee) According to the variable heavy chain sequence of SEQ ID NO: 846 and/or according to the variable light chain sequence (TPP-27478) of SEQ ID NO: 850,

ff) According to the variable heavy chain sequence of SEQ ID NO: 866 and/or according to the variable light chain sequence (TPP-27479) of SEQ ID NO: 870,

(gg) According to the variable heavy chain sequence of SEQ ID NO: 886 and/or according to the variable light chain sequence (TPP-27480) of SEQ ID NO: 890,

hh) Based on the variable heavy chain sequence of SEQ ID NO: 906 and/or based on the variable light chain sequence (TPP-29367) of SEQ ID NO: 910,

ii) The variable heavy chain sequence according to SEQ ID NO: 926 and/or the variable light chain sequence (TPP-29368) according to SEQ ID NO: 930, or

jj) The variable heavy chain sequence according to SEQ ID NO: 946 and/or the variable light chain sequence according to SEQ ID NO: 950 (TPP-29369).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment comprises a heavy chain sequence and/or a light chain sequence having at least 90%, 95%, 98%, or 100% sequence identity with the following sequences.

a) The heavy chain according to SEQ ID NO: 273 and the light chain according to SEQ ID NO: 274 (TPP-16966),

b) The heavy chain according to SEQ ID NO: 291 and the light chain according to SEQ ID NO: 292 (TPP-17575),

c) The heavy chain according to SEQ ID NO: 309 and the light chain according to SEQ ID NO: 310 (TPP-17576),

d) The heavy chain according to SEQ ID NO: 327 and the light chain according to SEQ ID NO: 328 (TPP-17577),

e) The heavy chain according to SEQ ID NO: 345 and the light chain according to SEQ ID NO: 346 (TPP-17578),

f) The heavy chain according to SEQ ID NO: 363 and the light chain according to SEQ ID NO: 364 (TPP-17579),

g) The heavy chain according to SEQ ID NO: 381 and the light chain according to SEQ ID NO: 382 (TPP-17580),

h) The heavy chain according to SEQ ID NO: 399 and the light chain according to SEQ ID NO: 400 (TPP-17581),

i) The heavy chain according to SEQ ID NO: 417 and the light chain according to SEQ ID NO: 418 (TPP-18205),

j) The heavy chain according to SEQ ID NO: 435 and the light chain according to SEQ ID NO: 436 (TPP-18206),

k) The heavy chain according to SEQ ID NO: 453 and the light chain according to SEQ ID NO: 454 (TPP-18207),

l) The heavy chain according to SEQ ID NO: 471 and the light chain according to SEQ ID NO: 472 (TPP-19546),

m) The heavy chain according to SEQ ID NO: 489 and the light chain according to SEQ ID NO: 490 (TPP-20950),

n) The heavy chain according to SEQ ID NO: 507 and the light chain according to SEQ ID NO: 508 (TPP-20955),

o) The heavy chain according to SEQ ID NO: 525 and the light chain according to SEQ ID NO: 526 (TPP-20965),

p) The heavy chain according to SEQ ID NO: 543 and the light chain according to SEQ ID NO: 544 (TPP-21045),

q) The heavy chain according to SEQ ID NO: 561 and the light chain according to SEQ ID NO: 562 (TPP-21047),

r) The heavy chain according to SEQ ID NO: 579 and the light chain according to SEQ ID NO: 580 (TPP-21181),

s) the heavy chain according to SEQ ID NO: 597 and the light chain according to SEQ ID NO: 598 (TPP-21183),

t) The heavy chain according to SEQ ID NO: 615 and the light chain according to SEQ ID NO: 616 (TPP-21360),

u) The heavy chain according to SEQ ID NO: 633 and the light chain according to SEQ ID NO: 634 (TPP-23411),

v) The heavy chain according to SEQ ID NO: 676 and the light chain according to SEQ ID NO: 677 (TPP-29596),

w) The heavy chain according to SEQ ID NO: 696 and the light chain according to SEQ ID NO: 697 (TPP-29597),

x) The heavy chain according to SEQ ID NO: 718 and the light chain according to SEQ ID NO: 719 (TPP-18429),

y) the heavy chain according to SEQ ID NO: 738 and the light chain according to SEQ ID NO: 739 (TPP-18430),

z) The heavy chain according to SEQ ID NO: 758 and the light chain according to SEQ ID NO: 759 (TPP-18432),

aa) The heavy chain according to SEQ ID NO: 778 and the light chain according to SEQ ID NO: 779 (TPP-18433),

bb) the heavy chain according to SEQ ID NO: 798 and the light chain according to SEQ ID NO: 799 (TPP-18436),

cc) the heavy chain according to SEQ ID NO: 818 and the light chain according to SEQ ID NO: 819 (TPP-19571),

(dd) The heavy chain according to SEQ ID NO: 842 and the light chain according to SEQ ID NO: 843 (TPP-27477),

(ee) The heavy chain according to SEQ ID NO: 862 and the light chain according to SEQ ID NO: 863 (TPP-27478),

ff) The heavy chain according to SEQ ID NO: 882 and the light chain according to SEQ ID NO: 883 (TPP-27479),

(gg) The heavy chain according to SEQ ID NO: 902 and the light chain according to SEQ ID NO: 903 (TPP-27480),

(hh) The heavy chain according to SEQ ID NO: 922 and the light chain according to SEQ ID NO: 923 (TPP-29367),

ii) The heavy chain according to SEQ ID NO: 942 and the light chain according to SEQ ID NO: 943 (TPP-29368), or

jj) The heavy chain according to SEQ ID NO: 962 and the light chain according to SEQ ID NO: 963 (TPP_29369).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains...

a) The heavy chain according to SEQ ID NO: 273 and the light chain according to SEQ ID NO: 274 (TPP-16966),

b) The heavy chain according to SEQ ID NO: 291 and the light chain according to SEQ ID NO: 292 (TPP_17575),

c) The heavy chain according to SEQ ID NO: 309 and the light chain according to SEQ ID NO: 310 (TPP_17576),

d) The heavy chain according to SEQ ID NO: 327 and the light chain according to SEQ ID NO: 328 (TPP-17577),

e) The heavy chain according to SEQ ID NO: 345 and the light chain according to SEQ ID NO: 346 (TPP-17578),

f) The heavy chain according to SEQ ID NO: 363 and the light chain according to SEQ ID NO: 364 (TPP-17579),

g) The heavy chain according to SEQ ID NO: 381 and the light chain according to SEQ ID NO: 382 (TPP-17580),

h) The heavy chain according to SEQ ID NO: 399 and the light chain according to SEQ ID NO: 400 (TPP-17581),

i) The heavy chain according to SEQ ID NO: 417 and the light chain according to SEQ ID NO: 418 (TPP-18205),

j) The heavy chain according to SEQ ID NO: 435 and the light chain according to SEQ ID NO: 436 (TPP-18206),

k) The heavy chain according to SEQ ID NO: 453 and the light chain according to SEQ ID NO: 454 (TPP-18207),

l) The heavy chain according to SEQ ID NO: 471 and the light chain according to SEQ ID NO: 472 (TPP-19546),

m) The heavy chain according to SEQ ID NO: 489 and the light chain according to SEQ ID NO: 490 (TPP-20950),

n) The heavy chain according to SEQ ID NO: 507 and the light chain according to SEQ ID NO: 508 (TPP_20955),

o) The heavy chain according to SEQ ID NO: 525 and the light chain according to SEQ ID NO: 526 (TPP-20965),

p) The heavy chain according to SEQ ID NO: 543 and the light chain according to SEQ ID NO: 544 (TPP-21045),

q) The heavy chain according to SEQ ID NO: 561 and the light chain according to SEQ ID NO: 562 (TPP-21047),

r) The heavy chain according to SEQ ID NO: 579 and the light chain according to SEQ ID NO: 580 (TPP-21181),

s) the heavy chain according to SEQ ID NO: 597 and the light chain according to SEQ ID NO: 598 (TPP-21183),

t) The heavy chain according to SEQ ID NO: 615 and the light chain according to SEQ ID NO: 616 (TPP-21360),

u) The heavy chain according to SEQ ID NO: 633 and the light chain according to SEQ ID NO: 634 (TPP-23411),

v) The heavy chain according to SEQ ID NO: 676 and the light chain according to SEQ ID NO: 677 (TPP-29596),

w) The heavy chain according to SEQ ID NO: 696 and the light chain according to SEQ ID NO: 697 (TPP-29597),

x) The heavy chain according to SEQ ID NO: 718 and the light chain according to SEQ ID NO: 719 (TPP-18429),

y) the heavy chain according to SEQ ID NO: 738 and the light chain according to SEQ ID NO: 739 (TPP-18430),

z) The heavy chain according to SEQ ID NO: 758 and the light chain according to SEQ ID NO: 759 (TPP-18432),

aa) The heavy chain according to SEQ ID NO: 778 and the light chain according to SEQ ID NO: 779 (TPP-18433),

bb) the heavy chain according to SEQ ID NO: 798 and the light chain according to SEQ ID NO: 799 (TPP-18436),

cc) the heavy chain according to SEQ ID NO: 818 and the light chain according to SEQ ID NO: 819 (TPP-19571),

(dd) The heavy chain according to SEQ ID NO: 842 and the light chain according to SEQ ID NO: 843 (TPP-27477),

(ee) The heavy chain according to SEQ ID NO: 862 and the light chain according to SEQ ID NO: 863 (TPP-27478),

ff) The heavy chain according to SEQ ID NO: 882 and the light chain according to SEQ ID NO: 883 (TPP-27479),

(gg) The heavy chain according to SEQ ID NO: 902 and the light chain according to SEQ ID NO: 903 (TPP-27480),

(hh) The heavy chain according to SEQ ID NO: 922 and the light chain according to SEQ ID NO: 923 (TPP-29367),

ii) The heavy chain according to SEQ ID NO: 942 and the light chain according to SEQ ID NO: 943 (TPP-29368), or

jj) The heavy chain according to SEQ ID NO: 962 and the light chain according to SEQ ID NO: 963 (TPP-29369).

The present invention also provides a variety of antibodies, which contain a random arrangement of CDRs of anti-CCR8 antibodies provided with the sequence listing.

Preferably, the antibody or antigen-binding fragment according to the present aspect is unfucosylated, as described elsewhere herein. Preferably, the antibody or antigen-binding fragment according to the present aspect induces ADCC and/or ADCP, as described elsewhere herein. Preferably, the antibody or antigen-binding fragment according to the present aspect is a non-internalizing or low-internalizing antibody, as described elsewhere herein.

Aspect 18 - Anti-mouse CCR8 antibody (sequence definition)

According to aspect eighteen, which may be the same as or different from aspects six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fourteenth, fifteenth and/or sixteen, an isolated anti-CCR8 antibody or its antigen-binding fragment is provided. Preferably, the antibody according to the present aspect binds to mouse CCR8, particularly to the isolated polypeptide according to SEQ ID NO: 45 and/or SEQ ID NO: 48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated. The CDRs of the antibody according to the present aspect are preferably human-derived CDRs. Furthermore, the antibodies are characterized by properties that make them particularly suitable for treatment, for example, antibodies according to at least one or more of aspects 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In summary, the antibody according to the present aspect can be used as an alternative antibody for recognizing mouse CCR8 and has the superior therapeutic properties as discussed, for example in Example 12.

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains at least one, two, three, four, five, or six CDR sequences having at least 90%, 95%, 98%, or 100% sequence identity with the following sequences:

a) SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 207 and SEQ ID NO: 208 (TPP-14095),

b) SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 222 (TPP-14099),

c) SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 235 and SEQ ID NO: 236 (TPP-15285), or

d) SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 249 and SEQ ID NO: 250 (TPP-15286).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains an HCDR3 sequence having at least 90%, 95%, 98%, or 100% sequence identity with any of SEQ ID NO: 204, SEQ ID NO: 218, SEQ ID NO: 232, or SEQ ID NO: 246.

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains at least one, two, preferably three, four, five, or six CDR sequences.

a) SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 207 and SEQ ID NO: 208 (TPP-14095),

b) SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 222 (TPP-14099),

c) SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 235 and SEQ ID NO: 236 (TPP-15285), or

d) SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 249 and SEQ ID NO: 250 (TPP-15286),

Optionally, at most one, two, three, four or five mutations have been introduced into at least one CDR.

For example, tyrosine can be exchanged with positively charged amino acids such as histidine, and vice versa. Similarly, positively charged amino acids can be exchanged with different positively charged amino acids, negatively charged amino acids with different negatively charged amino acids, polar amino acids with different polar amino acids, polar uncharged amino acids with different polar uncharged amino acids, small amino acids with different small amino acids, amphoteric amino acids with different amphoteric amino acids, and aromatic amino acids with different aromatic amino acids. As understood by those skilled in the art, it is possible to exchange amino acids that do not alter the specific interaction between the antibody and the sulfated TRD of CCR8. Introducing histidine-based amino acid exchanges is preferred.

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment comprises a variable heavy chain sequence and/or a variable light chain sequence having at least 90%, 95%, 98%, or 100% sequence identity with the following sequences.

a) Based on the variable heavy chain sequence of SEQ ID NO: 201 and the variable light chain sequence (TPP-14095) of SEQ ID NO: 205,

b) Based on the variable heavy chain sequence of SEQ ID NO: 215 and the variable light chain sequence (TPP-14099) of SEQ ID NO: 219,

c) The variable heavy chain sequence according to SEQ ID NO: 229 and the variable light chain sequence (TPP-15285) according to SEQ ID NO: 233, or

d) The variable heavy chain sequence according to SEQ ID NO: 243 and the variable light chain sequence according to SEQ ID NO: 247 (TPP-15286).

According to some preferred embodiments, the anti-CCR8 antibody or its antigen-binding fragment comprises e) the variable heavy chain sequence according to SEQ ID NO: 201 and the variable light chain sequence (TPP-14095) according to SEQ ID NO: 205.

f) The variable heavy chain sequence according to SEQ ID NO: 215 and the variable light chain sequence (TPP-14099) according to SEQ ID NO: 219.

g) The variable heavy chain sequence according to SEQ ID NO: 229 and the variable light chain sequence (TPP-15285) according to SEQ ID NO: 233, or

h) The variable heavy chain sequence according to SEQ ID NO: 243 and the variable light chain sequence according to SEQ ID NO: 247 (TPP-15286).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment comprises a heavy chain sequence and/or a light chain sequence having at least 90%, 95%, 98%, or 100% sequence identity with the following sequences.

a) The heavy chain according to SEQ ID NO: 211 and/or the light chain according to SEQ ID NO: 212 (TPP-14095),

b) The heavy chain according to SEQ ID NO: 225 and/or the light chain according to SEQ ID NO: 226 (TPP-14099),

c) The heavy chain according to SEQ ID NO: 239 and/or the light chain according to SEQ ID NO: 240 (TPP-15285), or

d) The heavy chain according to SEQ ID NO: 253 and/or the light chain according to SEQ ID NO: 254 (TPP-15286).

According to some preferred embodiments, the isolated anti-CCR8 antibody or its antigen-binding fragment contains...

a) The heavy chain according to SEQ ID NO: 211 and/or the light chain according to SEQ ID NO: 212 (TPP-14095),

b) The heavy chain according to SEQ ID NO: 225 and/or the light chain according to SEQ ID NO: 226 (TPP-14099),

c) The heavy chain according to SEQ ID NO: 239 and/or the light chain according to SEQ ID NO: 240 (TPP-15285), or

d) The heavy chain according to SEQ ID NO: 253 and/or the light chain according to SEQ ID NO: 254 (TPP-15286).

Based on the preferred combination of "all aspects"

The following are particularly preferred aspects of aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 (according to “All Aspects” in this section).

In all preferred embodiments, the antibody or antigen-binding fragment, the isolated antibody or its antigen-binding fragment, specifically binds to the sulfated tyrosine-rich domain of CCR8. In all preferred embodiments, the antibody or antigen-binding fragment is characterized in that the HCDR3 region contains 10% to 34% tyrosine and/or 2% to 20% histidine. In all preferred embodiments, the antibody or antigen-binding fragment is non-internalized or characterized in that it internalizes into cells expressing endogenous targets at a rate of 1.5, 2, 3, 4, 5, 6, 7, or 10 times lower than that of isotype controls.

In all preferred embodiments, the antibody or antigen-binding fragment comprises a human (derived) CDR. For example, the antibody may be a human anti-human CCR8 antibody. In all preferred embodiments, the antibody or antigen-binding fragment is cross-reactive to CCR8 from at least two species, preferably selected from humans, monkeys, macaques (cynomolgus monkeys), rhesus monkeys, rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels, and even more preferably selected from humans, cynomolgus monkeys, and mice. According to some of the most preferred embodiments of these embodiments, the antibody or antigen-binding fragment is cross-reactive to human and cynomolgus monkey CCR8.

In all preferred embodiments, the antibody or antigen-binding fragment binds to CCR8 from a first species with a first dissociation constant KD and to CCR8 from a second species with a second dissociation constant KD, wherein the first and second dissociation constants are on the same order of magnitude.

In all preferred embodiments, the antibody or antigen-binding fragment binds with a KD value of <5E-8M, <4E-8M, <3E-8M, <2E-8M, <1E-8M, <9E-9M, <8E-9M, <7E-9M, <6E-9M, <5E-9M, <4E-9M, <3E-9M, <2.5E-9M, <2E-9M, <1.5E-9M, <1E-9M, <9E-10M, <8E-10M, <7E-10M, <6E-10M, <5E-10M, <4E-10M, <3E-10M, <2.5E-10M, <2E-10M, <1.5E-10M, <1E-10M, or <9E-11M.

a) The polypeptide isolated according to SEQ ID NO: 43 and/or SEQ ID NO: 46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

b) The polypeptide isolated according to SEQ ID NO: 44 and/or SEQ ID NO: 47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

c) The polypeptide isolated according to SEQ ID NO: 45 and/or SEQ ID NO: 48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

In all preferred embodiments, the antibody or antigen-binding fragment (specifically) binds at an EC50 of <15 nM, <10 nM, <5 nM, <1 nM, or <0.6 nM.

a) Human CCR8 and/or polypeptides isolated according to SEQ ID NO: 43 and/or SEQ ID NO: 46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

b) Cynomolgus monkey CCR8 and/or polypeptides isolated according to SEQ ID NO: 44 and/or SEQ ID NO: 47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

c) Mouse CCR8 and/or polypeptides isolated according to SEQ ID NO: 45 and/or SEQ ID NO: 48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

It can also optionally bind to activated human regulatory T cells with EC50 values of <50 nM, <25 nM, <15 nM, or <10 nM.

According to some of the most preferred embodiments of these embodiments, the EC50 of the antibody or antigen-binding fragment binding to human CCR8 or the isolated polypeptide according to SEQ ID NO: 46 (wherein at least two or all of Y3, Y15 and Y17 have been sulfated) is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the EC50 of the cynomolgus monkey CCR8 or the isolated polypeptide according to SEQ ID NO: 47 (wherein at least two or all of Y3, Y15 and Y17 have been sulfated) is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

As disclosed, for example, in Example 10.1.1, the antibodies according to the present invention exhibit excellent affinity for their respective targets. For example, the cross-reactive antibodies TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bind to human CCR8 with EC50 values of 4.8 nM, 1.7 nM, 0.8 nM, 0.6 nM, ~0.9 nM, or 1.7 nM, respectively. Furthermore, TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bind to cynomolgus monkey CCR8 with EC50 values of 1.8 nM, 1 nM, 0.5 nM, 0.7 nM, ~0.55 nM, or 0.9 nM. In addition, TPP-17578, TPP-19546, TPP-18206, and TPP-21360 bind to human regulatory T cells with EC50 values of 25 nM, 15 nM, 23 nM, or 10 nM, respectively. Furthermore, the anti-mouse CCR8 antibody TPP-14099 binds to CHO cells expressing mouse CCR8 with an EC50 of 3 nM and to mouse iTreg cells with an EC50 of 13.2 nM (see Table 10.1.1.5).

In all preferred embodiments, the antibody or antigen-binding fragment is preferably at a concentration of 10 nM.

a) Does not, substantially does not, or blocks CCL1-induced β-arrestin signaling to a degree less than that of antibodies L263G8 and 433H, and/or

b) Does not, substantially does not, or induces ERK1/2 phosphorylation to a degree less than that of antibodies L263G8 and 433H and/or

c) AKT phosphorylation is not induced, or is substantially not induced, or is induced to a degree less than that of antibodies L263G8 and 433H.

In all preferred embodiments, the antibody or antigen-binding fragment blocks G protein-dependent signaling of the chemokine receptor.

In all preferred embodiments, the antibody or antigen-binding fragment is unfucosylated.

In all preferred embodiments, the antibody or antigen-binding fragment

a) Binding to human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) below 530 nM, 500 nM, 450 nM, 400 nM, 300 nM, or 200 nM, and/or

b) Bind human FcγRIIA (CD32a) with a dissociation constant (KD) of less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

In all preferred embodiments, the antibody or antigen-binding fragment

a) Inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells (e.g., human NK cells), and/or

b) Inducing antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells (e.g., human macrophages).

In all preferred embodiments, the antibody or antigen-binding fragment induces ADCC and/or ADCP and

a) The maximum exhaustion of ADCC-induced activated human regulatory T cells is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, and/or

b) The maximum exhaustion of ADCP-induced activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%, and/or

c) The maximum depletion of intratumoral regulatory T cells, either in vitro or in subjects, is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

In all preferred embodiments, the EC50 of the antibody or antigen-binding fragment

a) For ADCC-induced depletion of activated human regulatory T cells, below 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM or 5 pM and/or

b) For ADCP-induced depletion of activated human regulatory T cells, below 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM or 25 pM.

Preferably, in all preferred embodiments, an effective dose of antibody or antigen-binding fragment is used.

a) In vitro or in subjects, reducing the number of activated or intratumoral regulatory T cells to less than 30%, 25%, 20%, 10%, 5%, or 1% and/or

b) In vitro or in subjects, increasing the ratio of intratumoral CD8+ T cells to intratumoral Tregs to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or higher and/or

c) Reduce the percentage of regulatory T cells in tumor CD4+ T cells to <30%, <20%, <10%, or <5% in vitro or in subjects.

In all preferred embodiments, the antibody or antigen-binding fragment increases the absolute number of immune cells in a defined tumor volume by at least 1.5, 2, 2.5, 3, 3.5, or 4 times, preferably wherein the immune cells are selected from...

a) CD45+ cells (within the tumor)

b) CD8+ T cells (within the tumor)

c) CD4+ T cells (within the tumor)

d) Macrophages (within the tumor), such as M1 or M2 macrophages.

e)(tumor-intratumor) NK cells,

f)(tumor-bearing) B cells,

g)(tumor-intratumor) dendritic cells,

h)(tumor-intratumoral) γδT cells.

i)(tumor-intratumor) iNKT cells,

Or any combination thereof.

For example, immune cells are

a) CD8+ T cells, CD4+ T cells, and macrophages, or

b) CD8+ T cells, CD4+ T cells, NK cells, and macrophages, or

c) CD8+ T cells, ACOD1+ macrophages (M1 macrophages) and B cells.

In all preferred embodiments, three or more effective doses of the antibody or antigen-binding fragment increase the activity in the tumor.

a) The number of CD8+ T cells within the tumor is at least 150%, 200%, 250%, 300%, or 350%.

b) The ratio of intratumoral CD8+ T cells to intratumoral regulatory T cells is at least 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or higher.

c) The number of macrophages within the tumor is at least 2, 3, 4, or 5 times the normal value.

d) The number of ACOD1+ macrophages (M1 macrophages) within the tumor is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher.

e) The ratio of ACOD1+ macrophages (M1 macrophages) to MRC1+ macrophages (M2 macrophages) is at least 1.5, 2, 3, 4, 5, 10 or higher.

f) The number of NK cells within the tumor must be at least 140% or 200%.

g) The number of CD3+ T cells within the tumor must be at least 150%, 200%, 300%, or 400%.

h) The number of B cells within the tumor is at least 2, 5, 10, 20, 30, or 40 times greater.

i) The number of CD45+ T cells within the tumor is at least 150%, 200%, or 300%, and/or

j) The number of CD4+ T cells within the tumor must be at least 150%, 200%, 300%, or 400%.

Preferably, the tumor is characterized by tumor-infiltrating lymphocytes, such as tumor-infiltrating T cells.

In all preferred embodiments, the antibody or antigen-binding fragment induces the formation of tertiary lymphoid structures.

In all preferred embodiments, the antibody is an IgG antibody, preferably human IgG1 or mouse IgG2a.

In all preferred embodiments, the antibody or antigen-binding fragment binds at an EC50 of <15 nM, <10 nM, <5 nM, <1 nM, or <0.6 nM.

a) Human CCR8 or the isolated polypeptide according to SEQ ID NO: 46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

b) Cynomolgus monkey CCR8 or the polypeptide isolated according to SEQ ID NO: 47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

c) Mouse CCR8 or a polypeptide isolated according to SEQ ID NO: 48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

In all preferred embodiments, the EC50 of the antibody or antigen-binding fragment binding to human CCR8 or the isolated polypeptide according to SEQ ID NO: 46 (wherein at least two or all of Y3, Y15 and Y17 have been sulfated) is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the EC50 of the antibody or antigen-binding fragment binding to cynomolgus monkey CCR8 or the isolated polypeptide according to SEQ ID NO: 47 (wherein at least two or all of Y3, Y15 and Y17 have been sulfated) is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

In all preferred embodiments, the isolated antibody or antigen-binding fragment binds to activated human regulatory T cells at an EC50 of <50 nM, <25 nM, <15 nM, or <10 nM.

In all preferred embodiments, the dissociation constant of the antibody binding to the first isolated non-sulfated peptide is higher than 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM, or 3 μM, or undetectable. Preferably, the dissociation constant of the antibody binding to the first isolated non-sulfated peptide is higher than 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or undetectable.

The optimal combination of "all aspects"

According to preferred embodiment I, a separated antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment is non-internalized or characterized by internalization into cells expressing endogenous targets at a rate of 1.5, 2, 3, 4, 5, 6, 7 or 10 times lower than that of isotype control internalization.

According to preferred embodiment II, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or fragment is characterized in that the HCDR3 region contains 10 to 34% tyrosine and/or 2 to 20% histidine.

According to preferred embodiment III, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody comprises a human CDR.

According to preferred embodiment IV, a separate antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment is cross-reactive to CCR8 from at least two species (preferably selected from humans, cynomolgus monkeys, and mice), and most preferably, wherein the antibody or antigen-binding fragment is cross-reactive to human and cynomolgus monkey CCR8.

According to preferred embodiment V, a separated antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment,

a. Does not block CCL1-induced β-arrestin signaling and/or

b. Does not induce ERK1/2 phosphorylation and/or

c. Does not induce AKT phosphorylation.

According to preferred embodiment VI, a separated antibody or antigen-binding fragment thereof that specifically binds to CCR8 is provided, wherein the antibody or antigen-binding fragment is non-fucosylated and

a. Induction of antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 by human effector cells, such as human NK cells, and

b. Inducing antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 using human effector cells, such as human macrophages.

c. The in vitro depletion of target cells expressing human CCR8 induced by the largest ADCC and ADCP was at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%.

According to preferred embodiment VII, an isolated antibody or antigen-binding fragment according to any one of preferred embodiments II to VI is provided, wherein the antibody or antigen-binding fragment is non-internalized or characterized by internalization into cells expressing endogenous targets at a rate of 1.5, 2, 3, 4, 5, 6, 7 or 10 times lower than that of the isotype control.

According to preferred embodiment VIII, an isolated antibody or antigen-binding fragment according to any one of preferred embodiments I or III to VII is provided, wherein the antibody or antigen-binding fragment is characterized in that the HCDR3 region contains 10 to 34% tyrosine and/or 2 to 20% histidine.

According to preferred embodiment IX, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to II or IV to VIII is provided, wherein the antibody comprises a human-derived CDR.

According to preferred embodiment X, an isolated antibody or antigen-binding fragment is provided according to any one of preferred embodiments I to III or V to IX, wherein the antibody or antigen-binding fragment is cross-reactive to CCR8 from at least two species (preferably selected from humans, cynomolgus monkeys and mice), and most preferably wherein the antibody or antigen-binding fragment is cross-reactive to human and macaque CCR8.

According to preferred embodiment XI, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to X is provided, wherein the antibody or antigen-binding fragment binds to CCR8 from a first species with a first dissociation constant KD and binds to CCR8 from a second species with a second dissociation constant KD, wherein the first and second dissociation constants are of the same order of magnitude.

According to preferred embodiment XII, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to IV or VI to XI is provided, wherein the antibody or antigen-binding fragment,

a. Does not block CCL1-induced β-arrestin signaling and/or

b. Does not induce ERK1/2 phosphorylation and/or

c. Does not induce AKT phosphorylation.

According to preferred embodiment XIII, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to V or VII to XII is provided, wherein the antibody or antigen-binding fragment

a. Induction of antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 using human effector cells (e.g., human NK cells), and

b. Inducing antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells (e.g., human macrophages), and optionally...

c. The in vitro depletion of target cells expressing human CCR8 induced by the largest ADCC and ADCP was at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%.

According to preferred embodiment XIV, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to XIII is provided, wherein the antibody or antigen-binding fragment specifically binds to the sulfated tyrosine-rich domain of CCR8.

According to preferred embodiment XV, an isolated antibody or antigen-binding fragment according to any one of preferred embodiments I to XIV is provided, wherein the antibody or antigen-binding fragment specifically (i) binds with an EC50 of <15 nM, <10 nM, <5 nM, <1 nM, or <0.6 nM.

a. Human CCR8 and/or the polypeptide isolated according to SEQ ID NO: 46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

b. Cynomolgus monkey CCR8 and/or the polypeptide isolated according to SEQ ID NO: 47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and/or

c. Mouse CCR8 and/or the polypeptide isolated according to SEQ ID NO: 48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

and/or (ii) human regulatory T cells activated with EC50 < 50 nM, < 25 nM, < 15 nM or < 10 nM.

According to preferred embodiment XVI, an isolated antibody or antigen-binding fragment thereof according to any one of preferred embodiments I to XV is provided.

a. Wherein the antibody or antigen-binding fragment binds to human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) of less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM, and/or

b. The antibody or antigen-binding fragment binds to human FcγRIIA (CD32a) with a dissociation constant (KD) of less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

According to preferred embodiment XVII, an isolated antibody or antigen-binding fragment thereof according to any one of preferred embodiments I to XVI is provided, wherein the antibody or antigen-binding fragment is unfucosylated.

According to preferred embodiment XVIII, an isolated antibody or antigen-binding fragment thereof according to any one of preferred embodiments I to XVII is provided, wherein the antibody or antigen-binding fragment thereof

a. Induction of antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells (e.g., human NK cells), and/or

b. Inducing antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 using human effector cells (e.g., human macrophages).

Preferably, wherein

c. The maximum exhaustion of ADCC-induced activated human regulatory T cells is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, and/or

d. The maximum exhaustion of ADCP-induced activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%, and/or

e. The in vitro exhaustion of activated human regulatory T cells induced by the largest ADCC and ADCP was at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%.

According to preferred embodiment XIX, a separated antibody or antigen-binding fragment according to any one of preferred embodiments I to XVIII is provided, wherein...

a. Antibody or antigen-binding fragments inhibiting ADCC-induced activation of human regulatory T cells with an EC50 of less than 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM, or 5 pM and/or

b. Antibody or antigen-binding fragments that induce depletion of human regulatory T cells by ADCP at EC50 values below 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, or 25 pM.

According to preferred embodiment XX, an isolated antibody or antigen-binding fragment according to any of the foregoing preferred embodiments, wherein the effective dose of said antibody or antigen-binding fragment...

a. Reducing the number of activated or intratumoral regulatory T cells to less than 50%, 40%, 30%, 25%, 20%, 10%, 5%, or 1% in vitro or in subjects and/or

b. In vitro or in subjects, increasing the ratio of intratumoral CD8+ T cells to intratumoral Tregs to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or higher and/or

c. Reduce the percentage of regulatory T cells within the tumor CD4+ T cell population to <30%, <20%, <10%, or <5%, either in vitro or in subjects.

According to a preferred embodiment XXI, an isolated antibody or antigen-binding fragment according to any of the foregoing preferred embodiments is provided, wherein an effective dose of the antibody or antigen-binding fragment induces the formation of tertiary lymphoid structures in a tumor.

According to preferred embodiment XXII, an isolated antibody or antigen-binding fragment according to any of the foregoing preferred embodiments is provided, wherein the antibody is an IgG antibody, preferably human IgG1 or mouse IgG2a, and/or wherein the antigen-binding fragment is a fragment to scFv, Fab, Fab′ or F(ab′)2.

According to preferred embodiment XXIII, a conjugate comprising an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII is provided, preferably wherein the conjugate comprises

a. Radioactive elements,

b. Cytotoxic agents, such as auristatin, maytansin alkaloids, kinin spindle protein inhibitors, nicotinamide phosphoribosyltransferase inhibitors, or pyrrolobenzodiazepine derivatives.

c. Further antibody or antigen-binding fragments, or

d. Chimeric antigen receptor.

According to preferred embodiment XXIV, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII, or a conjugate according to preferred embodiment XXIII.

According to preferred embodiment XXV, a pharmaceutical composition according to preferred embodiment XXIV is provided, comprising one or more further therapeutically active compounds, preferably selected from...

a. An antibody or compound targeting a checkpoint protein, such as PD1, PD-L1, or CTLA-4, preferably wherein the antibody or compound targeting the checkpoint protein is Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, or Ipilimumab.

b. Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

c. Antibodies or small molecules targeting HER2 and/or EGFR,

d. Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma and esophageal tumors, melanoma, bladder cancer, liver cancer and/or prostate cancer.

e. Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine.

e.B cell depleting agents, such as anti-CD19 antibodies or anti-CD20 antibodies and/or

f. Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1.

According to preferred embodiment XXVI, an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII, or a conjugate according to preferred embodiment XXIII, or a pharmaceutical composition according to preferred embodiment XXIV or XXV is provided for use as a drug.

According to preferred embodiment XXVII, an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII, or a conjugate according to preferred embodiment XXIII, or a pharmaceutical composition according to preferred embodiment XXIV or XXV, is provided for treating tumors or diseases characterized by CCR8 positive cells, such as CCR8 positive regulatory T cells.

According to preferred embodiment XXVIII, an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII, or a conjugate according to preferred embodiment XXIII, or a pharmaceutical composition according to preferred embodiment XXIV or XXV, is provided for (i) preferably in combination with one or more additional therapeutically active compounds selected from the group consisting of, separately from, or sequentially thereof.

a. An antibody or compound targeting a checkpoint protein, such as PD1, PD-L1, or CTLA-4, preferably wherein the antibody or small molecule targeting the checkpoint protein is Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, or Ipilimumab.

b. Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

c. Antibodies that target proteins specifically expressed by tumor cells.

d. Antibodies or small molecules targeting HER2 and/or EGFR,

e. Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma and esophageal tumors, melanoma, bladder cancer, liver cancer and/or prostate cancer.

f. Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin or gemcitabine, and/or

g. Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1.

(ii) in combination with radiotherapy, and/or (iii) in combination with depleted intratumoral B cells, to treat tumors or diseases characterized by CCR8-positive cells, such as CCR8-positive regulatory T cells.

According to preferred embodiment XXIX, antibodies, fragments, conjugates, or pharmaceutical compositions for use in preferred embodiments XXVII or XXVIII are provided, wherein the tumor is selected from T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, head and neck squamous cell carcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic cancer, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer, B-cell lymphoma, T-cell lymphoma, or any other cancer involving CCR8-expressing cells, preferably wherein the tumor is selected from head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, prostate cancer, B-cell lymphoma, and T-cell lymphoma. According to preferred embodiment XXX, antibodies, fragments, conjugates, or pharmaceutical compositions for use in preferred embodiments XXVI to XXIX are provided, wherein the use includes determining

a. The presence or number of tumor-infiltrating lymphocytes

b. Presence or number of macrophages and/or NK cells

c. The presence or number of CCR8-positive or FOXP3-positive regulatory T cells.

d. Tumor mutation burden,

e. Cancer staging

f. The presence, level, or activation of interferon-stimulated genes or proteins.

g.CCR8 expression,

h. The presence or quantity of complement factor proteins, serine protease inhibitors, and/or MHC components.

i. The presence or quantity of cytokines, such as inflammatory or inhibitory cytokines.

j. Activation of immune gene expression, and/or

k. Expression of immune checkpoint (proteins), such as PD-(L)1 or CTLA4 expression.

1. The presence or number of tumor-infiltrating CD19+ B cells

m. The presence or number of tumor-infiltrating CD8+ T cells;

To predict or monitor treatment success rates.

According to preferred embodiment XXXI, a polynucleotide encoding an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII is provided.

According to preferred embodiment XXXII, a vector comprising a polynucleotide according to preferred embodiment XXXI is provided.

According to preferred embodiment XXXIII, an isolated cell is provided which is arranged to produce an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII.

According to preferred embodiment XXXIV, a method is provided for generating an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII, comprising culturing cells according to preferred embodiment XXXIII and optionally purifying the antibody or antigen-binding fragment.

According to preferred embodiment XXXV, an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII is provided for use as an in vivo or in vitro diagnostic agent.

According to preferred embodiment XXXVI, a kit is provided comprising an antibody or antigen-binding fragment according to any one of preferred embodiments I to XXII, or a conjugate according to preferred embodiment XXIII, or a pharmaceutical composition according to preferred embodiments XXIV or XXV, and instructions for use.

Aspect 19-antibody conjugate

In addition to naked antibodies, various other antibody- or antibody-fragment-based conjugates can be designed using the antibodies or antigen-binding fragments disclosed herein. These conjugates can be for diagnostic, therapeutic, research applications, and various other purposes. Antibodies or antigen-binding fragments conjugated to, for example, radionuclides, cytotoxic agents, organic compounds, protein toxins, immunomodulators such as cytokines, fluorescent moieties, and cells are provided, as well as additional antibodies or antigen-binding fragments thereof.

The conjugates disclosed herein, such as antibody-drug conjugates (ADCs), targeted thorium conjugates (TTCs), and bispecific antibodies, are essentially "modules." Throughout the disclosure, various specific embodiments of the various "modules" constituting the conjugates are described. Specific embodiments of antibodies or fragments thereof, linkers, and cytotoxic agents and/or cell inhibitors that can constitute an ADC are described as concrete, non-limiting examples. It is intended that all described specific embodiments can be combined with each other as if each specific combination were explicitly described separately.

According to the nineteenth aspect, a conjugate or combination thereof comprising antibody or antigen-binding fragments according to aspects six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, and/or eighteenth is provided. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect six. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect seven. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect eight. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect nine. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect ten. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect eleven. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect twelfth. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect thirteen. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect fourteen. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect fifteen. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect sixteen. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect seventeen. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect eighteen.

According to some preferred embodiments, the conjugate comprises (a) a radioactive element, (b) a cytotoxic agent, such as auristatin, maytansine alkaloid, kinin-spindle protein (KSP) inhibitor, nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, or pyrrolobenzodiazepine derivative, (c) a further antibody or antigen-binding fragment, and (d) a chimeric antigen receptor.

Radioactive conjugates

According to some first embodiments of the nineteenth aspect, the conjugate comprises or is configured to comprise a radionuclide, such as a beta particle, an alpha particle, or an Auger electron emitter. Suitable beta emitters according to the invention are, for example, yttrium-90, iodine-131, strontium-89-chloride, lutetium-177, holmium-166, rhenium-186, rhenium-188, copper-67, promethium-149, gold-199, and rhodium-105. Suitable Auger electron emitters according to the invention are, for example, bromine-77, indium-11, iodine-123, and iodine-125. Suitable alpha emitters according to the invention are, for example, thorium-227, bismuth-213, radium-223, actinium-225, and astatine-211.

For example, thorium-227 (227Th) can be effectively complexed with an octate 3,2-hydroxypyridinone (3,2-HOPO) chelating agent conjugated with the antibody of the present invention to produce highly stable targeted thorium-227 conjugates (TTCs), as described in Example 13. The targeted thorium conjugate (TTC) comprises three main structural units. The first component, the emission α-particle of the radionuclide ^227Th , is purified by ion-exchange chromatography after the β-particle decay of thorium-227. The second component is a chelating agent, such as a siderophore-derived chelating agent containing a HOPO group, which has four 3-hydroxy-N-methyl-2-pyridinone moieties on a symmetrical polyamine scaffold (functionalized with carboxylic acid linkers for bioconjugation). Conjugation to the targeting moieties can be achieved by forming amide bonds with the ε-amino groups of lysine residues. These octate 3,2-HOPO chelating agents can be effectively labeled with 227Th and exhibit high yield, purity, and stability under ambient conditions. Compared to tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators, which often require heating, HOPO chelators offer advantages due to their efficient radiolabeling at ambient temperatures and the high stability of the resulting complex. The third component is the targeting portion, which is a chemokine receptor antibody or its antigen-binding portion according to any of aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

ADC

According to some second embodiments of the nineteenth aspect, the conjugate contains a cytotoxic agent, for example, to form an antibody-drug conjugate (ADC).

In some preferred embodiments, the cytotoxic agent is auristatin, maytansin alkaloid, kinin-spindle protein (KSP) inhibitor, nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, or pyrrolobenzodiazepine derivative. Conjugates containing maytansin alkaloids may be generated as described in Chari, Ravi VJ, et al. "Immunoconjugates containing novel maytansinoids: promising anticancer drugs." Cancer research 52.1 (1992): 127-131 or EP2424569 B1, the entire contents of which are incorporated herein by reference. Conjugates containing kinin-spindle protein (KSP) inhibitors may be generated as described in WO2019243159 A1, the entire contents of which are incorporated herein by reference. The preparation of conjugates containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors can be described as in WO2019149637 A1, the entire contents of which are incorporated herein by reference. The preparation of conjugates containing pyrrolobenzodiazepines can be described as in EP3355935 A1, the entire contents of which are incorporated herein by reference.

Cytotoxic agents and/or cell inhibitors that are anti-chemokine receptors or anti-CCR8 ADCs can be any agent known to inhibit cell growth and/or replication and/or kill cells. Many agents with cytotoxic and/or cell growth-inhibiting properties are known in the literature. Non-limiting examples of cytotoxic agents and/or cell growth inhibitors include, for example, but not limited to, cell cycle regulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, alkylating agents, DNA cross-linking agents, intercalating agents, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents.

Connectors that link cytotoxic agents and/or cell inhibitors to the antigen-binding portion of anti-chemokine receptors or anti-CCR8 ADCs can be long, short, flexible, rigid, hydrophilic, or hydrophobic in nature, or can comprise fragments with different properties, such as flexible fragments, rigid fragments, etc. Connectors can be chemically stable to the extracellular environment, for example, chemically stable in bloodstream, or can include unstable connectors and release cytotoxic agents and/or cell inhibitors into the extracellular environment. In some embodiments, the connector includes a link designed to release the cytotoxic agent and/or cell inhibitor upon internalization of the anti-chemokine receptor or anti-CCR8 ADC within the cell. In some specific embodiments, the connector includes a link designed to be specifically or non-specifically cleaved and/or sacrificed or otherwise disintegrated within the cell. A variety of connectors for linking drugs to antigen-binding portions (e.g., antibodies in the context of ADCs) are known in the art. Any of these and other connectors can be used to link cytotoxic agents and/or cell growth inhibitors to the antigen-binding portion of anti-chemokine receptors or anti-CCR8 ADCs.

The number of cytotoxic agents and/or cell inhibitors (drug-antibody ratio: DAR) linked to the antigen-binding moiety of an anti-chemokine receptor or anti-CCR8 ADC can vary and is limited only by the number of available linker sites on the antigen-binding moiety and the number of reagents linked to a single linker. Typically, the linker links a single cytotoxic agent and/or cell inhibitor to the antigen-binding moiety of the anti-chemokine receptor or anti-CCR8 ADC. In embodiments of anti-chemokine receptor or anti-CCR8 ADCs containing more than one cytotoxic agent and/or cell inhibitor, each reagent may be the same or different. Considering the anti-chemokine receptor or anti-CCR8 ADC, the DAR is 20 or higher, provided that the anti-chemokine receptor or anti-CCR8 ADC does not exhibit unacceptable levels of aggregation under use and/or storage conditions. In some embodiments, the DAR of the anti-chemokine receptor or anti-CCR8 ADC described herein may be in the range of about 1-10, 1-8, 1-6, or 1-4. In certain specific embodiments, the DAR of the anti-chemokine receptor or anti-CCR8 ADC may be 2, 3, or 4. In some implementations, the anti-chemokine receptor or anti-CCR8 ADC is a compound according to structural formula (I):

[D-L-XY]n-Ab (I)

Or its salts, wherein each “D” represents a cytotoxic agent and/or cell growth inhibitor independently of the others; each “L” represents a linker independently of the others; “Ab” represents an anti-chemokine receptor binding moiety, such as the anti-CCR8 antibody according to the invention; each “XY” represents a connection formed between a functional group Rx on the linker and a “complementary” functional group Ry on the anti-chemokine receptor binding moiety; and n represents the DAR of the anti-chemokine receptor ADC.

In one specific exemplary embodiment, the anti-chemokine receptor ADC is a compound according to structural formula (I), wherein each "D" is identical and is a cell-permeable auristatin (e.g., dolastatin-10 or MMAE) or a cell-permeable minor groove binding DNA cross-linker; each "L" is identical and is a linker that can be cleaved by lysosomal enzymes; each "XY" is a bond formed between a maleimide and a thiol group; "Ab" is an antibody or fragment thereof comprising six CDRs corresponding to six CDRs of an anti-chemokine receptor or CCR8 antibody according to the present invention; n is 2, 3, or 4. In one specific embodiment, "Ab" is a fully human antibody comprising human CDRs.

Cytotoxic agents and cell growth inhibitors are known agents that inhibit cell growth and/or replication and/or kill cells, particularly tumor cells or intratumoral Treg cells. These compounds can be used in combination therapy with anti-chemokine receptor antibodies such as CCR8 antibodies, or as part of an anti-chemokine receptor ADC, as described below:

In some embodiments, the drug portion of the antichemokine receptor or anti-CCR8 ADC is selected from the following cell growth inhibitors: radionuclides, alkylating agents, DNA cross-linking agents, DNA intercalating agents (e.g., groove binding agents such as minor groove binding agents), cell cycle regulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents.

In some embodiments, the drug portion of the anti-chemokine receptor or anti-CCR8 ADC is an alkylating agent selected from: asaley (an alkylating agent of L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanine]-ethyl ester); AZQ (1,4-cyclohexadiene-1,4-dicarboxylic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-diethyl ester); BCNU (N,N′-bis(2-chloroethyl)-N-nitrosourea); sulfolane (1,4- Butylene glycol dimethyl sulfonate); (carboxyphthalic acid)platinum; CBDCA (cis-(1,1-cyclobutanedicarboxy)diamineplatin(II)); CCNU (N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea); CHIP (isopropylplatinum; NSC 256927); chloramphenicol; chlorzoxacin (2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-pyranose); cisplatin (cisplatin); chloramphenicol; cyanomorpholinodoxorubicin; cyclodiketone; dihydrogalactitol (5 6-Dihydroxyduroitol); Fluorodopa ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil); Heptylsulfamethoxazole; Cantharidin; Indomethacin dimer DGN462; Melphalan; Methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea); Mitomycin C; Mitozodamine; Nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride); PCNU ((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidinyl)- 1-Nitrourea); Piperazine alkylating agent (1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine hydrochloride); Piperazine dione; Piperazine (N,N′-bis(3-bromopropionyl)piperazine); Boswelliacin (N-methylmitomycin C); Spiral hydantoin; Tarcenosol (triglycidyl isocyanurate); Tetra-Latin; Thiophosphate (N,N′,N″-tri-1,2-ethylenedimethylthiophosphoramide); Triethanolamine; Uracil nitrogen mustard; Yoshi-864 (bis(3-methoxypropyl)amine hydrochloride).

In some embodiments, the drug portion of the anti-chemokine receptor or anti-CCR8 ADC is a DNA alkylating agent selected from the following: cisplatin; carboplatin; nedaplatin; oxaliplatin; saxaplatin; triplatin tetranitrate; procarbazine; hexamethylamine; dacarbazine; mirtazolamide; temozolomide.

In some embodiments, the drug portion of the antichemokine receptor or anti-CCR8 ADC is selected from the following alkylated antitumor agents: carboquinone; carmustine; chlornaphthazine; chlorzoxacin; docamycin; isophosphoramide; formustine; glucosamine; lomustine; mannitol; nimustine; phenanthreneplatin; bromide; ranimustine; semastine; streptozotocin; thiotetrafluoroethylene; endosulfan; triquinone; triethylene melamine; triplatin tetranitrate.

In some embodiments, the drug portion of the anti-chemokine receptor or anti-CCR8 ADC is selected from the following DNA replication and repair inhibitors: hexamethasone; bleomycin; dacarbazine; styromycin; mitobronitol; mitomycin; bleomycin; propramycin; procarbazine; temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281; AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.

In some embodiments, the drug portion that is anti-chemokine receptor or anti-CCR8 ADC is a cell cycle regulator, such as paclitaxel; Nab-paclitaxel; docetaxel; vincristine; vinca alkaloid; ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific kinase inhibitor; Aurora B-specific kinase inhibitor and pan-Aurora kinase inhibitor; AZD-5438; BMI-1040; BMS-032; BMS-387; CVT-2584; flavonoid pyridinol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib (CYC-202, R-roscovitines); ZK-304709; AZD4877; ARRY-520: GSK923295A.

In some embodiments, the drug portion that inhibits chemokine receptors or CCR8 ADCs is an apoptosis regulator, such as AT-101 ((-)gossypol); G3139 or oblimersen (Bcl-2-targeting antisense oligonucleotide); IPI-194; IPI-565; N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-ylbenzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylthio)methyl)propyl)amino)-3-nitrobenzenesulfonamide; N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexyl-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylthio)methyl) (3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-)); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; Venetoclax; Drugs targeting TRAIL or death receptors (e.g., DR4 and DR5), such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; Drugs targeting caspase, caspase modulators, BCL-2 family members, death domain proteins, TNF family members, Toll family members, and/or NF-kappa-B proteins.

In some implementations, the drug component that inhibits chemokine receptors or CCR8 ADCs is an angiogenesis inhibitor, such as ABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547,632; IM-862; pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034); vatalanib (PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap; vandetanib; ABT-165; ZD-6474; and DLL4 inhibitors.

In some embodiments, the drug portion that is anti-chemokine receptor or anti-CCR8 ADC is a proteasome inhibitor, such as bortezomib; carfilzomib; cyclooxygenin; ixazomib; halosporin A.

In some implementations, the drug portion that inhibits chemokine receptors or CCR8 ADCs is a kinase inhibitor, such as afatinib; axitinib; bosutinib; crizotinib; dasatinib; erlotinib; fostatinib; gefitinib; ibrutinib; imatinib; lapatinib; lenvatinib; mubutinib; nilotinib; pazopanib; peragatanib; sorafenib; sunitinib; SU6656; vandetanib; vemurafenib; CEP-701 (lesatinib); XL019; INCB018424 (ruxotetinib); ARRY-142886 (celetinib); ARRY -438162 (Binimetinib); PD-325901; PD-98059; AP-23573; CCI-779; Everolimus; RAD-001; Rapamycin; Tessiromolimus; ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242, PP30, Torin 1; LY294002; XL-147; CAL-120; ONC-21; AEZS-127; ETP-45658; PX-866; GDC-0941; BGT226; BEZ235; XL765.

In some embodiments, the drug portion that inhibits chemokine receptors or CCR8 ADCs is a protein synthesis inhibitor, such as streptomycin; dihydrostreptomycin; neomycin; flammycin; paromomycin; ribostamycin; kanamycin; amikacin; abekacin; bekanamycin; dibekacin; tobramycin; spectinomycin; hygromycin B; paromomycin; gentamicin; netilmicin; sisomicin; isipamicin; cordycepsmycin; astromycin; tetracycline; doxycycline; chlortetracycline; chloramphenicol; demethylchlortetracycline; lysine; methoxycycline; methylcycline; minocycline; oxytetracycline; penicillin; rolitetracycline; tetracycline; glycylcycline; tigecycline. Oxazolidinones; Piperazolamide; Linezolid; Oxazolamide; Redizolamide; Lanbezolid; Sultezolid; Tedizolamide; Peptidyl transferase inhibitors; Chloramphenicol; Azidamico; Thiamphenicol; Florfenicol; Pleurotus ostreatus; Retamoline; Tiamulin; Vonimulin; Azithromycin; Clarithromycin; Dierythromycin; Erythromycin; Fluerythromycin; Josamycin; Midecamycin; Micardiitis; Pyruvicin; Rotamycin; Roxithromycin; Spiramycin; Pyruvicin; Ketoconazole; Telithromycin; Cyerythromycin; Solithromycin; Clindamycin; Lincomycin; Pirimibromycin; Streptomycin; Primalmycin; Quinupertin/Dalfopristin; Virginiamycin.

In some embodiments, the drug portion that inhibits chemokine receptors or CCR8 ADCs is a histone deacetylase inhibitor, such as vorinostat; romidesin; chidamide; pabisostat; valproic acid; belistat; moxistat; abexinostat; entecavir; SB939 (pracinostat); remistat; gilvestartar; quinacrinestat; thiourea-butyronitrile (Kevetrin ^™ ); CUDC-10; CHR-2845 (tefilstat); CHR-3996; 4SC-202; CG200745; ACY-1215 (roxilinstat); ME-344; sulforaphane.

In some embodiments, the drug fraction that inhibits chemokine receptors or CCR8 ADCs is a topoisomerase I inhibitor, such as camptothecin; various camptothecin derivatives and analogs (e.g., NSC 100880, NSC 603071, NSC107124, NSC 643833, NSC 629971, NSC 295500, NSC 249910, NSC 606985, NSC...). 74028, NSC176323, NSC 295501, NSC 606172, NSC 606173, NSC 610458, NSC 618939, NSC 610457, NSC610459, NSC 606499, NSC 610456, NSC 364830, and NSC 606497); Morphyrin isoxastatin; SN-38.

In some embodiments, the drug portion that resists chemokine receptors or CCR8 ADCs is a topoisomerase II inhibitor, such as doxorubicin; amonafide (benzisoquinolinedione); m-AMSA (4′-(9-acridylamino)-3′-methoxymethanesulfonylaniline); anthraquinone derivative ((NSC 355644); etoposide (VP-16); pyrazoloacridin ((pyrazolo[3,4,5-kl]acridin-2(6H)-propylamine, 9-methoxy-N,N-dimethyl-5-nitro-,monosulfonate); piracetam hydrochloride; daunorubicin; deoxydaunorubicin; mitoxantrone; minogalin; N,N-dibenzyldaunorubicin; orantrone; rubidazine; teniposide.

In some embodiments, the drug portion that is anti-chemokine receptor or anti-CCR8 ADC is a DNA intercalating agent, such as anthramycin; zithromycin A; tomatine; DC-81; sibromycin; pyrrolodiazapyridine derivatives; SGD-1882((S)-2-(4-aminophenyl)-7-methoxy-8-(3S)-7-ethoxy-2-(4-methoxyphenyl)-5-oxo-5,11-dihydro-1H-benzo[e]pyrrolo[1,2-a][1] ,4] diazaoct-8-yl)oxy)propoxy)-1H benzo[e]pyrrolo[1,2-a][1,4] diazaoct-5(11aH)-one); SG2000(SJG-136; (11a S, 11a′S)-8,8′-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4] diaza-5(11aH)-one)).

In some embodiments, the drug portion that inhibits chemokine receptors or CCR8 ADCs is an RNA/DNA antimetabolite, such as L-adenosine; 5-azacytidine; 5-fluorouracil; acevir; aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid; aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridyl)methyl]amino]benzoyl]L-aspartic acid monohydrate; anti- Folic acid PT523 ((Nα-(4-amino-4-deoxypteroyl)-Nγ-hemiphthaloyl-L-ornithine)); Baker's soluble antifolic acid (NSC 139105); dichloroallylloxacin ((2-(3,3-dichloroenyl)-3-hydroxy-1,4-naphthoquinone); Brefinar; flutifu ((prodrug; 5-fluoro-1-(tetrahydro-2-furanyl)-uracil); 5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivative (N-[[4-[[(2,4-diamino-6-pterodinyl)methyl]methylamino]-1-naphthyl]carbonyl]-L-glutamic acid); PALA ((N-(phosphonyl)-L-aspartic acid); pyrazolylphosphonate; methotrexate.

In some embodiments, the drug portion of the antichemokine receptor or anti-CCR8 ADC is a DNA antimetabolite, such as 3-HP; 2′-deoxy-5-fluorouridine; 5-HP; α-TGDR (α-2′-deoxy-6-thioguanosine); afedipine glycinate; araC (cytarabine); 5-aza-2′-deoxycytidine; β-TGDR (β-2′-deoxy-6-thioguanosine); cyclocytidine; guanidine; hydroxyurea; inosylglycodialdehyde; maculin II; pyrazoimidazole; thioguanine; thiopurine.

In some embodiments, the drug component that inhibits chemokine receptors or CCR8 ADCs is a mitochondrial inhibitor, such as trypsin; benpanstatin; rhodamine-123; senna; d-α-tocopherol succinate; compound 11β; aspirin; elutin; berberine; azuril; GX015-070(1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-); tripterygium oleate; metformin; bright green; ME-344.

In some embodiments, the drug portion that inhibits chemokine receptors or CCR8 ADCs is an antimitotic agent, such as allocolchicine; auristatin, such as MMAE (monomethyl auristatin E) and MMAF (monomethyl auristatin F); squalane B; simadotine; colchicine; colchicine derivatives (N-benzoyl-deacetylated benzamide); slug toxin 10; slug toxin 15; maytansine; maytansine alkaloids, such as DM1 (N2′-deacetylated-N2′-(3-mercapto-1-oxopropyl)-matansine); rosocine; paclitaxel; paclitaxel derivatives ((2′-N-[3-(dimethylamino)propyl]glutaric acid paclitaxel); docetaxel; thiocolchicine; triphenylmethylcysteine; vincristine sulfate; vincristine sulfate.

In some embodiments, the drug portion that is anti-chemokine receptor or anti-CCR8 ADC is a nuclear export inhibitor, such as callystatin A; lactamycin; KPT-185 (propyl-2-yl(Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]propyl-2-enoate); cassumycin A; leptin; leptofuran A; leptomycin B; rajadone; Verdinexor ((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin-2-ylpropyl-2-enoate).

In some implementations, the drug portion that inhibits chemokine receptors or CCR8 ADCs is a hormonal therapeutic agent, such as anastrozole; exemestane; azoxifen; bicalutamide; cetrorex; degarelix; dilorelin; trichloroethane; dexamethasone; flutamide; raloxifene; fazodone; toremifene; fulvestrant; letrozole; formetanin; glucocorticoids; doxorubicin; sevelamer carbonate; lasoxifene; leuprorelin acetate; medroxyprogesterone acetate; mifepristone; nilumet; tamoxifen citrate; abalinic acid; prednisone; finasteride; lilosterone; buterelin; luteinizing hormone-releasing hormone (LHRH); histamine; trelosterone or modrastan; frenelin; goserelin.

Any of these reagents includes, or can be modified to include, a linking site with an antibody and/or binding fragment, which may be included in an anti-chemokine receptor ADC, such as in an anti-CCR8 ADC.

CAR T cells

According to some third embodiments of the nineteenth aspect, the conjugate is a CAR T cell conjugate engineered for targeting a chemokine receptor or CCR8. Recently, CAR T cells have gained attention due to their clinical success and accelerated FDA approval (see WO2020102240, the entire contents of which are incorporated herein by reference). In the CAR T cell approach, T cells are collected from a patient's blood and then genetically engineered to express a CAR that is specific to antigens present on tumor cells. These engineered T cells are then re-administered to the same patient. After injection, the CAR T cells recognize the target antigen on the target cells to induce target cell death. Thus, T cells expressing chimeric antigen receptors (CAR T cells) constitute a novel modality for medical applications such as cancer therapy. A chimeric antigen receptor (CAR) is a genetically engineered receptor designed to target specific antigens, such as tumor antigens. This targeting can lead to cytotoxicity against tumors, for example, enabling CAR T cells expressing CARs to target and kill tumors via specific tumor antigens.

According to the present invention, antibodies or antigen-binding fragments provided for CCR8 or chemokine receptor recognition can be used to engineer CAR T cells to specifically recognize CCR8-expressing cells or cells expressing the corresponding chemokine receptors. The CAR according to the present invention may include...

a) A recognition region, such as a single-chain variable region (scFv) derived from a provided anti-CCR8 or anti-chemokine receptor antibody, for recognizing and binding to the CCR8 or chemokine receptor expressed on target cells, and

b) Activation signaling domains, such as the CD3 chain of T cells, can serve as T cell activation signals in CARs.

Preferably, the CAR according to the invention comprises a co-stimulatory domain (e.g., CD137, CD28, or CD134) to achieve prolonged activation of T cells in vivo. The addition of the co-stimulatory domain enhances the in vivo proliferation and survival of CAR-containing T cells, and preliminary clinical data suggest that such constructs are promising therapeutic agents for diseases such as cancer. According to the invention, CAR T cells can be used to treat any disease in which locally or systemically abnormal cells expressing target chemokine receptors (particularly CCR8-expressing cells, such as Treg cells) are present.

Bispecific

According to some fourth embodiments of the nineteenth aspect, the conjugate is a bispecific antibody or a multispecific antibody. In some preferred embodiments, the bispecific antibody comprises at least one Fc domain.

In some preferred embodiments of the nineteenth aspect, the first binding portion of the bispecific antibody is an antibody or antigen binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18.

In some embodiments A of the fourth embodiment of the nineteenth aspect, the first binding portion of the bispecific antibody is an antibody or antigen binding fragment based on aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, and the second binding portion of the bispecific antibody is the same or different antibody or antigen binding fragment based on aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18.

In some embodiments B of the fourth embodiment of the nineteenth aspect, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment binding to human CCR8 according to the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th antibodies, and the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment binding to different chemokine receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1. The second binding portion of the bispecific antibody is Mogamulizumab or its antigen-binding fragment.

In some embodiments C of the fourth embodiment of the nineteenth aspect, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that binds to cell surface proteins, such as cell surface proteins expressed on immune cells or tissues—or cell type-specific antigens. In some embodiments, the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment targeting a checkpoint protein, such as an anti-PD1 antibody, an anti-PD-L1 antibody, or a CTLA-4 antibody. Other examples include Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, or Ipilimumab. In some other embodiments of these embodiments, the second binding portion of the bispecific antibody is a HER2-targeting antibody, Trastuzumab, Pertuzumab, and/or Margetuximab.

In some embodiments D of the fourth embodiment of the nineteenth aspect, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment of the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th, preferably binding to CCR8, and the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that binds to cell surface molecules associated with T cell activation, preferably selected from CD25, CTLA-4, PD-1, LAG3, TIGIT, ICOS, TNF receptor superfamily members, 4-1BB, OX-40, GITR.

Techniques for preparing bispecific or multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello. "Hybrid hybrids and their use in immunohistochemistry." Nature 305.5934(1983): 537-540.; WO1993008829 A1, and Traunecker, André, Antonio Lanzavecchia, and Klaus Karjalainen. "Bispecific single chain mol Ecules (Janusins) target cytotoxic lymphocytes on HIV-infected cells. "The EMBO Journal 10.12 (1991): 3655-3659.), and chemical conjugation of two different monoclonal antibodies (see Staerz, Uwe D., Osami Kanagawa, and Michael J. Bevan. "Hybrid antibodies can target sites for attack by T cells. "Nature 314.6012 (1985): 628-631). Multispecific antibodies can also be prepared by crosslinking two or more antibodies or fragments (see, for example, US Patent No. 4,676,980, and Brennan, Maureen, Peter F. Davison, and Henry Paulus. "Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments." Science 229.4708(1985): 81-83), using leucine zippers to generate bispecific antibodies (see, for example, Kostelny, Sheri A., M.S. Cole, and J. Yun Tso. "Formation o f a bispecific antibody by the use of leucine zippers. "The Journal of Immunology 148.5 (1992): 1547-1553.), bispecific antibody fragments were prepared using biantibody technology (see, for example, Holliger, Philipp, Terence Prospero, and Greg Winter. "Diabodies: small bivalent and bispecific antibody fragments. "Proceedings of the National Academy of Sciences 90.14 (1993): 6444-6448), using single-chain Fv ( The sFv) dimer (Gruber, Meegan, et al. "Efficient tumor cell lysis mediated by a bispecific single-chain antibody expressed in Escherichia coli." The Journal of Immunology 152.11 (1994): 5368-5374.) was prepared by the preparation of trispecific antibodies as described (e.g., in Tutt, Alison, G.T. Stevenson, and M.J. Glennie. "Trispecific F(ab′)3 derivatives that use cooperative signaling via t The TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells. "The Journal of Immunology 147.1 (1991): 60-69.), and according to Labrijn, Aran F., et al. "Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange." Proceedings of the National Academy of Sciences 110.13 (2013): 5145-5150.

Conjugates for diagnostic and research applications

According to some fifth embodiments of the nineteenth aspect, the conjugate includes a detectable portion. Examples of detectable portions include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and reactive portions. The detectable substance can be directly or indirectly coupled or conjugated to an antibody or a fragment thereof, for example, through a connector or another portion known in the art, using techniques known in the art. Examples of enzyme-labeled enzymes include luciferases (e.g., firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucosylamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc.

Examples of suitable cofactor complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and jellyfish luminescent protein; examples of suitable radioactive substances include 125I, 131I, 111In, or 99mTc.

Detecting the expression of chemokine receptor or CCR8 typically involves contacting a biological sample (an individual's tumor, cells, tissue, or body fluid) with one or more antibodies or fragments according to the invention (optionally conjugated to a detectable portion) and detecting whether the sample is positive for chemokine receptor or CCR8, or whether the expression of the sample has changed (e.g., decreased or increased) compared to a control sample.

Aspect 20 - Pharmaceutical Composition

According to aspect 20, a pharmaceutical composition comprising an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, is provided. Preferably, the pharmaceutical composition comprises a therapeutically effective amount of an antibody or a fragment or conjugate thereof, or a combination thereof, according to any of the embodiments described herein, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

Pharmaceutical compositions can be prepared by mixing an antibody, fragment, or conjugate of desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer (Remington, Joseph Price. Remington: The science and practice of pharmacy. Vol. 1. Lippincott Williams & Wilkins, 2006). Pharmaceutical compositions can be in the form of, for example, lyophilized formulations or aqueous solutions.

Carrier, excipient, stabilizer

Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used, including buffers such as phosphate-buffered saline (e.g., PBS), citrate buffer, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight ( Examples of examples include polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

Multiple therapeutically active compounds

According to the present invention, a pharmaceutical composition may contain more than one active compound, for example, which is necessary or beneficial for a specific indication being treated.

According to some first embodiments of aspect 20, the pharmaceutical composition comprises one or more additional therapeutically active compounds.

In some preferred embodiments of the first embodiment of aspect 20, the pharmaceutical composition comprises an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or aspect 18, or a conjugate according to aspect 19, and

a) Antibodies or small molecules targeting checkpoint proteins, such as PD1, PD-L1, or CTLA-4, and/or

b) Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1, and/or

c) Antibodies and/or proteins specifically expressed by tumor cells.

d) Antibodies or small molecules targeting HER2 and/or EGFR, and/or

e) Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer, and/or

f) Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, paclitaxel or gemcitabine, and/or

g) Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1.

Combined with checkpoint inhibitors

According to a preferred embodiment A of the first embodiment of aspect 20, the pharmaceutical composition further includes an antibody or small molecule targeting checkpoint proteins such as PD1, PD-L1, or CTLA-4. Suitable checkpoint-targeting antibodies include Nivolumab (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Atezolizumab (PD-L1; humanized IgG1), Avelumab (PD-L1; human IgG1), Durvalumab (PD-L1; human IgG1), Cemiplimab, cemiplimab-rwlc (PD-1; human mAb), Dostarlimab (TSR-042) (PD-1; humanized IgG4), or Ipilimumab (CTLA-4; human IgG1).

In some implementations, antibodies or small molecules targeting checkpoint proteins target CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof.

Combination with chemokine receptor antibodies

According to some preferred embodiments B of the first embodiment of aspect 20, the pharmaceutical composition further comprises an antibody or small molecule targeting additional chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1. Suitable antibodies targeting additional chemokine receptors include antibodies provided according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and/or 18, or Mogamulizumab.

Combination with HER2 or EGFR-targeting antibodies or molecules

According to some preferred embodiments C of the first embodiment of aspect 20, the pharmaceutical composition further comprises an antibody or small molecule targeting HER2 and/or EGFR. Suitable antibodies targeting HER2 are Trastuzumab (HER2; humanized IgG1), Pertuzumab (HER2; humanized IgG1), Ado-trastuzumab emtansine (HER2; humanized IgG1; ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki (HER2; humanized IgG1 ADC), Sacituzumab govitecan; sacituzumab govitecan-hziy (TROP-2; humanized IgG1 ADC) and/or Margetuximab (HER2; chimeric IgG1). Suitable antibodies targeting EGFR are Cetuximab (EGFR; chimeric IgG1), Panitumumab (EGFR; human IgG2), and Necitumumab (EGFR; human IgG1).

Combination with therapeutic antibodies

According to some embodiments D of the first embodiment of aspect 20, the pharmaceutical composition comprises an additional therapeutic antibody selected from the following: Muromonab-CD3 (CD3; mouse IgG2a), Efalizumab (CD11a; humanized IgG1), Tositumomab-I131 (CD20; mouse IgG2a), Nebacumab (Endotoxin; human IgM), Edrecolomab (EpCAM; mouse IgG2a), Catumaxomab (EPCAM/CD3; rat/mouse bispecific mAb), Daclizumab (IL- 2R (humanized IgG1), Abciximab (GPIIb/IIIa (chimeric IgG1Fab), Rituximab (CD20 (chimeric IgG1)), Basiliximab (IL-2R (chimeric IgG1)), Palivizumab (RSV (humanized IgG1)), Infliximab (TNF (chimeric IgG1)), Trastuzumab (HER2 (humanized IgG1)), Adalimumab (TNF (human IgG1)), Ibritumomab tiuxetan (CD20 (mouse IgG1)), Omalizu mab (IgE; humanized IgG1), Cetuximab (EGFR; chimeric IgG1), Bevacizumab (VEGF; humanized IgG1), Natalizumab (α4 integrin; humanized IgG4), Panitumumab (EGFR; human IgG2), Ranibizumab (VEGF; humanized IgG1 Fab), Eculizumab (C5; humanized IgG2/4), Certolizumab pegol (TNF; humanized Fab, PEGylated), Ustekinumab (IgE; humanized IgG1), Cetuxima ... L-12/23 (human IgG1), Canakinumab (IL-1β; human IgG1), Golimumab (TNF; human IgG1), Ofatumumab (CD20; human IgG1), Tocilizumab (IL-6R; humanized IgG1), Denosumab (RANK-L; human IgG2), Belimumab (BLyS; human IgG1), Ipilimumab (CTLA-4; human IgG1), Brentuximab vedotin (CD30; chimeric IgG1; ADC), Pertuzumab (HER2; humanized IgG1), Ado-trastuzumab emtansine (HER2; humanized IgG1; ADC), Raxibacumab (B. anthrasis PA; human IgG1), Obinutuzumab (CD20; humanized IgG1 γ-coengineered), Siltuximab (IL-6; chimeric IgG1), Ramucirumab (VEGFR2; human IgG1), Vedolizumab (α4β7 integrin; humanized IgG1), Nivoluma b (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Blinatumomab (CD19, CD3; mouse bispecific tandem scFv), Alemtuzumab (CD52; humanized IgG1), Evolocumab (PCSK9; human IgG2), Idarucizumab (Dabigatran; humanized Fab), Necitumumab (EGFR; human IgG1), Dinutuximab (GD2; chimeric IgG1), Secukinumab (IL-17a; human IgG1) Mepolizumab (IL-5; humanized IgG1), Alirocumab (PCSK9; human IgG1), Daratumumab (CD38; human IgG1), Elotuzumab (SLAMF7; humanized IgG1), Ixekizumab (IL-17a; humanized IgG4), Reslizumab (IL-5; humanized IgG4), Olaratumab (PDGFRα; human IgG1), Bezlotoxumab (Clostridium difficile enterotoxin B; human IgG1), Ateaolizumab (PD-L1; human humanized IgG1), Obiltoxaximab (B.anthrasis PA; chimeric IgG1), Brodalumab (IL-17R; human IgG2), Dupilumab (IL-4Rα; human IgG4), Inotuzumabozogamicin (CD22; humanized IgG4; ADC), Guselkumab (IL-23p19; human IgG1), Sarilumab (IL-6R; human IgG1), Avelumab (PD-L1; human IgG1), Emicizumab (Factor Ixa, X; Humanized IgG4 (bispecific), Ocrelizumab (CD20; humanized IgG1), Benralizumab (IL-5Rα; humanized IgG1), Durvalumab (PD-L1; human IgG1), Gemtuzumab ozogamicin (CD33; humanized IgG4; ADC), Erenumab, erenumab-aooe (CGRP receptor; human IgG2), Galcanezumab, galcanezumab-gnlm (CGRP; humanized IgG4), Burosumab, burosumab-twza (FGF23; human IgG1), lanadelumab, lanadelumab-flyo (Plasma kallikrelin; human IgG1), mogamulizumab, mogamul izumab-kpkc (CCR4; humanized IgG1), Tildrakizumab; tildrakizumab-asmn (IL-23p19; humanized IgG1), Fremanezumab, fremanezumab-vfrm (CGRP; human Humanized IgG2), Ravulizumab, ravulizumab-cwvz (C5; humanized IgG2/4), Cemiplimab, cemiiplimab-rwlc (PD-1; human mAb), Ibalizumab, ibalizumab-uiyk (CD4; humanized IgG4), Emapalumab, emapalumab-lzsg (IFNg; human IgG1), Moxetumomabpasudotox, moxetumomab pasudotox-tdfk (CD22; mouse IgG1 dsF) V immunotoxin), Caplacizumab, caplacizumab-yhdp (von Willebrand factor; humanized nanobody), Risankizumab, risankizumab-rzaa (IL-23p19; humanized IgG1), Polatuzumab vedotin, polatuzumab vedotin-piiq (CD79b; humanized IgG1 ADC), Romosozumab, romosozumab-aqqg (Sclerostin; humanized IgG2), Brolu cizumab, brolucizumab-dbll (VEGF-A; humanized scFv), crizanlizumab; crizanlizumab-tmca (CD62 (aka P-selectin); humanized IgG2), Enfortuma b vedotin, enfortumab vedotin-ejfv (Nectin-4; human IgG1ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki ( HER2 (humanized IgG1 ADC), Teprotumumab, teprotumumab-trbw (IGF-1R; human IgG1), Eptinezumab, eptinezumab-jjmr (CGRP; humanized IgG1), Isatuximab, isatuximab-irfc (CD38; chimeric IgG1), Sacituzumab govitecan; sacituzumab govitecan-hziy (TROP-2; humanized IgG1 ADC), Inebilizumab (CD38; chimeric IgG1), 19; humanized IgG1), Leronlimab (CCR5; humanized IgG4), Satralizumab (IL-6R; humanized IgG2), Narsoplimab (MASP-2, human IgG4), Tafasitamab (CD19; humanized IgG1), REGNEB3 (Ebola virus; mixture of 3 human IgG1s), Naxitamab (GD2; humanized IgG1), Oportuzumab monatox (EpCAM; humanized scFv immunotoxin), Belantambab Mafodotin (B-cell maturation antigen; humanized IgG1 ADC), Margetuximab (HER2; chimeric IgG1), Tanezumab (nerve growth factor; humanized IgG2), Dostarlimab (TSR-042) (PD-1; humanized IgG4), Teplizumab (CD3; humanized IgG1), Aducanumab (β-amyloid protein; human IgG1), Sutimlimab (BIVV009) (C1s; humanized IgG4), Evinacumab (angiopoietin-like 3; human IgG4).

Combination with cytotoxic agents or cell growth inhibitors

According to some embodiments E of the first embodiment of aspect 20, the pharmaceutical composition further comprises a cell growth inhibitor and/or cytotoxic agent selected from the following: radionuclides, alkylating agents, DNA cross-linking agents, DNA intercalating agents (e.g., groove binding agents such as minor groove binding agents), cell cycle regulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents, as described in the second embodiment of aspect 19.

In some preferred embodiments, the cytotoxic agent is auristatin, maytansine alkaloid, kinin-spindle protein (KSP) inhibitor, nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, or pyrrolobenzodiazepine derivative.

Section 21 - Drug Uses/Treatment Methods

According to aspect 21, an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, is provided for use as a medicine.

In addition, the use of antibodies or antigen-binding fragments provided according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or conjugates according to aspect 19, or pharmaceutical compositions according to aspect 20, for the preparation of a medicament, for example, for the treatment of tumors or diseases involving cells expressing chemokine receptors, particularly CCR8.

In addition, a method for treating a disease is provided, the method comprising administering an effective dose of an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, to a patient in need.

For therapeutic applications, the antibodies or antigen-binding fragments or conjugates or pharmaceutical compositions according to the invention can be administered to patients or subjects, for example, to human or non-human subjects in pharmaceutically acceptable dosage forms. For example, they can be administered intravenously in pellet form or by continuous infusion over a period of time via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes. The antibodies, fragments, conjugates, and pharmaceutical compositions according to the invention are particularly suitable for administration via intratumoral, peritumoral, intralesional, or perilesional routes to achieve local and systemic therapeutic effects.

Possible routes of administration include parenteral (e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous), intrapulmonary, and intranasal. Furthermore, antibodies, fragments, conjugates, and pharmaceutical compositions can be administered via pulsatile infusion, such as dose-decrease administration of antibodies, fragments, or conjugates. Preferably, administration is by injection, most preferably intravenous or subcutaneous, depending in part on whether the administration is short-term or long-term. The amount administered may depend on a variety of factors, such as the patient's or subject's clinical symptoms, weight, and whether other medications are being administered. Those skilled in the art will recognize that the route of administration will vary depending on the condition or disease to be treated.

Antibody dosing frequencies range from once every 3–6 months to once weekly, once every two weeks (BIW), or daily. Similarly, dose levels range from low fixed mg doses (daily, weekly, once every two weeks, or monthly, depending on the antibody) to approximately 1 g doses. Dosing frequency depends on a variety of factors, including target concentration and turnover, target biodistribution, the half-life of the antibody, fragment, or conjugate, and potential pharmacodynamic effects that may enhance the biological effects of the antibody, fragment, or conjugate, and pharmaceutical compositions present beyond their pharmacologically relevant levels.

In some embodiments, the anti-CCR8 antibody is administered at a dose of 1 mg/kg, for example, daily, weekly, q2w, q3w, or q4w. In some embodiments, the dose of the anti-CCR8 antibody is 1 to 30 mg/kg, preferably 5 to 10 mg/kg, for example, daily, weekly, q2w, q3w, or q4w. In some embodiments, the dose of the anti-CCR8 antibody is 4 to 8 mg/kg, preferably 5 to 6 mg/kg, for example, daily, weekly, q2w, q3w, or q4w.

The appropriate dosage of an antibody for prevention or treatment will depend on the type, severity, and course of the disease, whether the antibody is administered for prevention or treatment, prior treatment, the subject's clinical history and response to antibody variants, and the judgment of the attending physician or healthy veterinary professional. The antibody may be administered to the subject once or as part of a series of treatments.

As illustrated in Example 12.4.2, it was surprisingly observed that even a single dose could be sufficient to establish a significant therapeutic response. According to the invention, an anti-CCR8 antibody for treating tumors that induces ADCC and/or ADCP is thus provided, whereby only a single dose of the anti-CCR8 antibody is administered to the subject, eliminating the need to administer further doses of the same or different anti-CCR8 antibody to the subject. This approach is not only logically superior and particularly convenient for patients, but also avoids patient compliance issues.

When administered intravenously, a pharmaceutical composition comprising an antibody, fragment, or conjugate can be administered by infusion over a period of about 0.5 to about 5 hours. In some embodiments, the infusion may last for about 0.5 to about 2.5 hours, about 0.5 to about 2.0 hours, about 0.5 to about 1.5 hours, or about 1.5 hours, depending on the antibody, fragment, conjugate, and pharmaceutical composition administered, and the amount of the antibody, fragment, or conjugate administered.

Aspect 22 - Secondary Medical Uses/Treatment Methods

Treatment with the anti-CCR8 antibody according to the invention has shown significant efficacy in various syngeneic tumor models. In some cases, administration of an effective dose of the antibody or antigen-binding fragment to a group of diseased subjects resulted in

a. At least 15%, 20%, or 25% of the subjects had a complete response, and/or

b. Overall response rate of at least 40%, 50%, 60%, or 70% of the subjects.

According to aspect 22, antibodies or antigen-binding fragments according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or conjugates according to aspect 19, or pharmaceutical compositions according to aspect 20, are provided for treating tumors or diseases characterized by chemokine receptor-positive cells, preferably CCR8-positive cells, such as CCR8-positive regulatory T cells. Treatment may occur as discussed in aspect 21.

Preferably, an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, is provided for treating a tumor or disease, characterized in that it involves cells expressing a targeted seven-transmembrane receptor or chemokine receptor.

For the antibodies, antigen-binding fragments, or conjugates according to the present invention, various modes of action are conceivable. One mode of action is to conjugate the anti-chemokine receptor antibody of the present invention with a drug to form an antibody-drug conjugate (ADC). Another mode of action is the ability of the antibody to target the chemokine receptor and induce ADCC. A third mode of action lies in the ability of the antibody to target the chemokine receptor and induce ADCP.

Specific CCR8 antibodies or antigen-binding fragments according to aspects 8, 11, 12, 13 and 14 are particularly suitable for ADCC/ADCP-based methods, for example because they are characterized as having particularly low internalization or being non-internalizing antibodies, thus residing on the surface of target cells expressing CCR8 and causing their effective killing, as demonstrated in vitro by activated human Tregs (Example 10.3.3 ff, Example 10.3.4 ff) or in vivo (Example 12 ff).

Regulatory T cells (Tregs) promote tumor growth by suppressing the function of effector T cells, including tumor reactive T cells in the tumor microenvironment. In fact, Tregs are one of the key resistance mechanisms that hinder the efficacy of immune checkpoint inhibitors (ICIs) in many indications.

CCR8 is a surface receptor of the GPCR family of chemokine receptors, primarily expressed on activated immunosuppressive Tregs commonly found in tumors. Unlike other approaches targeting these suppressor T cells, targeting CCR8 offers the opportunity to deplete intratumoral Tregs without affecting resting Tregs or other systemic immune cells. Therefore, targeting CCR8 may be superior in both efficacy and safety. Consequently, this mode of action can be used for tumors with intratumoral Tregs, but also for other diseases characterized by the presence of abnormally activated Tregs, i.e., CCR8 expression.

Specific indications

In principle, antibodies, fragments, conjugates, and pharmaceutical compositions can be used to treat any cancer involving CCR8-expressing cells. For example, cancer may include tumor cells expressing CCR8, such as B-cell lymphoma and T-cell lymphoma. Alternatively, cancer may contain intratumoral Tregs expressing CCR8. As shown in Example 11, CCR8 is upregulated in a variety of cancer indications, such as T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, head and neck squamous cell carcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic cancer, ovarian or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer. According to some preferred embodiments, the use as a medicine is for treating head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumors, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

According to some first embodiments of aspect 22, the tumor is selected from adrenal carcinoma (e.g., adrenocortical carcinoma or pheochromocytoma), bladder cancer (e.g., transitional cell carcinoma, transitional cell carcinoma-papillary), brain cancer (e.g., glioma-astrocytoma, glioma-astrocytoma-glioblastoma, glioma-oligodendroglioma, glioma-oligodendroglioma), breast cancer (e.g., ADC, ADC-duct, ADC-duct-TNBC, ADC-duct-TPBC, ADC-lobule), colorectal cancer (e.g., ADC), esophageal cancer (e.g., ADC), esophageal cancer (e.g., SCC), gastric cancer (e.g., ADC, ADC-diffuse, ADC-intestinal type, ADC-intestinal-tubular), head and neck cancer (e.g., laryngeal cancer-SCC, SCC, oral cancer-SCC), kidney cancer (e.g., ccRCC, chromophobe, papillary, papillary type I, papillary type II), liver cancer, etc. Cancers such as HCC, lung cancer (e.g., NSCLC-ADC, NSCLC-ADC-mixed, NSCLC-SCC, SCLC), mesothelioma (e.g., epithelioid), ovarian cancer (e.g., ADC-cystadenocarcinoma-serous papillary carcinoma), pancreatic cancer (e.g., ADC ductal), prostate cancer (e.g., ADC-acinoid), sarcoma (e.g., leiomyosarcoma, dedifferentiated liposarcoma, malignant fibrous histiocytoma), skin cancer (e.g., melanoma), testicular cancer (e.g., germ cell tumor-seminomatous tumor), thymoma, thyroid cancer (e.g., follicular carcinoma, papillary carcinoma-classic variant), or uterine cancer (e.g., cervical-SCC, cervical-SCC-keratotic, cervical-SCC-nonkeratotic, endometrial-ADC-endometrioid, endometrial-ADC-papillary serous, endometrial-carcinosarcoma-malignant Müllerian mixed tumor), B-cell lymphoma, and T-cell lymphoma (see Table 11.1.2).

According to some second embodiments of aspect 22, the tumor is selected from T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, head and neck squamous cell carcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic cancer, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer, or any other cancer involving cells expressing chemokine receptors or CCR8. Preferably, the tumor is selected from head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumors, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

Combination therapy

According to some third embodiments of aspect 22, it can and is suggested to be combined with the first and/or second embodiments of aspect 22, the use being simultaneous, alone or sequentially used with one or more other therapeutically active compounds. Examples of combination therapy are provided in Examples 12.6, 12.6.1, 12.6.2, 12.6.3, 12.6.4, 12.6.5, 12.6.6, 12.6.7, 12.6.8 and 12.6.9, demonstrating the broad applicability of the anti-CCR8 antibody of the present invention in combination therapy with general anti-CCR8 antibodies.

Antichemokine receptor antibodies, such as CCR8 antibodies or ADCs, can be used adjunctive or in conjunction with other agents or treatments that have anticancer properties. When used adjunctive, antibodies, fragments or conjugates, and other agents can be formulated together in a single combination pharmaceutical composition or formulation, as described in aspect 20, or preferably formulated and administered separately on a single coordinated dosing regimen or different dosing regimens.

Sequential dosing: Combination partners after anti-CCR8 antibody therapy

According to the present invention, the administration of an anti-CCR8 antibody as a first agent and the subsequent administration of one or more other therapeutically active compounds have increased efficacy in an unpredictable manner.

According to the preferred treatment regimen, treatment begins with the administration of an anti-CCR8 antibody (e.g., daily, weekly, q2w, q3w, or q4w as described above), followed optionally by the administration of one or more additional therapeutically active compounds. As described in Example 12.6ff, initiating administration of a second therapeutic agent or therapy after the initial dose of the anti-CCR8 antibody is associated with further improved efficacy. Without being bound by theory, the time between initial doses should be chosen to allow for at least 30%, 40%, 50%, or 60% depletion of intratumoral Tregs. One or more additional therapeutically active compounds or combination partners are suggested for any of those described for this purpose. For example, one or more additional therapeutically active compounds may include checkpoint inhibitors (e.g., anti-PD1/anti-PD-L1/anti-CTLA4 antibodies), antibodies targeting proteins specifically expressed by tumor cells, or chemotherapeutic agents described herein.

In different implementations, treatment begins with the administration of chemotherapy agents, followed by the administration of anti-CCR8 antibodies (e.g., on a weekly or bi-weekly schedule).

Other therapeutically active compounds for combination therapy

Agents used as adjunctive therapy with anti-chemokine receptor antibodies or ADCs (e.g., anti-CCR8 antibodies or ADCs) may have complementary activity to the anti-chemokine receptor antibodies or ADCs, thus the antibody/ADC and other drugs will not adversely affect each other. Agents that can be used as adjunctive therapy with the anti-chemokine receptor or anti-CCR8 antibodies or ADCs according to the present invention can be alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T-cell conjugate) antibodies, antibody-drug conjugates, biological response modifiers, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase 2 inhibitors, DVD, leukemia virus oncogene homologue (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunology, etc. Inhibitors of apoptosis protein inhibitors (IAPs), embedded antibiotics, kinase inhibitors, kinase inhibitors, Jak2 inhibitors, mammalian rapamycin target inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly(adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum-based chemotherapy drugs, pololf-like kinase (Plk) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoic acid/deltaic alkaloids, inhibitory small RNAs (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, etc., as well as one or more combinations of these agents.

According to some highly preferred embodiments of the third embodiment of aspect 22, an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, is provided for use in conjunction with, separately from, or sequentially combined with the following.

(i) one or more other therapeutically active compounds, preferably selected from...

a) Antibodies or small molecules that target checkpoint proteins, such as PD1, PD-L1, or CTLA-4.

b) Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

c) Antibodies that target proteins specifically expressed by tumor cells.

d) Antibodies or small molecules targeting HER2 and/or EGFR,

e) Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer, and/or

f) Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine.

g) Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1, and/or

(ii) Radiation therapy, and/or

(iii) Depletion of B cells within the tumor

For example, in the treatment of tumors or diseases, cells characterized by chemokine receptor positivity, such as CCR8 positive cells, such as CCR8 positive regulatory T cells, are preferred.

Suitable taxanes can be selected, such as those selected from paclitaxel, albumin-bound paclitaxel, cabazitaxel, or docetaxel or their derivatives.

The embodiments in which anti-CCR8 antibodies are combined with doxorubicin, taxanes, cisplatin or carboplatin, the embodiments in which anti-CCR8 antibodies are combined with trastuzumab or pertuzumab, and the embodiments in which anti-CCR8 antibodies are combined with PD-L1 antibodies such as atezolizumab are particularly suitable for the treatment of breast cancer, such as stage IV breast cancer.

The embodiments in which anti-CCR8 antibodies are combined with cis/carboplatin, paclitaxel, docetaxel or gemcitabine, the embodiments in which anti-CCR8 antibodies are combined with avastin, EGFR inhibitors (e.g. Tarceva or Iressa), ALK inhibitors, ROS inhibitors, BRAF inhibitors or NTRK inhibitors, and the embodiments in which anti-CCR8 antibodies are combined with nivolumab, pembrolizumab or atezolizumab or derivatives of any of these are particularly suitable for the treatment of NSCLC.

Embodiments in which anti-CCR8 antibodies are combined with nivolumab, pembrolizumab or ipilimumab (CTLA4), anti-IL2 antibodies or any derivatives thereof, embodiments in which anti-CCR8 antibodies are combined with BRAF inhibitors or MEK inhibitors, and embodiments in which anti-CCR8 antibodies are combined with dacarbazine, paclitaxel, carboplatin or cisplatin are particularly suitable for the treatment of melanoma.

Combination with checkpoint inhibitors

If the tumor responds to immune checkpoint inhibition, or if the tumor shows high or moderate immune infiltration, and if there are a large number of CD8+ T cells and/or NK cells or they can be induced (e.g., in addition to a high CCR8/FoxP3 ratio), the tumor is found to be particularly sensitive to anti-CCR8 antibody therapy (see Example 12.6ff, see Response Isotype Model).

According to some particularly preferred embodiments of the third embodiment of aspect 22, the one or more additional therapeutically active compounds comprise antibodies or small molecules targeting checkpoint proteins such as PD1, PD-L1, or CTLA-4. Suitable checkpoint-targeting antibodies include Nivolumab (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Atezolizumab (PD-L1; humanized IgG1), Avelumab (PD-L1; human IgG1), Durvalumab (PD-L1; human IgG1), Cemiplimab, cemiplimab-rwlc (PD-1; human mAb), Dostarlimab (TSR-042) (PD_1; humanized IgG4), or Ipilimumab (CTLA-4; human IgG1). In some embodiments, antibodies or small molecules targeting checkpoint proteins, specifically CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B7 family ligands, or combinations thereof, are used. Example 12.6ff demonstrates excellent therapeutic effects obtained by combining an antibody targeting the checkpoint protein PD-L1 with the antibodies of the present invention according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18. Example 12.6ff demonstrates that combinations of the antibodies of the present invention according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18 with antibodies targeting the checkpoint protein CTLA4 also exhibit excellent efficacy in tumor models.

Combination with chemokine receptor antibodies

According to some preferred embodiments B of the third embodiment of aspect 22, the one or more additional therapeutically active compounds comprise antibodies or small molecules targeting additional chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1. Suitable antibodies targeting additional chemokine receptors include antibodies provided according to aspect 6, such as CCR5 antibody, CXCR5 antibody, CCR4 antibody, or Mogamulizumab.

Combination with HER2 or EGFR-targeting antibodies or molecules

According to some preferred embodiments C of the third embodiment of aspect 22, the one or more additional therapeutically active compounds comprise antibodies or small molecules targeting HER2 and/or EGFR. Suitable antibodies targeting HER2 are Trastuzumab (HER2; humanized IgG1), Pertuzumab (HER2; humanized IgG1), Ado-trastuzumabemtansine (HER2; humanized IgG1; ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki (HER2; humanized IgG1 ADC), Sacituzumab govitecan; sacituzumab govitecan-hziy (TROP-2; humanized IgG1 ADC) and/or Margetuximab (HER2; chimeric IgG1). Suitable antibodies targeting EGFR are Cetuximab (EGFR; chimeric IgG1), Panitumumab (EGFR; human IgG2), and Necitumumab (EGFR; human IgG1).

Combination with therapeutic antibodies

According to some embodiments D of the third embodiment of aspect 22, the one or more additional therapeutically active compounds comprise additional therapeutic antibodies selected from: Muromonab-CD3 (CD3; mouse IgG2a), Efalizumab (CD11a; humanized IgG1), Tositumomab-I131 (CD20; mouse IgG2a), Nebacumab (Endotoxin; human IgM), Edrecolomab (EpCAM; mouse IgG2a), Catumaxomab (EPCAM/CD3; rat/mouse bispecific mAb), Dac lizumab (IL-2R; humanized IgG1), Abciximab (GPIIb/IIIa; chimeric IgG1 Fab), Rituximab (CD20; chimeric IgG1), Basiliximab (IL-2R; chimeric IgG1), Palivizumab (RSV; humanized IgG1), Infliximab (TNF; chimeric IgG1), Trastuzumab (HER2; humanized IgG1), Adalimumab (TNF; human IgG1), Ibritumomab tiuxetan (CD20; mouse I) IgG1), Omalizumab (IgE; humanized IgG1), Cetuximab (EGFR; chimeric IgG1), Bevacizumab (VEGF; humanized IgG1), Natalizumab (α4 integrin; humanized IgG4), Panitumumab (EGFR; human IgG2), Ranibizumab (VEGF; humanized IgG1Fab), Eculizumab (C5; humanized IgG2/4), Certolizumab pegol (TNF; humanized Fab, PEGylated), Ust ekinumab (IL-12/23; human IgG1), Canakinumab (IL-1β; human IgG1), Golimumab (TNF; human IgG1), Ofatumumab (CD20; human IgG1), Tocilizumab (IL-6R; humanized IgG1), Denosumab (RANK-L; human IgG2), Belimumab (BLyS; human IgG1), Ipilimumab (CTLA-4; human IgG1), Brentuximab vedotin (CD30; chimeric IgG1; ADC), P ertuzumab (HER2; humanized IgG1), Ado-trastuzumab emtansine (HER2; humanized IgG1; ADC), Raxibacumab (B. anthrasis PA; human IgG1), Obinutuzumab (CD20; humanized IgG1 Glycoengineered), Siltuximab (IL-6; chimeric IgG1), Ramucirumab (VEGFR2; human IgG1), Vedolizumab (α4β7 integrin; humanized IgG1), Nivolumab (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Blinatumomab (CD19, CD3; mouse bispecific tandem scFv), Alemtuzumab (CD52; humanized IgG1), Evolocumab (PCSK9; human IgG2), Idarucizumab (Dabigatran; humanized Fab), Necitumumab (EGFR; human IgG1), Dinutuximab (GD2; chimeric IgG1), Secukinumab (IL-17) a; human IgG1), Mepolizumab (IL-5; humanized IgG1), Alirocumab (PCSK9; human IgG1), Daratumumab (CD38; human IgG1), Elotuzumab (SLAMF7; humanized IgG1), Ixekizumab (IL-17a; humanized IgG4), Reslizumab (IL-5; humanized IgG4), Olaratumab (PDGFRα; human IgG1), Bezlotoxumab (Clostridium difficile enterotoxin B; human IgG1), Ateaolizumab (PD-L1; humanized IgG1), Obiltoxaximab (B. anthrasis PA; chimeric IgG1), Brodalumab (IL-17R; human IgG2), Dupilumab (IL-4Rα; human IgG4), Inotuzumabozogamicin (CD22; humanized IgG4; ADC), Guselkumab (IL-23p19; human IgG1), Sarilumab (IL-6R; human IgG1), Avelumab (PD-L1; human IgG1), Emicizumab (Factor) Ixa, X; humanized IgG4, bispecific), Ocrelizumab (CD20; humanized IgG1), Benralizumab (IL-5Rα; humanized IgG1), Durvalumab (PD-L1; human IgG1), Gemtuzumab ozogamicin (CD33; humanized IgG4; ADC), Erenumab, erenumab-aooe (CGRP receptor; human IgG2), Galcanezumab, galcanezumab-gnlm (CGRP; humanized IgG4), Burosumab, burosumab-twza (FGF23; human IgG1), Lanadelumab, lanadelumab-flyo (Plasma kallikrelin; human IgG1), Mogamulizumab, m ogamulizumab-kpkc (CCR4; humanized IgG1), Tildrakizumab; tildrakizumab-asmn (IL-23 p19; humanized IgG1), Fremanezumab, fremanezumab-vfrm ( CGRP (humanized IgG2), Ravulizumab, ravulizumab-cwvz (C5; humanized IgG2/4), Cemiplimab, cemiiplimab-rwlc (PD-1; human mAb), Ibalizumab, ibalizumab-uiyk (CD4; humanized IgG4), Emapalumab, emapalumab-lzsg (IFNg; human IgG1), Moxetumomabpasudotox, moxetumomab pasudotox-tdfk (CD22; mouse I gG1dsFv immunotoxin), Caplacizumab, caplacizumab-yhdp (von Willebrand factor; humanized nanobody), Risankizumab, risankizumab-rzaa (IL-23p19; humanized IgG1), Polatuzumab vedotin, polatuzumab vedotin-piiq (CD79b; humanized IgG1 ADC), Romosozumab, romosozumab-aqqg (Sclerostin; humanized IgG2), Brolucizumab, brolucizumab-dbll (VEGF-A; humanized scFv), crizanlizumab; crizanlizumab-tmca (CD62 (aka P-selectin); humanized IgG2), Enfo rtumab vedotin, enfortumab vedotin-ejfv (Nectin-4; human IgG1 ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-n xki (HER2; humanized IgG1 ADC), Teprotumumab, teprotumumab-trbw (IGF-1R; human IgG1), Eptinezumab, eptinezumab-jjmr (CGRP; humanized IgG1), Isatuximab, isatuximab-irfc (CD38; chimeric IgG1), Sacituzumab govitecan; sacituzumab govitecan-hziy (TROP-2; humanized IgG1 ADC), Inebilizumab (CD19; humanized IgG1), Leronlimab (CCR5; humanized IgG4), Satralizumab (IL-6R; humanized IgG2), Narsoplimab (MASP-2, human IgG4), Tafasitamab (CD19; humanized IgG1), REGNEB3 (Ebola virus; mixture of 3 human IgG1s), Naxitamab (GD2; humanized IgG1), Oportuzumab monatox (EpCAM; humanized scFv immunotoxin), Belantama bmafodotin (B-cell maturation antigen; humanized IgG1 ADC), Margetuximab (HER2; chimeric IgG1), Tanezumab (nerve growth factor; humanized IgG2), Dostarlimab (TSR-042) (PD-1; humanized IgG4), Teplizumab (CD3; humanized IgG1), Aducanumab (β-amyloid protein; human IgG1), Sutimlimab (BIVV009) (C1s; humanized IgG4), Evinacumab (angiopoietin-like 3; human IgG4).

Combination therapy with any of these additional therapeutic antibodies is particularly preferred for tumors that specifically express the target of the selected additional therapeutic antibody.

According to some embodiments E of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise additional therapeutic antibodies targeting CD16a, ILDR2, PSMA, or mesothelin.

Combination with cytotoxic or cell growth inhibitors

Several chemotherapy drugs, such as doxorubicin and oxaliplatin, induce immunogenic cell death (ICD). However, ICD has been found to be associated with the response to anti-CCR8 antibodies, suggesting that combination therapy could add further benefits to the application of monotherapy.

According to some embodiments F of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise a cell growth inhibitor selected from: radionuclides, alkylating agents, DNA cross-linking agents, DNA intercalating agents (e.g., groove binding agents such as minor groove binding agents), cell cycle regulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents.

Alkylating agents

According to some embodiments F1 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise an alkylating agent selected from: asaley (an alkylating agent of L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanine]-ethyl ester); AZQ (1,4-cyclohexadiene-1,4-dicarboxylic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-diethyl ester); BCNU (N,N′-bis(2-chloroethyl)-N-nitrosourea). ; sulfolane (1,4-butanediol dimethylsulfonate); (carboxyphthalic acid)platinum; CBDCA (cis-(1,1-cyclobutanedicarboxy)diamineplatin(II)); CCNU (N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea); CHIP (isopropylplatinum; NSC 256927); chloramphenicol; chlorzolam (2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-pyranose); cisplatin (cisplatin); chloramphenicol; cyanomorpholinodoxorubicin; cyclodione; dihydrogen monoxide Lactitol (5,6-dihydroxydurotol); Fluorodopa (5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil); Heptylsulfamethoxazole; Cantharidin; Indomethacin dimer DGN462; Melphalan; Methyl CCNU (1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea); Mitomycin C; Mitozodamine; Nitrogen mustard (bis(2-chloroethyl)methylamine hydrochloride); PCNU (1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidinyl) )-1-nitrosourea); piperazine alkylating agent (1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine hydrochloride); piperazine dione; piperazine (N,N′-bis(3-bromopropionyl)piperazine); blepharomycin (N-methylmitomycin C); spiral hydantoin; teroselanone (triglycidyl isocyanurate); tetra-Latin; thiophosphate (N,N′,N″-tri-1,2-ethylenedimethylthiophosphoramide); triethanolamine; uracil nitrogen mustard; Yoshi-864 ((bis(3-methoxypropyl)amine hydrochloride).

DNA alkylation agent

According to some embodiments F2 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise a DNA alkylating agent selected from the following: cisplatin; carboplatin; nedaplatin; oxaliplatin; saxaplatin; triplatin tetranitrate; procarbazine; hexamethylamine; dacarbazine; mirtazolamide; temozolomide.

Alkylated antitumor agents

According to some embodiments F3 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise alkylated antitumor agents selected from: carboquinone; carmustine; chlornaphthazine; chlorzoxazone; docamycin; isophosphoramide; formustine; glucosamine; lomustine; mannitol; nimustine; phenanthreneplatin; bromide; ranimustine; semastine; streptozotocin; thiotetrafluoroethylene; endosulfan; triquinone; triethylene melamine; triplatin tetranitrate.

DNA replication and repair inhibitors

According to some embodiments F4 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise a DNA replication and repair inhibitor selected from the following: hexamethasone; bleomycin; dacarbazine; styromycin; mitobronitol; mitomycin; bleomycin; propramycin; procarbazine; temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281; AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.

Cell cycle regulators

According to some embodiments F5 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise cell cycle regulators, such as paclitaxel; Nab-paclitaxel; docetaxel; vincristine; vinca alkaloid; ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific kinase inhibitor; Aurora B-specific kinase inhibitor and pan-Aurora kinase inhibitor; AZD-5438; BMI-1040; BMS-032; BMS-387; CVT-2584; flavonoid pyridinol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib (CYC-202, R-roscovitines); ZK-304709; AZD4877; ARRY-520: GSK923295A.

Apoptosis regulators

According to some embodiments F6 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise an apoptosis regulator, such as AT-101 ((-)gossypol); G3139 or oblimersen (Bcl-2-targeted antisense oligonucleotide); IPI-194; IPI-565; N-(4-(((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-ylbenzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylthio)methyl)propyl)amino)-3-nitrobenzenesulfonamide; N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexyl-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-(( (Phenylthio)methyl)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide; GX-070(1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-)); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; Venetoclax; Drugs targeting TRAIL or death receptors (e.g., DR4 and DR5), such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; Drugs targeting caspase, caspase modulators, BCL-2 family members, death domain proteins, TNF family members, Toll family members, and/or NF-kappa-B proteins.

Angiogenesis inhibitors

According to some embodiments F7 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise angiogenesis inhibitors, such as ABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547, 632; IM-862; pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034); vatalanib (PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap; vandetanib; ABT-165; ZD-6474; DLL4 inhibitors.

Proteasome inhibitors

According to some embodiments F8 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise a proteasome inhibitor, such as bortezomib; carfilzomib; cyclooxygenin; ixazomib; or halosporin A.

kinase inhibitors

According to some embodiments F9 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise kinase inhibitors, such as afatinib; axitinib; bosutinib; crizotinib; dasatinib; erlotinib; fostatinib; gefitinib; ibrutinib; imatinib; lapatinib; lenvatinib; mubutinib; nilotinib; pazopanib; peragatanib; sorafenib; sunitinib; SU6656; vandetanib; vemurafenib; CEP-701 (lesatinib); XL019; INCB018424 (ruxotetinib); ARRY-142886 (celecinib); ARRY-4381 62 (binimetinib); PD-325901; PD-98059; AP-23573; CCI-779; everolimus; RAD-001; rapamycin; tesiromolimus; ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242, PP30, Torin 1; LY294002; XL-147; CAL-120; ONC-21; AEZS-127; ETP-45658; PX-866; GDC-0941; BGT226; BEZ235; XL765, regorafenib, and MEKi-1.

Protein synthesis inhibitors

According to some embodiments F10 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise protein synthesis inhibitors, such as streptomycin; dihydrostreptomycin; neomycin; flammycin; paromomycin; ribostamycin; kanamycin; amikacin; abekacin; bekanamycin; dibekacin; tobramycin; spectinomycin; hygromycin B; paromomycin; gentamicin; netilmicin; sisomicin; isipamicin; cordycepsmycin; astromycin; tetracycline; doxycycline; chlortetracycline; chloramphenicol; demethylchlortetracycline; lysine; methoxycycline; methylcycline; minocycline; oxytetracycline; penicillin; rolitetracycline; tetracycline; glycylcycline. Tigecycline; Oxazolidinones; Piperazolyl ester; Linezolid; Oxazolamide; Redizolamide; Lanbezolid; Sultezolid; Tedizolamide; Peptidyl transferase inhibitors; Chloramphenicol; Azidamico; Thiamphenicol; Florfenicol; Pleurotus ostreatus; Retamoline; Tiamulin; Vonimulin; Azithromycin; Clarithromycin; Dierythromycin; Erythromycin; Fluerythromycin; Josamycin; Midecamycin; Micardiitis; Pyruvicin; Rotamycin; Roxithromycin; Spiramycin; Pyruvicin; Ketoconazole; Telithromycin; Cypermethrin; Solithromycin; Clindamycin; Lincomycin; Pirimibacin; Streptomycin; Primalmycin; Quinupertin/Dalfopristin; Virginiamycin.

Histone deacetylase inhibitors

According to some embodiments F11 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise histone deacetylase inhibitors, such as vorinostat; romidesin; chidamide; pabisostat; valproic acid; belistat; moxistat; abexinostat; entecavir; SB939 (pracinostat); remistat; gilvestartar; quinacrinestat; thiourea-butyronitrile (Kevetrin ^™ ); CUDC-10; CHR-2845 (tefilstat); CHR-3996; 4SC-202; CG200745; ACY-1215 (roxilinstat); ME-344; sulforaphane.

Topoisomerase I inhibitors

According to some embodiments F12 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise a topoisomerase I inhibitor, such as camptothecin; various camptothecin derivatives and analogs (e.g., NSC100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500, NSC 249910, NSC60698). 5, NSC 74028, NSC 176323, NSC 295501, NSC 606172, NSC 606173, NSC 610458, NSC618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830, and NSC606497); Morphyrin isoxastatin; SN-38.

Topoisomerase II inhibitors

According to some embodiments F13 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise topoisomerase II inhibitors, such as doxorubicin; amonafide (benzisoquinolinedione); m-AMSA (4′-(9-acridylamino)-3′-methoxymethanesulfonylaniline); anthraquinone derivative ((NSC 355644); etoposide (VP-16); pyrazoloacridin ((pyrazolo[3,4,5-kl]acridin-2(6H)-propylamine, 9-methoxy-N,N-dimethyl-5-nitro-,monosulfonate); piracetam hydrochloride; daunorubicin; deoxydaunorubicin; mitoxantrone; minogalin; N,N-dibenzyldaunorubicin; orantrone; rubidazine; teniposide.

DNA intercalating agent

According to some embodiments F14 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise a DNA intercalating agent, such as anthramycin; zithromycin A; tomatine; DC-81; sibromycin; pyrrolodiazapyridine derivatives; SGD-1882((S)-2-(4-aminophenyl)-7-methoxy-8-(3S)-7-ethoxy-2-(4-methoxyphenyl)-5-oxo-5,11-dihydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazaoct-8-yl)oxy)propoxy)-1Hbenzo[e]pyrrolo[1,2-a][1,4]azaoct-5(11aH)-one); SG2000(SJG-136; (11a S, 11a′S)-8,8′-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5(11aH)-one)).

RNA/DNA antimetabolites

According to some embodiments F15 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise gemcitabine; L-adenosine; 5-azacytidine; 5-fluorouracil; acetivorin; aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid; aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridyl)methyl]amino]benzoyl]L-aspartic acid monohydrate; anti- Folic acid PT523 ((Nα-(4-amino-4-deoxypteroyl)-Nγ-hemiphthaloyl-L-ornithine)); Baker's soluble antifolic acid (NSC 139105); dichloroallylloxacin ((2-(3,3-dichloroenyl)-3-hydroxy-1,4-naphthoquinone); Brefinar; flutifu ((prodrug; 5-fluoro-1-(tetrahydro-2-furanyl)-uracil); 5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivative (N-[[4-[[(2,4-diamino-6-pterodinyl)methyl]methylamino]-1-naphthyl]carbonyl]-L-glutamic acid); PALA ((N-(phosphonyl)-L-aspartic acid); pyrazolylphosphonate; methotrexate.

DNA antimetabolites

According to some embodiments F16 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise DNA antimetabolites, such as 3-HP; 2′-deoxy-5-fluorouridine; 5-HP; α-TGDR (α-2′-deoxy-6-thioguanosine); afenidimycin glycinate; ara C (cytarabine); 5-aza-2′-deoxycytidine; β-TGDR (β-2′-deoxy-6-thioguanosine); cyclocytidine; guanidine; hydroxyurea; inosine dialdehyde; macrocin II; pyrazoimidazole; thioguanine; thiopurine.

Mitochondrial inhibitors

According to some embodiments F17 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise mitochondrial inhibitors, such as trypsin; benpanstatin; rhodamine-123; senna; d-α-tocopherol succinate; compound 11β; aspirin; elutin; berberine; azuril; GX015-070(1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-); tripterygium oleate; metformin; bright green; ME-344.

Antimitotic agents

According to some embodiments F18 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise an antimitotic agent, such as allocolchicine; auristatin, such as MMAE (monomethyl auristatin E) and MMAF (monomethyl auristatin F); squalene B; simadotin; colchicine; colchicine derivative (N-benzoyl-deacetylated benzamide); slug toxin 10; slug toxin 15; maytansine; maytansine alkaloids, such as DM1 (N2′-deacetylated-N2′-(3-mercapto-1-oxopropyl)-matansine); rosocine; paclitaxel; paclitaxel derivative ((2′-N-[3-(dimethylamino)propyl]glutaric acid paclitaxel); docetaxel; thiocolchicine; triphenylmethylcysteine; vincristine sulfate; vincristine sulfate.

Nuclear output inhibitor

According to some embodiments F19 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise nuclear export inhibitors, such as callystatin A; lactamycin; KPT-185 (propyl-2-yl(Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]propyl-2-enoate); cassumycin A; leptin; leptofuran A; leptomycin B; rajadone; Verdinexor ((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin-2-ylpropyl-2-enoylhydrazine).

Hormone therapy

According to some embodiments F20 of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise hormonal therapeutic agents, such as anastrozole; exemestane; azoxifen; bicalutamide; cetrorex; degarelix; dilorelin; trichloroethane; dexamethasone; flutamide; raloxifene; fazodone; toremifene; fulvestrant; letrozole; formetanin; glucocorticoids; doxorubicin; sevelamer carbonate; lasoxifene; leuprorelin acetate; medroxyprogesterone acetate; mifepristone; nilumet; tamoxifen citrate; abalinix; prednisone; finasteride; lilosterone; buterelin; luteinizing hormone-releasing hormone (LHRH); histamine; trelosterone or modrastan; frenelin; goserelin.

According to some embodiments G of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise antibodies that target myeloid suppressor cells.

According to some embodiments H of the third embodiment of aspect 22, one or more additional therapeutically active compounds contain an agent that induces antigen-presenting cells (APCs), which in turn can activate T cells.

According to some embodiments of the third embodiment of aspect 22, one or more additional therapeutically active compounds comprise a BiTE antibody. The BiTE antibody is a bispecific antibody that can guide T cells to attack cancer cells by simultaneously binding to two types of cells. The T cells then attack the target cells, i.e., Tregs. For example, the BiTE method complements the naked CCR8 antibody method based on ADCC.

Standard therapy

According to some embodiments J of the third embodiment of aspect 22, the combination is a combination with a standard treatment for a tumor or disease characterized by chemokine receptor positivity (e.g., CCR8 positive cells). This standard treatment may be a standard treatment for any of the following: T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), testicular cancer, gastric cancer, head and neck squamous cell carcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic cancer, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer.

According to some preferred embodiments, standard treatment is the standard treatment for head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer. In case of doubt, the standard treatment is defined as "S3 Leitlinie" for the corresponding tumor, such as anal cancer, actinic keratosis and squamous cell carcinoma of the skin, chronic lymphocytic leukemia (CLL), endometrial cancer, follicular lymphoma, bladder cancer, skin cancer prevention, hepatocellular carcinoma (HCC), testicular tumors, Hodgkin's lymphoma, colorectal cancer, laryngeal cancer, lung cancer, gastric cancer, breast cancer, melanoma, oral cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, palliative care, pancreatic cancer, penile cancer, prostate cancer, psycho-oncology, supportive care, and cervical cancer, effective June 26, 2020, cf. https://www.leitlinienprogramm-onkologie.de/leitlinien/, the entire contents of which are incorporated herein by reference on January 6, 2021.

Radiation therapy

According to some embodiments K of the third embodiment of aspect 22, it can and is suggested to be combined with embodiments of the first and/or second embodiments of aspect 22, for use in conjunction with, separate from, or in combination with radiotherapy.

For example, radiotherapy can be a therapy involving radiation protocols known in the art, such as external beam radiation therapy via X-rays, typically with a total dose of 40 Gy divided into 15 daily treatments, or a total dose of 70 Gy divided into 37 daily treatments. For example, radiotherapy can be brachytherapy. Example 12.6.8 illustrates a combination therapy of anti-CCR8 antibody and radiotherapy.

NK cell therapy

According to some embodiments L of the third embodiment of aspect 22, it can and is suggested to be combined with embodiments of the first and/or second embodiments of aspect 22, for use in conjunction with NK cell therapy, either simultaneously, separately, or sequentially. For example, NK cell therapy may include the use of engineered NK cell lines as described in EP1771471, or may include the use of isolated primary NK cells, possibly including modifications such as chimeric receptors, see WO2006052534.

Targeting cell surface molecules associated with T cell activation

According to some embodiments M of the third embodiment of aspect 22, conjugates according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or conjugates according to aspect 19, or pharmaceutical compositions according to aspect 20, are provided for use simultaneously, separately or sequentially in combination with one or more additional therapeutically active compounds that target cell surface molecules associated with T cell activation, selected from CD25, CTLA-4, PD-1, LAG3, TIGIT, ICOS and TNF receptor superfamily members, 4-1BB, OX-40 and GITR.

B cell depletion

According to some embodiments N of the third embodiment of aspect 22, it can and is suggested to be combined with embodiments of the first and/or second embodiments of aspect 22, the use being simultaneous, separate, or sequentially combined with intratumoral B cell depletion. Intratumoral B cell depletion can occur by administering antibodies or other molecules or any additional treatment specifically targeting intratumoral B cells (preferably CD19+ B cells). Example 12.3.2 shows a treatment in which an anti-CCR8 antibody is administered and intratumoral CD19+ B cells are depleted. While CD8+ T cell depletion negates the beneficial effects of anti-CCR8 antibody treatment, it was surprisingly found that B cell depletion improves the beneficial effects of anti-CCR8 antibody treatment. Without limitation, B cells can be depleted by using antibodies targeting CD19 or CD20 or another suitable B cell marker.

Stratification/Prediction and Monitoring Scheme

The stratification steps described in the following embodiments are also provided as standalone methods, such as stratification, diagnosis, or monitoring of treatment success for anti-CCR8 antibody therapy.

According to some fourth embodiments of aspect 22, it may and is suggested to be combined with embodiments according to the first, second, and/or third embodiments of aspect 22, the use including determining certain parameters to predict or monitor treatment success, for example, for stratifying a group of subjects or patients to predict tumor response or monitor treatment success. For example, subjects may be human or non-human, such as mice, rodents, or cynomolgus monkeys.

According to some embodiments A of the fourth embodiment of aspect 22, the use includes determining

a. The presence or number of tumor-infiltrating lymphocytes

b. Presence or number of macrophages and/or NK cells

c. The presence or number of CCR8-positive or FOXP3-positive regulatory T cells.

d. Tumor mutation burden,

e. Cancer staging

f. The presence, level, or activation of interferon-stimulated genes or proteins.

g.CCR8 expression,

h. The presence or quantity of complement factor proteins, serine protease inhibitors, and/or MHC components.

i. The presence or quantity of cytokines, such as inflammatory or inhibitory cytokines.

j. Activate immune gene expression,

k. Expression of immune checkpoint proteins, such as PD-(L)1 or CTLA4.

1. The presence or number of tumor-infiltrating CD19+ B cells, and/or

m. The presence or number of tumor-infiltrating CD8+ T cells

Used to stratify a group of subjects or patients to predict tumor response, or to predict or monitor treatment success.

According to some embodiments A1 of the fourth embodiment of aspect 22, the use includes determining the presence or number of (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells, and/or (c) CCR8-positive or FOXP3-positive regulatory T cells. Assessing the presence or number of cell types, such as (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells, and/or (c) CCR8-positive regulatory T cells, can be performed via biopsy, such as skin biopsy, surgical biopsy, endoscopic biopsy, or needle biopsy, such as fine-needle aspiration biopsy, core needle biopsy, vacuum-assisted biopsy, or image-guided biopsy, followed by staining. The number or relative amount of (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells, and/or (c) CCR8-positive regulatory T cells can then be compared with a reference, such as a reference sample or reference value. The relative presence or number of (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells and/or (c) CCR8-positive regulatory T cells can be used to predict a favorable response to the antibody or fragment therapy of the present invention. Alternatively, a scoring system can be used, for example, to determine the number or relative amount of tumor-infiltrating lymphocytes, such as the scoring system described in Zhang, Dachuan, et al. "Scoring system for tumor-infiltrating lymphocytes and its prognostic value for gastric cancer." Frontiers in Immunology 10 (2019): 71.

According to some embodiments A2 of the fourth embodiment of aspect 22, this use includes determining tumor mutational burden to predict and monitor tumor response. Tumor mutational burden (TMB) is a biomarker used to measure the number of somatic mutations present in a cancer patient's tumor and quantifies it as megabase mutations per 1,000 bases (mut/Mb). This indicator can be used to stratify patients, for example, to predict or monitor response to treatment with an antibody or conjugate according to the invention. An FDA-approved test is present and can be used, for example, a reference value ≥10 mut/Mb. Alternatively, microsatellite instability (MSI), which is caused by a failure of the DNA mismatch repair system, can be used for stratification.

According to some embodiments A3 of the fourth embodiment of aspect 22, this use includes determining cancer staging to predict and monitor tumor response. Cancer staging can be performed as is known in the art, for example using the TNM classification system or FIGO staging, and can be used to stratify patients, for example to predict or monitor response to antibody or conjugate treatment of the present invention.

According to some embodiments A4 of the fourth embodiment of aspect 22, this use includes determining the presence, level, or activation of interferon or interferon-stimulated genes or proteins for the purpose of predicting and monitoring tumor responses. Interferon-stimulated genes are genes whose expression is stimulated by interferon, particularly IFNg. Suitable interferon-stimulated genes or proteins include, but are not limited to, ACOD1, ACTG1, ACTR2, ACTR3, ADAMTS13, AIF1, AQP4, ASS1, B2M, BST2, C9JQL5, CALCOCO2, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CASP1, CCL1, CCL11, CCL13, CCL14, CCL15, CCL15, CCL14, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL 8. CD40, CD44, CD58, CDC42EP2, CDC42EP4, CIITA, CITED1, CLDN1, CX3CL1, CXCL16, CYP27B1DAPK1, DAPK3, EDN1, EPRS, EVL, FCGR1A, FCGR1B, FLNB, GAPDH, GBP1, GBP2, GB P4, GBP5, GBP6, GCH1, GSN, HCK, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRAHLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DR B5, HLA-E, HLA-F, HLA-G, HLA-H, ICAM1, IFI30, IFITM1, IFITM2, IFITM3, IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1IL23R, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, JAK1, JAK2, KIF16B, KIF5B, KYNU, LGALS9, MEFV, MID1, MRC1, MT2A, MYO1C, NCAM1, NMI, NOS2, NUB1, OAS1, OAS2, OAS3, OASL, PDE12, PML, PRKCD, PTAFR, RAB12, RAB2 0, RAB43, RAB7B, RPL13A, RPS6KB1RYDEN, SEC61A1SLC11A1SLC26A6SLC30A8 SNCA, SP100, STAR, STAT1, STX4, STX8, STXBP1, STXBP2, STXBP3, STXBP4, SYNCRIPTDGF1, TL R2, TLR3, TLR4, TRIM21, TRIM22, TRIM25, TRIM26, TRIM31, TRIM34, TRIM38, TRIM5, TRIM62, TRIM68, TRIM8, UBD, VAMP3, VCAM1, VIM, VPS26B, WAS, WNT5A, XCL1, XCL2, ZYX.

According to some embodiments A5 of the fourth embodiment of aspect 22, this use includes determining CCR8 expression for predicting and monitoring tumor response. CCR8 expression levels can be determined, for example, based on biopsy samples, as known in the art, such as using IHC, PCR, or ELISA-based methods, such as using antibodies, fragments, or conjugates disclosed herein.

According to some embodiments A6 of the fourth embodiment of aspect 22, the use includes determining the presence, level, or activation of a gene or protein selected from (a) complement factor proteins, (b) serine protease inhibitors, (c) MHC components, or (d) Arg2, or (e) another biomarker disclosed herein (see, for example, Examples 12.7.1 or 12.7.2) for predicting and monitoring tumor response. Suitable complement factor proteins include, but are not limited to, C1R, C1S, C4A, C5ar2, F12, and MASP2, or their analogues from various species for therapeutic purposes. Suitable serine protease inhibitors include, but are not limited to, Serpina1a, Serpina1b, Serpina1d, Serpina3i, or their analogues from other species. Suitable MHC components include, but are not limited to, H2-K2, H2-T24, H2-Q10, H2-B1, H2-Q1, H2-Q5, and H2-DMb2, or their analogues from other species.

As discussed in Example 12.1.2, the inventors, based on genome-wide RNA-seq data from early-stage tumors in syngeneic mouse models, found that increased levels of the following genes were closely associated with tumor response: Eif3j2, Eno1b, Ifi44l, Hist1h2al, Ifi202b, Hmga1b, Amd2, Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpina1b, Prss41, C1rb, Cyld, C cnblipl, Masp2, Acaa1b, C4a, Snord93, Abhd1, Serpina3h, H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2, Igsf21, Olfr373, C1s2, Crym, Arvl, Hddc3, Plppr4, Ppplr11, Rps3a2, Zfp459, Rnd1, Serpinala, Vcpkmt, Atp10d , Gbp2b, H2-T24, Tlcd2, Ctse, H2-Q10, Cyp2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d, Cacna1a, Kcnj14 , Ttc13, Farsa, Olfr1217, Jaml, H2-Bl, Tnpo2, Rims3, Dock9, Car5b, Atpla4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel The list includes: l, Selenbp2, Nsd3, Wt1, Nap112, Ranbp9, Gtpbp3, AY761185, Rnaset2a, Serpina3i, El12, Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agptpbp1, Msh5, Cox18, Zfp330, Ttc37, Klk4, H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2, and H2-DMb2. Interestingly, this set is highly enriched for early complement factors, complement regulators (such as serine protease inhibitors), and MHC components. In particular, the presence of high levels of complement C1/C4 may contribute to Treg cleavage. When complement factor depletion/consumption reduces the efficacy of Treg depletion, complement system supplementation, such as in combination therapy, may be an option.

According to some preferred embodiments of the fourth embodiment of aspect 22, the use includes determining the presence of a protein selected from Eif3j2, Eno1b, Ifi441, Hist1h2a1, Ifi202b, Hmga1b, Amd2, Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpinalb, Prss41, C1rb, Cyld, Ccnblip1, Massp2, Acaa1b, C4a, Snord93, Ab hd1, Serpina3h, H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2, Igsf21, Olfr373, C1s2, Crym, Arv1, Hddc 3. Plppr4, Ppp1rl1, Rps3a2, Zfp459, Rnd1, Serpina1a, Vcpkmt, Atp10d, Gbp2b, H2-T24, Tlcd2, Ctse, H2-Q10, Cy p2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d, Cacnala, Kcnj14, Ttc13, Farsa, Olfr1217, Jaml, H2-B1, Tnpo2, Rims3, Dock9, Car5b, Atp1a4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel1, Selenb2, Nsd3, Wt1, Nap112, Ranbp9, The presence, level, or activation of genes or proteins such as Gtpbp3, AY761185, Rnaset2a, Serpina3i, Ell2, Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agptpbp1, Msh5, Cox18, Zfp330, Ttc37, Klk4, H2-Q5, Cxc111, Rab39, Pm20d1, Nod2, H2-DMb2, or any combination thereof are used to predict and monitor tumor responses. As will be immediately understood by those skilled in the art, for human subjects, stratification, prediction, or monitoring methods include determining the presence, level, or activation of human counterparts of the provided genes or proteins.

According to some embodiments A7 of the fourth embodiment of aspect 22, this use includes determining the presence or quantity of inflammatory or inhibitory cytokines to predict and monitor tumor response. The assessment of the presence or quantity of inflammatory or inhibitory cytokines can be performed as is known in the art.

According to some embodiments A8 of the fourth embodiment of aspect 22, this use includes predicting or monitoring tumor response based on the activation of immune gene expression. The activation of immune gene expression can be measured using methods known in the art, such as gene set enrichment analysis or biomarker-based assays known in the art. Furthermore, stratification, prediction, or monitoring can occur based on the gene signature characteristics of activated Tregs or immune cells.

According to some highly preferred embodiments A9 of the fourth embodiment of aspect 22, the use includes stratifying a group of subjects or patients to predict tumor response based on the expression of immune checkpoint proteins, such as PD-L1 expression, PD-1 expression, or CTLA4 expression, or the use includes determining the expression level of immune checkpoint proteins, such as PD-L1 expression, PD-1 expression, or CTLA4 expression, to predict and monitor tumor response.

Programmed death-ligand 1 (PD-L1) is an immune-related biomarker that can be expressed on the surface of many tissue types, including tumor cells. PD-L1 protein expression can be determined using tumor proportion score (TPS) or combined positive score (CPS). According to the invention, it was unexpectedly found that subjects with ICI-responsive tumors, particularly those with (high) PD-L1 expression, showed improved response to (mono) therapy with anti-CCR8 antibodies (see Example 12.7). Based on this observation, the inventors found that FoxP3+Treg cell infiltration was also associated with high PD-L1 expression. Therefore, the inventors propose using PD-L1 as an alternative marker for stratification to overcome the problem that CCR8 is difficult to analyze and therefore cannot be easily used to select subject populations most likely to benefit from anti-CCR8 antibody therapy. In a particularly preferred embodiment, PD-L1 expression is determined based on CPS or TPS. If PD-L1 expression is present or high, for example, as shown by CPS≥1, preferably CPS≥5, most preferably CPS≥10, or as shown by TPS≥1%, preferably TPS≥10%, most preferably TPS≥50%, the subject or patient is determined to be suitable for treatment with anti-CCR8 antibody.

Aspect 23 - Further and Diagnostic Uses

The anti-chemokine receptor or anti-CCR8 antibodies, fragments, and conjugates described herein can be used for a variety of purposes, such as aiding in the purification or immobilization of chemokine receptor or CCR8-expressing cells, such as activated or intratumoral Tregs, for in vitro, in vivo, and ex vivo applications or diagnostics. As a specific example, antibodies can be used for immunoassays to qualitatively and/or quantitatively measure the levels of chemokine receptors or chemokine receptor-expressing cells in biological samples; see, for example, Harlow et al., Antibodies: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1988).

For example, anti-chemokine receptor antibodies, anti-CCR8 antibodies, or their antigen-binding fragments can be used to detect the presence of tumors expressing chemokine receptors or CCR8. The presence or level of chemokine receptor or CCR8-expressing cells or shed chemokine receptors or CCR8 can be analyzed in various biological samples (including serum and tissue biopsy samples). Furthermore, anti-chemokine receptor or anti-CCR8 antibodies can be used in various imaging methods, such as immunoscintigraphy using 99Tc (or different isotopes) conjugated antibodies. For example, imaging protocols similar to those described using 111In conjugated anti-PSMA antibodies can be used to detect cancer (Sodee, D. Bruce, et al. "Preliminary Imaging Results Using In-111 Labeled CYT-356 (Prostascint) in the Detection of Recurrent Prostate Cancer." Clinical nuclear medicine 21.10 (1996): 759-767.). Another detection method that can be used is positron emission tomography (PET), for example by conjugating the antibody of the present invention with a suitable isotope (see Herzog, Hans, et al. "Measurement of pharmacokinetics of yttrium-86 radiopharmaceuticals with PET and radiation dose calculation of analogous yttrium-90 radiopharmaceuticals." Journal of Nuclear Medicine 34.12 (1993): 2222-2226).

According to aspect 23, an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, is provided for diagnostic applications or diagnosis.

In some first embodiments of aspect 23, an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, is provided for use in in vivo diagnostic methods. This use may be for diagnosing tumors or diseases characterized by chemokine receptor-positive cells, such as CCR8-positive cells, like CCR8-positive regulatory T cells. For example, tumors or diseases characterized by chemokine receptor-positive cells are selected from T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, head and neck squamous cell carcinoma, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic cancer, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer. According to some preferred embodiments, the use in in vivo diagnostic methods is for the diagnosis of head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumors, melanoma, bladder cancer, liver cancer and/or prostate cancer, or any other tumors described herein.

According to some preferred embodiments, an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, is used to treat a subject or patient stratification, for example, for treating the disease described herein.

DNA/RNA of 24-CCR8 antibody

According to aspect 24, a polynucleotide encoding an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18 is provided. Preferably, the polynucleotide according to this aspect is a polynucleotide provided in the sequence listing.

Aspect 25 - Antibody Vector

According to aspect 25, a vector comprising the polynucleotide according to aspect 24 is provided. Various suitable vector systems are known in the art or described herein.

Aspect 26 - Antibody Production Cells

According to aspect 26, an isolated cell is provided, which is arranged to produce an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18. Preferably, the isolated cell according to the present aspect is a eukaryotic cell, such as CHO or HEK cells containing a vector according to aspect 25. In another preferred embodiment, the cell is a rat myeloma YB2/0 cell.

Aspect 27 - Antibody Production Methods

According to aspect 27, a method is provided for producing an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, comprising culturing cells according to aspect 26 and optionally purifying the antibody or antigen-binding fragment. In some highly preferred embodiments, the method includes defucosylation of the antibody, as described elsewhere herein; in some embodiments, the method includes purification of the antibody.

For example, the antibodies of the present invention can be purified by the purification method described below. The described purification process is applicable to a wide range of monoclonal IgG antibodies and is optimized for bioreactor titers up to 8 g/L, with a anticipated harvest volume of 2000 L from a disposable or stainless steel bioreactor. Antibody cultures with other titer ranges can be processed with appropriate modifications. Cell cultures are collected and clarified by depth filtration and/or electrofiltration. The clarified harvest is stored in disposable bags or, optionally, in a cooled stainless steel tank.

General process conditions

Closed-loop processing and single-use systems are preferred; stainless steel chutes or systems are also possible with appropriate cleaning and conversion procedures. Single-use bags typically use low-density or ultra-low-density polyethylene as the contact layer. Sodium acetate/acetic acid or Tris/Tris-HCl buffer systems may be used, among others, typically with a total concentration of 50 mM, including 50 mM NaCl, unless otherwise specified. Buffers and intermediates for individual unit operations (e.g., loading, elution, or flow-through) are typically filtered in-line at 0.2 μm or using filter assemblies such as PES membranes and/or wool filters. These solutions are typically stored in single-use bags or stainless steel containers at ambient temperature (e.g., 18–26 °C) or 2–8 °C.

capture

Antibody capture is performed using protein A-based chromatography, such as with Cytiva MabSelect SuRe, MabSelect SuRe LX, Mab Select PrismA, or Purolite A50 resin. Loading is performed after clarification, with or without pre-concentration via ultrafiltration. Loading densities are typically 45–75 g/L, and the clarified harvest is applied to the column using a single or variable flow rate (120–400 cm/h). Chromatography can be performed in batch mode using one or more columns, or in continuous (MCC/SMB) mode using up to eight columns. For batch mode, the typical bed height is 20 cm for pre-packed or self-packed columns. For continuous mode, the target contact time between the clarified harvest and the resin is 1–4 minutes. Depending on the antibody titer, capture is performed in one or more cycles. Before loading, equilibrate the column with Tris buffer (e.g., pH 7); washing is performed in at least two wash steps; the first wash buffer has a high NaCl concentration in acetate buffer, followed by a second wash buffer with a low NaCl concentration (e.g., first wash: 1M NaCl, pH 5.5; second wash: 50mM NaCl, βH 6.0). Elution can be achieved, for example, with a low pH (e.g., 3.5) acetate buffer containing 0–50mM NaCl, typically ≤5 column volumes (CV), and eluent collection can be performed using UV or volume control. The eluent can be pooled and thoroughly mixed at this step or after virus inactivation. The affinity column is regenerated with ≥3CV of acetic acid (e.g., 0.1M, including 500mM NaCl), sterilized with 0.5–1.0M NaOH (wash in place, CIP) for 30 minutes, and reequilibrated with ≥4CV. The column is stored, for example, in 20% EtOH or 2% BnOH.

Alternatively, affinity capture from clarified harvest can be performed using affinity ligands on cellulose structures (e.g., FibroPrismA) using the same loading scheme and buffering system described above. Regeneration or CIP blocks can be skipped to achieve optimal processing time. Contact times of 5–20 seconds are applicable to various sizes of Fibro units (e.g., 0.4 mL to 2.4 L). Typically, loading capacities of 25–35 g/L per unit volume can be achieved. A single unit can be used to process a single batch or multiple batches, depending on the size of the Fibro unit and bioreactor, as well as the titer of the antibody culture. Affinity-loaded material can be further clarified before loading through an electrostatic filter at a throughput of >300 L/m² to maximize usability and minimize contamination.

Low pH virus inactivation

The capture eluent is adjusted to pH 3.7–3.9 with HOAc or HCl and incubated at ≥18°C in the same or separate bags for 120–240 minutes to inactivate any present viruses. Alternatively, virus inactivation is performed at pH 3.4–3.6 for >30 minutes. The process intermediate is then adjusted to storage or further processing conditions, for example, by adjusting to pH 4–7 using a 1–2M Tris/-HCl titrant stock solution.

Polishing 1

The antibody is further purified by anion exchange chromatography (AEX), typically using a membrane capsule or cartridge (e.g., Sartorius Sartobind STIC PA 4mm or 3M MA ST Polisher) at a flow rate ≤350 mV/h, to remove process-related and/or product-related impurities and particles. Depending on the amount of antibody, the entire quantity from a batch can be processed in one or more cycles. Before loading, the intermediate is adjusted to the target conductivity, e.g., 8-20 mS/cm, and the target pH, e.g., 7.0 for antibodies with a basic isoelectric point. For antibodies with an isoelectric point below pH 7.0, the conductivity can be 5-20 mS/cm, and the pH can be between its isoelectric point and pH 5.0. The membrane is equilibrated with Tris buffer pH 7.0 or acetate buffer pH 5-6, and the loading density is generally between 0.5-10 g antibody/mL membrane volume (MV). Washing with equilibration buffer can be performed and combined with the collected effluent, the collection criteria of which are controlled by, for example, ultraviolet light or volume. The membrane adsorber can be used once or multiple times, for which it is regenerated (for 25 mV, 1 M NaCl) and reequilibrated (Tris buffer, pH 7.0).

Refined 2

Final purification can be achieved, for example, by cation exchange chromatography (CEX). In principle, the order of AEX and CEX unit operations can be reversed. CEX unit operations are typically performed in binding and elution mode at flow rates between 100 and 200 cm/h. Pre-packed or self-packed columns with resin, such as Cytiva Capto S ImpAct or Capto SP ImpRes, can be used at a bed height of 20 cm. Depending on the amount of antibody, the purification step is performed in one or more cycles. If necessary, the loading can be adjusted to a target conductivity between 5 and 9 mS/cm and a pH of 4.5–7.5 using HOAc, Tris titrant, or WFI. The loading is applied to a CEX column equilibrated with ≥5 CV acetate buffer (pH and conductivity matched to the CEX loading) at a typical density between 40 and 105 g/L, followed by washing with the same buffer at ≥5 CV. Elute the antibody with acetate buffer containing an appropriate NaCl concentration (>50 mM; <500 mM), and collect the eluent under UV control, with the collected eluent volume typically ≤10 CV. Regenerate the column with 0.5–2 M NaCl acetate buffer, reequilibrate (≥5 CV using CEX equilibration buffer), sterilize (0.5–1.0 M NaOH for ≥30 minutes), reequilibrate (≥5 CV using CEX equilibration buffer), and finally store in, for example, 20% EtOH or 2% BnOH.

Other refined

When additional purification is required to remove residual product or process-related impurities, mixed-mode chromatography (MMC) can be used instead of or in conjunction with the CEX step. Examples of mixed-mode chromatography resins are Capto adhere and Capto MMC ImRes. This unit operation is typically performed in flow-through mode, but binding-elution mode is also possible. The column can be self-packed or pre-packed, with a bed height of 5–20 cm, various diameters, and is operated at a flow rate of 100–500 cm/h in flow-through mode or 100–220 cm/h in binding-elution mode.

If necessary, the loading is typically adjusted to a target conductivity between 3-12 mS/cm and a pH between 4.2-7.5 using HOAc, Tris titrant, or WFI. The loading is applied at a typical density between 75-300 g/L onto an MMC column equilibrated with ≥5 CV acetate buffer (pH and conductivity matched to the MMC loading). Washing with equilibration buffer can be performed and combined with the collected effluent, the collection criteria of which are controlled by, for example, UV or volume. The MMC column can be used once or multiple times, depending on the amount of antibody to be purified. The column is regenerated using 0.5-2 M NaCl in acetate buffer, reequilibrated (≥5 CV, using equilibration buffer), sterilized (0.5-1.0 M NaOH ≥30 min), and finally stored in, for example, 20% EtOH or 2% BnOH.

nanofiltration

Residual foreign viruses may be removed by PDVF-based nanofiltration, such as using Planova BioEX with or without a pre-filter. The load is applied to a pre-equilibrated nanofilter (≥5 L/ ^m² acetate buffer, formulation matched to MA AEX or CEX elution conditions, for example) at a loading density of 1500–5000 g antibody/ ^m² under constant pressure (e.g., 2.0–3.4 bar). The filter can be tracked using the same buffer, and the filter integrity is typically tested after use. This step can be repeated if the integrity test fails. Alternatively, foreign viruses can be removed by cellulose-based nanofiltration with or without a pre-filter. The load is applied to a pre-equilibrated nanofilter (≥5 L/ ^m² acetate buffer, formulation matched to MA AEX or CEX elution conditions, for example) at a loading density of 500–2000 g antibody/ ^m² under constant pressure (e.g., 0.8–1.2 bar).

Concentration and buffer exchange

The intermediate is concentrated to 15-110 g/L by ultrafiltration and percolated with ≥6 percolation volumes of percolation buffer (DFB) (e.g., 10 mM histidine, 10 mM methionine, 30 mM arginine, pH 5.3), further concentrated to 200 g/L if necessary. This is typically performed using a tangential flow filtration system and a suitable membrane with a cutoff of 30-50 kDa (e.g., Millipore Pellicon, Sartorius Hydrosart, Pall Omega), with a loading typically of 300-1000 g antibody/ ^m² . The process is usually controlled by feed or retention flow rate (e.g., 2-7 L/min/ ^m² ), or by feed pressure (2-4 bar) and TMP (0.7-2 bar). The membrane can be washed with DFB, and the combined retentates are diluted with DFB to the target concentration. The TFF system and membrane are sterilized with 0.5 M NaOH and equilibrated with acetate buffer or DFB. Membrane integrity or permeability should be tested before use.

Stablize

In an optional stabilization step, excipient concentrate is added to produce the drug substance (BDS). Bioburden reduction filtration (e.g., 0.2 μm) is performed using, for example, a pre-equilibration filter (e.g., excipient concentrate diluted in DFB), followed by an integrity test after use. If the integrity test fails, the filtration step can be repeated.

Fill and freeze

BDS are packaged in suitable bags with an LDPE contact layer (e.g., 5 or 10 L) and preferably aseptically connected and disconnected i.d. sampling chambers. These bags are individually protected by optional vacuum sealing (LDPE outer packaging bag) and placed in a protective shell (e.g., RoS S shell). After storage at an optional intermediate temperature of 2–8 °C, the shell bags are frozen (using a plate or passive freezer) and subsequently stored at ≤-30 or ≤-60 °C, e.g., +/-5 °C, or ≤-25 or ≤-60 °C, e.g., +/-5 °C.

Section 28 - Kits or portions containing antibodies defined by antigens

According to aspect 28, a kit is provided comprising an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or a pharmaceutical composition according to aspect 20, and instructions for use.

The antibodies, fragments, conjugates, or pharmaceutical compositions of the present invention can be provided in a kit, i.e., a combination of reagents packaged in a predetermined amount in one or more containers along with instructions for use. For example, when the antibody, fragment, or conjugate is a therapeutic antibody, fragment, or conjugate, the instructions for use may include a packaging insert. For example, the packaging insert may include information describing advantageous modes of administration or combination therapies as described herein.

For example, when antibodies are labeled with enzymes, the kit may include the substrate and cofactors required for the enzyme (e.g., providing a substrate precursor that can detect chromophores or fluorophores). Additionally, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents can be varied widely to provide reagent solution concentrations that substantially optimize the sensitivity of the assay. In particular, reagents may be provided as dry powders, typically lyophilized, including excipients that provide a reagent solution of appropriate concentration upon dissolution.

Aspect 29 - Combination therapy including anti-CCR8 antibodies

According to one aspect, an anti-CCR8 antibody for treating tumors and a further therapeutically active compound or therapy are provided, wherein said further therapeutically active compound is

a) Chemotherapy agents, preferably taxane, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine.

b) Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

c) Antibodies that target proteins specifically expressed by tumor cells.

d) Antibodies or small molecules targeting HER2 and/or EGFR,

e) Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

f) Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1, and/or

g) Radiotherapy.

In other words, an anti-CCR8 antibody, preferably an anti-CCR8 antibody that induces ADCC and/or ADCP as described elsewhere herein, is provided for the treatment of tumors, wherein this use includes administration of non-target checkpoint proteins and of other therapeutically active compounds or therapies selected from the following.

h) Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine.

i) Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

j) Antibodies that target proteins specifically expressed by tumor cells.

k) Antibodies or small molecules targeting HER2 and/or EGFR,

l) Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

m) Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1,

n) Radiation therapy, and/or

o)(compound or antibody used for) depletion of tumor B cells, preferably CD19+ B cells.

The anti-CCR8 antibody may be the anti-CCR8 antibody of the present invention as described herein or any other therapeutically suitable anti-CCR8 antibody known in the art. The tumor may be any tumor described herein, for example, for aspect 22. Preferably, the tumor or disease is characterized by chemokine receptor-positive cells, such as CCR8-positive cells, such as CCR8-positive regulatory T cells or CCR8-positive tumor cells. Preferably, the tumor is an ICI-responsive tumor. Based on the examples described herein (e.g., Example 12.6ff., see responsive mouse model), it seems reasonable that, in particular, tumors that are responsive to or initially responsive to immune checkpoint inhibition will benefit from anti-CCR8 antibody therapy, whether alone or in combination. Additional therapeutically active compounds may be therapeutically active compounds as described elsewhere herein. Suitable taxanes may be selected, such as those derived from paclitaxel, albumin-bound paclitaxel, cabazitaxel, or docetaxel or derivatives thereof. According to a preferred embodiment, this use also includes administration of checkpoint inhibitors, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or CTLA4 antibodies, as described elsewhere herein.

Aspect 30 - Triple combination therapy including anti-CCR8 antibody

According to one aspect, anti-CCR8 antibodies, checkpoint inhibitors such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or CTLA4 antibodies, and additional therapeutically active compounds or therapies are provided for treating tumors, wherein the additional therapeutically active compounds or therapies are preferably...

a) Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

b) Antibodies that target proteins specifically expressed by tumor cells.

c) Antibodies or small molecules targeting HER2 and/or EGFR,

d) Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

e) Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine.

f) Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1, and/or

g) Radiotherapy,

h)(compounds or antibodies used for) the depletion of tumor B cells, preferably CD19+ B cells.

In other words, an anti-CCR8 antibody that induces ADCC and/or ADCP for the treatment of tumors is provided, wherein this use includes administration of a non-target checkpoint protein and selected from other therapeutically active compounds or therapies.

a) Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine.

b) Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

c) Antibodies that target proteins specifically expressed by tumor cells.

d) Antibodies or small molecules targeting HER2 and/or EGFR,

e) Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1,

f) Radiation therapy, and/or

The use of this method also includes the administration of checkpoint inhibitors, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or CTLA4 antibodies, as described elsewhere herein.

Aspect 31 - Sequential combination therapy including anti-CCR8 antibodies

According to one aspect, an anti-CCR8 antibody for treating tumors and a further therapeutically active compound or therapy are provided, wherein a dose of the further therapeutically active compound or therapy is administered after a first dose of the anti-CCR8 antibody, for example...

a) After anti-CCR8 antibodies increase the ratio of intratumoral CD8 cells to T reg cells by at least 2, 3, 4, or 5 times, or

b) After the anti-CCR8 antibody depletes at least 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the intratumoral Treg cells.

In other words, an anti-CCR8 antibody, preferably an anti-CCR8 antibody that induces ADCC and/or ADCP as described elsewhere herein, is provided for the treatment of tumors, wherein this use includes the administration of additional therapeutically active compounds or therapies, wherein an additional therapeutically active compound or therapy is administered after a first dose of the anti-CCR8 antibody, preferably.

a) After anti-CCR8 antibody induces an increase of at least 2, 3, 4, or 5 times in the ratio of CD8 cells to T reg cells within the tumor, or

b) After the anti-CCR8 antibody depletes at least 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the tumor's intratumoral Treg cells.

According to the preferred treatment regimen, treatment begins with the administration of a first dose of the anti-CCR8 antibody (e.g., once daily, once weekly, q2w, q3w, or q4w as described above), followed by the administration of additional therapeutically active compounds or therapies. Without being bound by theory, the time between the initial dose of the anti-CCR8 antibody and the additional therapeutically active compound should be chosen to allow at least 40, 50, or 60% depletion of intratumoral Tregs. The anti-CCR8 antibody may be the anti-CCR8 antibody of the present invention as described herein, but may also be any other therapeutically suitable anti-CCR8 antibody. The tumor may be any tumor described herein, such as for aspect 22. Preferably, the tumor or disease is characterized by chemokine receptor-positive cells, such as CCR8-positive cells, such as CCR8-positive regulatory T cells or CCR8-positive tumor cells. Preferably, the tumor is or is initially an ICI-responsive tumor. Because acquired resistance to PD(L)-1 antibodies can be strongly influenced by tumor-activated regulatory T cells (characterized by CCR8 expression), sequential therapy combining anti-CCR8 antibodies with checkpoint inhibitors is beneficial in overcoming this acquired resistance. Administering an additional therapeutically active compound or therapy after the first dose of anti-CCR8 antibody has been found to significantly improve treatment efficacy, even in highly challenging tumor models. The time between the first dose of anti-CCR8 antibody and the dose of the additional therapeutically active compound should be sufficient to induce a primary anti-tumor immune response. Based on the data provided herein (see Example 12.6ff), this manifests as an increase of at least 2, 3, 4, or 5-fold in the ratio of intratumoral CD8 cells to T reg cells, or (temporarily) depletion of at least 40%, 45%, 50%, 55%, 60%, 65%, or 70% of intratumoral T reg cells.

The additional therapeutically active compound or therapy may be any compound or therapy disclosed elsewhere herein. The therapeutically active compound may or may not be a checkpoint inhibitor, such as an anti-PD-L1 antibody, an anti-PD1 antibody, or an anti-CTLA4 antibody. Although the second therapeutic agent may be a non-targeted agent, targeted agents have been found to be highly preferred for this purpose. A targeted agent, as defined herein, is a therapeutically active compound that specifically recognizes tumor cells rather than all rapidly dividing cells or immune cell populations. While drugs that target all dividing cells or affect immune cell populations can be used in combination and may improve efficacy (see Examples 12.6.8, 12.6.9), they may interfere with the immune cell populations required for a sustained immune-tumor response. This problem is addressed by using antibodies that target proteins specifically expressed by tumor cells. Preferred targeted therapies are antibodies that bind to proteins specifically expressed by tumor cells.

Preferably, the additional therapeutically active compounds or therapies are selected from...

a) Antibodies or small molecules targeting checkpoint proteins, such as PD-(L)1 or CTLA-4,

b) Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine.

c) Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1.

d) Antibodies targeting proteins specifically expressed by tumor cells.

e) Antibodies or small molecules targeting HER2 and/or EGFR,

f) Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1,

g) Standard treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

h) Radiation therapy, and/or

i) For use with tumor B cells, preferably compounds or antibodies that deplete CD19+ B cells.

Suitable taxanes can be selected, such as those derived from paclitaxel, albumin-bound paclitaxel, cabazitaxel, or docetaxel or their derivatives.

Preferably, the dose of the additional therapeutically active compound or therapy is the first dose of the additional therapeutically active compound or therapy.

Aspect 32 - Stratification Methods/Diagnostic Methods

As discussed elsewhere herein, the layering steps described for the fourth embodiment according to aspect 22 can be used as part of the treatment method according to aspect 22, or provided separately as...

a) The method for selecting subjects most likely to benefit from anti-CCR8 antibody treatment.

b) Diagnostic methods for diagnosing tumors that are sensitive to anti-CCR8 antibody therapy, or

c) A method for monitoring the success of anti-CCR8 antibody therapy.

For the present invention, the antibody may be the antibody according to the present invention, but may also be any therapeutically suitable anti-CCR8 antibody.

Biomarkers for anti-CCR8 antibody therapy

To date, no biomarker has been available for predicting or monitoring the success rate of treatment with anti-CCR8 antibodies. While the demonstrated mode of action suggests that CCR8 itself could serve as a potential biomarker, the inventors have found CCR8 levels somewhat difficult to assess in clinical settings or clinical studies. To address this pressing issue, the inventors have proposed hypotheses, some of which can be verified as suitable biomarkers with excellent predictive capabilities, see, for example, Examples 12.7.1 and 12.7.2. Biomarker expression levels are typically determined using assays or methods known in the art, and then compared to reference values described elsewhere herein, and subsequently used to predict or monitor treatment response.

Biomarkers are provided for methods of diagnosing/grading subjects with tumors sensitive to anti-CCR8 antibody therapy, the uses of which include

a. Determine the levels of biomarkers in tumor samples or tumor samples containing regulatory T cells.

b. Compare the levels of biomarkers with reference samples or values, and

c. If the biomarker level is higher than or equal to the reference sample or value, the subject will be diagnosed/classified as having a tumor sensitive to anti-CCR8 antibody therapy.

Among them, biomarkers are

d. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4.

e. Granulase or immune cell markers, preferably markers of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof.

f. Treg infiltration markers, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9.

g. T cell markers or cytotoxic T cell markers are preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40, or GITR.

h. Interferon or interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα,

i. Complement factors,

j. serine protease inhibitors, and/or

k. Molecules derived from genes in Tables 12.7.2.1, 12.7.2.2, or 12.7.2.3.

Preferably, the biomarker is an immune checkpoint protein, most preferably PD-1, PD-L1, or CTLA4.

It also provides the use of biomarkers for predicting or monitoring treatment success, including the administration of anti-CCR8 antibodies, which includes

a. Determine the levels of biomarkers in the sample, and

b. Compare the levels of biomarkers with reference samples or values.

Among them, biomarkers are

c. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4.

d. Granzymes or immune cell markers, preferably lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof.

e. Treg infiltration markers, preferably selected from FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, CXCL9.

f. T cell markers or cytotoxic T cell markers, preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5 and TNF receptor superfamily members, such as 4-1BB, OX-40 or GITR.

g. Interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα,

h. Complement factors,

i. serine protease inhibitors and/or

j. Molecules derived from genes in Tables 12.7.2.1, 12.7.2.2, or 12.7.2.3.

Preferably, the biomarker is an immune checkpoint protein, most preferably PD-1, PD-L1, or CTLA4.

In addition, a method is provided for diagnosing/grading a subject with a tumor sensitive to anti-CCR8 antibody therapy using a molecule that binds to the biomarker. This use includes determining the level of the biomarker in a tumor or tumor sample using the molecule that binds to the biomarker, wherein the biomarker is (preferably)

a. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4.

b. Granulase or immune cell markers, preferably markers of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof.

c. Treg infiltration markers, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9.

d. T cell markers or cytotoxic T cell markers are preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40, or GITR.

e. Interferon or interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα,

f. Complement factors,

g. serine protease inhibitors, and/or

h. Molecules derived from genes in Tables 12.7.2.1, 12.7.2.2, or 12.7.2.3.

The molecule that binds to the biomarker is preferably an antibody or antigen-binding fragment, or a conjugate thereof. Most preferably, the molecule that binds to the biomarker is an anti-PD-1, PD-L1, or CTLA4 antibody, such as Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, or Ipilimumab.

Furthermore, the use of molecules that bind biomarkers for predicting or monitoring treatment success, including the administration of anti-CCR8 antibodies, is provided. This use includes using molecules that bind biomarkers to determine the level of a biomarker in a tumor or tumor sample, wherein the biomarker is (preferably)

a. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4.

b. Granulase or immune cell markers, preferably markers of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof.

c. Treg infiltration markers, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9.

d. T cell markers or cytotoxic T cell markers are preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40, or GITR.

e. Interferon or interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα,

f. Complement factors,

g. serine protease inhibitors, and/or

h. Molecules derived from genes in Tables 12.7.2.1, 12.7.2.2, or 12.7.2.3, and

The molecule that binds the biomarker is preferably an antibody or antigen-binding fragment, or a conjugate thereof. Most preferably, the molecule that binds the biomarker is an anti-checkpoint, anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, such as Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, or Ipilimumab.

In a preferred embodiment of the present invention, the biomarker is an immune cell marker, and the molecule binding the biomarker is preferably an antibody that binds to a marker of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or a combination thereof.

In a preferred embodiment of the present aspect, the molecule that binds the biomarker is a molecule that binds the Treg infiltration marker, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9.

In a preferred embodiment of the present aspect, the molecule that binds the biomarker is an antibody that binds a T-cell marker, which is preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5 and members of the TNF receptor superfamily, including 4-1BB, OX-40, or GITR.

In a preferred embodiment of the present aspect, the molecule that binds the biomarker is an antibody that binds to interferon or interferon-induced protein, preferably selected from INFγ, IL10, IL12p70, IL1β, IL2 and TNFα.

Immune checkpoint proteins as biomarkers for anti-CCR8 antibody therapy

According to the present invention, it has been surprisingly found that, for subjects who initially responded to ICI therapy, particularly to anti-PD-(L)1 or CTLA4 antibody therapy, the response to (monotherapy) using anti-CCR8 antibodies was improved, see, for example, Table 12.7.1. Since PD-L1 expression is a predictor of response to anti-PD-L1 antibody therapy, the inventors hypothesized that PD-L1 expression and checkpoint protein expression themselves may also be suitable for directly predicting response to anti-CCR8 antibody therapy. This hypothesis can be confirmed by correlation data between PD-L1 expression and response to anti-CCR8 antibody therapy (Table 12.8.1). PD-L1 expression can be determined as is known in the art, for example, but not limited to using tumor proportion score (TPS), combined positive score (CPS), or mRNA expression. Therefore, the inventors propose immune checkpoint biomarkers, particularly PD-L1, as alternative biomarkers for stratification to overcome the difficulty in analyzing and therefore implementing CCR8 in order to select the patient population most likely to benefit from anti-CCR8 antibody therapy.

Therefore, a method is provided for diagnosing/grading subjects with tumors sensitive to anti-CCR8 antibody therapy, comprising a molecule that binds to immune checkpoint proteins.

a. Determine the expression levels of immune checkpoint proteins in tumor (samples),

b. Compare the expression levels of immune checkpoint proteins with reference samples or values, and

c. If the expression level of immune checkpoint proteins is higher than or equal to the reference sample or value, the subject will be diagnosed/graded as having a tumor sensitive to anti-CCR8 antibody therapy.

Furthermore, the use of molecules that bind to immune checkpoint proteins for monitoring the therapeutic success of anti-CCR8 antibody therapy is provided, the method comprising:

a. Determine the expression levels of immune checkpoint proteins in tumor (sample)/sample, and

b. Compare the expression levels of immune checkpoint proteins with reference samples or values.

The molecule that binds to the immune checkpoint protein is preferably an antibody. The immune checkpoint protein is preferably PD-1, PD-L1, or CTLA4. The molecule that binds to the immune checkpoint protein is preferably an antibody that binds to PD-1, PD-L1, or CTLA4. For example, the antibody may be PD-L128-8, PD-L1 22C3, PD-L1 SP142, PD-L1 SP263, or any other suitable antibody known in the art as a companion diagnostic. In a particularly preferred embodiment, the checkpoint protein expression level is determined based on a combined positive score (CPS) or a tumor proportion score (TPS). If immune checkpoint protein expression is present or high, such as CPS ≥ 1, preferably CPS ≥ 5, most preferably CPS ≥ 10, or such as TPS ≥ 1%, preferably TPS ≥ 10%, most preferably TPS ≥ 50%, then the subject's or patient's tumor is diagnosed/stratified as sensitive to anti-CCR8 antibody treatment. Therefore, in some preferred embodiments, the reference value is CPS = 1, more preferably CPS = 5, or most preferably CPS = 10. In some other or similar preferred embodiments, the reference value is TPS = 1%, more preferably TPS = 10%, or most preferably TPS ≥ 50%.

In some preferred embodiments, PD-L1 expression is determined by CPS≥1, preferably by CPS≥5, most preferably by CPS≥10 or by TPS≥1%, preferably by TPS≥10%, and most preferably by TPS≥50%.

Treg infiltration markers as biomarkers for anti-CCR8 antibody therapy

A molecule that binds to a Treg invasion marker is provided for a method of diagnosing/grading subjects with tumors sensitive to anti-CCR8 antibody therapy, the method comprising:

a. Determine the expression levels of Treg invasion markers in tumor (samples).

b. Compare the expression levels of the Treg infiltration marker with reference samples or values, and

c. If the level of the Treg invasion marker is higher than or equal to the reference sample or value, the subject will be diagnosed/classified as having a tumor sensitive to anti-CCR8 antibody therapy.

Treg infiltration markers are markers highly expressed on activated Tregs. Preferably, the Treg infiltration markers are selected from CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, and CXCL9 (see Tables 11.1.1 and 12.8.1). Treg infiltration markers may differ from CCR8. The molecule binding the Treg infiltration marker is preferably an antibody, such as an antibody binding to one or more Treg infiltration markers.

The Treg infiltration markers disclosed herein can also be used as biomarkers for controlling treatment success, as described elsewhere herein. Treatment success is considered to be achieved if at least 40% or 50% (temporary) Treg exhaustion is achieved, for example by reducing Treg infiltration markers by a factor of 2.

Immune cell markers as biomarkers for anti-CCR8 antibody therapy

A method is provided for diagnosing/grading a subject with a molecule that binds to an immune cell marker for diagnosing/classifying a tumor sensitive to anti-CCR8 antibody therapy, the method comprising:

a. Determine the expression levels of immune cell markers in tumor (samples),

b. Compare the expression levels of immune cell markers with reference samples or values, and

c. If the level of immune cell markers is higher than or equal to the reference sample or value, the subject will be diagnosed/classified as having a tumor sensitive to anti-CCR8 antibody therapy.

Immune cell markers can be markers of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof. Suitable markers for these immune cell populations are described throughout this disclosure. For example, suitable T cell markers include CD3 (e.g., CD3E, D, and/or G). For example, suitable cytotoxic T cell markers include CD8 (e.g., CD8A and/or B). For example, suitable macrophage markers include MS4A7. For example, suitable M1 macrophage markers include Acod1. For example, suitable M2 macrophage markers include Mrc1. For example, suitable B cell markers include CD19, CD20, CD22, and/or MSA41.

The immune cell markers disclosed herein can also be used as biomarkers for controlling treatment success, as described elsewhere herein. Treatment is considered successful if a (temporary) increase in immune cells occurs, for example, at least 50%, or as indicated by an increase of at least 2-fold in immune cell infiltration markers.

T-cell markers as biomarkers for anti-CCR8 antibody therapy

A method is provided for diagnosing/grading subjects with tumors sensitive to anti-CCR8 antibody therapy using a molecule that binds to T-cell markers, the method comprising:

a. Determine the expression levels of T cell markers in tumor (samples),

b. Compare the expression levels of T cell markers with reference samples or values, and

c. If the level of T-cell markers is higher than or equal to the reference sample or value, the subject will be diagnosed/classified as having a tumor sensitive to anti-CCR8 antibody therapy.

T-cell markers can be markers highly expressed on T cells, such as activated T cells. Preferably, T-cell markers are selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, and members of the TNF receptor superfamily, including 4-1BB, OX-40, or GITR. Molecules that bind to T-cell markers are preferably antibodies, such as antibodies that bind to one or more T-cell markers disclosed herein.

The T-cell markers disclosed herein can also be used alone or in combination with Treg infiltration markers (such as those described elsewhere herein) as biomarkers for controlling treatment success. Treatment is considered successful if a (transient) increase in T cells occurs, such as at least 50%, or as indicated by an increase in T-cell markers of at least 2-fold.

Interferon or interferon-induced protein as biomarkers for anti-CCR8 antibody therapy

A method is provided for diagnosing/grading subjects with tumors sensitive to anti-CCR8 antibody therapy or for monitoring treatment success, the method comprising:

a. Determine the level of interferon or interferon-induced protein in the tumor (sample).

b. Compare the levels of interferon or interferon-induced proteins with reference samples or values, and

c. If the level of interferon or interferon-induced protein is higher than or equal to the reference sample or value, the subject will be diagnosed/stratified as having a tumor sensitive to anti-CCR8 antibody therapy.

Interferon or interferon-induced proteins are preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2, and TNFα. According to the present invention, the correlation between CCR8 mRNA expression and the inflammatory marker IFNγ was found to be significant for 50 tumor indications (see Table 11.1.2). It can be concluded that IFNγ levels are suitable as biomarkers disclosed herein. This has been confirmed, for example, in Example 12.6.2, where IFNγ and TNFα levels were associated with treatment response. In different experiments (Example 12.6.6), increased IFNγ, IL-1β, and IL-2 levels were associated with improved efficacy. Suitable interferon-stimulated genes or proteins include, but are not limited to, ACOD1, ACTG1, ACTR2, ACTR3, ADAMTS13, AIF1, AQP4, ASS1, B2M, BST2, C9JQL5, CALCOCO2, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CASP1, CCL1, CCL11, CCL13, CCL14, CCL15, CCL15, CCL14, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, and CCL8. , CD40, CD44, CD58, CDC42EP2, CDC42EP4, CIITA, CITED1, CLDN1, CX3CL1, CXCL16, CYP27B1 DAPK1, DAPK3, EDN1, EPRS, EVL, FCGR1A, FCGR1B, FLNB, GAPDH, GBP1, GBP2, GB P4, GBP5, GBP6, GCH1, GSN, HCK, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRAHLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DR B5, HLA-E, HLA-F, HLA-G, HLA-H, ICAM1, IFI30, IFITM1, IFITM2, IFITM3, IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1IL23R, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, JAK1, JAK2, KIF16B, KIF5B, KYNU, LGALS9, MEFV, MID1, MRC1, MT2A, MYO1C, NCAM1, NMI, NOS2, NUB1, OAS1, OAS2, OAS3, OASL, PDE12, PML, PRKCD, PTAFR, RAB12, RAB2 0,RAB43,RAB7B,RPL13A,RPS6KB1RYDEN,SEC61A1SLC11A1SLC26A6SLC30A8SNCA,SP100,STAR,STAT1,STX4,STX8,STXBP1,STXBP2,STXBP3,STXBP4,SYNCRIPTDGF1,TLR 2. TLR3, TLR4, TRIM21, TRIM22, TRIM25, TRIM26, TRIM31, TRIM34, TRIM38, TRIM5, TRIM62, TRIM68, TRIM8, UBD, VAMP3, VCAM1, VIM, VPS26B, WAS, WNT5A, XCL 1, XCL2, ZYX.

Example

Example 1: Comparison of CC chemokine receptor and CXC chemokine receptor

Human CC and CXC chemokine receptor sequences were retrieved from Uniprot and aligned using ClustalOmega. The results were imported into Jalview for visualization (Figure 1). Negatively charged amino acid residues E and D (gray), as well as tyrosine residues (Y, dark gray) and cysteine residues (C, light gray) are highlighted. Although the N-terminal domains are sequence-biased, they are characterized by a relatively large number of negatively charged amino acid and tyrosine residues.

Example 2: Comparison of CCR8 in humans, cynomolgus monkeys, and mice

Human, cynomolgus monkey, and mouse CCR8 sequences were retrieved from Uniprot and aligned using Clustal Omega. The results were imported into Jalview for visualization and computation of sequence identity (see Figure 2a). While pairwise alignment of human and cynomolgus monkey CCR8 yielded 94.37% sequence identity, the percentages between mouse and human CCR8 (percentage ID = 70.99) and between mouse and cynomolgus monkey CCR8 (percentage ID = 71.55) were significantly lower. Substantial differences were observed in the N-terminal (TRD) and C-terminal regions.

The cynomolgus monkey TRD showed 68% sequence homology with the human TRD, while the mouse TRD showed 52% sequence homology. However, acidic and tyrosine-sulfated TRDs exhibited negatively charged clusters, which were successfully used to generate cross-reactive antibodies (see Example 6). At least the MDYT and YYPD motifs were found to be conserved across species.

Example 3: Evaluation of existing-technology antibodies that specifically bind to CCR8

FACS experiments were performed using cell lines expressing CCR8 to evaluate a variety of commercially available, existing-technology antibodies that specifically bind to CCR8 and CCR8 staining. The mouse T-lymphoma cell line BW5147.4 expresses mouse CCR8 and human cutaneous T-lymphoma, while HuT78 expresses low levels of human CCR8. Existing antibodies against chemokine receptors, particularly those against CCR8, often exhibit low specificity. For example, Xing et al., Appl IHC Mol Morphol (2015): “Based on our experience and as previously reported by others, we are unable to assess CCR8 expression by immunohistochemistry due to the lack of specificity of available antibodies.” Chenivesse et al., JI (2012): “We are unable to assess the attractiveness of CCL18 to CD4 ⁺ CCR8 ⁺ cells because all commercially available anti-CCR8 antibodies lack specificity, which does not allow for cell sorting, at least in our hands.” or Pease, Biochem J (2011): “The latter has been problematic due to the lack of seemingly reliable antibodies against human CCR8. For example, in a study examining CCR8 expression in a CD4+CD25+ Treg population, CCR8 surface staining was undetectable using commercially available antibodies, despite these cells clearly expressing functional CCR8 as assessed by CCL1-driven microfilament polymerization.” (cf. Xing, Xiaoming, et al. "Expression of the chemokine receptor gene, CCR8, is associatedWith DUSP22 rearrangements in anaplastic large cell lymphoma. Appliedimmunohistochemistry&molecular morphology: AIMM/official publication of the Society for Applied Immunohistochemistry 23.8(2015):580; Chenivesse, Cécile, et al. Pulmonary CCL18 recruits human regulatory T cells. "The Journal of Immunology 189.1 (2012): 128-137.; and Pease, James E. "Targeting chemokinereceptors in allergic disease." Biochemical Journal 434.1 (2011): 11-24.).

Table 3.1 lists three commercially available antibodies that were analyzed but did not specifically stain CCR8 in the inventors' hands (see Figure 3). Rat IgG2B clone #191704 (MAB1429, R&D Systems) did not show specific binding to human CCR8, consistent with the observations described in Example 6 of WO2007044756. Abcam E77 showed a very weak signal and high background (data not shown). MM0068-4G19 (Abcam), 191704 (R&D Systems), 4G 19 (Bioaol (Genetex)), and 11n24 and 10k39 (all from antikoerper-online.de) did not show specific binding to human CCR8 in the inventors' hands.

Table 3.1: Selected existing technology antibodies, cf. Figure 3.

The inventors have discovered only two reliable monoclonal antibodies that specifically bind to human CCR8 in the prior art: clone 433H, developed by ICOS and initially disclosed in WO2007044756, and L263G8, supplied and marketed by Biolegend. Both antibodies are produced using cells overexpressing human CCR8 as immunogens. L263G8 does not cross-react with mouse CCR8 and does not bind to mouse CCR8 in BW5147.3 cells or HEK 293T cells transfected with mCCR8.

Monoclonal rat IgG2b,κ anti-mouse CCR8 clone SA214G2 (Biolegend catalog number 150302) showed positive staining on BW5147.4 cells expressing mouse CCR8. This antibody was produced based on cells transfected with mouse CCR8.

In summary, antibodies that specifically recognize the human CC chemokine receptor are rare for some members of this target class. This low rate is consistent with results from other commercially available antibodies targeting the human chemokine receptor.

Example 4: Antigens of CC and CXC chemokine receptors

Because the literature on tyrosine sulfation is incomplete and partially contradictory (e.g., across species), the inventors used a variety of available tools, literature searches, and their own experience to predict sulfation sites. Table 4.1 summarizes the resulting peptides suitable as antigens to obtain improved antibodies against various chemokine receptors. These antigens have been successfully used for antibody production, for example, as shown in the following examples, either as antigens or for off-target screening. The underlined sulfation sites were confirmed with an E-cutoff value of 55 by applying the algorithm according to Monigatti F. et al. (Monigatti, Flavio, et al. "The Sulfinator: predicting tyrosine sulfation sites in protein sequences." Bioinformatics 18.5 (2002): 769-770.). These sequences were not previously included in the training set of the Hidden Markov Model.

The sulfation of tyrosine residues in parentheses can be omitted. For human CCR3 and human CCR8, additional sulfation sites are predicted for Y172 and Y353, respectively. For monkey CCR3 and monkey CCR2, additional sulfation sites are predicted for Y172 and Y188, respectively. The sulfation sites shown in bold have been previously described in the literature (cf. Liu, Justin, et al. "Tyrosine sulfation is prevalent in human chemokine receptors important ant in lung disease." American Journal of Respiratory Cell and Molecular Biology 38.6 (2008): 738-743; Millard; Christopher J., et al. "Structural basis of receptor sulfotyrosine recognition by a CC chemokine: the N-terminal region of CCR3 bound to CCL11/eotaxin-1." Structure 22.11 (2014): 1571-1581).

Peptide design

The N-terminus of CCR8 is formed by amino acids 1 to 35 of the full-length human and cynomolgus monkey CCR8 protein and amino acids 1 to 33 of the full-length mouse CCR8 protein. The N-terminus consists of a TRD (amino acids 1-24 of human and cynomolgus monkey CCR8 and amino acids 1-22 of mouse CCR8) linked by cysteine residues and an LID domain (amino acids 26-35 of human and cynomolgus monkey CCR8 and amino acids 24-33 of mouse CCR8). For the synthesis of peptides containing the N-terminus, cysteine residues are replaced with serine residues to avoid peptide aggregation. Table 5.1 shows the respective IDs of the peptides used to generate anti-CCR8 antibodies via phage display panning as described in Example 6. To test whether post-translational modifications of the protein lead to difficulties in antibody production, unmodified or sulfated peptides containing the TRD and/or N-terminal sequence were used. Sulfation was introduced on at least 50% of the tyrosine residues of the TRD in cynomolgus monkey and human CCR8, for example at positions 3, 15, and 17 of the corresponding peptides. For mouse TRD and N-terminal peptides, sulfation is introduced at tyrosine residues 3, 14, and 15 of the corresponding peptides (see Table 6.1). Additionally, peptides containing LIDs engineered by introducing C-terminal biotinylation linked via a TTDS (trioxadecane-succinamide) linker are also included. Other tags and linkers can also be used. TRD or N-terminal peptides are modified by introducing N-terminal biotinylation linked by a TTDS-lysine linker. Similarly, other known linkers and/or tags can also be used to facilitate peptide immobilization.

To test whether post-translational modifications of proteins lead to difficulties in antibody production, unmodified or sulfated peptides containing TRD and/or N-terminal sequences were used. Sulfation was introduced on at least 50% of the tyrosine residues of the TRD in cynomolgus monkeys and human CCR8, for example, at positions 3, 15, and 17 of the corresponding peptide. For mouse TRDs and N-terminal peptides, sulfate was introduced on tyrosine residues 3, 14, and 15 of the corresponding peptide (see Table 6.1). Additionally, peptides containing LIDs engineered by introducing C-terminal biotinylation linked via a TTDS (trioxadecane-succinamide) linker were also used. Other tags and linkers can also be used. TRD or N-terminal peptides are modified by introducing N-terminal biotinylation linked by a TTDS-lysine linker. Similarly, other known linkers and/or tags can also be used to facilitate peptide immobilization.

Peptide synthesis

Peptides are available from various commercial sources and can be prepared as is known in the art, for example using standard Fmoc solid-phase peptide synthesis (SPPS) chemistry. The efficient synthesis of sulfated peptides is technically challenging because tyrosine sulfated peptides are stable under alkaline conditions, while sulfotyrosine undergoes desulfurization under acidic conditions, making it unsuitable to simply incorporate FmocTyr(SO3Na)OH stepwise into the grown peptide. Whole-scale sulfation of the peptide is possible, for example using sulfur trioxide-pyridine, but specific sulfation of selected tyrosines is not permitted. However, Fmoc-based SSPS can still be used to synthesize sulfated peptides. For this purpose, orthogonally protected tyrosine derivatives such as fluorosulfated tyrosine, azidomethyl-protected tyrosine, or neopentyl-protected tyrosine can be used for incorporation. The protected tyrosine can then be revealed selectively and quantitatively.

Chen et al., Angew Chem, 2016, reported a one-step synthesis of Fmoc-fluorosulfated tyrosine. An efficient Fmoc solid-phase peptide synthesis strategy was then introduced to incorporate fluorosulfated tyrosine residues into the target peptide. Standard simultaneous peptide resin cleavage and removal of acid-labile side-chain protecting groups provided crude peptides containing fluorosulfated tyrosine. Basic ethylene glycol was used as both solvent and reactant to convert the fluorosulfated tyrosine peptide to sulfotyrosine peptides in high yield.

The resulting peptides can then be purified as is known in the art, for example by HPLC, such as on a BEH C-18 (Waters) or Kinetex C-18 (Phenomenex) column, and analyzed by mass spectrometry (IonSpray and positive ion detector), for example for quality control.

In the literature, the two disulfide bridges in the full-length human CCR8 protein have been described as connecting extracellular loop 1 (ECL1) and extracellular loop 2 (ECL2) via cysteine residues at positions 106 and 183. Another disulfide bond between cysteine residues 25 and 272 is hypothesized to connect the 7th transmembrane helix to the N-terminus. However, the peptides of the anticipated embodiments do not form any disulfide bonds between cysteine residues.

Example 6: Phage display and screening of human anti-CCR8 antibodies

A fully human antibody phage display library (BioInvent n-CoDeR Fab library) was used to isolate human monoclonal antibodies by selection against soluble biotinylated peptides. The peptides were provided by Pepscan. Peptide synthesis was performed as described in Example 5. The following protocol was applied for the panning process.

Table 6.1: List of modified peptides used for antibody selection via phage display and ELISA screening.

Streptomyces-conjugated Dynabeads M-280 (Invitrogen ^™ ) were coated with biotinylated peptides (1 tube) and biotinylated off-target peptides (3 tubes) for one hour at room temperature (RT). The Dynabeads were washed and then blocked end-to-end by spin-coating at room temperature for one hour.

To remove off-target bindings, a blocked phage library was added to blocked off-target loaded Dynabeads and incubated end-to-end at room temperature for 10 minutes. This removal step was repeated twice. The removed phage library was then added to blocked target loaded Dynabeads and incubated end-to-end at RT for 60 minutes.

After rigorous washing (3 times with blocking buffer (PBS-T, 3% milk powder) and 9 times with PBS-T (150mM NaCl; 8mM Na2HPO4; 1.5mM KH2PO4; adjusted to pH 7.4-7.6, 0.05% Tween-20), Dynabeads carrying the Fab-phage-specific binding target were directly used to infect Escherichia coli strain HB101. The phage was then amplified in E. coli strain HB101 using the M13KO7 helper phage (Invitrogen).

In subsequent selection rounds, the target concentration was reduced to increase the selection pressure on high-affinity conjugates. Two different selection strategies were implemented during library panning (Figure 4). This strategy aimed to identify antibodies exhibiting binding activity against the N-terminal region of human CCR8 containing TRD and/or the N-terminal region of cynomolgus monkey CCR8 containing TRD. All peptides used as antigens or for off-target panning were sulfated at at least one position, see Tables 4.1 and 6.1.

For both strategies, a removal step using biotinylated human CCR4 N-terminal region containing TRD (TPP-14829, SEQ ID NO: 22, Y22 is sulfated) as an off-target peptide is added.

Strategy I involves a first round of panning on the N-terminal sulfated peptide of human CCR8 (TPP-14827, SEQ ID NO: 46, Y3, Y15, Y17 are sulfated), followed by a third round of panning on the same peptide (TPP-14827, SEQ ID NO: 46, Y3, Y15, Y17 are sulfated) or on the N-terminal sulfated peptide of cynomolgus monkey CCR8 (TPP-14828, SEQ ID NO: 47, Y3, Y15, Y17 are sulfated).

Strategy II involves a first round of panning for cynomolgus monkey CCR8 N-terminal sulfated peptides (TPP-14828, SEQ ID NO: 47, Y3, Y15, and Y17 are sulfated), followed by three rounds of panning for the same peptide (TPP-14828, SEQ ID NO: 47, Y3, Y15, and Y17 are sulfated) or human CCR8 N-terminal sulfated peptides (TPP-14827, SEQ ID NO: 46, Y3, Y15, and Y17 are sulfated), as shown in Figure 4.

For the initial qualitative evaluation, 88 randomly selected phage-on-phage clones from each clone library were cultured and expressed as monoclonal samples, and binding to the clones with the various targets previously used for panning was then tested. Furthermore, binding to human and cynomolgus monkey CCR8-expressing cells was determined by flow cytometry (FACS).

Certain conjugates are defined as molecular displays in a specific context.

(i) In ELISA assays, the signal intensity of the target is at least 10 times higher than the off-target signal intensity.

(ii) In FACS assays, the signal of cell lines expressing CCR8 was at least 2-fold higher than that of control cell lines not expressing CCR8, and

(iii) In FACS assays, the signal on the control cell line is less than 10,000.

Plasmids from 12 libraries that showed significant hit rates in at least one of the two assays were submitted for gene III removal and screened for soluble Fab in high-throughput ELISA and FACS screening.

Example 7: High-throughput screening of human anti-human CCR8 antibodies

From 12 clone libraries generated during phage display, 17,000 distinct sFab clones were screened in high-throughput screening using ELISA (HTS-ELISA) to determine their binding to peptides previously used for phage display panning: (i) TPP-14827, SEQ ID NO: 46, Y3, Y15, Y17 sulfated; (ii) TPP-14828, SEQ ID NO: 47, Y3, Y15, Y17 sulfated; and (iii) the corresponding off-target peptide TPP-14829, SEQ ID NO: 22, Y22 sulfated. For ELISA screening, the peptides were immobilized at a concentration of 0.1 μg/ml in coating buffer (Carbonat-Basis, Candor 121125) at 4°C on streptomycin-coated plates (Greiner bio-one 781997). After washing the plate three times with 60 μl PBS 0.05% Tween and blocking it for 1 hour at 20 °C with 50 μl Smart (Candor 113500), 10 μl of sFab sample was added to the plate and incubated at 20 °C for 1 hour. Following this, the plate was washed three times with 60 μl PBS 0.05% Tween, and 20 μl of anti-c-Myc HRP antibody was added. The plate was incubated at 20 °C for 1 hour, then washed three times with 60 μl PBS 0.05% Tween, and 20 μl of Amplex Red solution (Invitrogen A12222, 1:1000 in 50 mM NaP buffer, pH 7.6, containing 1:10000 of 30% H2O2) was added. After a final incubation of 20 minutes at 20 °C, the signal was determined using an emission wavelength of 595 nm and an excitation wavelength of 530 nm. For analysis, the signal-to-background ratio of a single sFab clone was used, with the background defined by the average of 48 wells on each plate that did not contain any sFab culture but contained the corresponding sample culture medium.

A total of 2193 different sFab clones showed significant binding to both human and cynomolgus monkey N-terminal sulfated peptides TPP-14827 (SEQ ID NO: 46, Y3, Y15, Y17 sulfated) and TPP-14828 (SEQ ID NO: 47, Y3, Y15, Y17 sulfated), as indicated by a signal-to-background ratio greater than 5, while no significant binding was shown to off-target TPP-14829 (SEQ ID NO: 22, Y22 sulfated). These sFab clones were reconstructed into full-length human IgG1 antibodies and used for FACS screening of human and cynomolgus monkey CCR8-expressing cell lines.

Based on binding to both cynomolgus monkey and human CCR8-expressing cells, and the absence of significant nonspecific binding to parental cell lines that do not express CCR8, ten antibody clones were selected for generation and further characterization. These initial hits were TPP-17575 to TPP-17581 and TPP-18205 to TPP-18207, and CDRs are shown in Table 7.1. Further manual optimization of the antibodies for therapeutic use resulted in the antibody sets shown in Tables 7.2a and 7.2b, see also the sequence listing.

Furthermore, to obtain the antibody TPP-27495, the sequence of TPP-23411 was engineered to include the YTE mutations M252Y, S254T, and T256E. TPP-27495 is 70% fucosylated-free.

Furthermore, to obtain the antibody TPP-27496, the sequence of TPP-23411 was engineered to include the LS mutations M428L and N434S. TPP-27496 is 70% fucosylated-free.

Example 8: Production of anti-mouse CCR8 antibody

Phage display and antibody screening

A fully human antibody phage display library (BioInvent n-CoDeR Fab lambda library) was used to isolate human monoclonal antibodies recognizing mouse CCR8 by selecting soluble biotinylated peptides. The peptides were provided by Pepscan. Peptide synthesis was performed as described elsewhere in this document. The following protocol was used for the panning process.

Streptomycin-conjugated Dynabeads M-280 (Invitrogen ^™ ) were coated with biotinylated peptides (1 tube) and biotinylated off-target peptides (3 tubes) for one hour at room temperature (RT). The Dynabeads were washed and then blocked end-to-end by rotation at room temperature for 1 hour. To remove off-target conjugates, a blocked phage library was added to the blocked off-target loaded Dynabeads and incubated end-to-end by rotation at room temperature for 10 minutes. This removal step was repeated twice. The removed phage library was then added to the blocked target loaded Dynabeads and incubated end-to-end by rotation at RT for 60 minutes.

After rigorous washing (3 times with blocking buffer (PBS-T, 3% milk powder) and 9 times with PBS (150mM NaCl; 8mM Na2HPO4; 1.5mM KH2PO4; adjusted to pH 7.4-7.6) and 0.05% Tween-20), Dynabeads carrying Fab-phage-specific binding targets were directly used to infect Escherichia coli strain HB101. The phage was then amplified in E. coli strain HB101 using the M13KO7 helper phage (Invitrogen ^™ ).

In subsequent selection rounds, the target concentration is reduced to increase the selection pressure on high-affinity binders.

To generate antibodies binding to mouse CCR8, a selection strategy was designed to identify antibodies exhibiting binding activity against the tyrosine-rich domain (TRD) of mouse CCR8 in the N-terminal region. For positive selection of mouse CCR8 TRD peptides, a mixture containing 50% normal peptide and 50% sulfated peptide was used (Seq ID No: 45 non-sulfated (TPP-13792) or Y3, Y14, Y15 sulfated (TPP-13793)). A removal step involving the human CCR4 TRD in the N-terminal region of the off-target peptide was also included. Furthermore, a human CCR4 removal step was performed on a mixture of sulfated and non-sulfated peptides (Seq ID No: 19 non-sulfated (TPP-13799) or Y19 and Y22 sulfated (TPP-13800)).

The screening strategy involves a total of four rounds of screening, each round targeting a specified mixture of sulfated and non-sulfated peptides, as shown in Figure 5.

For each cloning library, 88 randomly selected phage-on-phage Fab clones were cultured and expressed as monoclonal samples, and binding of the clones to the corresponding targets previously used for panning was then tested. However, sulfated and non-sulfated peptides were measured separately by ELISA.

Furthermore, the binding to cells expressing human, mouse, and cynomolgus monkey CCR8 was determined by flow cytometry (FACS).

Certain conjugates are defined as molecular displays in a specific context.

(i) In ELISA assays, the target signal intensity is at least 10 times higher than the off-target signal intensity, and

(ii) In FACS assays, the signal of cell lines expressing CCR8 was at least 10 times higher than that of control cell lines not expressing CCR8.

In these measurements, antibodies TPP-14095 and TPP-14099 were identified as binding to mouse CCR8-expressing cells, but not to control cells, human CCR8-expressing control cells, or cynomolgus monkey CCR8-expressing control cells. Furthermore, the antibodies were found to bind to sulfated TRD peptides, but not to non-sulfated peptides.

Example 9: Structural characterization of CCR8 antibody

Among the six CDR rings, the H3 ring exhibits the greatest structural diversity and is located at the center of the binding site. It also acquires the most mutations through affinity maturation and has the highest average number of contacts with the antigen. Therefore, it plays a crucial role in antigen binding.

Analysis of the specific structures of the antibodies obtained in the preceding examples revealed a surprising structural difference between the HCDR3 domain and the typical human HCDR3 domain. More specifically, the number of tyrosine residues in the HCDR3 domain of the human anti-human CCR8 antibody was significantly higher than expected for a random whole-human HCDR3 of matching length (~10%, see Zemlin, Michael, et al. "Expressed murine and human HCDR3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures." Journal of Molecular Biology 334.4 (2003): 733-749).

These data suggest that the presence of tyrosine has a beneficial effect on the specific binding of sulfated antigens. The optimized antibodies are also characterized by a significant increase in histidine frequency. For these antibodies, the average histidine content is 10%. The average occurrence of histidine in matched-length HCDR3 is approximately 2% in mouse HCDR3 and even lower in human HCDR3; see Zemlin, Michael, et al. (2003).

Because the antigens used for selection are characterized by a negative charge provided in the form of sulfated tyrosine and negatively charged amino acids, the inventors hypothesized that the presence of a positive charge in HCDR3 enhances binding to sulfated antigens, but is less necessary for binding to non-sulfated antigens. While the frequency of positively charged residues (H, K, R) in HCDR3 of the initially obtained antibody group was approximately 7%, the improved antibodies have an average of 15% positively charged amino acids per HCDR3 (e.g., up to approximately 31%).

The antibodies according to the invention are characterized by a higher tyrosine frequency than that of typical fully human HCDR3. While these high tyrosine frequencies are more readily obtained using mouse CDR, this may explain why previous attempts to obtain human anti-human antibodies that specifically recognize CC chemokine receptors such as CCR8 have been unsuccessful.

Example 10: Functional characterization of anti-CCR8 antibody

cell lines

Stable HEK 293 and CHO cell lines expressing human, cynomolgus monkey, or mouse CCR8 were generated using Inscreenex, a technology known in the art. The thymocyte cell line Hut-78 and the lymphoma cell line TALL-1 were obtained from ATCC and confirmed to show endogenous CCR8 expression. Because CCR8 expression can vary, expression levels were monitored.

Human regulatory T cells

FACS-sorted CD4+ and CD25+ cells from healthy donor PBMCs were purchased from BioIVT, AllCells, or StemCell Technologies. After thawing and overnight recovery in culture, cells were activated with anti-CD3 and anti-CD28 beads from Miltenyi, designed for Treg amplification, following the Treg amplification kit protocol, and supplemented with IL-2 (R&D Systems). Typically, CCR8 expression peaks between days 5 and 7 after activation of immature PBMCs, and cells can be reactivated 7–9 days after initial activation. Following restimulation, CCR8 expression peaks between days 2 and 4.

Example 10.1.1: Assessing the affinity for binding CCR8 from different species

FACS Experiment

The affinity of the antibodies for human, cynomolgus monkey, and mouse CCR8 and other chemokine receptors was determined using FACS known in the art. Briefly, CHO cells were engineered to express CCR8 from the respective species. Alternatively, activated human Tregs from human donors were used to determine the antibody affinity for activated Tregs. EC50 values were determined by FACS. All candidate antibodies showed not only high affinity for human CCR8 with EC50s in the low nanomolar range, but also similarly high affinity for cynomolgus monkey CCR8. This similar affinity across these species is crucial for advancing research to predict future safety concerns in humans. This is a major challenge in the search for therapeutic antibodies in immuno-oncology, as mouse models have significant limitations in predicting immunological side effects in humans. For the antibodies of the present invention shown in Table 10.1.1.1, the determined EC50s for binding to human Tregs were between 10 and 25 nM.

The affinity of the antibody of the present invention in CHO cells expressing human CCR8, CHO cells expressing cynomolgus monkey CCR8, or activated human Tregs (donor 1) is shown in Figures 6, 7, and 8, respectively.

Table 10.1.1.1: EC50 values of the antibodies of this invention in nM. FACS staining was performed on CHO cells stably expressing human CCR8 or cynomolgus monkey CCR8, or on activated human CD4+CD25+ T cells. These cells were activated using Miltenyi's Treg amplification kit.

Antibodies TPP-23411 and TPP-21360 are distinguished by the addition of a lysine residue at the C-terminus of the heavy chain; specifically, TPP-23411 contains lysine. TPP-23411 and TPP-21360 were tested for binding to human and cynomolgus monkey CCR8 in separate FACS experiments. The EC50 curves for binding to human and cynomolgus monkey CCR8 were perfectly correlated between the two antibodies (data not shown; IC50 values are shown in Table 10.1.1.2).

Table 10.1.1.2: Candidate EC50 values measured in nM. CHO cells stably expressing human CCR8 or cynomolgus monkey CCR8 were stained.

TPP-21360 TPP-23411 CHO with hCCR8:EC50 in nM 1.4 1.7 CHO with cynoCCR8:EC50 in nM 0.8 0.9

TPP-21181 and TPP-23411 were tested for binding to human and cynomolgus monkey CCR8 in separate FACS experiments (see Figures 9 and 10). Their respective EC50 values are listed in Table 10.1.1.3.

Table 10.1.1.3: Antibody EC50 values measured in nM. CHO cells stably expressing human CCR8 or cynomolgus monkey CCR8 were stained.

TPP-21181 TPP-23411 CHO with hCCR8:EC50 in nM 4.8 4.4 CHO with cynoCCR8:EC50 in nM 1.8 1.3

In further FACS experiments, prior art antibodies L263G8 (Biolegend) and 433H (ICOS, BD) were compared with TPP-21360 in terms of binding to human and cynomolgus monkey CCR8, see Figures 11, 12 and Table 10.1.1.4. Prior art antibodies showed only very low or virtually no overall binding to cynomolgus monkey CCR8 (low saturation, MFI << 1000). Only the antibody according to the invention specifically recognized human and cynomolgus monkey CCR8 with the same order of magnitude affinity, i.e., even in the sub-nanomolar range in some cases. Table 10.1.1.4: EC50 values of TPP-21360 compared to L263G8 and 433H. CHO cells stably expressing human or cynomolgus monkey CCR8 were stained.

TPP-21360 L263G8 433H CHO with hCCR8:EC50 in nM 0.58 0.31 2.8 CHO with cynoCCR8:EC50 in nM 0.51 132 12.1

These results demonstrate that the use of sulfated antigens promotes the generation of cross-reactive antibodies specific to their intended targets, such as cynomolgus monkey/human antibodies, against difficult targets (e.g., CCR8). As a proof of concept, activated human Tregs were stained with the antibody of this invention, TPP-23411, or the prior art antibody, L263G8, as shown in Figure 13. The antibody of this invention recognizing mouse CCR8 also exhibits excellent affinity for its target, as shown in Table 10.1.1.5. Table 10.1.1.5: EC50 values of TPP-14095 and TPP-14099. FACS staining was performed on CHO cells expressing mouse CCR8.

TPP-14095 TPP-14099 EC50 with nM 0.7 3

Table 10.1.1.6 shows the EC50 values of various other anti-human CCR8 antibodies of the present invention binding to CHO cells transfected with human CCR8 or cynomolgus monkey CCR8, or Hut78 cells expressing endogenous human CCR8. Interestingly, antibodies with the G50A mutation exhibit higher binding affinity to CHO cells expressing cynomolgus monkey CCR8. Therefore, anti-CCR8 antibodies containing this scaffold mutation are preferred.

Table 10.1.1.6 shows the EC50 values, expressed in M, characterizing the binding of the anti-human CCR8 antibody of the present invention to CHO cells transfected with human CCR8 or cynomolgus monkey CCR8 (as described elsewhere herein) or to the HuT78 cell line endogenously expressing human CCR8.

Example 10.1.2: SPR assay for characterizing the binding affinity of antibodies to unmodified TRD domains of different species

To analyze the affinity of antibodies for their unmodified (i.e., non-sulfated) antigens, surface plasmon resonance (SPR) binding assays were performed on a Biacore T200 instrument at 25°C using assay buffer HBS-EP+1x, 1 mg/ml BSA, and 300 mM NaCl. IgG was captured using anti-human Fc IgG covalently coupled to the CM5 sensor chip. N-terminal His6-labeled human CCR8 TRD (TPP-19950: MDYTLDLSVTTVTDYYYPDIFSSP, SEQ ID NO: 43), cynomolgus monkey CCR8 TRD (TPP-19952: MDYTLDPSMTTMTDYYYPDSLSSP, SEQ ID NO: 44), or mouse CCR8 TRD (TPP-19951: MDYTMEPNVTMTDYYPDFFTAP, SEQ ID NO: 45) were fused at the C-terminus with human serum albumin (HSA) to generate CCR8-HSA fusion proteins. The respective CCR8-HSA fusion proteins were used as analytes in a multi-cycle kinetic model at concentrations ranging from 1.56 to 200 nM. The obtained sensor maps were adapted for a 1:1 Langmuir binding model. The binding to human anti-human CCR8 antibodies TPP-23411 and TPP-21360 is very low (160 nM) and even lower with cynomolgus monkey CCR8 TRD, for example in the μM range. These support the importance of sulfation for antibody recognition of the antibodies of the present invention.

Table 10.1.2.1: Binding affinity of the antibodies of the present invention to His6-labeled HSA fusion TRDs of human, cynomolgus monkey, or mouse CCR8, as determined by SPR. Binding to the cynomolgus monkey CCR8 peptide is in the μM range and is an approximation because the highest concentration tested was 200 nM. No binding was shown for HSA alone or for the isotype control (data not shown).

Example 10.1.3: SPR Experiment for System Characterization of Binding Affinity

SPR binding assays were performed to systematically evaluate the binding of antibodies to a) non-sulfated v. sulfated antigen, b) TRD v. N-terminus, and c) human v. cynomolgus monkey CCR8. SPR binding assays were performed on a Biacore T200 instrument (Cytiva) at 25°C using assay buffer 1xHBS-EP+ (order no. BR100826, Cytiva). Peptides were used as ligands and captured onto a streptomycin-coated sensor chip (“Sensor Chip SA”, order no. BR100398, Cytiva) using their C-terminal biotinylated label. For this purpose, a terminal lysine (K) was added to the C-terminus of each peptide for biotinylation. For the N-terminus of CCR8, the naturally occurring cysteine between the TRD and LID domains was replaced with a serine. Antibodies were used as analytes (TPP-19546, TPP-21181, TPP-23411, hIgG1 isotype control TPP-9809, and mIgG2a isotype control TPP-10748). The concentration of analytes in the assay buffer ranged from 0.05 to 10 nM, and measurements were performed in a multi-cycle kinetic mode. The sensor surface was regenerated with glycine at pH 2.0 after each cycle. The obtained sensor maps were dual-referenced (subtracting the reference flow cell signal and buffer injection) and adapted to a 1:1 Langmuir binding model to export kinetic data using Biacore T200 evaluation software.

Table 10.1.3.1: List of peptides used to characterize antibody epitopes. Peptides containing the TRD sequence (#1 to 4) or N-terminus (#5 to 8) of CCR8, derived from cynomolgus monkey (#1, 2, 5, 6) or human (#3, 4, 7, 8) CCR8, sulfated (#2, 4, 6, 8) or non-sulfated (#1, 3, 5, 7). SO3: Sulfate residues. Biot: Biotin modification for immobilization.

For the analyzed antibodies of this invention, TPP-19546, TPP-21181, and TPP-23411, no binding to the non-sulfated TRD or non-sulfated N-terminus of CCR8 was detected, regardless of species. In contrast, sulfated TRD and sulfated N-term, such as cynomolgus monkey and/or human CCR8, showed excellent affinity with measured _KD values in the pM range. In some cases, the _KD values for binding to the human CCR8 TRD and N-terminus were essentially the same, i.e., within the error range. In other cases, binding to the N-terminus was slightly improved.

In summary, these results indicate that sulfated tyrosine is the cause of binding. They further suggest that sulfated TRD is both necessary and sufficient for binding, but the presence of the intact N-terminus as an epitope may further improve binding in certain situations. Finally, these results also demonstrate the cross-reactivity of CCR8 in humans and cynomolgus monkeys.

Table 10.1.2.3: Inventive antibody binding affinity to the TRD or N-terminus SPR of cynomolgus monkey or human CCR8. Odd-numbered peptides are characterized by unmodified tyrosine residues, and even-numbered peptides are characterized by sulfated tyrosine residues (Ys). No binding to any peptide was observed in the isotype control (data not shown). 1) mp = polyphase: approximate value.

Example 10.2: Binding Specificity

FACS analysis was performed as described elsewhere in this article to determine nonspecific binding to the target-negative human cell line. No nonspecific binding of the anti-human CCR8 antibody TPP-23160 was observed in CHO cells with mimic transfectants (Figure 14).

In addition, HEK cells were transiently transfected with human CCR1 or human CCR4. Most of the antibodies of this invention exhibited low off-target binding (Figures 15, 16).

Cell groups from cell lines derived from different tumor tissues were used to characterize nonspecific binding in these tissues (Table 10.2.1). The overall profile of most antibodies was favorable, while some antibodies showed some degree of multireactivity. Generally, the degree of multireactivity was within acceptable limits for the therapeutic application of all the invented antibodies. However, TPP-20950, TPP-18206, TPP-18429, TPP-18430, and TPP-18433 exhibited higher degrees of multireactivity and showed some degree of binding in at least two or three of the seven cell lines shown below.

Table 10.2.1: Multireactivity of the antibodies of the present invention in cell groups. EC50 is shown as M, if applicable.

The first ELISA-based assay was performed to analyze the nonspecific binding of antibodies TPP-18206, TPP-17578, TPP-19546, and TPP-23411, as well as two reference antibodies, to BVP, insulin, or DNA. The nonspecific binding of the present invention's antibodies to insulin, DNA, and/or BVP was lower than that of the first reference antibody (Gantenerumab, Roche) and also lower than that of the second reference antibody (Remicade, Janssen Biotech). Among the present invention's antibodies tested, TPP-23411 showed the lowest multireactivity to each of DNA, BVP, and insulin.

A second ELISA-based assay was performed to analyze antibodies TPP-18206 (wt or afuco), TPP-21360 (afuco), TPP-23411 (wt or afuco), TPP-20955, TPP-21047, and reference TPP-9809 (isotype control), Gantenerumab (TPP-12151), Lenzilumab (TPP-12166), Remicade (TPP-12160), and Avastin (TPP-17586). The nonspecific binding of the tested antibodies of the present invention to insulin, DNA, and BVP was lower than that of the first reference antibody (Gantenerumab, Roche). TPP-20955 and TPP-21047 showed generally higher nonspecific binding than other antibodies of the present invention (data not shown). Overall, the degree of multireactivity was within acceptable limits for the therapeutic applications of all the antibodies of the present invention, independent of their unfucosylated state.

A third ELISA-based assay was performed to analyze the nonspecific binding of antibodies TPP-27495 (YTE), TPP-27496 (LS), TPP-18429, TPP-18430, TPP-18432, TPP-18433, TPP-18436, TPP-27479, TPP-27480, and a control antibody to BVP, insulin, or DNA. Overall, the degree of multireactivity was within acceptable limits for the therapeutic applications of all the invented antibodies, independent of YTE or LS mutations.

The undesirable binding of the CCR8 antibody of the present invention to immune cell populations different from human Tregs was analyzed by staining these cell populations, see Figure 17.

Figure 18 shows the upregulation of CCR8 in human Tregs after activation.

Example 10.3: Evaluation of ADCC and ADCP induction

Example 10.3.1: Fucosylation-free antibody

The antibody according to the invention is glycoengineered using GlymaxX technology, see US8642292, to improve the interaction between the antibody's FC region and the FC receptor expressed on effector cells. Briefly, this technology is based on the heterologous cytosol co-expression of the bacterial enzyme GDP-6-deoxy-D-lyxo-4-hexylose reductase, which redirects the de novo fucose synthesis pathway to the glyconucleotide GDP-rhamnose, which cannot be metabolized by eukaryotic cells. This depletes the fucose library and results in the production of non-fucosylated antibodies on both the core portion (coreless fucose) and the variable portion (antennaeless fucose) of the N-glycan. It also inhibits the fucosylation of O-glycans and protein-O-fucosylation. Removal of fucose from the N-glycan on N297 results in increased affinity for FcγR, as illustrated in Example 10.3.2.

Example 10.3.2: SPR-based human Fcγ receptor binding analysis

To determine the ability of the antibody of the present invention and its non-fucosylated form to bind to the corresponding FC receptor expressed on human effector cells involved in ADCC or ADCP, SPR binding assays were performed on a T200 instrument at 25°C using a CM5 sensor chip and assay buffer HBS-EP + 500 mM NaCl. The FcγR variant was captured by an amine-conjugated anti-His capture antibody (ab), with IgG used as the analyte at concentrations up to 25 μM. KD values were derived from steady-state affinity analysis or kinetic data conforming to the 1:1 Langmuir isotherm. The non-fucosylated form of the antibody showed increased binding to both human and cynomolgus monkey FcgRIIIa, indicating improved ADCC induction in the non-fucosylated form of the antibody of the present invention in both species, thus leading to Treg depletion (Table 10.3.2.1).

Furthermore, comparable affinity for both cynomolgus monkey and human FcyRIII is important for mimicking ADCC efficacy in cynomolgus monkey animal models. These model systems are particularly important in tumor immunology because rodent models are generally unsuitable for reflecting the immune side effects of therapeutic antibodies. The non-fucosylated antibody of the present invention exhibits highly comparable affinity for the respective FC receptors in cynomolgus monkeys and humans.

Table 10.3.2.1: SPR analysis of the affinity of the antibody of this invention for different FC receptors. Italics: approximate value at the highest concentration of 25 μM and before saturation; n.e.: not evaluable; n.b.: no binding; res.bdg.: residual binding, not evaluable quantitatively; KD > 25 μM: fitted value exceeds saturation. curve.TPP-9809: isotype control.

Example 10.3.3: CCR8 antibody-mediated ADCC induction

Example 10.3.3.1: Anti-human CCR8 antibody-mediated ADCC induction

For the functional ADCC assay, HEK cells expressing human CCR8 or activated human Tregs were used as target cells, and NK92v-GPF-CD16 176V (NantKwest) were used as effector cells. In 96-well tissue culture plates, target cells and effector cells expressing CCR8 were incubated at a 4:1 ratio in the presence of various concentrations of therapeutic antibodies in RPMI 1640 containing 1% heat-inactivated FBS, 100 U/ml Pen/Strep, 1 mN sodium pyruvate, and 1x NEAA. Digitalis saponins were used for maximum lysis, and CytoTox-Glo (Promega) was added to the assay at the end of a 2-hour incubation. The raw luminescence value was determined using the CytoTox-gloPromega procedure. The mean maximum background, MMB = (target maximum) - (target spontaneous), was used to calculate % lysis. % Lysis = (raw luminescence - no ab) / MMB x 100.

For the candidate antibodies TPP-19546 (top inset) and TPP-21360 (bottom inset), in HEK cells expressing human CCR8, the absence of fucosylation increased ADCC-induced cytotoxicity to ~50% and ~70%, respectively (see Figure 19). In activated human Tregs with approximately 85% CCR8 expression, ADCC-derived cytotoxicity from the absence of fucosylated TPP-21360 or TPP-23411 reached as high as 59% or 52% (see Figure 20, Table 10.3.3.1.3). For the absence of fucosylated TPP-21360, using NK92v as effector cells, the mean EC50 for cytotoxicity % in activated human Tregs with 85% CCR8 expression was approximately ~20 pM. Tables 10.3.3.1.1 to 10.3.3.1.6 show results for different inventive antibodies and/or different donors.

Table 10.3.3.1.1: Summary of ADCC assays: EC50 and maximum response using activated Tregs as target cells and NK92v cell line as effector cells.

Table 10.3.3.1.2: Summary of ADCC Assay: EC50 and maximum response were measured using activated Tregs as target cells and the NK92v cell line as effector cells. All TPPs used in this assay were unfucosylated.

Table 10.3.3.1.3: Summary of ADCC assays: EC50 and maximum response were measured using activated Tregs as target cells and the NK92v cell line as effector cells. The activated human Tregs used in this study had approximately 85% CCR8 expression.

Table 10.3.3.1.4: Summary of ADCC assays: Tregs were used as target cells and the NK92v cell line as effector cells for EC50 and maximum response. The activated human Tregs used in this study showed only approximately 31% CCR8 expression.

Table 10.3.3.1.5: ADCC assay: EC50 and maximum response were measured using HEK cells expressing human CCR8 (>95% CCR8) as target cells and NK92v cell line as effector cells.

Table 10.3.3.1.6: ADCC assay: EC50 and maximum response were measured using HEK cells expressing human CCR8 (>95% CCR8) as target cells and NK92v cell line as effector cells.

Example 10.3.3.2: Anti-mouse CCR8 antibody (alternative antibody) mediated ADCC induction

To compare the behavior of the human anti-human/cynomolgus monkey CCR8 antibody of the present invention with that of the human anti-mouse CCR8 alternative antibody of the present invention, the latter also characterizing ADCC induction, primary mouse NK cells were used as effector cells, and mouse CCR8-expressing HEK293 cells and BW5417.3 (data not shown) were used as target cells for functional ADCC assays of the alternative antibody. A medium containing ultra-low IgG was used during co-culture to avoid non-specific binding of the CCR8 antibody to serum IgG. Furthermore, to more accurately reflect in vivo conditions, the target cells were pre-incubated with the alternative CCR8 antibody before adding effector cells, thereby allowing FcγRIII to better aggregate on the effector cells, an early step in the ADCC action mode. Effector cells and target cells were co-cultured for 20 hours at a 10:1 ratio in RPMI 1640 containing 1% Ultra-low IgG FBS One Shot medium in U-bottom 96-well plates. The alternative anti-mouse CCR8 antibody TPP-15285 and the isotype control antibody TPP-10748 were used for various concentration steps. Cytotoxicity was determined using the CytoTox-Glo ^™ Cytotoxicity Assay (Promega) (Cytotoxicity % = [(Experimental value - Control without antibody / (Maximum lysis of target cells - Spontaneous lysis of target cells)] x (100%). Control values were determined by co-culturing target cells with effector cells after adding the alternative CCR8 antibody. Control values were also determined by co-culturing target cells with effector cells without adding the antibody of the present invention. The maximum lysis value of target cells was determined for cases where digitalis saponins were added at the start of the assay. Spontaneous lysis values were determined by measuring spontaneous lysis of target cells in the assay medium without adding any other reagents. 92% to 97% of target cells showed expression of mouse CCR8 (determined by FACS). The ADCC-related cytotoxicity of TPP-15285 was approximately 39%, with a mean EC50 of approximately 111 pM (Table 10.3.3.2.1). The highest antibody concentration of the present invention resulted in a hook effect, further supporting the proposed ADCC-based mode of action. The optimized functional ADCC assay thus confirms that ADCC is the relevant mode of action for the in vivo efficacy of the alternative CCR8 antibody of the present invention, and therefore confirms that the anti-mouse CCR8 antibody TPP-15285 is suitable as an alternative antibody to the anti-human CCR8 antibody.

Table 10.3.3.2.1: Summary of ADCC assays using fucosylation (wild-type) as an alternative to CCR8 antibody.

Table 10.3.3.2.2: ADCC assay showing the percentage of cytotoxicity induced by wild-type alternative CCR8 antibody TPP-15285 (4 replicates).

Example 10.3.4: Anti-CCR8 antibody-mediated ADCP induction

Example 10.3.4.1: Anti-human CCR8 antibody-mediated ADCP induction

To generate macrophage (M2c) effector cells, CD14+ cells were negatively selected (Miltenyi) from PBMCs of healthy donors (AllCells). M2c macrophage differentiation was performed according to the eight-day differentiation protocol provided by StemCell Tech (see Figure 21). On assay day, M2c cells were stained with FACS to assess the expression of CD16, CD32, CD64, CD163, CD206, CD11b, CD80, and CD14. Target cells were HEK cells expressing human CCR8 or activated human Treg cells. Briefly, target cells were labeled with carboxyfluorescein succinimide (CFSE) (ThermoFisher) according to the manufacturer's instructions. Target cells were co-cultured in 96-well plates with varying concentrations of antibody and effector cells at a target cell to effector cell ratio of 4:1. In these assays, 1 μg/ml of anti-CD47 (positive control) was added; this enhances the response as it blocks the "don't eat me" signal. After incubation for 3–4 hours, samples were run on a MACSQuant or Attune flow cytometer. Phagocytosis percentage was determined by CD206+CFSE+ double-positive cells. CD206 is a marker expressed on macrophages.

M1 macrophages were similarly generated from the same source material (fresh PBMCs). Briefly, CD14+ cells were isolated by negative selection and cultured for four days in serum-free medium (StemCell TechImmnocult) containing 50 ng/mL M-CSF. However, on day 5, M1 macrophages were polarized by the addition of 10 ng/mL LPS + 50 ng/mL IFN-γ, while M2c macrophages were polarized by the addition of 10 ng/mL IL-10.

Figure 22 shows HEK cells expressing human CCR8 as target cells and in vitro differentiated M2c macrophages as effector cells inducing phagocytosis. Both wild-type and fucosylated forms of the present invention's antibodies TPP-19546, TPP-21360, and TPP-23411 induced ADCP. Figures 23 and 24, and Tables 10.3.4.1.1 to 10.3.4.1.7, show ADCP induction characterized by EC50 and maximum response to various antibodies of the present invention, various donors, and different macrophage populations. A considerable degree of ADCP was observed for all antibodies.

Table 10.3.4.1.1: Summary of ADCP Assay: EC50 and maximum response were measured using activated Tregs as target cells and M2c macrophages as effector cells. All TPPs used in this assay were unfucosylated. TPP-9809 was an isotype control. The human Treg donor 1212CCR8 was expressed in 40% of the cells.

Table 10.3.4.1.2: Summary of ADCP assays: EC50 and maximum response were measured using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 was an isotype control. The human Treg donor 1163CCR8 was expressed in 67% of the cells.

Table 10.3.4.1.3: Summary of ADCP assays: EC50 and maximum response were measured using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 was an isotype control. The human Treg donor 1163CCR8 was expressed in 67% of the cells.

Table 10.3.4.1.4: Summary of ADCP assays: EC50 and maximum response were measured using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 was an isotype control. Human Treg donor 1212CCR8 was expressed in 40% of cells.

Table 10.3.4.1.5: Summary of ADCP assays: EC50 and maximum response were measured using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 was an isotype control. Human Treg donor 1212CCR8 was expressed in 40% of cells.

Table 10.3.4.1.6: Summary of ADCP assay: EC50 and maximum response were measured using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 was an isotype control.

Table 10.3.4.1.7: Summary of ADCP assay: EC50 and maximum response were measured using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 was an isotype control.

Table 10.3.4.1.7: Summary of ADCP assays using Incucyte real-time imaging: EC50 and maximum response using macrophages as effector cells and HEK cells expressing human CCR8 as target cells at a ratio of 4:1. Human CCR8 expression was 80%. TPP-9809 was used as an isotype control. M2c macrophages were seeded into ultra-low attachment 96-well plates. After adding different concentrations of therapeutic antibody, target cells labeled with pHrodo red dye (Sartorius #4649) were added, and the plates were incubated at 37°C and 5% CO2. Phase contrast and red fluorescence images were automatically acquired and analyzed every 30 minutes using a 20x objective lens in the Incucyte S3 for 12 hours (4 images/well, 3 replicates). ADCP induction varied depending on the therapeutic wild-type antibody tested.

Example 10.3.4.2: ADCP induction mediated by anti-mouse CCR8 antibody (alternative antibody)

To compare the behavior of the human anti-human/cynomolgus monkey CCR8 antibody of the present invention with that of the human anti-mouse CCR8 alternative antibody of the present invention, the latter also characterized ADCP induction. For the functional ADCP assay, primary mouse bone marrow-derived M2 macrophages were used as effector cells, and mouse CCR8-expressing HEK293 cells and BW 5417.3 cells (data not shown) were used as target cells. To generate effector cells, mouse bone marrow-derived macrophages were seeded into 24-well plates and polarized into M2 macrophages by adding 20 ng/ml M-CSF, 0.05 μg/ml IL-4, and 0.05 μg/ml IL-13. Target cells were labeled with carboxyfluorescein succinimide dye (CFSE, ThermoFisher) and added to effector cells at an effector cell to target cell ratio of 2:1. Finally, different concentrations of the alternative anti-mouse CCR8 antibody TPP-15285 and the isotype control TPP-10748 were added. After co-culturing for 2 hours, cells were stained with the mouse M2 macrophage marker F4/80, and the fractionation of F4/80+ and CFSE+ double-positive macrophages was determined by flow cytometry to ascertain the percentage of phagocytosis. 92% to 97% of target cells showed expression of mouse CCR8 (as determined by FACS). TPP-15285-induced phagocytosis was ~11%, with a mean EC50 of ~402 pM (Table 10.3.4.2.1). The highest antibody concentration resulted in a hook effect, further supporting the proposed mode of action. Optimized functional ADCP assays further confirmed that ADCP is the relevant mode of action for the in vivo efficacy of the CCR8 antibody of this invention, thus demonstrating that the anti-mouse CCR8 antibody TPP-15285 is suitable as an alternative antibody to anti-CCR8 antibodies.

Table 10.3.4.2.1: Summary of ADCP assays using fucosylation (wild-type) as an alternative to CCR8 antibody.

TPP-15285 TPP-10748 EC50(pM) 402 NA Maximum response (%) 11 5 <![CDATA[R²]]> 0.93 NA

Table 10.3.4.2.2: ADCP assay using alternative CCR8 antibody.

Example 10.4: Regulation of CCR8 signaling by CCR8 antibody

CCL1 is a specific ligand for CCR8. Upon binding to CCR8, CCL1 can induce calcium (Ca) flux, chemotaxis, and receptor internalization via β-arrestin signaling, the latter potentially independent of G protein signaling. Antibodies can regulate GPCRs in at least six different ways:

1. The inverse agonist quells the potential endogenous activation of GPCRs upon binding. For CCR8, endogenous activation can be, for example, ~10%.

2. Antagonists that favor β-arrestin or G proteins block G protein-dependent signaling or β-arrestin signaling without affecting their other pathways.

3. Neutral antagonists can block the G protein and β-arrestin signaling pathways.

4. Full agonists activate G protein and β-arrestin signaling pathways.

5. Agonists that favor β-arrestin or G proteins activate either the G protein signaling pathway or the β-arrestin signaling pathway, but cannot activate both simultaneously.

6. Agonists that promote dimerization crosslink two GPCRs, such as different arms of an antibody, leading to various signaling activities.

For methods to eliminate intratumoral Tregs based on ADCC and ADCP, it is ideal to exclude any potential signaling effects that antibodies might produce on CCR8. To assess this, the inventors used Ca flux assays as a readout of G protein-dependent signaling and β-arrestin activation and phosphate signaling as readouts of G protein-independent signaling pathways. Phosphorylation of Erk1/2 is involved in the MAP kinase pathway and regulates a variety of cellular responses, while phosphorylation of AKT is known to be associated with the PI3 kinase pathway and has been shown to be involved in promoting chemotaxis in CCR8. The AKT pathway is also involved in promoting cell survival and growth, and recently, CCL1 has been shown to promote the survival of CCR8-expressing cells (Barsheshet, Yiftah, et al. "CCR8+FOXp3+Treg cells as master drivers of immune regulation." Proceedings of the National Academy of Sciences 114.23 (2017): 6086-6091.).

Example 10.4.1: DiscoverX Measurement for Monitoring β-arrestin Signal Transduction

In response to stimuli, i.e., ligand binding—for example, CCL1 and CCR8-GPCR can activate G protein-independent signals, such as β-arrestin signaling. This leads to the internalization of chemokine receptors (Fox, James M., et al. "Structure/Function Relationships of CCR8 Agonists and Antagonists. Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist." Journal of Biological Chemistry 281.48(2006): 36652-36661). β-arrestin assays were purchased from DiscoverX. In short, CCR8 is fused in-frame with a small enzyme donor fragment, ProLink ^™ (PK), and co-expressed in cells with a fusion protein that stably expresses a larger N-terminal deletion mutant of β-arrestin and β-galactosidase (called the enzyme acceptor or EA). Activation of CCR8 stimulates β-arrestin to bind to PK-labeled CCR8, forcing the two enzyme fragments to complement each other, thereby forming an active β-galactosidase. This interaction leads to increased enzyme activity, which can be measured using the chemiluminescent PathHunter assay.

In the first experiment, the antibody or CCL1 was incubated with CHO cells co-expressing human CCR8 and EA labeled with ProLink for 90 minutes, followed by the addition of the detection reagent. Neither the prior art antibody nor the antibody of this invention induced β-arrestin signaling (Figure 25).

In the next experiment, cells were stimulated with CCL1 at EC80 to activate β-arrestin signaling, and the antibodies of the present invention or prior art antibodies were added to evaluate their ability to block activated β-arrestin signaling. Surprisingly, β-arrestin signaling was blocked by prior art antibodies L263G8 and 433H, while the antibody of the present invention TP-23411 showed no significant effect on β-arrestin signaling at a concentration of at least 100 nM (Figure 26).

Table 10.4.1.1: IC50 values of TPP-23411 and prior art antibodies L263G8 and 433H for inhibiting CCL1-induced β-arrestin signaling, in nM. CHO cells stably expressing ProLink-labeled human CCR8 were stained. The IC50 of the antibody of the present invention could not be determined, indicating that TPP-23411 does not block CCL1-induced β-arrestin signaling.

Example 10.4.2: Phosphate ELISA

Phosphorylated AKT signaling promotes survival and growth, while phosphorylated Erk1/2 signaling regulates a variety of cellular responses. Phosphorylated Erk1/2 and phosphorylated AKT antibodies were purchased from CellSignaling and assayed by ELISA according to the manufacturer's protocol. CHO cells expressing human CCR8 or activated human Tregs were treated with CCL1, TPP-23411, Biolegend L263G8, or BD antibody 433H, or left untreated as a negative control. CCL1 served as a positive control. Interestingly, both the prior art antibodies Biolegend L263G8 and BD antibody 433H induced a significant increase in phosphorylated Erk1/2 levels (Figs. 27, 28) and phosphorylated AKT levels (Figs. 29, 30), for example, after 15 minutes in activated human Tregs, which was not the case with the antibodies of the present invention.

In summary, both existing antibody technologies have agonistic effects on G protein-independent pathways such as AKT phosphorylation or ERK1/2 phosphorylation.

Example 10.4.3: Calcium flux determination

Intracellular calcium mobilization measurement is a reliable detection method that can be performed in a high-throughput manner to study the effects of compounds or antibodies on potential drug targets such as CCR8. For this purpose, the activity of receptors that emit signals by releasing Ca2+ can be monitored using a fluorescence-based FLIPR calcium assay (Molecular Devices, Sunnyvale, CA). Briefly, CHO cells expressing human CCR8 are seeded into assay plates and incubated overnight. The cells are then loaded with the Ca+-sensitive dye BUV396/496.

For the measurement of antagonistic function, Ca flux signal was monitored using FLIPR Tetra, which was initiated simultaneously with the addition of different concentrations of antibody or reference compound (MC148) to the cells. After incubating the cells with the antibody or reference compound for 1 minute, the CCL1 agonist was added, and the calcium flux signal was additionally recorded using FLIPR Tetra for 3 minutes. To measure agonistic function, the antibody/reference compound was added in parallel to monitor the Ca flux signal using FLIPR for 3 minutes.

Tables 10.4.3.1 to 10.4.3.4 show the calcium flux assay results for anti-mouse or anti-human CCR8 antibodies. The IC50 values for TPP-14099 and TPP-21047 could not be determined. The IC50 values for other antibodies of this invention differ. In general, most antibodies of this invention, as well as prior art antibodies, effectively block CCL1-induced G protein-dependent signaling.

Table 10.4.3.1: Ca flux assay. CHO cells expressing mouse CCR8 were used to test the ability of the antibody to block CCL1-induced G protein-dependent Ca signaling. 40 ng/ml of CCL1 was used for induction. Both TPP-14095 and TPP-14099 bound to mouse CCR8. No agonistic effect on G protein-dependent Ca signaling was observed with either the antibody of this invention or prior art antibodies (data not shown).

Table 10.4.3.2: Ca flux assay. CHO cells expressing human CCR8 were used to test the ability of the antibody to block CCL1-induced G protein-dependent Ca signaling. CCL1 was used at a concentration of 80 ng/ml.

TPP-9809 TPP-18205 TPP-18206 TPP-17577 TPP-17578 IC50(nM) N/A 1.6 3 93.8 4.53

Table 10.4.3.3: Ca flux assay. CHO cells expressing human CCR8 were used to test the ability of the antibody to block CCL1-induced G protein-dependent Ca signaling. CCL1 was used at a concentration of 60 ng/ml.

TPP-9809 TPP-20955 TPP-21045 TPP-21047 TPP-21360 IC50(nM) N/A 0.52 0.29 N/A 0.50

Table 10.4.3.4: Ca flux assay. CHO cells expressing human CCR8 were used to test the ability of the antibody to block CCL1-induced G protein-dependent Ca signaling. CCL1 was used at a concentration of 60 ng/ml.

Example 10.5: Internalization of the antibody of the present invention

To assess CCR8 internalization, imaging techniques were used to visualize the process. Specific anti-CCR8 antibodies TPP-21360 and TPP-23411, along with the corresponding isotype control antibody TPP-5657, were labeled with fluorescent dyes. Additionally, commercial antibodies L263G8 and 433H, as well as the mouse antibody SA214G2, were conjugated with the permanent dye BODIPY. These antibodies were lysine-conjugated with 2 to 6 moles of excess FL dye (ester, D2184, Thermo Fisher) at pH 8.3. Following conjugation, the reaction mixture was purified by chromatography (PD10 desalting column, 1–2.5 mL; GE Healthcare) to remove excess dye and adjust the pH. The protein solution was then concentrated (VIVASPIN 500, Fa. Sartoriusstedim Biotec). The dye loading of the antibodies was determined using a spectrophotometer (NanoDrop) and calculated using the formula D: P = Adye εprotein: (A ₂₈₀ - 0, 16Adye) εdye. The dye loadings of the target-specific antibodies TPP-21360 and TPP-23411, as well as the isotype control antibody, showed similar ranges (dye loading/ab: 4.0 and 4.1). The commercial antibodies elicited dye loading/antibody ratios of 3.8 and 3.9. Conjugation can negatively impact antibody affinity; therefore, testing conjugated antibodies in a cell binding assay (FACS) is crucial to ensure that the label does not alter binding to CCR8 (CD198) (see Figures 31a, 32a, 34a, and b). The conjugated antibodies were then used in an internalization assay using the human CCR8-positive cell line HuT78 and the mouse CCR8-positive cell line BW5147.3. Prior to treatment, CCR8-expressing cell lines (2 x ^10⁴ cells/well) were seeded in 100 μl of 96-MTP (CellCarrier Ultra, PerkinElmer) medium. After incubation at 37°C/5% CO₂ for ₁₈ hours, the medium was replaced and different concentrations of labeled antibody (2.5, 1 μg/ml; repeated three times) were added. The same treatment protocol was applied to labeled isotype control antibodies (negative controls). Additionally, the parental CHO-K1 cell line (CCR8-negative cells) was used as a counter for selection (specific control) and similar treatment was performed. Target-specific antibody internalization was studied kinetically. Images were captured at different time points (0 h, 0.5 h, 2 h, 6 h, and 24 h), and internalization efficacy was determined by measuring total internalized fluorescence intensity/cell. Measurements were performed using Operetta CLS (PerkinElmer), and image data were analyzed using Harmony high-content analysis software (PerkinElmer).

Figures 31b and 32b show extensive internalization of commercial anti-human CCR8 antibodies 433H and L263G8 in HuT78 cells and commercial anti-mouse CCR8 antibody SA214G2 in BW5147.3 cells, while Figure 33 shows low or no internalization of the antibodies of the present invention TPP-21360 and TPP-23411, which are essentially equivalent to the isotype control.

Table 10.5.1: Summary of internalization data for inventive and prior art antibodies. n.a: Not tested.

To confirm that low internalization is indeed characteristic of all the anti-CCR8 antibodies of the present invention obtained using the sulfated peptide antigen described herein, the antibodies of the present invention listed in Table 10.5.2 were analyzed for their internalization behavior using vesicle regions/cells as reads (see also Figure 89). Anti-CD71 antibodies known to exhibit high internalization were used as positive controls. In fact, all the anti-human CCR8 antibodies of the present invention tested were non-internalizing antibodies, i.e., antibodies that did not show a significant degree of internalization. Interestingly, TPP-17577, the only tested human anti-human anti-CCR8 antibody that did not contain histidine in HCDR3, showed higher internalization than all the tested human anti-human anti-CCR8 antibodies that did contain histidine in HCDR3.

Table 10.5.2: Summary of internalization data for the antibodies of this invention. n.a: Not tested.

Example 10.6.1: FcRn affinity chromatography of anti-CCR8 antibody

Neonatal Fc receptors (FcRn) are involved in the regulation of IgG antibody metabolism in vivo. Binding of FcRn to the acidic environment of vascular endothelial cell nuclei protects IgG from lysosomal degradation. IgG release from FcRn into the bloodstream occurs at physiological pH 7.4. Therefore, FcRn binds to IgG at pH < 6.5 and does not bind at physiological pH 7.4. The interaction between IgG and FcRn affects the in vivo pharmacokinetics of IgG. Characterizing pH-dependent IgG binding to FcRn by immobilizing FcRn to mimic antibody cycling interactions can reveal IgG properties that contribute to its pharmacokinetic properties.

FcRn affinity assays were performed on a Pure 25 chromatography system at 25°C. The FcRn column (Roche, order number 8128057001; pre-packed with approximately 1 mL of resin; binding capacity: ≥100 μg IgG) was equilibrated with run buffer A: 20 mM Tris/HCl, pH 5.5, 140 mM NaCl. 30 μL of each antibody (1 mg/mL in equilibration buffer) was loaded onto the column. A linear pH gradient was generated by increasing the percentage of run buffer B (20 mM Tris/HCl, pH 8.8, 140 mM NaCl) as follows: 0 min - 20% B, 10 min - 20% B, 80 min - 100% B, 90 min - 100% B, 93 min - 20% B, 103 min - 20% B. The flow rate was 0.5 mL/min. Monitor the pH value at the elution point and read it from the chromatogram generated by Unicorn 7.1.

As shown in Table 10.6.1.1, the prior art antibody Ustekinumab was used as a standard pharmacokinetic control, and the prior art antibody Briakinumab was used as a rapid pharmacokinetic control (see Schoch, Angela, et al. "Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics." Proceedings of the National Academy of Sciences 112.19 (2015): 5997-6002). TPP-17577, TPP-18205, TPP-27495, TPP-21047, and TPP-27496 showed elution pH values greater than pH 7.5 and can be considered to show binding to FcRn at pH 7.4 to pH 7.5. The measured pH values can be used to predict antibody clearance and half-life.

Table 10.6.1.1: FcRn affinity chromatography for different antibodies: pH during elution. The two pH values represent biphasic peaks.

TPP Nr. elution pH Ustekinumab 7.22 27478 7.25 27477 7.25 19571 7.25 23411 7.38 20955 7.38 18436 7.38 18432 7.38 27479 7.43 23480 7.5 18433 7.5 18430 7.5 18429 7.5 18206 7.5 17578 7.5 17577 7.50/7.69 18205 7.50/7.77 Briakinumab 7.85 27495 7.88 21047 8 27496 8.13

Example 10.6.2: SPR-based FcRn binding analysis

To assess the binding affinity of anti-CCR8 antibody to FcRn, binding was investigated using SPR at pH 6.0 and pH 7.4. Binding assays were performed on a Biacore T200 instrument at 25 °C using a CM5 sensor chip and assay buffer PBST. For FcRn binding assays, human, mouse, or cynomolgus monkey FcRn amines were coupled to the surface of the CM5 sensor chip (~300 RU), and IgG was injected into PBST at concentrations of 15.6–2,000 nM at pH 6. Regeneration was performed using PBST at pH 7.4. In another experiment conducted only at pH 7.4, IgG was tested at a concentration of 2 μM, thus qualitative assessment was only performed upon binding. KD values were derived from steady-state affinity analysis and kinetic analysis by fitting 1:1 binding isotherms.

As shown in Table 10.6.2.1, as previously described, Ustekinumab was used as a positive control and Briakinumab as a negative control, as described by Schoch et al., 2015. These two antibodies did not show a significant difference in affinity for FcRn, but their binding responses to FcRn at pH 7.4 differed. Therefore, it can be hypothesized that the antibody exhibiting a binding response at pH 7.4 may have an accelerated in vivo half-life.

Example 10.7: Heparin affinity chromatography of anti-CCR8 antibody

For the antibodies of this invention that recognize human CCR8, it is likely that the interaction is charge-mediated, as the antibodies recognize charged antigen tyrosine motifs and have specific structural compositions, such as HCDR3. However, apart from binding FcRn at pH 7.4, charge-mediated interactions are a driving force for IgG degradation. Binding to the negatively charged glycocalyx of monocytes or macrophages may lead to pinocytosis and subsequent proteolytic degradation. Kraft et al. showed that heparin affinity chromatography, when performed in conjunction with FcRn chromatography, can more effectively and accurately predict antibody behavior in vivo (Kraft, Thomas E., et al. "Heparin chromatography as an in vivo predictor for antibody clearance rate through pinocytosis." MAbs. Vol. 12. No. 1. Taylor & Francis, 2020).

Table 10.6.2.1: SPR analysis of the affinity of the antibodies of the present invention for different FCRN receptors.

Small binding response is defined as a response <30RU, and binding response is defined as >30RU.

High affinity, high levels of interaction, and therefore high retention times can predict a short half-life of the test antibody. Heparin affinity tests were performed on a Pure 25 chromatography system at 25°C. Heparin columns (Tosoh) were equilibrated with run buffer A: 50 mM Tris, pH 7.4. 50 μL of each antibody (1 mg/mL at 20 mM histidine, pH 5.5) was loaded onto the column. A linear salt gradient was generated by increasing the percentage of run buffer B (20 mM Tris, pH 7.4, 1 M NaCl) as follows: 2 min post-injection - 0% B, 0-55% B over 16.5 min, 100% over 0.5 min, 100% B over 4 min. The flow rate was 0.8 mL/min. Elution times were monitored and read from the chromatograms generated by the Unicorn 7.1.

As can be seen from Table 10.7.1, and as previously stated, Ustekinumab was used as a positive control and Briakinumab as a negative control. Most of the antibodies tested showed retention times around 15 to 18 minutes. Therefore, these antibodies may exhibit shortened half-lives if electrostatic interactions are at play in vivo.

Table 10.7.1: Heparin affinity chromatography for different antibodies: pH during elution.

Example 10.8: Toxicity Study and CCR8 Depletion in Crab-Eating Macaques

Crab-eating macaques were treated with 50 mg/kg of TPP-23411 for four weeks. These animals showed no signs of toxicity. One week after the last treatment, thymus tissue (which has the highest levels of CCR8 in healthy tissue) was analyzed using RNA-seq. A significant decrease in CCR8 mRNA levels was found in animals treated with anti-CCR8 antibodies. No significant decrease in CCR8 levels was found at lower doses of TPP-23411.

Example 11: Therapeutic Applicability of the Target

For therapeutic applications of specific targets in immuno-oncology, target-specific expression is crucial for minimizing systemic side effects. Targets should be specific to the target tissue or cell; for example, for intratumoral Tregs, they should not be expressed in healthy tissues. Furthermore, increased expression of a target in specific tumor tissues may indicate medical use for that indication. For example, CCR8 is also expressed in B-cell lymphoma and T-cell lymphoma tumor cell lines.

Systemic depletion of Treg cells can stimulate and enhance not only tumor immunity but also autoimmunity, as demonstrated in patients with Immune Dysfunction-Multi-Endocrine Disorder-Enteropathy-X-Linked (IPEX) syndrome. In this disease, patients lack Tregs due to genetic alterations and, without appropriate immunosuppressive therapy, will die within the first two years of life due to systemic autoimmunity. Therefore, high specificity of targets aimed at depleting or inhibiting intratumoral Tregs is considered crucial to avoiding the side effects of therapeutic antibodies, such as those observed with CCR4-targeting antibodies.

Example 11.1: Specificity of the target CCR8

In the first analysis, the inventors assessed the expression of CCR8 mRNA in different human tissues and cell types. Significant expression of CCR8 mRNA was observed only in activated regulatory T cells and tumor-infiltrating lymphocytes (Figure 35).

In the second analysis, the inventors evaluated CCR8 mRNA expression in 50 different tumor indications. Indications with the highest CCR8 expression included, for example, breast cancer, lung adenocarcinoma (ADC), testicular cancer, gastric cancer and squamous cell carcinoma (SCC), head and neck malignancies, and esophageal tumors, but high expression was also found in colorectal cancer, ovarian cancer, and cervical cancer. In all indications except pancreatic cancer and melanoma, higher expression was found in tumors compared to the corresponding normal tissues (Figure 36).

In the third analysis, the inventors assessed the co-expression of CCR8 mRNA and FOXP3 mRNA across various tumor indications. Co-expression with the Treg marker FOXP3 was important in demonstrating that CCR8 is indeed primarily expressed on Tregs (see Table 11.1.1). IHC staining confirmed these findings (see Figure 37).

Table 11.1.1: CCR8 and the regulatory T cell defining marker FOXP3 were the most closely associated genes in all 9588 TCGA tumor samples (excluding thymoma as an indication), indicating that these genes are co-expressed in the same cell type. This table shows the 25 genes (out of 35021 genes) most closely associated with CCR8 and FOXP3 mRNA expression levels and their respective Pearson correlation coefficients r.

Furthermore, the correlations between CCR8 mRNA and pan-T cell marker CD3, cytotoxic T cell marker CD8, macrophage marker MS4A7, general inflammatory marker IFNg, and B cell markers CD19, CD20, and CD22 were evaluated for 50 oncology indications (see Table 11.1.2). Interestingly, the correlations were highly significant for almost all indications, indicating increased CCR8 expression when immune cells infiltrate the tumor itself, particularly T-reg infiltration. Therefore, it can be expected that immune cell infiltration, especially T-cell infiltration, is also a valuable biomarker that can be used for patient stratification to identify patients more likely to benefit from anti-CCR8 therapy.

Table 11.1.2: Pearson correlation coefficients (r) between the log2 mRNA levels of CCR8 and Treg markers FOXP3, pan-T cell marker CD3, cytotoxic T cell marker CD8, macrophage marker MS4A7, general inflammatory marker IFNg, or B cell markers CD19, CD20, and CD22. The results indicate that in 48 out of 50 indications for TCGA, CCR8 is highly significantly co-expressed with immune cells, particularly T reg infiltration into tumors. Therefore, it can be expected that, for example, T cell infiltration constitutes a valuable stratified biomarker for identifying patients who may benefit from anti-CCR8 therapy.

Example 11.2: CCR8's specificity against intratumoral Tregs compared to T effector cells

Several issues need to be considered regarding Treg depletion in human cancer immunotherapy. Tregs and activated effector T cells often share the expression of the same types of cell surface molecules, including CD25 and CTLA-4, making it difficult to selectively deplete Tregs with molecules-specific antibodies without affecting these effector T cells.

Foxp3+CD25+CD4+ Treg cells in tumors (tumor-infiltrating or intratumoral Tregs) express higher levels of cell surface molecules associated with T cell activation, such as CD25, CTLA-4, PD-1, LAG3, TIGIT, ICOS, and TNF receptor superfamily members including 4-1BB, OX-40, and GITR, compared to Tregs in lymphoid or non-lymphoid tissues or blood. Furthermore, tumor-infiltrating Tregs express high levels of specific chemokine receptors, including CCR4 and CCR8.

CD4+CD25+ sorted and unsorted PBMCs were activated for 6 days with anti-CD3 and anti-CD28 beads + IL2. FACS analysis showed that CCR8 protein expression was preferentially expressed on stimulated Tregs (CD4+CD25+FoxP3+CD127dim) (see Figure 38). Alternative targets OX40, GITR, and CD25, but not CCR8, were significantly expressed on stimulated CD8+ Teff cells and CD4+ T cells (CD4+CD25+Foxp3-). CCR4 showed Treg-specific expression but not intratumoral Treg specificity. These findings are consistent with observations of immunological side effects from CCR4 antibodies, attributed to systemic Treg depletion. In terms of specificity, CCR8 appears to be superior in supporting the specific depletion of intratumoral Tregs.

To confirm these findings, refer to Guo, Xinyi, et al. "Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing." Nature Medicine 24.7 (2018): 978-985, and Zhang, Yuanyuan, et al. "Deep single-cell RNA sequencing data of individual T cells from treatment-colorectal cancer patients." Scientific data 6. 1(2019): 1-15, and Zheng, Chunhong, et al. "Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 169.7(2017): 1342-1356". Raw data for NSCLC, CRC, and liver cancer were obtained to analyze CCR8 specificity, as shown in Figure 39. In fact, for these tumors, CCR8 mRNA expression was mainly limited to regulatory T cells present in tumor tissue (light gray box), while most of it was absent in regulatory T cells, CD4 helper T cells, and CD8 cytotoxic T cells in normal tissue (medium gray and dark gray boxes, respectively).

FACS analysis of human tumor lysate samples confirmed that the CCR8 protein is specifically expressed on human tumor-infiltrating Tregs in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), renal cell carcinoma (RCC), and lymph nodes (LNs, data not shown) (Figure 40). Up to 90% of tumor-infiltrating Tregs are CCR8 positive; for example, in NSCLC, 40-90% for stage III+, 40-70% for stage I/II, 20-40% for stage III+ in CRC, and 40-50% for stage III+ in RCC. Based on these data, it is believed that stratification according to stage would provide further benefit to patients receiving anti-CCR8 antibody therapy.

Example 12: In vivo experiment

Example 12.1.1: Alternative Antibodies and Homogeneous Tumor Models

In mouse experiments, alternative antibodies binding to mouse CCR8 were used to demonstrate antitumor efficacy and characterize the mechanism of action, namely that the consumption of Tregs within the tumor depends on the efficacy of ADCC (antibody-dependent cytotoxicity) and ADCP (antibody-dependent phagocytosis).

Table 12.1.1.1: List of alternative anti-mouse CCR8 antibodies used in in vivo studies. HEK293 cells overexpressing mouse CCR8 were used as target cells, and NK92 cells were used as effector cells to determine ADCC. ADCC activity was measured by lactate dehydrogenase (LDH) release.

The anti-mouse CCR8 antibody TPP-14099 bound CHO cells expressing mouse CCR8 at 3 nM EC50 and mouse iTregs at 13.2 nM EC50, as determined by FACS (data not shown).

Different syngeneic tumor models were used to simulate tumors with high immune infiltration and tumors with low levels of immune cell infiltration. Furthermore, the models used showed biased responses to known checkpoint inhibitor treatments, such as those with anti-CTLA4, anti-PD1, or anti-PD-L1 antibodies. Table 12.1.1.2 shows an overview of the tested tumor models, including efficacy data for the anti-CCR8 antibody of this invention. Notably, efficacy was demonstrated in various syngeneic tumor models.

In short, a suspension containing 500,000–1,000,000 tumor cells per 100 μl of PBS, culture medium, or 50% Matrigel mixture was subcutaneously injected into the flank of female immunized mice (e.g., Balb/c, C57B16). At a palpable tumor size of approximately 60–100 cubic millimeters (typically 100 cubic millimeters), CCR8 antibody administration was initially initiated with an intraperitoneal injection of 10 mg/kg at a volume of 5–10 ml/kg. Lower doses were tested in dose titration studies. Non-binding antibodies of their respective isotypes served as controls. Groups consisted of n = 10 mice. Treatment was administered twice weekly, typically on a q3/4-day schedule, with tumor size and body weight measured three times weekly. Tumor growth data were plotted as mean volume over time.

Twenty-four hours after secondary antibody therapy, tumor and tissue samples were obtained from satellite animals that had undergone the same treatment (n=5 per group) and analyzed by flow cytometry after dissociation into single cells and erythrocyte lysis, with particular attention paid to changes in the absolute number of regulatory T cells (Tregs) per 100 mg of tissue. The markers used to identify activated Tregs were CD45+CD4+CD25+FoxP3+. Other FACS markers included CD8, NKp46, 4-1BB, F4/80, CD11c, Gr1, and MHCII.

Table 12.1.1.2: Overview of different tumor models with parameters characterizing treatment outcomes. Efficacy was measured by Treg depletion, overall response rate, and tumor-to-control ratio based on the final tumor volume of the anti-CCR8 alternative antibody in the syngeneic mouse tumor model. ¹ Treg depletion was calculated as the percentage difference between intratumoral Tregs in the syngeneic control and intratumoral Tregs in the tumor treated with the therapeutic antibody. ² Overall response rate (ORR) and complete response (CR) were based on RECIST criteria for adaptation to rapid tumor growth in the mouse model (CR: complete response (<10% of initial tumor volume); PR: partial response (maximum 300% of initial tumor volume); NR: non-responder (>300% of initial tumor volume)). ³ T/C refers to the treated tumor volume/control tumor volume at the end of the study. nd: undetermined. ⁴ Mean values from several studies in this tumor model.

For each syngeneic mouse tumor model, 10 tumors from the isotype control group and the anti-CCR8 treatment group were harvested at the end of the treatment cycle, 24 hours after the last treatment (treated every two weeks until the tumors in either group reached an average volume of 3000 ^mm³ ), and whole-genome mRNA expression was measured by RNA-seq. Of the 17 models evaluated by RNA-seq, 13 showed increased CD8 mRNA levels in the anti-CCR8 treatment group compared to the control group, with fold changes shown in Table 12.1.1.3. For the CT26, H22, RM1, MBT2, and Hepa1-6 models, the increase in CD8 levels was significant according to the T-test, indicating a significant increase in cytotoxic T cell infiltration at the end of the treatment cycle. These data strongly suggest that CD8+ cells are involved in influencing tumor response.

Table 12.1.1.3: Changes in CD8 mRNA levels induced by anti-CCR8 treatment.

Model Multiple change T-test p-value CT26 8.604 8.7E-08 H22 7.160 1.7E-09 RM1 3.105 3.1E-03 MBT2 2.716 1.3E-09 WEHI-164 1.680 1.4E-01 Hepa 1-6 1.621 2.7E-02 LL2 1.315 1.3E-01 PANC02 1.242 5.4E-01 KLN205 1.194 5.2E-01 RENCA 1.188 6.7E-01 Colon26 1.137 7.6E-01 B16BL6 1.022 9.3E-01 J558 0.924 8.6E-01 MC38 0.898 6.2E-01 4T1 0.499 8.8E-02 EG7-OVA 0.449 4.3E-02

Example 12.1.2: Homogeneous model characterization for biomarker identification

Based on genome-wide RNA-seq data from early-stage tumors of an endogenetic model, elevated levels of the following genes were found to be closely associated with tumor response: Eif3j2, Eno1b, Ifi441, Hist1h2al, Ifi202b, Hmga1b, Amd2, Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpina1b, Prss41, C1rb, Cyld, Ccnb1ip1, Masp2, A caa1b, C4a, Snord93, Abhd1, Serpina3h, H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2, Igsf21, Olfr3 73, C1s2, Crym, Arv1, Hddc3, Plppr4, Ppp1rl1, Rps3a2, Zfp459, Rnd1, Serpina1a, Vcpkmt, Atp10d, Gbp2b, H2- T24, Tlcd2, Ctse, H2-Q10, Cyp2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d, Cacnala, Kcnj14, Ttc13, Farsa, Olfr1217, Jaml, H2-Bl, Tnpo2, Rims3, Dock9, Car5b, Atpla4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel1, Sel The biomarkers included enbp2, Nsd3, Wt1, Nap112, Ranbp9, Gtpbp3, AY761185, Rnaset2a, Serpina3i, Ell2, Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agptpbp1, Msh5, Cox18, Zfp330, Ttc37, Klk4, H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2, and H2-DMb2. Interestingly, this set was highly enriched for early complement factors, complement regulators (such as Serpins), and MHC components. These biomarkers, or combinations thereof, could be used to stratify, predict, or monitor tumor responses. In particular, the presence of high levels of complement C1/C4 may contribute to Treg lysis. When the depletion/consumption of complement factors reduces the effectiveness of Treg depletion, supplementing the complement system, for example in combination therapy, may be an option.

Different syngeneic models further characterized the absolute number of immune cell subsets and the frequency of total CD45+ immune cells in the tumor. For the models analyzed by flow cytometry (Table 12.1.2.1), B16F10 was characterized by the lowest number of immune cells—and the least reduction in tumor volume, see Table 12.1.2.1. Therefore, immune cell infiltration can be used to stratify and predict response to CCR8 antibody therapy. This hypothesis was later confirmed using B16Bl6.

Table 12.1.2.1: The tumor microenvironment was characterized by the absolute number of immune cell subsets and the frequency of total CD45+ immune cells in the tumors of the load-treated group analyzed at an early time point (24 hours after the second treatment). T/C vol describes the tumor volume ratio of the CCR8 antibody treatment group (10 mg/kg, twice weekly) to the control group at the end of the study.

Example 12.2: Efficacy in CT26 tumor-bearing mice

Treatment with anti-CCR8 antibodies TPP-15285 and TPP-15286 in CT26 tumor-bearing mice demonstrated potent antitumor efficacy (Figure 41). TPP-15285 showed reduced efficacy at the lowest dose of 0.1 mg/kg, with no significant differences at other dose-dependent levels. As expected, the anti-PD-L1 antibody (“PDL1”) exhibited moderate efficacy in the model. No efficacy was observed with any of the isotype-specific unbound antibodies.

Spider plots of the corresponding treatment groups (tumor growth of individual mice over time) (Figure 42) clearly demonstrate the efficacy with high homogeneity, with complete responses from both anti-CCR8 antibodies observed in all dose groups.

Treg analysis of CT26 tumor samples by flow cytometry 24 hours after secondary antibody treatment showed a significant reduction in the number of Tregs in the anti-CCR8 antibody treatment group compared to the isotype control group. In the case of TPP-15285, Treg depletion was clearly dose-dependent (Figure 43).

Table 12.2.1: Relevant data from Figure 43.

Example 12.3: Study on the mode of action

Example 12.3.1: Study on the ADCC/ADCP mechanism of action in mice burdened with CT26 tumors

Compared to glycosylated antibodies, non-glycosylated antibodies, which can be generated in prokaryotic hosts, exhibit nearly identical antigen-binding affinity, stability at physiological temperatures, and in vivo serum persistence. However, the absence of N-linked glycans virtually eliminates all FcγR binding affinity and immune effector functions, which are crucial for clearing antibody-opyrated target cells via ADCC or ADCP. To provide evidence for ADCC/ADCP-based modes of action, non-glycosylated versions of the antibodies were generated as negative controls.

TPP-18208 and TPP-18209 are glycosylated human IgG1 versions of anti-CCR8 alternative antibodies that cannot bind to effector cells, such as NK cells and/or macrophages, via Fc-γ receptors. These antibodies were tested for antitumor efficacy in mice burdened with CT26 tumors and compared to their corresponding wild-type/glycosylated counterparts containing the same sequences (TPP-14095, TPP-14099). Additionally, this study included the anti-CCR8 antibodies TPP-15285 and TPP-15286 (glycosylated, mIgG2 isotype).

Wild-type antibodies exhibited strong antitumor efficacy, while non-glycosylated antibodies showed no efficacy compared to the isotype control (Figure 44). Spider plots illustrate the results for individual mice in each treatment group (Figure 45). Thus, in vitro tumor analysis by flow cytometry demonstrated Treg depletion only after treatment with wild-type (glycosylated) antibodies, while non-glycosylated antibodies did not show Treg depletion compared to the corresponding unbound isotype control (Figure 46).

These results confirm that ADCC and/or ADCP are the main modes of action of anti-CCR8 antibody-depleted Tregs in CT26 tumors.

Example 12.3.2: The involvement of B cells or T cells and the effect of CD8+ cell depletion or CD19+ cell depletion on the anti-tumor efficacy of CCR8 antibody.

To test the correlation between the presence of tumor-infiltrating CD8+ T cells or CD19+ B cells and the antitumor efficacy of anti-CCR8 antibodies, the inventors used genetically modified C57BL6 mice that expressed diphtheria toxin receptor (DTR) under the control of lineage-specific Cd8a+ or lineage-specific Cd19+ promoters. Following diphtheria toxin (DT) injection, CD8+ T cells or CD19+ B cells were quantitatively depleted from mouse blood (data not shown) and subcutaneously carried syngeneic MC38 tumors (Table 12.3.2.1).

As expected, the depletion of CD8+ T cells completely eliminated the anti-tumor effect of anti-CCR8 therapy, resulting in tumor growth identical to that of the isotype control group (Figure 87). These results confirm that the anti-tumor efficacy of the anti-CCR8 antibody of the present invention depends on the presence of CD8+ T cells.

Conversely, quite surprisingly, depletion of CD19+ B cells significantly increased the efficacy of TPP15285 (Figure 88), resulting in a decrease in the T/C ratio from 0.35 to 0.16 (T-test p-value = 0.032). Therefore, it is recommended to combine anti-CCR8 therapy with B-cell depletion or B-cell depletion agents (such as anti-CD19 antibodies or anti-CD20 antibodies, such as rituximab) to further improve clinical cancer treatment.

Table 12.3.2.1: Depletion of CD8a and CD19 expression in diphtheria toxin-treated DTR mice.

Table 12.3.2.2: Treatment groups assessed for the effects of CD19+ B cell depletion or CD8+ T cell depletion.

Example 12.3.3: Time course of increased CD8/FOXP3 ratio in CT26 syngeneic tumor mice

To gain some mechanistic insights into the immune cell level, CD8 and FOXP3 mRNA levels were monitored in CT26 syngeneic tumor mice treated with TPP15285 or an isotype control. Ten tumors were harvested directly before the first dose and at 12, 24, 36, 48, and 72 hours after the first dose for RNA-seq analysis. Similarly, tumors were harvested 24 hours after the second, third, and fourth doses of TPP15285. RNA-seq analysis was performed on all tumors to measure mouse Foxp3, CD8a, and CD8b1 mRNA levels. The mean ratios of Cd8a and Cd8b1 to Fopx3 were calculated, and the significance of the difference in ratios between the control and TPP15285 treatment groups was determined using a t-test.

The ratio of CD8 to FOXP3 mRNA varied between approximately 0.9 and 2.0 at the earliest time point after TPP15285 treatment in the isotype control and TPP15285 groups. However, in the TPP15285 group, it significantly increased to 5.8, 3.9, and 4.4 at 72 hours after the first dose, 24 hours after the second dose, and 24 hours after the third dose, respectively, indicating that TPP15285 significantly depleted regulatory T cells. At 24 hours after the fourth dose, the ratio returned to 2.6.

Table 12.3.3.1: CD8/FOXP3 ratio of mRNA levels measured at different time points after administration of TPP15285 or isotype control.

Example 12.4: Efficacy of CCR8 antibody TPP-15285 in EMT6 tumor-bearing mice

Example 12.4.1: Efficacy of CCR8 antibody TPP-15285 in EMT6 tumor-bearing mice—Different dosages

The anti-CCR8 alternative antibody TPP-15285 demonstrated dose-dependent efficacy in EMT6 tumor-bearing mice, with the strongest effect observed at the 10 mg/kg dose. At this dose, TPP-15285 showed superior efficacy compared to the anti-CTLA4 antibody (Figures 47 and 48). Strong efficacy was also observed at the 1 mg/kg dose, while the 0.1 and 0.01 mg/kg doses showed almost no efficacy. Spider diagrams illustrate these results at the individual mouse level (Figure 49).

Treg analysis of EMT6-tumor samples by flow cytometry 24 hours after the second antibody treatment showed a significant reduction in the number of Tregs compared to the isotype control group. Treg depletion was clearly dose-dependent (Figure 50). Treg depletion of anti-CTLA4 was also observed, as previously described in the literature. Furthermore, a strong increase in CD8+ T cells was demonstrated in tumors sampled at the end of the study for effective doses of TPP-15285 (10 mg/kg) and anti-CTLA4 at the end of the study (Figure 51). Tables 12.4.1.1 and 12.4.1.2 show further changes in the immune cell population.

Table 12.4.1.1: The relative percentage of immune cell populations was determined by FACS analysis of immune cells 24 hours after the second treatment of tumor-bearing EMT6 mice with different doses of CCR8 antibody TPP-15285 or at the end of the study.

Table 12.4.1.2: The relative percentage of Tregs, CD8:Treg ratio, or CD4conv:Treg ratio were analyzed by FACS 24 hours after the second treatment of tumor-bearing EMT6 mice with different doses of the CCR8 antibody TPP-15285 or at the end of the study.

Example 12.4.2: Efficacy of CCR8 antibody TPP-15285 in EMT6 tumor-bearing mice—single or multiple administrations

The mouse breast cancer model EMT-6 was used in conjunction with TPP-15285 to test the effects of single versus multiple administrations. Four different treatment regimens were tested: single treatment, two BIW treatments, three BIW treatments, or four BIW treatments. Each group was treated with a load-controlled regimen or with TPP-15285 at 0.1 mg/kg, 1 mg/kg, or 5 mg/kg. In addition to efficacy studies, satellite animals were sacrificed at 24, 48, and 120 hours post-treatment for in vitro phenotypic analysis. Antibody administration occurred intraepithelially (i.p.), but other routes could also be used.

Efficacy data for single-dose, two-dose, three-dose, or four-dose BIW treatments are shown in Figure 85. Interestingly, single-dose treatment was most effective in achieving superior therapeutic results. At the end of the study, the frequency, absolute number, and activation of CD8+ intratumoral T cells were increased (see Table 12.4.2.1). However, there were no differences in the number and frequency of intratumoral Tregs or CCR8 expression at the end of the study (see Table 12.4.2.2). Furthermore, there were no differences in the number of intratumoral NK cells or macrophages at the end of the study or during the study (data not shown).

However, over time, a dose-dependent decrease in intratumoral Tregs (CD4+CD25+Foxp3+) was observed after the first and second treatments, but less pronounced after the third and fourth treatments (see Table 12.4.2.3). This was also observed in CCR8+Tregs (data not shown). Furthermore, the absolute number of CD8+ T cells increased over time (see Table 12.4.2.4). Therefore, compared to the isotype control, the CD8/Treg ratio increased after the first dose and remained high after the fourth dose. Characterization of blood samples did not show a significant decrease in Tregs or an increase in CD8 T cells in the blood across different treatment regimens and over time (data not shown). Additionally, no changes were observed in NK cells and macrophages in the blood (data not shown).

Table 12.4.2.1: Absolute CD8+ cells per 100 mg of tumor as determined by FACS at the end of the study. FACS analysis revealed an increase in the frequency, absolute number, and activation status of cytotoxic T cells.

Table 12.4.2.2: Absolute CD4+CD25+FoxP3+Treg cells per 100 mg tumor as determined by FACS at the end of the study.

Table 12.4.2.3: Absolute CD4+CD25+FoxP3+Treg cells/100mg tumor, as determined by FACS in satellite animals.

Table 12.4.2.4: Absolute CD8+ cells per 100 mg tumor as determined by FACS in satellite animals.

Example 12.5: Efficacy of anti-CCR8 antibody TPP-15285 in F9 tumor-bearing mice

The anti-CCR8 alternative antibody TPP-15285 showed dose-dependent efficacy in F9 tumor-bearing mice, exhibiting potent efficacy compared to the anti-PD-L1 antibody at 10 mg/kg and 1 mg/kg doses, but with decreased efficacy at 0.4 mg/kg dose (Figures 52 and 53). Spider diagrams illustrate these results on an individual mouse basis (Figure 54).

Treg analysis of F9 tumor samples by flow cytometry was performed 24 hours after the second antibody treatment. The results showed a significant reduction in the number of Tregs in the 10 mg/kg anti-CCR8 antibody treatment group compared with the isotype control group. Treg depletion was clearly dose-dependent (Figure 55). Treg depletion was also observed with anti-PD-L1 antibody treatment. Furthermore, a strong and dose-dependent increase in CD8+ T cells in the tumor was demonstrated in all dose groups of TPP-15285 and anti-PD-L1 antibody (Figure 55). Further effects on various immune cell populations are shown in Figures 55 and 56 and Table 12.5.1.

Table 12.5.1: Effects of anti-CCR8 or anti-PDL1 antibodies on immune cell populations and their ratios 24 hours after the second treatment.

Example 12.6: Efficacy of anti-CCR8 antibody in combination therapy

Various experiments were conducted to analyze the efficacy of anti-CCR8 antibodies alone or in combination with checkpoint-targeting antibodies (such as anti-PD-L1, anti-PD-1, and anti-CTLA4 antibodies). Combinations with these checkpoint-targeting molecules were found to provide additional efficacy. Furthermore, further combination therapies, such as chemotherapy drugs like oxaliplatin, doxorubicin, gemcitabine, or radiation therapy, were evaluated. Table 12.6.1 summarizes the results of different combination therapies. Additional benefit was observed if a second therapeutic agent was started only after the initiation of anti-CCR8 antibody therapy.

Table 12.6.1: Combination therapy with anti-CCR8 antibodies and results.

These results demonstrate the beneficial effects of combining anti-CCR8 antibody treatment with other therapeutic agents. The inventors also concluded that sequential administration of the therapeutic agents, if the anti-CCR8 antibody is administered first and further therapeutic agents are administered only after the initial reduction of Treg cells (e.g., at least 50%), provides additional benefits, as illustrated in Examples 12.6.5 and 12.6.7.

Based on these experiments and results, the inventors are convinced that combinations of triplet agents, such as anti-CCR8 antibodies, checkpoint protein-targeting antibodies, and targeted therapeutic agents (small molecules and antibodies), can further improve survival rates due to the differences in these therapeutic modes of action. Therefore, combinations of anti-CCR8 antibodies with checkpoint-targeting antibodies and tumor cell-targeting antibodies or agents are proposed for cancer treatment. In a preferred embodiment, this combination is a combination of a CCR8 antibody, an anti-PD(L)-1 antibody, and paclitaxel.

Example 12.6.1: Efficacy of anti-CCR8 antibody in C38 tumor-bearing mice and its combination therapy with anti-PD-L1 antibody

The anti-CCR8 alternative antibodies TPP-14099 and TPP-15285 showed potent efficacy in C38 tumor-bearing mice, comparable to that of the anti-PD-L1 antibody (Figure 57a). The combination of TPP-15285 with 3 mg/kg of the anti-PD-L1 antibody showed further enhanced efficacy. Both antibodies were separately formulated in phosphate-buffered saline and administered twice weekly via a total of four separate intraperitoneal injections, i.e., on the same day as the start of treatment.

Mice were monitored in a survival study exceeding 94 days (Figure 57b). Notably, 8 mice treated with a combination of anti-CCR8 and anti-PD-L1 antibodies survived for 94 days, 4 out of 10 mice treated with the CCR8 antibody TPP-15285 alone survived for 94 days, and mice not treated with anti-PD-L1 antibodies alone survived for 94 days, confirming the synergistic efficacy of the anti-CCR8 and PD-L1 antibodies. C38 is a low-invasive syngeneic mouse model that uses C38 colon cancer cells for tumor induction. This model is generally considered to respond to anti-PD-L1 antibody therapy. Given the available data described in this article, it is recommended that patients be stratified according to T-cell infiltration and/or response to PD-L1 therapy to provide additional benefit.

Treg analysis of C38 tumor samples by flow cytometry 24 hours after the second antibody treatment showed a significant reduction in the number of Tregs in the anti-CCR8 antibody treatment group compared to the isotype control group (Figure 58). The strongest Treg depletion was observed with the combination of TPP-15285 and the anti-PD-L1 antibody. Furthermore, the highest CD8+/Treg ratio was observed with the combination of TPP-15285 and the anti-PD-L1 antibody. Interestingly, macrophage analysis showed an increase at day 24 of the study (Figure 59).

Example 12.6.2: Efficacy of anti-CCR8 antibody in B16F10-OVA tumor-bearing mice & combination therapy with anti-CTLA4 antibody

The anti-CCR8 antibody TPP-15285 (10 mg/kg) showed comparable efficacy to the anti-CTLA4 antibody (10 mg/kg) in mice carrying B16F10-OVA (Figure 60). The combination of TPP-15285 and the anti-CTLA4 antibody showed synergistic efficacy, which was associated with increased CD8+ T cell and IFNg/TNFα levels.

Both antibodies were prepared in PBS and combined with the treatment that started at the same time on the same day, administered intraperitoneally for a total of three times, twice a week.

Treg analysis of B16F10-OVA tumor samples by flow cytometry 24 hours after the second antibody treatment (day 12) showed a significant reduction in Treg numbers in the CCR8 antibody treatment group compared to the isotype control group (Table 12.6.2.1). The strongest Treg depletion was observed with the combination of TPP-15285 and anti-CTLA4 antibody. Furthermore, the strongest increase in CD8+ T cells was observed with the combination of TPP-15285 and anti-CTLA4 antibody.

24 hours after secondary antibody treatment with TPP-15285+ isotype control, anti-CTLA4 antibody+ isotype control, or TPP-15285+ anti-CTLA4 antibody, FACS analysis of B16F10-OVA tumors showed a decrease in the frequency of intratumoral Tregs and an increase in the absolute number of CD8+ T cells.

Table 12.6.2.1: Intratumoral cell populations determined by FACS and IFNg determined by ELISA at the end of the study. *n = mean of 5 samples.

Example 12.6.3: Combined therapy with anti-PD-1 and anti-CCR8 antibodies in mice carrying EMT-6 tumors: synergistic efficacy

The therapeutic effects of TPP-15285 (1 mg/kg) alone, anti-mouse PD-1 antibody (CDRs: atezolizumab, 10 mg/kg) alone, or in combination with TPP-15285 (1 mg/kg) and anti-mouse PD-1 antibody (10 mg/kg) were evaluated in EMT-6 tumor-bearing mice. Both antibodies were prepared in PBS and administered intraperitoneally twice a week for a total of four times, in conjunction with concurrently initiated treatments, on the same day.

The anti-CCR8 antibody TPP-15285 showed weak efficacy in mice carrying EMT-6 at a low dose of 1 mg/kg, comparable to the weak efficacy of the anti-PD-1 antibody at 10 mg/kg (Figure 61). The combination of 1 mg/kg TPP-15285 and 10 mg/kg anti-PD-1 showed synergistic efficacy.

Treg analysis of EMT-6 tumor samples by flow cytometry 24 hours after the second antibody treatment showed that the number of Tregs in the CCR8 antibody treatment group was significantly reduced compared with the isotype control group or anti-PD-1 monotherapy (Figure 62).

Table 12.6.3.1: Intratumoral cell populations identified in EMT-6 tumors by FACS at the end of the study. *n = mean of 5 samples, cells/100 mg tumor.

Example 12.6.4: Combined treatment with anti-PD-1 and anti-CCR8 antibodies in C38 tumor-bearing mice

The therapeutic efficacy of TPP-15285 (5 mg/kg) alone, anti-mouse PD-1 antibody (aPD_1, CDR: atezolizumab, 5 mg/kg) alone, or in combination with TPP_15285 (5 mg/kg) and anti-mouse PD-1 antibody (5 mg/kg) was evaluated in C38 tumor-bearing mice. Both antibodies were prepared in PBS and administered intraperitoneally twice a week for a total of four times, on the same day in conjunction with concurrently initiated treatment.

The anti-CCR8 antibody TPP-15285 demonstrated potent efficacy in C38 tumor-bearing mice, exceeding that of the anti-PD-1 antibody administered at the same dose (Figure 63a). After 64 days, the combination of 5 mg/kg TPP-15285 and 5 mg/kg anti-PD-1 antibody showed improved survival efficacy with 10/10 complete responders, compared to 9/10 of those treated with anti-CCR8 monotherapy and 4/10 of those treated with anti-PD1 monotherapy (Figure 63b).

Treg analysis of C38 tumor samples by flow cytometry 24 hours after the second antibody treatment is shown in Figure 64 and Table 12.6.4.1.

Table 12.6.4.1: Intratumoral cell population measured by FACS in C38 tumors 24 hours after secondary antibody treatment. *n = mean of 5 samples, cells/100 mg tumor.

Example 12.6.5: Sequential combination therapy of anti-CCR8 antibody and anti-PD-1 antibody in MB49 tumor-bearing mice

The therapeutic efficacy of anti-mouse CCR8 antibody TPP-15285 (10 mg/kg) alone, anti-mouse PD-1 antibody (CDR: atezolizumab, 10 mg/kg) alone, and the combination of TPP-15285 (10 mg/kg) and anti-PD-1 antibody (10 mg/kg) was evaluated in MB49 tumor-bearing mice. In this study, the first dose of anti-PD-1 antibody was administered only after the anti-CCR8 antibody, i.e., after allowing the anti-CCR8 antibody to affect Tregs, resulting in an increase in the CD8+ T cell to Treg ratio. More specifically, anti-PD-1 antibody treatment was initiated 24 hours after the second anti-CCR8 antibody treatment.

The anti-CCR8 antibody TPP-15285 showed strong efficacy in mice carrying MB49 tumors, exceeding that of the anti-PD1 antibody (Figure 65). The combination of 10 mg/kg TPP-15285 with 10 mg/kg anti-PD-1 showed further improved efficacy.

At the end of the study, FACS analysis of MB49-derived tumor samples showed that, compared with the load control group or anti-PD-1 antibody monotherapy, the anti-CCR8 antibody treatment group had increased frequencies of CD45+ cells, CD8+ cells, and NK cells, and decreased frequencies of Tregs (Figure 66, Table 12.6.5.1).

MB49 is an intermediate-invasive syngeneic model using bladder cancer cells that have responded to immune checkpoint inhibitor (ICI) therapy. Based on these data, it is recommended that subjects be stratified by low immune infiltration and/or response to ICI treatment to provide additional benefit from this combination therapy.

In particular, it was found that initiating combination therapy after anti-CCR8 antibody treatment brought benefits. Unbound by theory, the inventors believe that the initial depletion of Tregs by CCR8 antibodies enhances the activity of antigen-presenting cells and the activation of tumor T cells, leading to increased sensitivity to checkpoint inhibitors such as PD-(L)1, because Tregs, as an external factor to tumor cells, play a role in the primary and adaptive resistance of cancer patients to checkpoint inhibitors.

Table 12.6.5.1: Intratumoral cell populations identified in MB49 tumors by FACS at the end of the study. *n = mean of 5 samples, cells/100 mg tumor.

Example 12.6.6: Combination therapy including anti-CCR8 antibody and chemotherapy in mice with EMT-6 tumors

The therapeutic efficacy of anti-CCR8 antibody TPP-15285 (5 mg/kg, q3/4d i.p.) alone, oxaliplatin (5 mg/kg, q4d i.p.) alone, doxorubicin (6 mg/kg, i.v. SD) alone, docetaxel (10 mg/kg, q2d x 5, i.v.) alone, or in combination with oxaliplatin, doxorubicin, or docetaxel was evaluated in EMT-6 tumor-bearing mice. The therapeutic agents were prepared in PBS, and combination therapy was initiated on the same day, with treatment starting simultaneously.

While the combination of anti-CCR8 antibodies with each chemotherapeutic agent provides benefits superior to monotherapy using that chemotherapeutic agent, the combination is not superior to anti-CCR8 monotherapy (Figure 67). The inventors are convinced that sequential administration of the therapeutic agents is beneficial, allowing the anti-CCR8 antibody to effectively deplete Tregs before applying the chemotherapeutic agent to deplete rapidly dividing cells.

At the end of the study, immune cell populations in tumors and blood were analyzed by flow cytometry, see Tables 12.6.6.1 and 12.6.6.2. For combination therapy, compared with each monotherapy and control, the intratumoral CD8/Treg ratio and the frequency of activated CD8+CD25+ T cells in the blood were increased.

In addition, IFN-γ, IL-10, IL-12p70, IL-1beta, IL-2, and TNFα were analyzed. Elevated levels of IFN-γ, IL-1β, and IL-2 were observed in both the single and combined groups. An increase in TNFα was observed only in the combined group (data not shown).

Table 12.6.6.1: Intratumoral cell populations determined by FACS at the end of the study. *Unless otherwise specified, n = mean of 5 samples, cells/100 mg tumor.

Table 12.6.6.2: Blood immune cell populations identified by FACS at the end of the study. *n = mean of 5 samples, cells/100 μl blood.

Example 12.6.7: Sequential combination therapy with anti-PD-(L)1 antibody and anti-CCR8 antibody in Lewis lung cancer mice

The therapeutic efficacy of anti-CCR8 antibody TPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs: atezolizumab, 10 mg/kg) alone, anti-PD-L1 antibody (10 mg/kg) alone, anti-CTLA4 antibody alone, a combination of TPP-15285 (10 mg/kg) and anti-PD-1 antibody (10 mg/kg), or a combination of TPP-15285 (10 mg/kg) and anti-PD-L1 antibody (10 mg/kg) was evaluated in Lewis lung tumor-carrying mice (Figure 69). Antibodies were prepared in PBS. For anti-CCR8 antibody, administration was performed on days 11, 14, 18, and 22 post-tumor inoculation. For combination therapy, anti-PD-(L)1 antibody was administered 24 hours after the second dose of anti-CCR8 antibody. At this time point, FACS analysis showed that intratumoral Treg consumption was 60% compared to the isotype control TPP-15285 single group (data not shown).

Lewis lung was used as an syngeneic model of a suppressive tumor microenvironment and is known to be unresponsive to anti-PD-(L)1 and anti-CTLA4 therapy. Although effective Treg depletion was measured 24 hours after the second treatment, no monotherapy efficacy of TPP-15285 was observed. However, combination therapy with an anti-mouse PD-1 antibody following anti-CCR8 antibody therapy showed improved efficacy, which was also associated with an increased CD8/Treg ratio at the end of the study (Table 12.6.7.1).

Table 12.6.7.1: Intratumoral cell populations identified by FACS at the end of the study. *n = mean of 5 samples.

Example 12.6.8: Combined treatment with anti-CCR8 antibody and radiotherapy in mice carrying EMT-6 tumors

The therapeutic effects of anti-CCR8 antibody TPP-15285 (3 mg/kg) or radiotherapy (RT, 3 x 2 Gy) alone or in combination were evaluated in EMT-6 tumor-bearing mice (Figure 70). Combination therapy began with 3 x 2 Gy fractionated irradiation, followed by 3 mg/kg biw x 2. Radiotherapy alone only slightly delayed tumor growth. The combination of anti-CCR8 antibody and radiotherapy showed only a slight improvement in efficacy, but Treg exhaustion was more pronounced at the end of the study than with either single therapy (data not shown).

Example 12.6.9: Combination therapy in MBT2 tumor-bearing mice containing anti-CCR8 antibody and administration of PD-1 antibody, PD-L1 antibody, or paclitaxel.

As shown in Table 12.6.9.1, the therapeutic efficacy of the anti-CCR8 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, or paclitaxel of the present invention was tested alone or in combination in MBT2 syngeneic tumor-bearing mice. Each group consisted of 10 tumor-bearing mice, and treatment began on day 10 post-inoculation, at which time the tumor had reached a size of approximately 100 ^mm³ . In the single-treatment group, anti-CCR8 showed the strongest and most significant antitumor effect. Combining these treatments with anti-PD-1 antibody, anti-PD-L1 antibody, or paclitaxel with anti-CCR8 therapy significantly increased the antitumor effect of these therapies. The antibodies were prepared in PBS. The T/C ratio was analyzed after three weeks of treatment, every two weeks.

Table 12.6.9.1: T/C ratios on day 24 in the treatment group and relative to the isotype control group.

Group treat dose way Dosing regimen T/C 01 Isotype control 10mg/kg i.p. BIW 1 02 Anti-CCR8 antibody TPP15285 10mg/kg i.p. BIW 0.50 03 Anti-PDL1 antibody 10mg/kg i.p. BIW 0.89 04 Anti-PD1 antibody 10mg/kg i.p. BIW 0.66 05 Paclitaxel 10mg/kg i.v.i.v. Q4D 0.83 06 Combination: TPP15285 + anti-PDL1 ab 10mg/kg i.p.+i.p. BIW+BIW 0.41 07 The combination of TPP15285 and anti-PD1 ab 10mg/kg i.p.+i.p. BIW+BIW 0.15 08 The combination of TPP15285 and paclitaxel 10mg/kg i.p.+i.v. BIW+Q4D 0.39

Table 12.6.9.2: Increase in CD8 mRNA levels after anti-CCR8 combination therapy. CD8 mRNA levels were obtained by RNA-seq of MBT2 tumors acquired 24 hours after the end of the treatment cycle. The mean CD8 expression level from 10 tumors in each treatment group was calculated. Compared with the isotype, anti-CCR8 therapy alone significantly increased CD8 levels, while PD1, PDL1, and paclitaxel had no significant effect. Combining anti-CCR8 therapy with the other three therapies further increased CD8 levels. The combination of anti-CCR8 and anti-PD1 therapy yielded the most significant and strongest increase, exceeding 8-fold.

Table 12.6.9.3: Tumor size on day 24 was significantly reduced by anti-CCR8 treatment, and further reduced by combining anti-CCR8 with PD-1 antibody (CrownVivoPremium, catalog number RMP1-14), PD-L1 antibody (CrownVivoPremium, catalog number CVP034), or paclitaxel. The largest and most significant reduction in tumor size compared to the isotype control was achieved by combining anti-CCR8 antibody with anti-PD-1 antibody.

Example 12.7.1: Correlation between anti-PD-L1 antibody response and anti-CCR8 antibody response

To compare the efficacy of anti-CCR8 antibody therapy with anti-PD-L1 antibody therapy, results from different syngeneic tumor models were compiled, as shown in Table 12.7.1.1. Surprisingly, a good response to anti-PD-L1 antibody therapy, as measured by T/C volume, also predicted a good response to anti-CCR8 antibody therapy. PD-L1 expression is a known predictor of response to anti-PD-L1 therapy. Based on these observations, the inventors hypothesized that PD-L1 expression might also be applicable for stratifying subjects to identify those most likely to benefit from anti-CCR8 antibody therapy. This hypothesis can be retrospectively verified by correlating PD-L1 expression levels with tumor volume.

Table 12.7.1.1 Comparison of responses to antiPD-L1 antibody and anti-CCR8 antibody in different syngeneic models.

Example 12.7.2: Correlation between antitumor responses and mRNA biomarkers for stratification or disease control

Early untreated tumors (100–200 ^mm³ , N=10 per model), larger untreated tumors (500 and 1000 ^mm³ ), or mean gene expression (RNA-seq) at the end of the study were associated with the T/C ratio in 21 syngeneic mouse models treated with TPP-14099 or TPP-15285, as described elsewhere in this article.

Top-ranking related genes were found to be significantly enriched in T cell and inflammatory markers (Pearson correlation values), see Table 12.7.2.1. Inflammatory markers IFNg and IFNg response genes (e.g., Gbp3/4/5/8/9, Cxcl9, Acod1), PDL1 (CD276), C1 complement factor, T cell genes such as Klra3/5 and Trav, and Treg markers CD25 (IL2RA), FOXP3, and CTLA4 were the genes most associated with the response.

Table 12.7.2.2 shows the fold change in expression between responding cell lines (T/C < 0.6) and non-responding cell lines (T/C > 0.6) in early-stage untreated tumors. The 100 genes with the largest fold change in expression between responders and non-responders are listed. IFNg and IFNg response genes (e.g., Gbp2/3/4/8/10/11, Cxc19/11, Ubd), cytotoxic T cell markers (e.g., granzyme, Prf1), mast cell markers (Tpsab1, Cma1, Tpsb2), and C1 complement factor are the most predictive genes for response.

Table 12.7.2.3 shows the fold change in expression between responsive cell lines (T/C < 0.6) and non-responsive cell lines (T/C > 0.6) for larger untreated tumors (500 and 1000 ^mm3 ).

Candidate biomarkers are listed in Tables 12.7.2.1, 12.7.2.2, and 12.7.2.3. Therefore, patients with high expression of the human corresponding genes of these genes, or combinations thereof, or traits can be expected to respond to anti-CCR8 therapy in clinical settings. Furthermore, these biomarkers, or combinations thereof, can also be used to monitor treatment success. Biomarkers derived from italicized/bold genes are particularly preferred.

Table 12.7.2.1 lists suitable biomarkers associated with the antitumor response achieved using the anti-CCR8 antibody of the present invention. These biomarkers have been found to be upregulated in early-stage untreated tumors and correlated with treatment response. Biomarkers derived from italicized/bold genes are particularly preferred. One hundred genes with the largest negative correlation coefficient (r value) between expression and T/C ratio are listed.

Table 12.7.2.2 Suitable biomarkers associated with the antitumor response achieved using the anti-CCR8 antibody of the present invention. Fold-change in expression between responding cell lines (T/C < 0.6) and non-responding cell lines (T/C > 0.6). The most relevant (pearson) gene was found to be significantly enriched in T cells and inflammatory markers.

Table 12.7.2.3 Suitable biomarkers for genes showing the greatest fold change between responders and non-responders in large untreated tumors (500 ^mm³ and 1000 ^mm³ ). Granase and other immune cell marker expression was higher in responder models than in non-responder models. Biomarkers derived from italicized/bold genes are particularly preferred.

Example 12.8: Changes in mRNA expression in a homogeneous tumor model after administration of anti-CCR8 antibody

For each syngeneic tumor model, 10 mice treated with the anti-CCR8 antibody TPP-14099 and 10 mice treated with an isotype control were sacrificed 24 hours after final treatment. Tumors were cut into small pieces and immersed in RNAlater at 4°C. Poly-A mRNA was extracted according to Illumina's Hi-seq protocol to generate cDNA libraries for subsequent RNA sequencing. The sequencing depth was approximately 40 million 150 bp paired-end reads per sample.

Figures 63 to 73 show the effects of treatment with an isotype control (TPP-9809) or anti-CCR8 antibody (TPP-14099) on the mRNA expression levels of different immune cell markers in different syngeneic tumor models. Treatment increased the expression of inflammatory markers lfng, macrophage markers Ms4a7, Acod1, and Mrc1, cytotoxic T cell markers Cd8a and Cd8b1, natural killer (NK) cell marker Ncrl, pan-T cell marker Cd3e/d/g, and B cell markers Cd19 and Cd22.

Notably, the inventors observed significant induction of LTta/b and Cxcr5 and its ligand Cxcl13 in the CT26, MBT2, and H22 tumor models, as well as overall upregulation of these genes, including RM1, Hepa1-6, and 4T1, in several other models. Unbound by theory, the induction of tertiary lymphoid structures by the anti-CCR8 antibodies TPP-14099 or TPP-15285 may contribute to the antitumor response induced by these antibodies.

Table 12.8.1: Inverse correlation between T cell markers, checkpoint proteins, Treg markers, NK cell markers, macrophage markers, B cell markers, and interferon-γ with response to anti-CCR8 antibody therapy.

Example 13: Preparation of a targeted thorium conjugate (TTC) containing an anti-CCR8 antibody

The entire contents of WO2016096843 are incorporated herein by reference, particularly relating to the production of conjugates as described in this embodiment.

The conjugation of 3,2-hydroxypyridinone (3,2-HOPO) chelating agent or any other suitable chelating agent with antibody TPP-23411, TPP-21360, or any other described anti-CCR8 antibody may be performed as previously described in patent application WO2016096843. Briefly, to activate the chelating agent, 3,2-HOPO chelating agent (dissolved in DMA, at a 1:1 ratio with 0.1M MES buffer pH 5.4), NHS, and EDC (both dissolved in 0.1M MES buffer pH 5.4) are mixed in a 1/1/3 ratio. For conjugation with the antibody, the activated chelating agent can be bound to the mAb at a molar ratio of 7.5/7.5/22.5/1 (chelating agent/NHS/EDC/mAb). After 20–60 minutes, the pH is adjusted to 5.5 with 12% v/v 0.3M citric acid to quench the reaction. Protein concentration was determined by HPLC, with integration of the peak area at 280 nm absorbance. The solution was then buffered at a constant volume using tangential flow filtration (TFF) to prepare a solution containing 30 mM citrate, 50 mg/ml x M sucrose, 2 mM EDTA, 0.5 mg/ml pABA, and pH 5.5. At the end of percolation, the solution was drained into a preparation container. The product was prepared from TFF buffer (30 mM citrate, x 50 mg/ml M sucrose, 2 mM EDTA, 0.5 mg/ml pABA, pH 5.5) and 7% w/v polysorbate 80 to obtain a corresponding CCR8 antibody-chelating agent conjugate (CCR8-ACC) at 2.5 mg/ml. CCR8-ACC could be filtered through a 0.2 μm filter into sterile vials.

As described in WO2016096843, CCR8-ACC is radiolabeled with thorium-227. In short, 5 μl of CCR8-ACC is mixed with 32 μl of thorium-227 (activity 3.875 MBq/ml) and 13 μl of citrate buffer to generate a CCR8-targeted thorium-227 conjugate (CCR8-TTC) with an activity of 10 kBq/µg. The sample can be incubated at room temperature for 60 minutes to allow for stable labeling of thorium-227 to the 3,2-HOPO chelating agent. Aliquots of the sample can be analyzed by instantaneous thin-layer chromatography (iTLC).

antibody sequence

Claims (15)

1. An anti-CCR8 antibody that induces ADCC and/or ADCP, used for the treatment of tumors, wherein said use includes administration of an additional therapeutically active compound or therapy, wherein a dose of the additional therapeutically active compound or therapy is administered after a first dose of said anti-CCR8 antibody, preferably. a. After the anti-CCR8 antibody has induced an increase in the ratio of CD8 cells to T reg cells in the tumor by at least 2, 3, 4, or 5 times, or b. After the anti-CCR8 antibody has depleted at least 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the intratumoral Treg cells. 2. The anti-CCR8 antibody according to claim 1, wherein the additional therapeutically active compound or therapy is selected from... a. Antibodies or small molecules that target checkpoint proteins, such as PD(L)1 or CTLA-4. b. Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine. c. Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1. d. Antibodies targeting proteins specifically expressed by tumor cells. e. Antibodies or small molecules targeting HER2 and/or EGFR, f. Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1. g. Radiation therapy, and/or h. Depletion of intratumoral B cells, preferably CD19+ B cells. 3. The use of an anti-CCR8 antibody that induces ADCC and/or ADCP for the treatment of tumors, wherein said use comprises administration of a non-checkpoint protein-targeting and further therapeutically active compound or therapy selected from the following. a. Chemotherapy agents, preferably taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine. b. Antibodies targeting other chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1. c. Antibodies that target proteins specifically expressed by tumor cells. d. Antibodies or small molecules targeting HER2 and/or EGFR, e. Targeted kinase inhibitors, such as sorafenib, regorafenib, or MEKi-1, f. Radiation therapy, and/or g. Deplete intratumoral B cells, preferably CD19+ B cells. 4. The anti-CCR8 antibody according to the preceding claims, wherein the use includes further administration of a checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or a CTLA4 antibody. 5. An anti-CCR8 antibody that induces ADCC and/or ADCP, used for the treatment of tumors, wherein a single dose of the anti-CCR8 antibody is administered to the subject, such that the same or different doses of the anti-CCR8 antibody are not administered to the subject. 6. The use of a biomarker for diagnosing/grading a subject with a tumor sensitive to anti-CCR8 antibody therapy, said use including a. Determine the levels of biomarkers in tumors or tumor samples containing regulatory T cells. b. Compare the levels of biomarkers with reference samples or values, and c. If the level of the biomarker is higher than or equal to the reference sample or value, the subject will be diagnosed/classified as having a tumor sensitive to anti-CCR8 antibody therapy. The biomarkers mentioned are d. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4. e. Granulase or immune cell markers, preferably markers of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof. f. Treg infiltration markers, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9. g. T cell markers or cytotoxic T cell markers, preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40, or GITR. h. Interferon or interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα, i. complement factors, and/or j. Serine protease inhibitors. 7. The use of a biomarker for predicting or monitoring treatment success, including administration of an anti-CCR8 antibody, said use comprising: a. Determine the levels of the biomarkers mentioned above in tumor samples, and b. Compare the levels of the biomarkers with reference samples or values. The biomarkers mentioned are c. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4. d. Granulase or immune cell markers, preferably lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof. e.Treg infiltration markers, preferably selected from FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, and CXCL9. f. T cell markers or cytotoxic T cell markers, preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5 and TNF receptor superfamily members, such as 4-1BB, OX-40 or GITR. g. Interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα, h. complement factors, and/or i. Serine protease inhibitors. 8. The use of the biomarker according to claim 6, or the use of the biomarker according to claim 7, wherein the biomarker is an immune checkpoint protein, preferably PD-1, PD-L1, or CTLA4. 9. Use of a molecule that binds to a biomarker for diagnosing/grading a subject as having a tumor sensitive to anti-CCR8 antibody therapy, the use comprising using the molecule that binds to the biomarker to determine the level of the biomarker in a tumor or tumor sample, wherein the biomarker is a. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4. b. Granulase or immune cell markers, preferably markers of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof. c. Treg infiltration markers, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9. d. T cell markers or cytotoxic T cell markers, preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40, or GITR. e. Interferon or interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα, f. complement factors, and/or g. Serine protease inhibitors, Furthermore, the molecule that binds the biomarker is preferably an antibody or an antigen-binding fragment, or a conjugate thereof. 10. The use of molecules that bind to biomarkers for predicting or monitoring treatment success, including the administration of anti-CCR8 antibodies, wherein the use includes using molecules that bind to said biomarkers to determine the level of the biomarker in a tumor or tumor sample, wherein said biomarker is a. Immune checkpoint proteins, preferably PD-1, PD-L1, or CTLA4. b. Granulase or immune cell markers, preferably markers of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or combinations thereof. c. Treg infiltration markers, preferably FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9. d. T cell markers or cytotoxic T cell markers, preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, and TNF receptor superfamily members such as 4-1BB, OX-40, or GITR. e. Interferon or interferon-induced protein, preferably selected from IFNγ, IL10, IL12p70, IL1β, IL2 and TNFα, f. complement factors, and/or g. Serine protease inhibitors, Furthermore, the molecule that binds the biomarker is preferably an antibody or an antigen-binding fragment, or a conjugate thereof. 11. Use of the molecule binding to a biomarker according to claim 9 or the molecule binding to a biomarker according to claim 10, wherein the molecule binding to a biomarker is an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, such as nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, dostarlimab, or ipilimumab. 12. Use of the molecule that binds a biomarker according to claim 8 or the molecule that binds a biomarker according to claim 9, wherein the biomarker is an immune cell marker, and wherein the molecule that binds a biomarker is preferably an antibody or a combination thereof that binds a marker of lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells. 13. The use of the molecule that binds a biomarker according to claim 12, wherein the molecule that binds a biomarker is a molecule that binds a Treg infiltration marker, preferably an antibody that binds to CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9. 14. The use of the biomarker-binding molecule according to claim 12, wherein the biomarker-binding molecule is an antibody that binds a T-cell marker preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5 and TNF receptor superfamily members including 4-1BB, OX-40 or GITR. 15. Use of the molecule binding biomarker according to claim 9 or the molecule binding biomarker according to claim 10, wherein the molecule binding biomarker is an antibody that binds interferon or interferon-induced protein, preferably selected from INFγ, IL10, IL12p70, IL1β, IL2 and TNFα.