TW202600601A - Conjugates of trop2-specific antigen binding proteins and cytokines - Google Patents

Abstract

The present invention relates to conjugates of TROP2-specific antigen binding proteins and muteins of the cytokines 4-1BB ligand (4-BBL) extracellular domain (ECD) and IL-21. The 4-1BBL ECD muteins and IL-21 muteins have reduced affinities for their cognate receptors 4-1BB and IL-21 receptor (IL-21R), respectively. The 4-1BBL ECD muteins can be present in homo- or heterotrimeric fusion protein comprising three 4-1BBL ECD monomers. The conjugates can further comprise an antigen-binding region that has affinity for a surface antigen expressed on NK cells, e.g. CD16A. The conjugates of the invention specifically redirect and activate NK cells to lyse targeted tumor cells that express TROP2. The invention further relates to the use of these conjugates thereof in the treatment of cancer, preferably a cancer expressing the TROP2.

Description

TROP2 specific antigen-binding protein and cytokine conjugate

This invention relates to the medical field, particularly to oncology, immunology, and immunotherapy for tumors. Specifically, this invention relates to an anti-TROP2 antibody conjugated to a mutant protein of the extracellular domain (ECD) of 4-1BB ligand (4-1BBL) and a mutant protein of IL-21. These conjugates are used in medical treatments, such as cancer treatment.

Today, we consider a medical milestone in immuno-oncology to be the application of redirecting a patient's immune system, primarily by manipulating alfa (α)-beta (β) T cells to overcome tolerance and attack tumor cells. This can be achieved through various means, such as (i) antibodies that direct the response of these T cells (called conjugates) and antibodies that overcome various immunosuppressive mechanisms by blocking immune checkpoint interactions. (ii) Alternatively, cell immunotherapy has proven highly effective for certain types of leukemia using chimeric antigen receptors (CARs), which are genetically introduced into the patient's own T cells, subsequently mass-produced in a commercial manufacturing environment, and administered intravenously to the recipient.

While both technologies initially achieved success, numerous challenges remain. For example, checkpoint inhibitors and binding agents can cause severe toxicity with a high incidence rate. Furthermore, it is currently impossible to predict why some, but not all, patients will respond to a particular antibody. Cell therapy also faces multifaceted difficulties. Because the cells are derived from severely ill patients, manufacturability is poor, manufacturing itself is time-consuming and costly, and there is a lack of predictive strategies to understand which patients will benefit from the expensive treatment. Finally, cell immunotherapy is currently limited to a few malignant indications.

To overcome these challenges, progress in this field has primarily focused on enhancing existing technologies to regulate αβ T cells, but efforts are gradually expanding to include members of the innate immune system, which coordinate a complete and natural immune response, involving cells and mechanisms beyond the direct action of therapeutics. Furthermore, innate lymphocytes are generally not restricted by the major histocompatibility complex (MHC), thus allowing for potential allogeneic use in multiple recipients without inducing graft-versus-host disease (GvHD). Antibody therapies and recurrent cell therapies targeting innate cells have shown significantly lower rates of therapy-related toxicities, such as cytokine release syndrome (CRS) and neurotoxicity. Candidates for these therapies include natural killer (NK) cells, induced NK (iNK) cells, macrophages, and gamma- and delta-type T cells.

Strategies for recruiting cytotoxic NK cells are currently under development. One such strategy employs multifunctional antibodies called natural killer cell engagers (NKCEs), which simultaneously target tumor-associated antigens (TAAs) and activate receptors on endogenous NK cells. Demaria et al. (Eur. J. Immunol. 2021. 51: 1934–1942) reviewed several NKCEs currently under development for clinical applications. NKCEs are designed to enhance the interaction between NK cells and targeted tumor cells and increase the effector function of NK cells on tumor cells. However, tumors contain a low number of NK cells and exhibit poor function (exhaustion phenotype). To date, NKCEs developed only address the interaction between NK cells and tumor cells, and in most cases do not improve their function, nor do they improve the number of NK cells in tumors under any circumstances.

Recently, WO2024/056862 and WO2024/056861 disclosed this type of multifunctional antigen-binding protein, which includes 4-1BB agonists such as 4-1BB ligand (4-1BBL) and IL-21 receptor agonists such as IL-21 as NK cell-activating cytokines. Multifunctional antigen-binding proteins in these applications may include an antigen-binding region that specifically binds to TROP2, for targeting tumor cells expressing TROP2. While these multifunctional antigen-binding proteins do address the issues of NK cell quantity, function, and location, the specificity of the 4-BB agonist and IL-21R agonist in these proteins is insufficient to exert their effects solely within tumors, potentially causing extratumor (e.g., peripheral) side effects.

Therefore, this technology requires a TROP2-targeted therapy to address these issues. Thus, one objective of this invention is to provide such a TROP2-targeted therapy.

Various terms used throughout this specification and the scope of the claims relate to the methods, compositions, uses, and other aspects of the invention. Unless otherwise stated, these terms shall have their ordinary meaning in the art to which the invention pertains. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein. While any methods and materials similar to or equivalent to those described herein may be used to practice the tests of the invention, preferred materials and methods are described herein.

"A(a)", "an", and "the": Unless otherwise expressly stated, these singular terms include plural references. Thus, the indefinite articles "a(a)" or "an" generally mean "at least one". Therefore, for example, referring to "a cell" includes combinations of two or more types of cells, and similar ones.

"Approximately" and "about": These terms, when referring to measurable values such as quantities, time intervals, and the like, are intended to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods. Furthermore, quantities, ratios, and other values are sometimes presented in range format herein. It should be understood that this range format is used for convenience and brevity, and should be flexibly interpreted to include values explicitly specified as range limits, as well as all individual values or subranges covered within that range, as if each value and subrange were explicitly specified. For example, the ratios in the range of about 1 to about 200 should be understood to include the explicitly stated limits of about 1 and about 200, but also to include individual ratios (such as about 2, about 3 and about 4) and sub-ranges (such as about 10 to about 50, about 20 to about 100, etc.).

"And/or": The term "and/or" means that one or more of the above situations may occur alone, or in combination with at least one of the above situations, up to and including all of the above situations.

"Include": This term should be interpreted as inclusive and open, not exclusive. Specifically, the term and its variations mean including the specified feature, step, or component. Such terms should not be interpreted as excluding the existence of other features, steps, or components.

"Illustrative": This term means "serving as an example, illustration, or illustration" and should not be construed as excluding other configurations disclosed herein.

As used in this article, "cancer" and "cancerous" refer to or describe the physiological condition in mammals characterized by unregulated cell growth. Cancer is also known as a malignant tumor.

As used herein, "combination" is intended to refer to all forms of administration in which a first drug is delivered together with another (second, third) drug. The drugs may be administered simultaneously, alone, or sequentially, and the order is arbitrary. The drugs administered in combination are biologically active to the recipient to whom they are delivered.

As used herein, “simultaneous” administration means administering more than one drug at the same time, but not necessarily via the same route of administration or in the form of a combination formulation. For example, during a patient’s visit, one drug may be administered orally while another drug may be administered intravenously. “Alone” includes administering a drug alone and/or at different times, but again not necessarily via the same route of administration. “Sequential” means administering a second drug immediately or promptly after administering the first drug.

As used herein, "composition," "product," or "combination" for use in the methods of this disclosure includes those suitable for various routes of administration, including but not limited to intravenous, subcutaneous, intradermal, subdermal, intrachorionic, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including cheek and sublingual), rectal, vaginal, aerosol, and/or non-intestinal or mucosal applications. Compositions, formulations, and products invented according to this disclosure generally comprise a pharmaceutical substance (alone or in combination) and one or more suitable pharmaceutically acceptable excipients.

As used herein, "effective dose" refers to the amount of medication required to improve the symptoms of a disease relative to an untreated patient. The effective dose of the active ingredient used in the practice of this invention and in the therapeutic treatment of cancer varies depending on the method of administration, the age, weight, and overall health of the patient. Ultimately, the attending physician or veterinarian will determine the appropriate dosage and regimen. This amount is referred to as the "effective" dose. Therefore, in the context of the present disclosure, the application of a drug that is “effective against a disease or condition” means that when administered in a clinically appropriate manner, it will produce beneficial effects on at least a statistically significant portion of patients, such as symptom improvement, cure, reduction of at least one disease sign or symptom, life extension, quality of life improvement, or other effects that are generally considered positive by a physician familiar with treating a particular type of disease or condition.

"Sequence identity" is defined herein as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences determined by sequence comparison. In this art, "identity" also implies the degree of sequence correlation between amino acid or nucleic acid sequences, as determined by matching such sequence strings. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated using known methods. The term "sequence consistency" or "sequence similarity" means that two (poly)peptide sequences or two nucleotide sequences share at least a certain percentage of sequence consistency, as defined elsewhere in this document, for optimal alignment, preferably across the entire length (at least the shortest sequence in the comparison), and maximize the number of matches and minimize the number of gaps, such as using default parameters by programs like ClustalW (1.83), GAP, or BESTFIT. GAP uses the Needleman and Wunsch global alignment algorithms to align two sequences across the entire length, maximizing the number of matches and minimizing the number of gaps. Typically, GAP default parameters are used, where the gap generation penalty = 50 (nucleotides) / 8 (protein) and the gap extension penalty = 3 (nucleotides) / 2 (protein). For nucleotides, the default scoring matrix used was nwsgapdna, and for proteins, the default scoring matrix was BLOSUM62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). The preferred multiple alignment program for aligning the protein sequence of this invention was ClustalW (1.83), using the BLOSUM matrix and default settings (gap opening penalty: 10; gap extension penalty: 0.05). Sequence alignment and percentage sequence consistency scores can be determined as follows: using computer programs, such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open-source software, such as the programs "needle" (using the global Needleman-Wunsch algorithm) or "water" (using the local Smith algorithm) in EmbossWIN version 2.10.0. The Waterman algorithm can be used with the same parameters as the GAP algorithm described above, or with default settings (for 'needle' and 'water' sequences, and for protein and DNA alignments, the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; the default scoring matrix for proteins is BLOSUM62, and the default scoring matrix for DNA is DNAFull). When the overall sequence lengths differ significantly, local alignments, such as peer-to-peer alignments using the Smith-Waterman algorithm, are preferred. Alternatively, percentage similarity or consistency can be determined by searching public databases using algorithms such as FASTA and BLAST.

Depending on the circumstances, when determining the degree of amino acid similarity, those skilled in the art may also consider so-called "conservative" amino acid substitutions, which will be clear to them. Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. The table below provides examples of categories of conservatively substituted amino acid residues. acidic residues Asp (D) and Glu (E) alkaline residues Lys (K), Arg (R), and His (H) Hydrophilic, non-electrolyte residue Ser (S), Thr (T), Asn (N) and Gln (Q) Aliphatic uncharged residues Gly (G), Ala (A), Val (V), Leu (L) and Ile (I) Nonpolar uncharged residual base Cys (C), Met (M), and Pro (P) Aromatic residues Phe (F), Tyr (Y), and Trp (W)

Substitution of conservative amino acid residues. 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

Physical and functional classification of amino acid residues. Residual groups containing alcohol groups S and T Aliphatic residues I, L, V and M Cycloalkenyl-related residues F, H, W and Y Hydrophobic residue A, C, F, H, I, L, M, R, T, V, W and Y Negatively charged residue D and E polar residues C, D, E, H, K, N, Q, R, S and T Positively charged residual base H, K and R Small Remnant Base A, C, D, G, N, P, S, T and V Very small disabled base A, G and S Residual bases involved in the formation of transition A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residual substrate Q, T, K, S, G, P, D, E, and R

The term "agent" generally refers to any entity that is not normally present or not present at the level of application to cells, tissues, or objects. An agent can be a compound or a combination thereof. Agents can be selected from, for example, the following groups: polynucleotides, polypeptides, small molecules, (multispecific) antigen-binding proteins, such as antibodies and their functional fragments.

The term "antigen-binding domain" or "antigen-binding region" refers to the portion of an antigen-binding protein that specifically binds to an antigen or antigenic determinant. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region, such as comprising an antibody light chain variable region ( _VL ) and an antibody heavy chain variable region ( _VH ). Examples of such antigen-binding regions include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region derived from a single-domain antibody that consists only of a heavy chain and has no light chain, as is known, for example, from camels, where the antigen-binding site is located on and formed from a single variable domain (also called an "immunoglobulin single variable domain" or "ISVD"). Examples of such ISVDs include the single variable domain ( _VHH ) of camel heavy-chain antibodies, also known as nanoantibodies, domain antibodies (dAbs), and the single domain (IgNAR domain) derived from shark antibodies. In other embodiments, the antigen-binding region includes a non-immunoglobulin-derived domain that can specifically bind to antigens or antigenic determinants, such as DARPpins, Affilins, and anticarrier proteins.

The term "antibody" is used in the broadest sense and specifically includes full-length monoclonal antibodies, multiclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, provided they exhibit the desired biological and/or immune activity. Various techniques related to antibody production are provided, for example, in Harlow et al., *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). Antibodies may be human and/or humanized. A "humanized" form of non-human ( e.g. , rodent) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human antibody.

The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this article to refer to antibodies whose structure is substantially similar to that of natural antibodies. "Natural antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG class antibodies are approximately 150,000 daltons of heterotetrameric glycoproteins composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy domain or heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light domain or light chain variable domain), followed by a light chain constant region (CL) (also called a light chain constant region). The heavy chain of an antibody can be designated as one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or m (IgM), some of which can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody can be designated as one of two types, called kappa (κ) and lamundar (λ), based on the amino acid sequence of its constant regions.

"Antibody fragments" include a portion of the full-length antibody, such as its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)s, Fv (typically the _VH and _VL domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the _VH and CH1 domains) and dAb (typically the _VH domain) fragments; _VH , _VL , _VHH and V-NAR domains; microantibodies, biantibodies, triantibodies, tetraantibodies and kappa bodies (see, for example, Ill et al. Protein Eng 1997;10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments and one or more isolated CDRs or functional complements, wherein the isolated CDRs or antigen-binding residues or peptides may be attached or linked together to form a functional antibody fragment. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, NY, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing rescue receptors that bind antigenic determinant residues and have an increased in vivo half-life, see U.S. Patent 5,869,046. Biantibodies are antibody fragments with two antigen-binding sites, and can be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triantibodies and tetraantibodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Various types of antibody fragments have been described or summarized in, for example, Heiliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136; WO2005/040219, US20050238646 and US20020161201. Antibody fragments can be produced by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., CHO, E. coli, or bacteriophages), as described herein.

As used herein, the term "monoclonal antibody" is not limited to antibodies produced via hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single pure line (including any eukaryotic, prokaryotic, or phage pure line) and not to the method of its production. Monoclonal antibodies can be prepared using a variety of techniques known in this art, including the use of hybridoma, recombinant, and phage presentation techniques, or combinations thereof. For example, hybridoma techniques can be used to produce monoclonal antibodies, including those known in this field and those taught, for example, in Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, N.Y. (1988); Hammerling et al., "Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, N.Y. (1981), pp. 563-681 (both are incorporated herein by reference in their entirety).

As used herein, a "monospecific" antibody means that the antibody portion of a conjugate containing the antigen-binding region as described herein has one or more antigen-binding sites, each binding to the same antigenic determinant of the same antigen. The term "bispecific" means that the antibody portion of a conjugate as described herein has at least two antigen-binding sites that specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, a bispecific antigen-binding molecule can bind two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different types of cells.

As used in this application, the terms "valence" or "valence number" indicate the presence of a specific number of binding sites or ligands in the antigen-binding molecule or conjugate described herein. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" indicate the presence of two, four, and six binding sites or ligands in the antigen-binding molecule or conjugate, respectively.

Antibodies that are immunoreactive to specific antigens can be generated by recombinant methods, such as selecting recombinant antibody libraries in phages or similar vectors, see, for example, Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech.14:309-314 (1996), or by immunizing animals with antigens or DNA encoding antigens. Methods for generating and screening specific antibodies using hybridoma techniques are conventional and well-known in this art. In a non-limiting example, mice can be immunized with an antigen of interest or cells expressing that antigen. Once an immune response is detected, such as the detection of antibodies specific to the antigen in mouse serum, mouse spleens are harvested and spleen cells are isolated. The spleen cells are then fused with any suitable myeloma cells using well-known techniques. Hybridomas are selected and colonized using restrictive dilution. These pure hybridoma lines are then analyzed for cells secreting antibodies capable of binding to the antigen using methods known in this art. Ascites fluid typically containing high antibody levels can be produced by intraperitoneal inoculation of mice with positive hybridoma pure lines.

Immunoglobulins typically consist of heavy and light chains. Each heavy and light chain contains constant and variable regions (also called "domains"). The variable regions of the light and heavy chains contain four "framework" regions, interrupted by three hypervariable regions, also known as "complementary determinant regions" or "CDRs". The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of antibodies are composed of recombinant framework regions that form the light and heavy chains, and are used for localization and alignment of CDRs in three-dimensional space.

When used in this article, the term "hypervariate region" refers to the amino acid residues in the antibody responsible for antigen binding. Hypervariable regions typically contain amino acid residues from complementary determinant regions (CDRs) or chain-dependent domains (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, USA) and/or residues from hypervariable rings (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (L3), and 91-96 (L3) in the heavy chain variable domain) and residues 26-32 (H1), 53-55 (L3), and 91-34 (L1), 50-56 (L2), and 91-96 (L3) in the heavy chain variable domain). (H2) and 96-101 (H3); Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the amino acid residues in this region are numbered using the method described by Kabat et al. (above). Phrases such as “Kabat position,” “variable domain residue numbering as Kabat,” and “according to Kabat” refer to this numbering system for heavy chain or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the variable domain of the heavy chain may include a single amino acid insertion (according to Kabat residue 52a) after residue 52 of CDR H2, and may include inserted residues (e.g., according to Kabat residues 82a, 82b, and 82c, etc.) after residue 82 of the heavy chain FR. The Kabat number of a given antibody residue can be determined by comparing it to a region in the antibody sequence that is homologous to the "standard" Kabat number sequence.

As used in this article, the term "frame" or "FR" refers to regions in the antibody variable domain other than those defined as CDRs. Each antibody variable domain frame can be further subdivided into continuous regions separated by CDRs (FR1, FR2, FR3, and FR4).

As defined herein, "constant region" refers to an antibody-derived constant region encoded by one of the constant region genes of the light or heavy chain immunoglobulin. As used herein, "constant light chain" or "light chain constant region" means an antibody region encoded by the kappa (Ck) or laminda (Cλ) light chain. A constant light chain typically contains a single domain and, as defined herein, refers to positions 108-214 of Cκ or Cλ, where the numbers are based on the EU index (Kabat et al., 1991, ibid. ).

As used in this article, "constant heavy chain" or "constant heavy chain region" refers to the antibody region encoded by the muse, delta, gamma, alfa, or epsilon genes, to define the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full-length IgG antibodies, the constant heavy chain, as defined in this article, refers to the region from the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus encompassing positions 118-447, where the numbers are based on the EU index.

Papain digests an intact antibody to produce two identical antigen-binding fragments, referred to as "Fab" fragments, each containing a variable domain of the heavy chain and a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab fragments can also be reassembled using methods known in this art. Therefore, as used herein, the term "Fab fragment" or "Fab region" refers to an antibody fragment containing a light chain segment comprising the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab can refer to the region alone, or to the region in the context of a polypeptide, conjugate, or antigen-binding region, or any other embodiment as outlined herein. The Fab' fragment differs from the Fab fragment by the addition of several residues, including one or more cysteines from the antibody hind chain region, at the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH is a Fab' fragment with a free thiol group at the cysteine residue of the constant domain. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

As used herein, "monoclonal Fv" or "scFv" refers to an antibody fragment containing the _VH and _VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. Generally, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding. Methods for producing scFv are well known in this art. For a review of methods used to produce scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Rosenburg and Moore, Springer-Verlag, NY, pp. 269-315 (1994).

"Scaffold antigen-binding proteins" are known in this technology. For example, fibronectin and ankyrin repeat protein (DARPin) have been used as alternative scaffolds for antigen-binding domains. See, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one embodiment of this invention, the scaffold antigen-binding protein is selected from the following groups: CTLA-4 (Evibody), Lipocalin (anti-carrier protein), monomers, centyrin, Kunitz domain, knottin, fynomer, lipocarrier protein, protein A-derived molecules such as the Z domain (affinity), A domain (high-affinity multimer/maxibody) of protein A, serum transferrin ( trans- body); designed anchoring protein repeat (DARPin), variable domains of antibody light or heavy chains (single-domain antibody, sdAb), variable domains of antibody heavy chains (nano-antibody, aVH), V _NAR fragments, fibronectin (AdNectin), C-type lectin domain (Tetranectin); variable domain of novel antigen receptor beta-lactamase (V _NAR fragment), human gamma-crystal protein or ubiquitin (human ubiquitin molecule); Kunitz domain of human protease inhibitors, microbodies (such as proteins from the knotting family), peptide aptamers, and fibronectin (adnectin).

CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a CD28 family receptor that primarily manifests on CD4 ⁺ T cells. The extracellular domain of CTLA-4 contains a variable domain (e.g., Ig folding). Different binding properties can be imparted by replacing the loop corresponding to the antibody's CDR with a heterologous sequence. Engineered CTLA-4 molecules with different binding specificities are also called epoesomes (e.g., US7166697B1). The size of the epoesome is approximately the same as the separable variable region of the antibody (e.g., domain antibody). For more details, see Journal of Immunological Methods 248 (1-2), 31-45 (2001).

Lipocarriers are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, choline, retinoids, and lipids. They possess a rigid β-sheet secondary structure with numerous loops at the open end of their cone-shaped structure, and can be engineered to bind to different target antigens. Anticarriers are 160 to 180 amino acids in size and are derived from lipocarriers. For more details, see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1, and US20070224633.

The affinity domain is a scaffold derived from protein A of Staphylococcus aureus, engineered to bind to the antigen. This domain consists of a triple helix of approximately 58 amino acids. The library is generated through randomization of surface residues. For more details, see Protein Eng. Des.Sel.17, 455-462 (2004) and EP1641818A1.

High-affinity polymers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids employ defined disulfide bond structures. Diversity arises from the rearrangement of natural variations exhibited by the A-domain family. For more details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

Transferrin is a monomeric serum transfer protein. Transferrin can be engineered to bind to different target antigens by inserting peptide sequences into permitted surface loops. Examples of engineered transferrin scaffolds include the trans form. For more details, see J. Biol. Chem 274, 24066-24073 (1999).

Designed anchorin repeats (DARPin) are derived from anchorin, a family of proteins that mediate the attachment of integrated membrane proteins to the cytoskeleton. A single anchorin repeat is a 33-residue motif consisting of two Alpha helices and a beta transition. By randomizing the residues in the first Alpha helix and beta transition of each repeat, they can be engineered to bind to different target antigens. Their binding interfaces can be increased by increasing the number of motifs (a method of affinity maturation). For more details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

A single-domain antibody is an antibody fragment composed of a single monomeric variable antibody domain. The first single variable domain is derived from the variable domain of the antibody heavy chain (nanoantibody or _VHH fragment) from camels. In addition, the term single-variable-domain antibody includes autonomous human heavy chain variable domain (aVH) or _VNAR fragment derived from sharks.

Fibronectin is an engineerable scaffold for binding to antigens. Adenosine consists of a backbone of the native amino acid sequence of domain 10 of 15 repeating units of human type III fibronectin (FN3). The engineerable three loops at one end of the β-segment allow adenettin to specifically identify therapeutic targets of interest. For more details, see Protein Eng. Des.Sel.18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1.

Peptide aptamers are combinatorial recognition molecules composed of a constant scaffold protein, typically thioredoxin (TrxA), which contains a restricted variable peptide ring inserted at the active site. For more details, see Expert Opin. Biol. Ther. 5, 783-797 (2005).

Microsomes are naturally occurring microbial proteins with a length of 25-50 amino acids, containing 3-4 cysteine bridges—examples of microbial proteins include KalataBI, conotoxin, and clotting agents. Microbial proteins possess rings that can be engineered to include up to 25 amino acids without affecting the overall folding of the microbial protein. For more details on engineered clotting agent domains, see WO2008098796.

As used in this article, the terms "Fv", "Fv fragment", or "Fv region" refer to a polypeptide that contains the _VH and _VL domains of a single antibody.

As used herein, the term "Fc" or "Fc region" refers to a polypeptide containing the constant region of an antibody (excluding the first constant region immunoglobulin domain). Fc may refer to the region alone or in the context of an Fc polypeptide, as described below. As used herein, "Fc polypeptide" or "Fc-derived polypeptide" means a polypeptide containing all or part of the Fc region. Fc polypeptides as used herein include, but are not limited to, antibodies, Fc fusions, and Fc fragments. Furthermore, the Fc region according to the invention includes variants containing at least one modification that alters (enhances or weakens) the function of an Fc-related effector. Additionally, the Fc region according to the invention includes chimeric Fc regions that contain different portions or domains of different Fc regions, such as antibodies derived from different isotypes or species. Therefore, Fc refers to the last two constant immunoglobulin domains of IgA, IgD, and IgG, and the last three constant immunoglobulin domains of IgE and IgM, as well as the flexible hinged N-terminus of these domains. For IgA and IgM, Fc may include a J-chain. For IgG, Fc includes the hinged chains between the immunoglobulin domains Cγ2 (CH2) and Cγ3 (CH3) and between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the commonly defined human IgG heavy chain Fc region includes the C226, P230, or A231 residues at its C-terminus, where the designation is based on the EU index. The "CH2 domain" of the human IgG Fc region typically extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain as used herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the extension of the C-terminal residue in the Fc region to the CH2 domain (i.e., from an amino acid residue at approximately position 341 to an amino acid residue at approximately position 447 of the IgG). The CH3 region as used herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain in which a "protrusion" ("pestle") is introduced in one chain and a corresponding "cavity" ("mortar") is introduced in the other chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Such variants of the CH3 domain can be used to promote heterodimerization of two non-uniform antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The "mortar and pestle" technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a protrusion ("mortar") at the interface of a first polypeptide and a corresponding cavity ("pot") at the interface of a second polypeptide, such that the protrusion can be located within the cavity, thereby promoting heterodimer formation and inhibiting homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensation cavities of the same or similar size to the protrusions are created at the interface of a second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). The protrusions and cavities can be created by altering the nucleic acids encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis. In specific embodiments, protrusion modification includes substitution of T366W for one of the two subunits of the Fc region, while mortar modification includes substitution of T366S, L368A, and Y407V for the other of the two subunits of the Fc domain. In another specific embodiment, the subunits of the mortar-modified Fc region additionally contain the amino acid substitution S354C, and the subunits of the mortar-modified Fc region additionally contain the amino acid substitution Y349C. The introduction of these two cysteine residues leads to the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Numbered according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Region equivalent to the Fc region of immunoglobulins" is intended to include naturally occurring allelic variants of the Fc region of immunoglobulins, as well as variants with the ability to produce substitutions, additions, or deletions that do not substantially reduce the function of immunoglobulin-mediated effectors (such as antibody-dependent cytotoxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulins without substantially losing biological function. Such variants may be selected according to general rules known in the art to minimize the impact on activity (see, for example, Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term "effective function" refers to biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

"Activated Fc receptors" are Fc receptors that trigger signal transduction events upon binding to the Fc region of an antibody, stimulating receptor-bearing cells to exert their effector functions. Activated Fc receptors include FcγRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89). The specific activated Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141), also known as CD16 or CD16A. In humans, CD16 consists of two isoforms, CD16A and CD16B, encoded by two highly homologous genes. CD16A is a transmembrane protein expressed by lymphocytes and some monocytes, while CD16B is attached to the plasma membrane via GPI anchors and is primarily expressed by neutrophils. Therefore, when CD16 is mentioned in the context of its expression on NK cells, it usually means CD16A, unless otherwise stated.

As used herein, "variable region" refers to a region of the antibody containing one or more Ig domains substantially encoded by either the _VL (including Vκ and Vλ) and/or _VH genes, which respectively constitute immunoglobulin gene loci in the light chain (including κ and λ) and heavy _chain . The light chain or heavy chain variable region ( _VL or _VH ) comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed _. , WHFreeman & Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

As used herein, the term "hypervariant region" or "HVR" refers to regions within the antibody's variable domains that exhibit sequence hypervariability and/or form structurally defined rings ("hypervariant rings"). Generally, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant rings and/or from the complementarity determining region (CDR), which has the highest sequence variability and/or participates in antigen recognition. Exemplary hypervariable rings are present at amino acid residues at 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The hypervariable region (HVR), also known as the complementary determining region (CDR), and these terms are used interchangeably herein, refers to the variable portion that forms the antigen-binding region. This specific region has been described by Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definition includes overlap or subsets of amino acid residues when compared with each other. However, the application of any definition means that the CDR of an antibody or its variant is intended to be within the scope of the terms defined and used herein. The appropriate amino acid residues covering the CDRs defined in the references cited above are illustrated in Table A below for comparison. The exact number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. Table A. CDR Definition ¹ CDR Kabat Chotia AbM ² V _H CDR1 31-35 26-32 26-35 V _H CDR2 50-65 52-58 50-58 V _H CDR3 95-102 95-102 95-102 V _L CDR1 24-34 26-32 24-34 V _L CDR2 50-56 50-52 50-56 V _L CDR3 89-97 91-96 89-97 ^1. The numbering of all CDR definitions in Table A follows the numbering convention described by Kabat et al. (see below). ^2. "AbM" with a lowercase "b" as used in Table A refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can explicitly assign the "Kabat numbering" system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat number" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise stated, the numbering of specific amino acid residue positions within the antibody variable region is based on the Kabat numbering system.

Besides CDR1 in VH, CDRs typically contain amino acid residues that form highly variable rings. CDRs also contain "specificity-determining residues" or "SDRs" that act as antigen-contacting residues. SDRs are contained within the regions of CDRs known as abbreviated CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDRL2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are indicated herein according to Kabat et al., ibid.

As used herein, the term "affinity-matured" in the context of antigen-binding molecules (e.g., antibodies) refers to an antigen-binding molecule derived from a reference antigen-binding molecule (e.g., through mutation) that binds to the same antigen as the reference antibody, preferably to the same antigenic determinant, and exhibits a higher affinity for the antigen than the reference antigen-binding molecule. Affinity maturation typically involves the modification of one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Generally, an affinity-matured antigen-binding molecule binds to the same antigenic determinant as the initial reference antigen-binding molecule.

The "class" of an antibody refers to the type of constant domains or regions possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are respectively called α, δ, ε, γ, and m.

"Blocking" antibodies or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigen they bind to. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. As used herein, "activator antibodies" are antibodies that mimic at least one functional activity of the polypeptide of interest.

The term "specific binding" refers to the number of different types of antigens or antigen determinants that a specific antigen-binding region or antigen-binding protein can bind to. The specificity of an antigen-binding protein can be determined based on affinity and/or cohesion. Affinity, expressed as the equilibrium constant (K_{_D} ) for the dissociation of the antigen and the antigen-binding protein, is a measure of the binding strength between the antigen determinant and the antigen-binding site on the antigen-binding protein. Alternatively, affinity can also be expressed as the affinity constant (K _{_A} ), which is 1/K_{_D}. Affinity can be determined in ways known per se, depending on the specific combination of the antigen-binding protein and the antigen of interest. In this context, affinity should be understood as the binding strength of a target molecule with multiple binding sites to a larger binding agent complex, i.e., the binding strength of a multivalent binding. Affinity is related to both the affinity between antigen determinants and antigen-binding sites on antigen-binding proteins, and valence (i.e., the number of binding sites on antigen-binding proteins). On the other hand, affinity refers to the simple monovalent receptor-ligand system.

Typically, the antigen-binding region of the conjugate of the present invention specifically binds to its target molecule (antigen), with a dissociation constant ( _KD ) of about ^10⁻⁶ to ^10⁻¹² M or less, and preferably ^10⁻⁸ to ^10⁻¹² M or less, and/or a binding affinity of at least ^10⁻⁶ M or ^10⁻⁷ M, preferably at least ^10⁻⁸ M, more preferably at least ^10⁻⁹ M, such as at least ^10⁻¹⁰ , ^10⁻¹¹ , ^10⁻¹² M or less. Any _KD value greater than ^10⁻⁴ M (i.e., less than 100 μM) is generally considered to indicate nonspecific binding. Therefore, the antigen-binding region of a "specifically binding" antigen is an antigen-binding domain that binds to the antigen with a _KD value not exceeding ^10⁻⁴ M, as can be determined as described below. Preferably, the antigen-binding region of the conjugate of the present invention will bind specifically to the target molecule with an affinity of less than 800, 400, 200, 100, 50, 10 or 5 nM, more preferably less than 1 nM, such as less than 500, 200, 100, 50, 10 or 5 pM. Various methods for measuring binding affinity are known in this art, any of which can be used for the purposes of this invention (see, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds. Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983)). Specific illustrative embodiments are described below.

" _KD " or " _KD value" can be measured using an ELISA as described herein in the embodiments or by using surface plasma resonance assays (using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ)). In an exemplary method, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl/min to achieve approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, antibodies or Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 μl/min in PBS (PBST) containing 0.05% Tween 20 at 25°C in two consecutive dilutions. The binding rate (k_{_on}) and dissociation rate (k_off) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting binding and _dissociation sensing maps. The equilibrium dissociation constant ( _KD ) is calculated as the ratio _koff / _kon . See, for example, Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the bonding rate is found to exceed ^{10⁶ M⁻¹ S⁻¹} by the above surface plasma resonance test, the bonding rate can be determined by using a fluorescence quenching technique. This technique measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at increasing concentrations of antigen at 25°C. The fluorescence emission intensity is measured in a spectrometer such as a stop-flow spectrophotometer (Aviv Instruments) or an 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirring cup.

The terms "humanized antibody" or "humanized immunoglobulin" refer to an immunoglobulin comprising the human framework, at least one and preferably all complement-determining regions (CDRs) derived from non-human antibodies, and wherein any constant regions present therein are substantially identical to constant regions of human immunoglobulins, i.e., at least approximately 85%, at least 90%, and at least 95% identical. Therefore, all portions of a humanized immunoglobulin (possibly excluding CDRs) are substantially identical to corresponding portions of one or more native human immunoglobulin sequences. Framework residues in the human framework region are often replaced by corresponding residues of the CDR donor antibody to alter, and preferably improve, antigen binding. These frame replacements are identified using methods well known in this art, such as identifying frame residues important for antigen binding by modeling the interaction between the CDR and the frame residue, and by performing sequence comparisons to identify uncommon frame residues at specific locations. See, for example, Queen et al., U.S. Patents 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (each incorporated herein by reference in its entirety). Antibodies can be humanized using a variety of techniques known in this field, including, for example, CDR transplantation (EP 239,400; PCT Publication WO 91/09967; US Patents 5,225,539; 5,530,101 and 5,585,089), veneering, or resurfacing (EP 592,106; EP 519,596; Padlan, Mol. Immunol., 28:489 498 (1991); Studnicka et al., Prot. Eng. 7:805 814 (1994); Roguska et al., Proc. Natl. Acad. Sci. 91:969 973). (1994), and Chain Reorganization (US Patent No. 5,565,332), the entirety of which are hereby incorporated by reference.

The antigen-binding region used in this invention comprises an immunoglobulin single variable domain (ISVD), the amino acid sequence of which corresponds to the amino acid sequence of a naturally occurring single variable domain, but has been "humanized," that is, by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring single variable domain with one or more amino acid residues appearing at the corresponding positions in the _VH domain of a known human 4-strand antibody. This can be done in ways that are already known, and is clear to those familiar with the technique, such as existing techniques based on humanization, including, for example, Jones et al. (Nature 321:522-525, 1986); Riechmann et al. (Nature 332:323-329, 1988); Presta (Curr. Op. Struct. Biol. 2:593-596, 1992); Vaswani and Hamilton (Ann. Allergy, Asthma and Immunol., 1:105-115 1998); Harris (Biochem. Soc. Transactions, 23:1035-1038, 1995); Hurle and Gross (Curr. Op. Biotech., 5:428-433, 1994), and with V _H Specific existing techniques related to the humanization of H include, for example, Vincke et al. (2009, J. Biol. Chem. 284:3273–3284). Again, it should be noted that such humanized single variable domains of the present invention can be obtained in any suitable manner known per se, and are therefore not strictly limited to peptides obtained using peptides containing naturally occurring single variable domains as starting materials.

"Frame" or "FR" refers to a variable-domain residual other than the high-variable region (HVR) residual. A variable-domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

For the purposes of this document, "receptor human framework" refers to a framework containing an amino acid sequence derived from the human immunoglobulin framework or the human common framework as defined below, specifically a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework. A receptor human framework "derived from" the human immunoglobulin framework or the human common framework may contain the same amino acid sequence as the human immunoglobulin framework or the human common framework, or may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the sequence of the VL receptor human framework is identical to the VL human immunoglobulin framework sequence or the human common framework sequence.

As an alternative to humanization, human antibodies can be generated. "Human antibodies" means antibodies containing a complete human light and heavy chain, as well as a constant region, produced by any known standard method. For example, genetically modified animals (e.g., mice) can be obtained that, upon immunization, produce a complete library of human antibodies without producing endogenous immunoglobulins. For instance, isotype deletion of the antibody heavy chain binding region PH gene in chimeric and germline mutant mice has been described as completely suppressing endogenous antibody production. Transferring a human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies after immunization. See, for example, Jakobovits et al., Proc.Nat. Acad.Sci.USA, 90:255 1 (1993); Jakobovits et al., Nature, 362:255-258 (1993). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from a donor library of variable (V) domain immunoglobulin genes. According to this technique, antibody V domain gene frames are selected into major or minor capsid protein genes such as M13 or fd of filamentous phages and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on antibody functional properties also leads to the selection of genes encoding antibodies that exhibit those properties. Therefore, phages mimic some properties of B cells. Phage display can occur in various forms, for a review of which see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-57 1 (1993). Human antibodies can also be produced by in vitro activated B cells or by SCID mice with an immune system reconstituted using human cells. Once human antibodies are obtained, their encoding DNA sequences can be isolated and selected, and introduced into a suitable expression system, namely a cell line (preferably from mammals). The cell line then expresses the antibodies and releases them into a culture medium from which the antibodies can be isolated.

Amino acid substitutions are denoted herein as A _o X _As , where A _o represents the original amino acid, X represents the position of the original amino acid in the original amino acid sequence, and _As represents the substituted amino acid present at that position in the modified amino acid sequence. For example, V153Q indicates that the original amino acid glutamic acid (V) at position 153 is changed to glutamic acid (Q).

Amino acid deletions are denoted as A _o X- in this paper, where A _o represents the original amino acid, X represents the position of the original amino acid in the original amino acid sequence, and the dash indicates that the original amino acid A _o is no longer present in the modified amino acid sequence. For example, N59- indicates the deletion of aspartic acid (N) at position 59 in the modified amino acid sequence.

Amino acid deletions are represented in this paper as A _o X insA _1-n , where A _o represents the original amino acid, X represents the position of the original amino acid in the original amino acid sequence, and insA _1-n represents amino acid 1-n substitution of the original amino acid A _o . For example, G84 insGGGGG indicates that the original glycine at position 84 in the modified amino acid sequence is replaced by a sequence of 5 glycines.

As used herein, the term "NK cell" refers to a subset of lymphocytes involved in innate immunity. NK cells can be identified by certain characteristics and biological properties, such as the expression of specific surface antigens targeting human NK cells (including CD56 and/or NKp46), the absence of alfa/beta or gamma/delta TCR complexes on their cell surface, the ability to recognize and kill cells that cannot express "self" MHC/HLA antigens by activating specific cytolysis mechanisms, the ability to kill tumor cells or other diseased cells that express ligands of NK activation receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit immune responses. Using methods well known in this art, any of these characteristics and activities can be used to identify NK cells. Any subset of NK cells will also be covered under the term NK cells. In this context, "active" NK cells refer to biologically active NK cells, including those capable of lysing target cells or enhancing the immune function of other cells. NK cells can be obtained using various techniques known in this art, such as isolation from blood samples, cytapheresis, tissue or cell collections. Useful protocols for the assay of NK cells can be found in Natural Killer Cells Protocols (2000, edited by Campbell KS and Colonna M). Humana Press, pp. 219-238).

As used in this article, "tumor-associated antigen" (TAA) means any antigen associated with cancer, including but not limited to proteins, glycoproteins, gangliosides, carbohydrates, and lipids. This antigen can be expressed on malignant tumor cells or in the tumor microenvironment (such as tumor-associated blood vessels, extracellular matrix, stromal matrix, or immune infiltrates). The term TAA explicitly includes homologs of wild-type TAAs that differ from wild-type TAAs due to tumor-specific mutations (which can be patient-specific or shared) and result in altered amino acid sequences, i.e., so-called neoantigens.

In this document, "nucleic acid construct" or "nucleic acid vector" should be understood to mean an artificial nucleic acid molecule produced using recombinant DNA technology. Therefore, the term "nucleic acid construct" does not include naturally occurring nucleic acid molecules, although a nucleic acid construct may include (parts of) a naturally occurring nucleic acid molecule. The terms "expression vector" or "expression construct" refer to nucleic acid molecules capable of expressing nucleotide sequences or genes in host cells or host organisms compatible with such expression vectors or constructs. These expression vectors typically include regulatory sequence elements operatively linked to the nucleotide sequence to be expressed to achieve its expression. Such regulatory elements typically include at least a suitable transcriptional regulatory sequence and, where appropriate, a 3' transcription termination signal. Additional elements necessary for or helpful in achieving expression, such as expression-enhancing sub-elements, may also be present. The expression will be introduced into suitable host cells and will be able to realize the expression of the encoded sequence in a live extracellular cell culture of the host cells. The expression vector will be suitable for replication in the host cells or organisms of the present invention, and the expression architecture will typically be integrated into the host cell genome for maintenance. The technology for introducing nucleic acids into cells is well-established in this field, and any suitable technique can be used depending on the specific circumstances. For eukaryotic cells, suitable techniques may include phosphate-dimethyltransfection, DEAE-glucan, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., adenovirus, AAV, lentivirus, or vaccinia virus). For microorganisms, such as bacterial cells, suitable techniques may include calcium chloride conversion, electroporation, and transfection using bacteriophages. The introduced nucleic acid may be located on an extrachromosomal vector within the cell, or the nucleic acid may be integrated into the host cell's genome. According to standard techniques, integration can be facilitated by including sequences within the nucleic acid or vector that promote recombination with the genome. Following introduction, the nucleic acid can be expressed to produce the encoded fusion protein. In some embodiments, host cells (potentially including actual transformed cells, although more likely to be progeny of transformed cells) can be cultured in vitro under conditions of nucleic acid expression to produce the encoded fusion protein peptide; when using an inducible promoter, expression may require activation of that inducible promoter.

As used herein, a "proton" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that controls the transcription of one or more coding sequences. It is located upstream of the transcription start site of the coding sequence and is structurally identified by the presence of binding sites for DNA-dependent RNA polymerases, transcription start sites, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the transcriptional amount of the promoter. A "configurable" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of chemical inducers.

The term "selectable marker" is familiar to those skilled in the art and is used herein to describe any genetic entity that, when a selectable marker is expressed, can be used to select cells containing that selectable marker. The term "reporter gene" is used interchangeably with "marker," although it primarily refers to visible markers such as green fluorescent protein (GFP). Selectable markers can be dominant, recessive, or bidirectional.

As used herein, the term "operably linked" refers to the functional linking of polynucleotide elements. A nucleic acid is "operably linked" when it has a functional relationship with another nucleic acid sequence. For example, if a transcription regulatory sequence affects the transcription of a coding sequence, it is operably linked to the coding sequence. Operable linking means that the linked DNA sequences are usually adjacent, and when it is necessary to link two protein coding regions, they are adjacent and within the reading frame.

The terms "protein" or "peptide" are used interchangeably and refer to molecules composed of amino acid chains, without specifying their mode of action, size, three-dimensional structure, or origin.

The term "signal peptide" (sometimes called a signal sequence) refers to a short peptide (typically 16-30 amino acids long) located at the N-terminus of most newly synthesized proteins destined for the secretory pathway. The terminal amino acid of a signal peptide is usually recognized and cleaved by a signal peptidase during or after translocation (from the cytoplasm into the secretory pathway, i.e., ER), resulting in the free signal peptide and the mature protein. Signal peptides are extremely heterogeneous, and many prokaryotic and eukaryotic signal peptides can be functionally interchangeable even between different species; however, the efficiency of protein secretion may depend on the signal peptide. Suitable signal peptides are generally known in this technique, for example from Käll et al. (2004 J. Mol. Biol. 338: 1027–1036) and von Heijne (1985, J Mol Biol. 184 (1): 99–105).

The term "gene" means a segment of DNA containing a region (transcriptional region) that is transcribed into an RNA molecule (e.g., mRNA) in the cell, which is operablely linked to a suitable regulatory region (e.g., a promoter). A gene typically contains several operablely linked segments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) containing polyadenylation sites. "Gene expression" refers to the process by which a DNA region operablely linked to a suitable regulatory region (especially a promoter) is transcribed into biologically active RNA (i.e., capable of being translated into biologically active proteins or peptides).

When the term "homologous" is used to describe the relationship between a given (recombined) nucleic acid or polypeptide molecule and a given host organism or host cell, it should be understood to mean that the nucleic acid or polypeptide molecule is produced in nature by a host cell or organism of the same species, preferably the same strain or variety. If homologous to the host cell, the nucleic acid sequence encoding the polypeptide will usually (but not necessarily) be operatively linked to another (heterologous) promoter sequence, and, where applicable, to another (heterologous) secretory signal sequence and/or terminator sequence, rather than in its natural environment. It should be understood that regulatory sequences, signal sequences, terminator sequences, etc., can also be homologous to the host cell. When used to indicate the correlation between two nucleic acid sequences, the term "homologous" means that a single-stranded nucleic acid sequence can hybridize with a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on many factors, including the amount of similarity between the sequences and the hybridization conditions (such as temperature and salt concentration, which will be discussed later).

When used in relation to nucleic acids (DNA or RNA) or proteins, the term "heterogeneous" refers to nucleic acids or proteins that are not naturally occurring as part of the organism, cell, genome, or DNA or RNA sequence in which they exist, or that exist at one or more locations in a cell, genome, or DNA or RNA sequence different from those found in nature. Heterogeneous nucleic acids or proteins are not endogenous to the cell in which they are introduced, but are acquired from another cell, or synthesized or recombined. Generally, although not always, proteins encoded by such nucleic acids are not typically produced by cells that transcribe or express DNA. Similarly, proteins encoded by foreign RNA are typically not expressed in the cell where the foreign RNA is located. Heterogeneous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Those skilled in this art will recognize that any nucleic acid or protein that is heterologous or foreign to the cell in which it is expressed is covered herein by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, that is, combinations in which at least two of the sequences are foreign to each other.

We previously described a multispecific antigen-binding protein that binds to tumor-associated antigens of interest, such as TROP2, and contains an NK cell-activating cytokine that triggers at least one of interleukin-21 receptors and 4-1BB receptors on NK cells, and is capable of inducing a superfunctional phenotype in NK cells (see WO2024/056862 and WO2024/056861). NK cells with a superfunctional proliferative phenotype are resistant to the tumor microenvironment and possess an enhanced ability to mediate target cell lysis even when exposed to target cells in the absence of primary tumor-associated antigens and with high secretion of cytokines (such as IFN-γ), and this ability can be prolonged over time. Therefore, agents that can induce a superfunctional phenotype in NK cells could be used to treat cancer and infectious diseases. However, systemic administration of agents containing pleiotropic cytokines (such as 4-1BB ligands (4-1BBL) or IL-21) remains challenging due to the risks of toxicity and other adverse side effects. Therefore, this technology still needs to reduce the systemic pleiotropic effects of these cytokines while maintaining their potential for 4-1BBL and IL-21 therapy when targeting tumors or pathogen infection sites. Therefore, this invention provides novel mutant proteins with altered affinity for the extracellular domain (ECD) of 4-1BBL. For example, the 4-1BBL ECD mutant protein with reduced affinity for 4-1BB described herein, and conjugates containing such 4-1BBL ECD mutant proteins described herein, will have reduced pleiotropic effects and adverse side effects when present in the bloodstream, while their affinity at the target site will ensure local efficacy. These novel 4-1BBL ECD mutant proteins combine with IL-21 mutant proteins with reduced affinity for IL-21R in conjugates of anti-TROP2 antigen-binding proteins. Unbound by theoretical constraints, the novel conjugates described herein are designed to leverage the immuno-enhancing activity of the combination of 4-1BBL ECD and IL-21 (which may be a prerequisite for addressing toxicity and off-target immunosuppression) to maximize efficacy at TROP2-targeted tumor sites, while simultaneously improving the feasibility of clinical conjugate administration. 4-1BB ligands and mutant proteins of the extracellular domain of 4-1BB ligands.

4-1BB is a member of the tumor necrosis factor receptor family. Its alternative name is tumor necrosis factor receptor superfamily member 9 (TNFRSF9), CD137, and it is induced by lymphocyte activation (ILA). 4-1BB is encoded by the TNFRSF9 gene (Entrez Gene ID: 3604). The amino acid sequence of human 4-1BB is described in NCBI accession number NP_001552, incorporated as SEQ ID NO: 175, where mature 4-1BB corresponds to positions 24-255. 4-1BB is referred to as a co-stimulatory immune checkpoint molecule. 4-1BB is expressed on activated T cells of the CD4+ and CD8+ lineages, as well as on activated NK cells. NK cell proliferation and activation require at least the binding of a co-stimulatory receptor (such as 4-1BB) to its ligand 4-1BBL. NK cells with increased 4-1BB expression are known to be highly active against target cells expressing the 4-1BB ligand (e.g., tumor cells). The 4-1BB ligand (4-1BBL), also known as TNFSF9 or CD137L, is a protein encoded by the TNFSF9 gene (Entrez Gene ID: 8744) in humans. The amino acid sequence of human 4-1BBL is described in NCBI accession number NP_003802, the contents of which are incorporated herein by reference. The 4-1BB/4-1BBL complex consists of three 4-1BB monomers bound to a 4-1BBL trimer. Each 4-1BB monomer binds to two 4-1BBLs via a cysteine-rich domain (CRD). The interaction between the 4-1BB and the second 4-1BBL is necessary to stabilize their interaction.

As used herein, a “4-1BB agonist” is an agent that has “agonist” activity at 4-1BB, meaning that the agent can induce or increase “4-1BB signaling.” “4-1BB signaling” refers to, for example, the ability of 4-1BB to activate or transduce intracellular signaling pathways when expressed on the surface of T cells, B cells, and NK cells and triggered by its natural ligand 4-1BBL. The term “natural 4-1BB ligand” in this context should be understood as the extracellular domain (ECD) of human wild-type 4-1BBL, which contains or is composed of the amino acid sequence of human 4-1BBL (i.e., SEQ ID NO: 37) at positions 71 to 254. Therefore, the extracellular domain (ECD) of 4-1BBL should be understood in this paper as containing the amino acid sequence at positions 71 to 254 of human 4-1BBL or fragments thereof with 4-1BB agonist activity or polypeptides thereof.

4-1BB agonist activity, i.e., changes in 4-1BB signaling activity, can be measured, for example, by tests designed to measure changes in 4-1BB signaling pathways, such as by monitoring phosphorylation of signal transduction components, measuring the binding of certain signal transduction components to other proteins or intracellular structures, or measuring the biochemical activity of components such as kinases, or indirectly through downstream effects mediated by 4-1BB (such as the production of specific cytokines). Suitable cell-based assays for the biological activity of in vitro 4-1BB agonists are described, for example, in Zhang et al. (Clin Cancer Res, 2007;13(9): 2758-2767), using a microtiter plate pre-coated with an anti-CD3 monoclonal antibody (pure 145-11C line) to measure IL-2 produced from spleen cells aseptically extracted from BALB/c mice. Other suitable cell-based assays for the biological activity of in vitro 4-1BB agonists are described in WO2016/075278, Example 6 (see, for example, Example 6.1). Natural 4-1BB ligands, such as 4-1BBL ECD trimers, as described by Fellermeier et al. (Oncoimmunol. 2016, 5(11): e1238540), including 4-1BBL ECD trimers containing the amino acid sequence of SEQ ID NO: 36, or anti-CD137 agonist antibodies (such as antibody 2A, Epstein et al., Tumor necrosis imaging and treatment of solid tumors. In: VP Torchilin, editor. Handbook of targeted delivery of imaging agents, Vol. 16, Boca Raton: CRC Press; 1995. Page 259 can be used as a positive control in the assay of 4-1BB agonist activity and as a reference for determining the amount of 4-1BB agonist activity in non-natural 4-1BB agonists (such as multispecific antigen-binding proteins containing 4-1BB agonists as described herein). The data provided herein support the use of well-designed 4-1BBL ECD mutant proteins to achieve 4-1BB signaling at appropriate times and locations and to improve the pharmacokinetics of therapies containing such 4-1BBL ECD mutant proteins. Furthermore, the 4-1BBL ECD mutant proteins described herein are designed to reduce potential adverse effects, such as hepatotoxicity and cytokine release, in the absence of target cells.

In the first embodiment, this disclosure provides a mutant protein of 4-1BBL ECD comprising at least one substitution, deletion, and/or insertion. The amino acid substitutions, deletions, and insertions in the 4-1BBL ECD mutant protein provided herein are those indicated relative to the wild-type human 4-1BBL ECD amino acid sequence (provided herein as SEQ ID NO: 37). Therefore, to allow for allelic variation, the wild-type human 4-1BBL ECD preferably comprises an amino acid sequence having (with increasing preference) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 37.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein SEQ ID NO: 49 is The 4-1BBL ECD mutant protein amino acid sequence differs from the wild-type human 4-1BBL ECD amino acid sequence (SEQ ID NO: 37) by at least one amino acid. The amino acid positions in the 4-1BBL ECD mutant protein amino acid sequence of SEQ ID NO: 49, as described herein, correspond to the amino acid positions in the full-length 4-1BBL amino acid sequence. Therefore, the first amino acid position in SEQ ID NO: 49 is designated as position 71, and subsequent amino acid positions are counted from this position up to the last amino acid at position 254.

In one embodiment, a 4-1BBL ECD mutant protein is provided, comprising at least one amino acid substitution, deletion, or insertion at a position selected from the group consisting of the following positions in SEQ ID NO: 37: 154, 153, 110, 227, 101, 230, 100, 114, 115, 116, and 171 (with decreasing preference). In one embodiment, the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution, deletion, or insertion at a position selected from the group consisting of the following positions in SEQ ID NO: 37: 154, 153, 110, 227, 101, 110, 230, 100, 114, 115, 116, and 171.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein SEQ ID NO: 49 and SEQ ID NO: 37 differ by at least one amino acid at the position specified by X in SEQ ID NO: 49. In one embodiment, the 4-1BBL ECD mutant protein comprising SEQ ID NO: 49 has (with increasing preference) at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.4% sequence identity with SEQ ID NO: 37.

In one embodiment, a 4-1BBL ECD mutant protein is provided, comprising an amino acid sequence having (with increasing preference) at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 37, and wherein the amino acid sequence comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L101D; L101R; L101K; Y110E; Y110N; Y110D; Y110R; Y11 0K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G114Q; G114D; G114E; G114N; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A11 6Q; A116N; A116Y; A116H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; G155Q; R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T and Q230A. In one embodiment, a 4-1BBL ECD mutant protein is provided. ECD mutant proteins contain at least one amino acid substitution selected from the following group: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L101D; L101R ;L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G114 Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L11 5E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V1 53N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; R 171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T and Q230A. In one embodiment, 4-1BBL The ECD mutant protein contains no amino acid sequence modifications other than at least one amino acid substitution selected from the following groups: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L 101D; L101R; L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G1 14R; G114Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L11 5D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116 H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A.

In one embodiment, a 4-1BBL ECD mutant protein is provided, comprising at least two, three, four, or five amino acid substitutions selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L101D; L 101R; L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G 114Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L 115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V 153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; R 171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A. In one embodiment, 4-1BBL ECD mutant proteins contain no amino acid sequence modifications other than substitutions of at least two, three, four, or five amino acids selected from the following groups: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L10 1Q; L101D; L101R; L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114 K; G114R; G114Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A 116H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A15 4H; R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y ; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A.

In one embodiment, a 4-1BBL ECD mutant protein is provided, comprising at least one amino acid substitution selected from the substitutions listed in Table B at the position of SEQ ID NO: 37. Table B. Additional substitutions, deletions, and insertions of the 4-1BBL ECD mutant protein. Location wild type Additional mutation preferences (preference gradually decreases from left to right) 100 V S, A, G, N, D, E > K, R > all others 101 L E, Q, D > R, K > all others 110 Y E, N, D, R, K > S, A, G, T > All others 114 G R, Q, D, E, N > S, A, G, T > all others 115 L K, Q, N, D, E > S, A, G, T > all others 153 V N, R, K, D, E > S, G > all others 171 R E, Q, N > S, T, G, A > Y > all others 227 Q K, D > Y, H > G, S, T > all others 230 Q R, N, D, E > G, S, T, A > All others 116 A E > R, K, Q, N > Y, H > all others 154 A E > R, K, Q, N > Y, H > all others

In one embodiment, a 4-1BBL ECD mutant protein is provided, the CD70 ECD mutant protein comprising at least one amino acid substitution at a position selected from the group consisting of the following in SEQ ID NO: 37: A154D, A154E, V153Q, Q227E, L101N, Y110Q, Q230K, and V100Q. In another embodiment, a 4-1BBL ECD mutant protein is provided, the CD70 ECD mutant protein comprising at least one amino acid substitution at a position selected from the group consisting of the following in SEQ ID NO: 37: A154D; A154E; V153Q; Q227E; L101N; Y110Q, and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the CD70 ECD mutant protein comprising at least one amino acid substitution at the position selected from the group consisting of SEQ ID NO: 37: A154D; A154E; V153Q; and Q227E. In another embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising at least one amino acid substitution at the position selected from the group consisting of SEQ ID NO: 37: A154D and A154E.

In one embodiment, a 4-1BBL ECD mutant protein is provided, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from that of the wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 154 corresponding to the full-length 4-1BBL amino acid sequence. In one embodiment, the 4-1BBL ECD mutant protein and the wild-type amino acid sequence do not differ from each other except for the difference at position 154. In one embodiment, the different amino acid at position 154 is aspartic acid (D) or glutamic acid (E), wherein aspartic acid (D) is preferred.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 154, and preferably wherein the 4-1BBL ECD mutant protein comprises A154D or A154E substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided, wherein the 4-1BBL... ECD mutant proteins comprise a combination of A154D or A154E substitutions with at least one, two, three, four, or five amino acid substitutions selected from the group consisting of: V100T; V100Q; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101N; L101E; L101Q; L101D; L101R; L101K; Y110Q; Y110E ; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G114Q; G114D; G114E; 114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115 G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V153Q; V153N; V153R; V153K; V153D; V153E; V153S; V153G; G155Q; R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R17 1Y; Q227E; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230K; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A, among which V153Q; Q227E; L101N; Y110Q; Q230K; and V100Q are preferred.

In one embodiment, a 4-1BBL ECD mutant protein is provided, comprising a combination of substitutions selected from the group consisting of: A154D and V153Q; A154D and G155Q; A154D and Q227E; A154D and L101N; A154D and Y110Q; A154D and Q230K; A154D and V100Q; A154D and V100T; A154 D and V100S; A154D and V100A; A154D and V100G; A154D and V100N; A154D and V100D; A154D and V100E; A154D and V100K; A154D and V100R; A154D and L101E; A154D and L101Q; A154D and L101D L101R; A154D and L101K; A154D and Y110E; A154D and Y110N; A154D and Y110D; A154D and Y110R; A154D and Y110K; A154D and Y110S; A154D and Y110A; A154D and Y110G; A154D and Y110T; A154D and G1 14K; A154D and G114R; A154D and G114Q; A154D and G114D; A154D and G114E; A154D and G114N; A154D and G114S; A154D and G114A; A154D and G114G; A154D and G114T; A154D and L115R; A154D and L115 K; A154D and L115Q; A154D and L115N; A154D and L115D; A154D and L115E; A154D and L115S; A154D and L115A; A154D and L115G; A154D and L115T; A154D and A116D; A154D and A116E; A154D and A116R; A154D and A116K; A154D and A116Q; A154D and A116N; A154D and A116Y; A154D and A116H; A154D and V153N; A154D and V153R; A154D and V153K; A154D and V153D; A154D and V153E; A154D and V153S; A1 54D and V153G; A154D and R171D; A154D and R171E; A154D and R171Q; A154D and R171N; A154D and R171S; A154D and R171T; A154D and R171G; A154D and R171A; A154D and R171Y; A154D and Q227R; A154 A154D and Q227K; A154D and Q227D; A154D and Q227Y; A154D and Q227H; A154D and Q227G; A154D and Q227S; A154D and Q227T; A154D and Q230S; A154D and Q230R; A154D and Q230N; A154D and Q230D; A154D and Q230E; A154D and Q230G; A154D and Q230S; A154D and Q230T; A154D and Q230A; A154E and V153Q; A154E and Q227E; A154E and L101N; A154E and Y110Q; A154E and Q230K; A154E and V100Q; A154E and V1 00T; A154E and V100S; A154E and V100A; A154E and V100G; A154E and V100N; A154E and V100D; A154E and V100E; A154E and V100K; A154E and V100R; A154E and L101E; A154E and L101Q; A154E and L101D L101R; A154E and L101K; A154E and Y110E; A154E and Y110N; A154E and Y110D; A154E and Y110R; A154E and Y110K; A154E and Y110S; A154E and Y110A; A154E and Y110G; A154E and Y110T; A154E and G114K; A154E and G114R; A154E and G114Q; A154E and G114D; A154E and G114E; A154E and G114N; A154E and G114S; A154E and G114A; A154E and G114G; A154E and G114T; A154E and L115R; A154E and L115K; A154E and L115Q; A154E and L115N; A154E and L115D; A154E and L115E; A154E and L115S; A154E and L115A; A154E and L115G; A154E and L115T; A154E and A116D; A154E and A116E; A154E and A116R; A154E and A116K; A154E and A116Q; A154 E and A116N; A154E and A116Y; A154E and A116H; A154E and V153N; A154E and V153R; A154E and V153K; A154E and V153D; A154E and V153E; A154E and V153S; A154E and V153G; A154E and R171D; A154E and R171E; A154E and R171Q; A154E and R171N; A154E and R171S; A154E and R171T; A154E and R171G; A154E and R171A; A15 4E and R171Y; A154E and Q227R; A154E and Q227K; A154E and Q227D; A154E and Q227Y; A154E and Q227H; A154E and Q227G; A154E and Q227S; A154E and Q227T; A154E and Q230S; A154E and Q230R; A154E and Q230N; A154E and Q230D; A154E and Q230E; A154E and Q230G; A154E and Q230S; A154E and Q230T; and A154E and Q230A.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises V100T or AV100Q substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided, comprising a combination of V153Q substitution and at least one, two, three, four, or five amino acid substitutions selected from the group consisting of: V100T; L101N; Y110Q; G114K; L115R; A116D; R171D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising combinations of substitutions selected from the group consisting of: V153Q and V100T; V153Q and L101N; V153Q and Y110Q; V153Q and G114K; V153Q and L115R; V153Q and A116D; V153Q and R171D; V153Q and Q227E; V153Q and Q227R; V153Q and Q230S; and V153Q and Q230K.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises an L101N substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided comprising an L101N substitution and a combination of at least one, two, three, four, or five amino acid substitutions selected from the group consisting of: Y110Q; G114K; L115R; A116D; R171D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising a combination of substitutions selected from the group consisting of: L101N and Y110Q; L101N and G114K; L101N and L115R; L101N and A116D; L101N and R171D; L101N and Q227E; L101N and Q227R; L101N and Q230S; and L101N and Q230K.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises a Y110Q substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided comprising a combination of a Y110Q substitution and at least one, two, three, four, or five amino acid substitutions selected from the group consisting of: G114K; L115R; A116D; R171D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising combinations of substitutions selected from the group consisting of: Y110Q and G114K; Y110Q and L115R; Y110Q and A116D; Y110Q and R171D; Y110Q and Q227E; Y110Q and Q227R; Y110Q and Q230S; and Y110Q and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising combinations of substitutions for Y110Q, A154D, and Q227E.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises a G114K substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided comprising a combination of a G114K substitution and at least one, two, three, four, or five amino acid substitutions selected from the group consisting of: L115R; A116D; R171D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising combinations of substitutions selected from the group consisting of: G114K and G114K; G114K and L115R; G114K and A116D; G114K and R171D; G114K and Q227E; G114K and Q227R; G114K and Q230S; and G114K and Q230K.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises an L115R substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided comprising an L115R substitution and a combination of at least one, two, three, four, or five amino acid substitutions selected from the group consisting of: A116D; R171D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising a combination of substitutions selected from the group consisting of: L115R and A116D; L115R and R171D; L115R and Q227E; L115R and Q227R; L115R and Q230S; and L115R and Q230K.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises an A116D substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided comprising a combination of an A116D substitution and at least one, two, three, or four amino acid substitutions selected from the group consisting of: R171D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising a combination of substitutions selected from the group consisting of: A116D and R171D; A116D and Q227E; A116D and Q227R; A116D and Q230S; and A116D and Q230K.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises an R171D substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided comprising an R171D substitution and a combination of at least one or two amino acid substitutions selected from the group consisting of: Q227E; Q227R; Q230S; and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising a combination of substitutions selected from the group consisting of: R171D and Q227E; R171D and Q227R; R171D and Q230S; and R171D and Q230K.

In one embodiment, the 4-1BBL ECD mutant protein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence of the 4-1BBL ECD mutant protein differs from the amino acid sequence of wild-type human 4-1BBL ECD (SEQ ID NO: 37) at least at position 100, and wherein preferably the 4-1BBL ECD mutant protein comprises a Q227E or Q227R substitution. In one embodiment, a 4-1BBL ECD mutant protein is provided comprising a combination of a Q227E substitution and at least one amino acid substitution selected from the group consisting of Q230S and Q230K. In one embodiment, a 4-1BBL ECD mutant protein is provided, comprising a combination of substitutions selected from the group consisting of: Q227E and Q230S; and Q227E and Q230K. In another embodiment, a 4-1BBL ECD mutant protein is provided, comprising a combination of a Q227R substitution and at least one amino acid substitution selected from the group consisting of Q230S and Q230K. In yet another embodiment, a 4-1BBL ECD mutant protein is provided, comprising a combination of substitutions selected from the group consisting of: Q227R and Q230S; and Q227R and Q230K.

In one embodiment, a 4-1BBL ECD mutant protein is provided, the 4-1BBL ECD mutant protein comprising a combination of substitutions at positions selected from the group consisting of SEQ ID NO: 37: Y110Q, V153Q and Q227E; L101N, Y110Q and V153Q; V100Q, Y110Q and V153Q; L101N, V153Q and Q227E; Y110Q, A154D and Q227E; Y110Q and V153Q; V153Q and Q227E; Y110Q and Q227E; and L101N and Q227E.

Aspartic acid substitution (e.g., A154D), when followed (in the N-terminal to C-terminal direction) by a G, A, or S residue (e.g., G155), may potentially introduce an aspartic acid isomerization site, which could lead to instability and/or increased degradation of the mutant protein containing such an isomerization site. Therefore, in one embodiment, aspartic acid substitution, when followed by a G, A, or S residue, is combined with subsequent G, A, or S residues to substitutions for any amino acid residue other than G, A, S, or T. Preferably, the subsequent G, A, or S residue is substituted with Q, N, Y, L, V, or F. Therefore, in one embodiment, a 4-1BBL ECD mutant protein is provided, which comprises a combination of substitutions selected from the group consisting of Y110D and S111X; L115D and A116X; A116D and G117X; V153D and A154X; A154D and G155X; R171D and S172X; and Q230D and G231X, wherein X is any amino acid residue other than G, A, S or T, and preferably X is Q, E, N, D, H, K, R or Y. In a preferred embodiment, any 4-1BBL ECD mutant protein containing an A154D substitution is combined with a G155X substitution, wherein X is any amino acid residue other than G, A, S, or T, wherein X is preferably Q, E, N, D, H, K, R, or Y, with Q being the most preferred.

In one embodiment, a 4-1BBL ECD mutant protein as defined above is provided, which binds to its homologous receptor 4-1BB with a reduced affinity relative to the wild-type 4-1BBL ECD for 4-1BB. In another embodiment, a 4-1BBL ECD mutant protein as defined above is provided, which binds to its homologous receptor human 4-1BB with a reduced affinity relative to the wild-type 4-1BBL ECD for human 4-1BB (e.g., human 4-1BB having the amino acid sequence contained in SEQ ID NO: 175).

It should be understood that when present in a homotrimeric fusion protein containing three 4-1BBL ECD mutant protein monomers linked by a polypeptide linker such as (GGGGS) ₄ , the 4-1BBL ECD mutant protein, which has a reduced affinity for its homologous receptor 4-1BB, will also have a reduced affinity for 4-1BB compared to the wild-type 4-1BBL ECD mutant protein. Generally, the affinity of the 4-1BBL ECD mutant for its homologous receptor 4-1BB is defined here as the affinity for 4-1BB determined when the 4-1BBL ECD mutant is present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant monomers linked by a (GGGGS) ₄ polypeptide linker, wherein the homotrimer may be present in a conjugate with an antibody (e.g., trastuzumab), which may further comprise IL-21.

In one embodiment, a 4-1BBL ECD mutant protein with reduced affinity for 4-1BB relative to wild-type 4-1BBL ECD is provided, wherein the 4-1BBL ECD mutant protein comprises at least one substitution selected from the group consisting of: V100T; V100Q; L101N; Y110Q; G114K; V153Q; R171D; Q227E; Q227R; Q230S; Q230K; A116D; A154D; and A154E; or 4-1BBL... ECD mutant proteins comprise combinations of substitutions selected from the following groups: Y110Q, V153Q, and Q227E; L101N, Y110Q, and V153Q; V100Q, Y110Q, and V153Q; L101N, V153Q, and Q227E; Y110Q, A154D, and Q227E; A154D and G155Q; Y110Q and V153Q; V153Q and Q227E; Y110Q and Q227E; and L101N and Q227E.

The 4-1BBL ECD mutant protein described herein binds to 4-1BB in a non-covalent and reversible manner. In one embodiment, the binding strength of the 4-1BBL ECD mutant protein to 4-1BB can be described by its affinity, which is a measure of the strength of the interaction between the binding site of the mutant protein and 4-1BB. In one embodiment, the 4-1BBL ECD mutant protein described herein has a low affinity for 4-1BB and therefore binds a smaller amount of 4-1BB than wild-type 4-1BBL. In one embodiment, the 4-1BBL ECD mutant protein provided herein has a balance binding constant KA, which is (with decreasing preference) at least ^{10³ M⁻¹} , at least ^{10⁴ M⁻¹} , at least ^{10⁵ M⁻¹} , at least ^{10⁶ M⁻¹} , at least ^{10⁷ M⁻¹} , at least ^{10⁸ M⁻¹} , at least ^{10⁹ M⁻¹} , or at least ^{10¹⁰ M⁻¹} . As will be understood by those skilled in the art, _KA can be affected by factors including pH, temperature, and buffer composition.

In one embodiment, the binding strength of the 4-1BBL ECD mutant protein and 4-1BB provided herein can be described by its affinity, i.e., _KD . _KD is the equilibrium dissociation constant between the 4-1BBL ECD mutant protein and 4-1BB, the _koff / _kon ratio. _KD and _KA are inversely correlated. The _KD value is related to the concentration of the mutant protein (the amount of mutant protein required for a specific experiment or application), and therefore the lower the _KD value (the lower the required concentration), the higher the affinity of the mutant protein. In one embodiment, the binding strength of the 4-1BBL ECD mutant protein and 4-1BB provided herein can be described by _KD . In one embodiment, the _KD of the 4-1BBL ECD mutant protein provided herein is about ^10⁻³ M, about ^10⁻⁴ M, about ^10⁻⁵ M, about ^10⁻⁶ M, or less. In one embodiment, the _KD of the 4-1BBL ECD mutant protein provided herein is micromolar, nanomolar, or pimole. In one embodiment, the _KD of the 4-1BBL ECD mutant protein provided herein is in the range of about ^10⁻³ to ^10⁻⁴ M, or about ^10⁻⁴ to ^10⁻⁵ M, or ^10⁻⁵ to ^10⁻⁶ M, or ^10⁻⁷ to ^10⁻⁸ M, or ^10⁻⁸ to ^10⁻⁹ M. In one embodiment, the 4-1BBL ECD mutant protein provided herein binds to human 4-1BB with a _KD greater than or about 140 nM. In one embodiment, the 4-1BBL ECD mutant protein provided herein binds to human 4-1BB at a _KD of approximately 100 nM to approximately 4,000 nM, 200 nM to 4,000 nM, 500 nM to 4,000 nM, 1,000 nM to 4,000 nM, 120 nM to 3,000 nM, 200 nM to 2,000 nM, 500 nM to 2,000 nM, or 1,000 nM to 2,000 nM.

In one embodiment, the 4-1BBL ECD mutant protein provided herein exhibits a reduced binding affinity for human 4-1BB. In one embodiment, the 4-1BBL ECD mutant protein provided herein is a mutant protein exhibiting a reduced binding affinity for 4-1BB relative to wild-type 4-1BBL ECD by at least approximately 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 times.

In one embodiment, the binding affinity of the 4-1BBL ECD mutant protein provided herein for 4-1BB is determined by SPR, for example as described in the embodiments herein. In one embodiment, the binding affinity of the 4-1BBL ECD mutant protein provided herein for 4-1BB is thus determined when the 4-1BBL ECD mutant protein is present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, wherein the homotrimer is present in a conjugate with a monoclonal antibody (e.g., trastuzumab), the conjugate further comprising IL-21.

In one embodiment, the 4-1BBL ECD mutant protein described herein exhibits a binding affinity for human 4-1BB, expressed as _pKD , which is at least 0.4 lower than _that of wild-type 4-1BBL ECD for human 4-1BB. _pKD should be understood herein as -log ₁₀ ( _KD ). In one embodiment, the 4-1BBL ECD mutant protein has a pK _D at least 0.4 lower than that of wild-type 4-1BBL ECD against human 4-1BB, and _is a 4-1BBL ECD mutant protein containing at least one substitution selected from the following groups: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; and A116D; or is a 4-1BBL ECD mutant protein that does not contain any amino acid sequence modifications other than at least one amino acid substitution selected from the following groups. ECD mutant proteins: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; and A116D.

In one embodiment, the 4-1BBL ECD mutant protein provided herein exhibits a binding affinity for human 4-1BB, expressed as pK _D , which is at least 0.5 lower than _that of wild-type 4-1BBL ECD for human 4-1BB. In one embodiment, the 4-1BBL ECD mutant protein has a pK _D at least 0.5 lower than that of wild-type 4-1BBL ECD against human 4-1BB, and _is a 4-1BBL ECD mutant protein containing at least one substitution selected from the following groups: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; and V100T; or is a 4-1BBL ECD mutant protein that does not contain any amino acid sequence modifications other than at least one amino acid substitution selected from the following groups. ECD mutant proteins: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; and V100T.

In one embodiment, the 4-1BBL ECD mutant protein provided herein exhibits a binding affinity for human 4-1BB, expressed as pK _D , which is at least 1.0 lower than _that of wild-type 4-1BBL ECD for human 4-1BB. In one embodiment, the 4-1BBL ECD mutant protein has a pK _D at least 1.0 lower than that of wild-type 4-1BBL ECD relative to human 4-1BB, and is _a 4-1BBL ECD mutant protein containing at least one substitution selected from the following groups: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; Q230K; and V100Q; or is a 4-1BBL ECD mutant protein that does not contain any amino acid sequence modifications other than at least one amino acid substitution selected from the following groups. ECD mutant proteins: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; Q230K; and V100Q.

In one embodiment, the 4-1BBL ECD mutant protein provided herein exhibits reduced activity relative to the wild-type 4-1BBL ECD under corresponding conditions, as measured by a suitable assay for measuring changes in the 4-1BB signaling pathway as described above. In one embodiment, the 4-1BBL ECD mutant protein provided herein exhibits a reduction in activity of at least about 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 times relative to the wild-type 4-1BBL ECD under corresponding conditions, as measured by a 4-1BB signaling assay.

In one embodiment, the 4-1BBL ECD mutant protein provided herein exhibits a reduction in activity relative to wild-type 4-1BBL ECD under corresponding conditions of at least approximately 2-, 5-, 10-, 20-, 50-, 100-, 200-, 500-, 1,000-, 2,000-, 5,000-, and 10,000-fold, as measured by an NK cell proliferation assay. In one embodiment, the NK cell proliferation assay is a short-term proliferation assay, measuring proliferation over a period of less than one week, such as 3, 4, 5, or 6 days, as described in the examples herein. In one embodiment, the NK cell proliferation assay is a long-term proliferation assay, measuring proliferation over a period of more than one week, such as at least 10, 12, or 14 days, as described in the examples herein.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, exhibits a pEC 50 inducing NK cell proliferation in a 5-day NK cell proliferation assay in the presence of TAA _{-expressing tumor cells (e.g., SKOV-3 cells) that is not less than 0.25 logs higher than the pEC 50 of} a corresponding control conjugate containing wild-type 4-1BBL ECD in the same assay. This homotrimer is present in a conjugate with a monoclonal antibody against TAA (e.g., trastuzumab), which further comprises IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein includes at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; Q227R; L101N; Y110Q; and V100Q; or the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; Q227R; L101N; Y110Q; and V100Q.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, exhibited a pEC ₅₀ inducing NK cell proliferation in the presence of SKOV-3 tumor cells at 5 days, which was not less than the pEC ₅₀ of the corresponding control conjugate containing wild-type 4-1BBL ECD by more than 0.10 logs. This homotrimer was present in a conjugate with trastuzumab, which further contained IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein includes at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; and V100Q; or the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; and V100Q.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, exhibited a pEC ₅₀ inducing NK cell proliferation in the presence of SKOV-3 tumor cells at 5 days, which was not less than the pEC ₅₀ of the corresponding control conjugate containing wild-type 4-1BBL ECD by more than 0.05 logs. This homotrimer was present in a conjugate with trastuzumab, which further contained IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein contains at least one amino acid substitution selected from the group consisting of: A154D; A154E; and V100Q; or the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; and V100Q.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, induced maximum NK cell proliferation at a saturation concentration of 25 nM in a normalized 5-day NK cell proliferation assay in the presence of SKOV-3 tumor cells. This proliferation was at least 75%, 80%, 85%, 90%, or 95% of the proliferation induced in the same assay by a corresponding control conjugate containing wild-type IL-21 and a wild-type 4-1BBL ECD trimer, the homotrimer being present in a conjugate with trastuzumab, the conjugate further comprising IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein includes at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; Q227R; L101N; Y110Q; Q230K; and V100Q; or the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; Q227R; L101N; Y110Q; Q230K; and V100Q.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, induced maximum NK cell proliferation at a saturation concentration of 25 nM in a normalized 5-day NK cell proliferation assay in the presence of SKOV-3 tumor cells. This proliferation was at least 75% of the proliferation induced in the same assay by a corresponding control conjugate containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD, the homotrimer being present in a conjugate with trastuzumab, the conjugate further comprising IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein contains at least one amino acid substitution selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; and V100Q; or the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; Q230K; and V100Q.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, induced maximum NK cell proliferation at a saturation concentration of 25 nM in the presence of SKOV-3 tumor cells in a normalized 5-day NK cell proliferation assay. This proliferation was at least 80% greater than that induced in the same assay by a corresponding control conjugate containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD, the homotrimer being present in a conjugate with trastuzumab, the conjugate further comprising IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein contains at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; and Q230K; or the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101N; Y110Q; and Q230K.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, induced maximum NK cell proliferation at a saturation concentration of 25 nM in a normalized 5-day NK cell proliferation assay in the presence of SKOV-3 tumor cells. This proliferation was at least 85% greater than that induced in the same assay by a corresponding control conjugate containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD, the homotrimer being present in a conjugate with trastuzumab, the conjugate further comprising IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein contains at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; and Q227E; or the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; and Q227E.

In one embodiment, the 4-1BBL ECD mutant protein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1BBL ECD mutant protein monomers linked by a (GGGGS) ₄ polypeptide linker, induced maximum NK cell proliferation at a saturation concentration of 25 nM in a normalized 5-day NK cell proliferation assay in the presence of SKOV-3 tumor cells. This proliferation was at least 95% greater than that induced in the same assay by a corresponding control conjugate containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD, the homotrimer being present in a conjugate with trastuzumab, the conjugate further comprising IL-21. Therefore, in one embodiment, the 4-1BBL ECD mutant protein includes at least one amino acid substitution selected from the group consisting of: A154D and A154E; a combination of A154D and G155Q; and the 4-1BBL ECD mutant protein does not contain any other amino acid sequence modifications other than at least one amino acid substitution selected from the group consisting of: A154D; a combination of A154D and G155Q; and A154E.

In one embodiment, a 4-1BBL ECD mutant protein as described herein is provided, which, in the absence of antigen or antigen-carrying cells, when the 4-1BBL mutant protein is present in a conjugate of an antigen-binding protein that specifically binds to the (target) antigen, produces reduced (little or no) agonist activity of 4-1BB expressed on the cell surface due to the reduced affinity of the 4-1BBL ECD mutant protein for 4-1BB. However, when the conjugate binds to the antigen or antigen-carrying cells, the 4-1BBL ECD mutant protein in the conjugate exhibits significant agonist activity. This activity is due to the higher local density of the 4-1BBL ECD mutant protein on the surface of target cells, leading to enhanced epigenetic affinity for 4-1BB on local NK cells (see Figure 1) primarily through an affinity mechanism. Therefore, the 4-1BBL ECD mutant protein, as described herein, in conjugates containing antigen-binding proteins broadens the therapeutic window compared to corresponding conjugates containing wild-type 4-1BBL ECD. The term "therapeutic window" is understood herein as the ratio (or fold difference) of _{EC50 values} obtained from functional tests (e.g., proliferation tests) comparing conditions where cancer cells are absent with conditions where cancer cells are present. A therapeutic window of 10 (or 1 log) means that the EC50 in the absence of cancer cells is 10 times higher than that in the presence of cancer cells (with lower efficacy). After systemic application, the conjugate containing the 4-1BBL ECD mutant protein has almost no effect on peripheral cells (including T cells, B cells, or NK cells), but it can still effectively stimulate these immune cells, especially NK cells, at the tumor site, site of infection, or site of inflammation. This is because the target antigen bound by the antigen-binding protein exists only in high local concentrations in these specific regions, thus generating an affinity effect.

Therefore, in one embodiment, a 4-1BBL ECD mutant protein as described herein is provided, wherein when the 4-1BBL ECD mutant protein is present in a conjugate with an antigen-binding protein that specifically binds to an antigen, it has an _EC50 in the NK cell proliferation assay in the presence of the antigen or cells expressing the antigen, which (with increasing preference) is at least 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, or 100,000 times lower than the _EC50 in the corresponding NK cell proliferation assay in the absence of the antigen or cells expressing the antigen, wherein the conjugate further comprises IL-21, if applicable.

In one embodiment, reference multispecific antigen-binding proteins (such as AVC1) and reference tumor cells (such as HER2-expressing SK-OV-3 cells) were used to determine the difference in NK cell proliferation induction between the presence and absence of the antigen. The AVC1 multispecific antigen-binding protein consists of a first trastuzumab heavy chain fused to the extracellular domain of a 4-1BB ligand (SEQ ID NO: 11), a second trastuzumab heavy chain fused to wild-type IL-21 (SEQ ID NO: 12), and a trastuzumab light chain (SEQ ID NO: 2), wherein the constant regions of the first and second heavy chains are distinguished using a club-and-mortar technique (see WO2024/056862). The wild-type 4-1BBL ECD amino acid sequence in the first heavy chain amino acid sequence of SEQ ID NO: 11 can be replaced by the amino acid sequence of the 4-1BBL ECD mutant protein to be tested, for example, because of its ability to induce NK cell proliferation in the presence and absence of tumor cells expressing HER2 (such as SK-OV-3 cells).

In one embodiment, a 4-1BBL ECD mutant protein as described herein is provided, wherein when the 4-1BBL ECD mutant protein is present in a multispecific antigen-binding protein comprising: i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 11, wherein the wild-type 4-1BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1BBL ECD mutant protein; ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 12; and iii) a light chain containing the amino acid sequence of SEQ ID NO: 2, it has an _EC50 in the NK cell proliferation assay in the presence of SK-OV-3 cells (with increasing preference) compared to the EC50 in the corresponding NK cell proliferation assay in the absence of SK-OV-3 cells. ₅₀ is at least 10, 25, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, or 100,000 times lower.

In one embodiment, a 4-1BBL ECD mutant protein as described herein is provided, wherein when the 4-1BBL ECD mutant protein is present in a multispecific antigen-binding protein comprising: i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 11, wherein the wild-type 4-1BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1BBL ECD mutant protein; ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 12; and iii) a light chain containing the amino acid sequence of SEQ ID NO: 2, EC is generated in NK cell proliferation assays in the presence and absence of SK-OV-3 cells. A difference of ₅₀ times, which (with increasing preference) is at least 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000 or 5,000 times higher than the difference in _EC50 in NK cell proliferation assays in the presence or absence of SK-OV-3 cells, such as those generated by a reference multispecific antigen-binding protein, which is composed of: i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 11; a second heavy chain containing the amino acid sequence of SEQ ID NO: 12; and iii) a light chain containing the amino acid sequence of SEQ ID NO: 2.

In the above embodiments, the NK cell proliferation assay is preferably performed using NK cells isolated from healthy donors. In the above embodiments, the _EC50 value in the NK cell proliferation assay is preferably determined based on the average value of NK cells isolated from at least five different healthy donors. In the above embodiments, the NK cell proliferation assay is preferably performed substantially as described in the embodiments herein.

When the 4-1BBL ECD mutant protein described herein is present in a conjugate of an antigen-binding protein, the reduced affinity of the 4-1BBL ECD mutant protein increases the therapeutic window compared to the corresponding conjugate containing wild-type 4-1BBL ECD, although it also increases the ability of the conjugate of the 4-1BBL ECD mutant protein to induce NK cell cytotoxicity against cells carrying antigens bound by antigen-binding proteins, but preferably remains substantially unaffected.

Therefore, in one embodiment, a 4-1BBL ECD mutant protein as described herein is provided, wherein when the 4-1BBL ECD mutant protein is present in a multispecific antigen-binding protein comprising: i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 11, wherein the wild-type 4-1BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1BBL ECD mutant protein; ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 12; and iii) a light chain containing the amino acid sequence of SEQ ID NO: 2, it has an _EC50 in an NK cell cytotoxicity assay in the presence of SK-OV-3 cells, which (with increasing preference) is at least equal to the _EC50 of a reference multispecific antigen-binding protein in the same assay. The reference multispecific antigen-binding protein, which is at least 2, 5, or 10 times higher than the reference multispecific antigen-binding protein, comprises: i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 11; ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 12; and iii) a light chain containing the amino acid sequence of SEQ ID NO: 2. NK cell cytotoxicity assays in the presence of SK-OV-3 cells are preferably performed using NK cells isolated from healthy donors. Preferably, the _EC50 value in the NK cell cytotoxicity assay is determined based on the average value of NK cells isolated from at least 5 different healthy donors. In the above embodiments, the NK cell cytotoxicity assay is preferably performed substantially as described in the embodiments herein.

Furthermore, in the presence of cells carrying antigens bound by antigen-binding proteins, the ability of the conjugate between the 4-1BBL ECD mutant protein and the antigen-binding protein, as described herein, to support the long-term proliferation of NK cells remains largely unaffected.

Therefore, in one embodiment, a 4-1BBL ECD mutant protein as described herein is provided, wherein the 4-1BBL ECD mutant protein is present in a multispecific antigen-binding protein comprising: i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 11, wherein the wild-type 4-1BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1BBL ECD mutant protein; ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 12; and iii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 12. When the light chain of the amino acid sequence of SEQ ID NO: 11 is incorporated, it induces a fold increase in NK cells in the presence of SK-OV-3 cells in an NK cell amplification assay, with a (continuously increasing preference) at least equal to, or at least 2-fold, 5-fold, or 10-fold higher than, the fold increase induced by a reference multispecific antigen-binding protein in the same assay. The reference multispecific antigen-binding protein comprises: i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 11; ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 12; and iii) a light chain containing the amino acid sequence of SEQ ID NO: 2. The NK cell amplification assay in the presence of SK-OV-3 cells is preferably performed using NK cells isolated from healthy donors. Preferably, the fold increase of NK cells in the assay is determined based on the average value of NK cells isolated from at least 5 different healthy donors. In the above embodiments, the NK cell amplification assay is preferably performed substantially as described in the embodiments herein.

Another advantage of the 4-1BBL ECD mutant proteins described herein is that their reduced affinity for 4-1BB improves the pharmacokinetics of therapies containing the 4-1BBL ECD mutant proteins. In vivo, numerous cells, including T cells, B cells, or NK cells, express 4-1BB molecules on their surface. These 4-1BB molecules act as sinks for therapies containing portions of the 4-1BB-affinity, such as the 4-1BBL ECD mutant protein. Once bound to the surface-expressed 4-1BB, the therapy containing the 4-1BBL portion is internalized and thus loses its therapeutic effect, for example, in the tumor microenvironment. Therefore, as described herein, the reduced affinity of the 4-1BBL ECD mutant protein for 4-1BB reduces or prevents their disappearance in this receptor, thereby improving the pharmacokinetics of therapies containing the 4-1BBL ECD mutant protein.

In another embodiment, this disclosure provides a trimeric fusion protein comprising three 4-1BBL ECD monomers, wherein one, two, or all three monomers are 4-1BBL ECD mutant proteins as described above herein, and these monomers are fused together in a single polypeptide chain, as described, for example, in Fellermeier et al. (2016, ibid .). In one embodiment, the three 4-1BBL ECD monomers are linked by a polypeptide linker. In one embodiment, the three 4-1BBL ECD monomers are linked by a polypeptide linker selected from the group consisting of: (GGGGS) ₁ , (GGGGS) ₂ , (GGGGS) ₃ , (GGGGS) ₄ , (GGGGS) ₅ , GGGSGGG, GGSGGGGGSGG and G, wherein (GGGGS) ₄ is preferred. Other suitable flexible polypeptide linkers are described below. In one embodiment, two or three 4-1BBL ECD mutant protein monomers in the trimeric fusion protein are identical mutant proteins. In one embodiment, two or three 4-1BBL ECD mutant protein monomers in the trimeric fusion protein are different mutant proteins. In the trimeric fusion protein, the 4-1BBL ECD monomer that is not a 4-1BBL ECD mutant protein as described above may be a wild-type 4-1BBL ECD monomer or a 4-1BBL ECD mutant protein not described herein.

In one embodiment, this disclosure provides a trimeric fusion protein comprising three monomers of the 4-1BBL ECD mutant protein as described above herein, these monomers being fused together in a single polypeptide chain, as described, for example, in Fellermeier et al. (2016, ibid. ). In one embodiment, the three 4-1BBL ECD monomers are linked by a polypeptide linker. In one embodiment, the three 4-1BBL ECD monomers are linked by a polypeptide linker selected from the group consisting of: (GGGGS) ₁ , (GGGGS) ₂ , (GGGGS) ₃ , (GGGGS) ₄ , (GGGGS) ₅ , GGGSGGG, GGSGGGGGSGG and G, wherein (GGGGS) ₄ is preferred. Other suitable flexible polypeptide linkers are described below. In one embodiment, the three monomers of the 4-1BBL ECD mutant protein in the trimer fusion protein are identical mutant proteins. In another embodiment, at least two of the three monomers of the 4-1BBL ECD mutant protein in the trimer fusion protein are different mutant proteins.

In one embodiment, a fusion protein comprising three monomers of the 4-1BBL ECD mutant protein, preferably as described herein, is fused together in a single polypeptide chain comprising three identical monomers of the 4-1BBL ECD mutant protein. In another embodiment of the fusion protein, at least one of the monomers differs from the other two, or all three monomers differ from each other.

This article should be understood to mean that when referring to the 4-1BBL ECD mutant protein as described herein, it can also refer to a trimeric fusion protein containing the three monomers of this type of 4-1BBL ECD mutant protein. Conjugates containing 4-1BBL ECD mutant proteins.

In the second embodiment, this disclosure provides a conyx comprising one or more 4-1BBL ECD mutant proteins as described herein, coupled to a heterologous portion. It should be understood that the term "conyx comprising 4-1BBL ECD mutant proteins as described herein" also includes a conyx comprising a trimeric fusion protein comprising three 4-1BBL ECD mutant proteins as described herein. As used herein, the term "heterologous portion" is synonymous with the term "conyx portion" and refers to any molecule (chemical or biochemical, naturally occurring or non-coding) that is different from the 4-1BBL ECD mutant proteins described herein. Exemplary conjugate portions that can be linked to any 4-1BBL ECD mutant protein described herein include, but are not limited to, heterologous peptides or polypeptides (including, for example, immunoglobulins or portions thereof (e.g., variable regions, CDRs, or Fc regions)), targets, diagnostic markers (such as radioisotopes, fluorescent groups, or enzyme-catalyzed markers), polymers (including water-soluble polymers), or other therapeutics or diagnostic agents. In some embodiments, a conjugate comprising the 4-1BBL ECD mutant protein of this disclosure and an immunoglobulin is provided. In some embodiments, the conjugate comprises one or more of the 4-1BBL ECD mutant proteins described herein and one or more of the following: peptides or polypeptides (which are different from the 4-1BBL ECD mutant proteins described herein), nucleic acid molecules, antibodies or fragments thereof, polymers, quantum dots, small molecules, toxins, diagnostic agents, carbohydrates, and amino acids.

In one embodiment, a conjugate is provided in which the heterologous portion is attached to the 4-1BBL ECD mutant protein as described herein via non-covalent or covalent bonding. In exemplary embodiments, the bonding between the 4-1BBL ECD mutant protein and the heterologous portion is achieved via covalent chemical bonds (e.g., peptide bonds, disulfide bonds, and the like) or via physical forces (e.g., electrostatic, hydrogen, ion, van der Waals, or hydrophobic or hydrophilic interactions). A variety of non-covalent coupling systems can be used, including, for example, biotin-avidin, ligand/receptor, enzyme/receptor, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cell adhesion molecule conjugates; or any conjugates or fragments thereof with affinity for each other.

In one embodiment, a conjugate is provided in which the 4-1BBL ECD mutant protein, as described herein, is linked to the conjugate moiety via direct covalent bonding by reacting a targeting amino acid residue of the 4-1BBL ECD mutant protein with an organic derivatizer capable of reacting with selected side-chain, N-terminal, or C-terminal residues of such targeting amino acids. Reactive groups on the 4-1BBL ECD mutant protein or the conjugate moiety include, for example, aldehyde, amino, ester, thiol, α-haloacetyl, cis-butenylidene imine, or hydrazine groups. Derivatizing agents include, for example, cis-butadiene-benzoylsulfonate succinimide (via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the conjugated portion may be indirectly linked to the 4-1BBL ECD mutant protein via an intermediate carrier, such as a polysaccharide or peptide carrier. Examples of polysaccharide carriers include aminoglucan. Suitable peptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixtures of these amino acids with other (e.g., serine) polymers to impart the desired solubility properties to the resulting carrier.

The most common method is to react the cysteamine acetyl residue with α-haloacetate (and the corresponding amine) (such as chloroacetic acid, chloroacetamide) to give carboxymethyl or carboxyamine methyl derivatives. The cysteamine acetyl group can also be derivatized by reaction with bromotrifluoroacetone, alfa-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylcis-butadieneimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidine amide residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5–7.0, as this agent is relatively specific to the histidine amide side chain. It is also suitable for bromobenzoylmethyl bromide; the reaction is preferably carried out in 0.1 M sodium dimethylarsinate at pH 6.0.

The ionamine amide group and its amino-terminal residue react with succinic anhydride or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of the ionamine amide residue. Other suitable reagents for derivatizing alfa-amine residues include amide esters, such as methyl pyridinium amide, pyridoxal phosphate, pyridoxal, borohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylates.

The arginine residue can be modified by reaction with one or more known reagents, particularly benzoxaldehyde, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, the derivatization of the arginine residue requires alkaline conditions. Furthermore, these reagents can react with lysine groups and arginine epsilon-amine groups.

Specific modifications can be made to the tyramine amide residue, with particular interest in introducing spectral labeling into the tyramine amide residue through reaction with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetylimidamine amides and 3-nitro derivatives, respectively.

The carboxyl side group (aspartic acid amide or glutamic acid amide) is selectively modified by reacting with carbodiimide (R-N=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholino-4-ethyl)carbodiimide or 1-ethyl-3-(4-aza-cation-4,4-dimethylpentyl)carbodiimide.

In addition, aspartic acid amide and glutamate amide residues are converted into aspartic acid amide and glutamate amide by reacting with ammonium ions.

Other modifications include hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine acetylated or thiamethoxamyl residues; methylation of the alfa-amine groups on the side chains of lysine, arginine, and histidine (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)); deacetylation of aspartic acid or glutamate; acetylation of N-terminal amines and/or acetylation or esterification of C-terminal carboxylic acid groups.

Another type of covalent modification involves the chemical or enzymatic coupling of glycosides with 4-1BBL ECD mutant proteins. Glycosides can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free hydrogen sulfide groups, such as the free hydrogen sulfide groups of cysteine, (d) free hydroxyl groups, such as the free hydroxyl groups of serine, threonine or hydroxyproline, (e) aromatic residues, such as the aromatic residues of tyrosine or tryptophan, or (f) amide groups of glutamic acid. These methods are described in WO87/05330 and Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981), published on 11 September 1987.

In one embodiment, the heterologous portion is attached to the 4-1BBL ECD mutant protein as described herein via a linker. In some embodiments, the linker comprises a chain of 1 to about 60 atoms, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. The chain atoms and the linker may be selected based on their intended solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides functional groups that can be cleaved by enzymes or other catalysts or hydrolytic conditions found in target tissues, organs, or cells. In some embodiments, the linker is long enough to reduce the possibility of steric hindrance. If the linker is covalent or peptide-bonded and the conjugate is a polypeptide, the entire conjugate can be a fusion protein. Such peptide-based linkers can be of any length. Exemplary peptide-based linkers are about 1 to 50 amino acids long, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids long, and are flexible or rigid. Flexible linkers are typically used when the linker domain requires a certain degree of mobility or interaction. They are typically composed of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows for the movement of the linker domain. The introduction of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing adverse interactions between the linker and the protein moiety. Preferred flexible linkers have a sequence consisting primarily of fragments of Gly and Ser residues (“GS” linkers). A preferred (and widely used) example of a flexible linker is the sequence (GGGGS) _n (SEQ ID NO: 30). By adjusting the replication number “n”, the length of this GS linker can be optimized to achieve proper separation of functional domains or maintain necessary inter-domain interactions. The replication number “n” of this GS linker can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Specific examples of GS linkers include (GGGGS) ₄ (SEQ ID NO: 31), GGGSGGG (SEQ ID NO: 32), GGSGGGGGSGG (SEQ ID NO: 33), and G. In addition to GS linkers, many other flexible linkers have been designed for recombining fusion proteins. These flexible linkers are also rich in small amino acids or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility, such as the flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID NO: 34) and EGKSSGSGSESKST (SEQ ID NO: 35), which have been used to construct biologically active scFvs.

In one embodiment, a conyzoide is a conyzoide having a 4-1BBL ECD mutant protein valence of more than one, which is understood to mean that the conyzoide contains more than one, such as two, three, four, or five 4-1BBL ECD mutant protein moieties. Preferably, more than one 4-1BBL ECD mutant protein moieties are uniform 4-1BBL ECD mutant protein moieties.

In one embodiment, the conyzot is a conyzot having a valence of more than one trimeric 4-1BBL mutant protein fusion protein, which is understood to mean that the conyzot contains more than one, such as two, three, four, or five, such trimeric 4-1BBL mutant protein fusion proteins. Preferably, the more than one trimeric 4-1BBL ECD mutant protein fusion protein is a homogeneous trimeric fusion protein.

Therefore, in one embodiment, a conjugate is provided, wherein the heterologous portion comprises a polypeptide. The polypeptide comprised in the heterologous portion is preferably a polypeptide different from any 4-1BBL ECD mutant protein described herein. In one embodiment, the conjugate is a fusion polypeptide, fusion protein, chimeric protein, or chimeric polypeptide comprising a 4-1BBL ECD mutant protein or a trimeric 4-1BBL ECD mutant protein fusion protein as described herein, and the heterologous portion comprises a polypeptide fused to a single polypeptide chain. Further description of such conjugates as fusion proteins is provided below.

In one embodiment, a conjugate is provided, wherein the heterologous portion comprises a polypeptide or a polypeptide chain of an antigen-binding protein as an antigen-binding protein.

In one embodiment, a conjugate is provided, wherein the heterologous portion comprises an antigen-binding protein or a polypeptide chain of an antigen-binding protein, the antigen-binding protein comprising at least one of: a) at least one of: i) a first antigen-binding region specifically binding to a tumor-associated antigen (TAA) and specifically binding to an NK cell activation receptor; and ii) a second antigen-binding region specifically binding to a TAA and specifically binding to an NK cell activation receptor; and b) a third antigen-binding region having or potentially having an affinity for surface antigens expressed on natural killer (NK) cells.

The antigen-binding region used in antigen-binding proteins conjugated with the 4-1BBL ECD mutant protein described herein can be derived from any of a variety of immunoglobulin or non-immunoglobulin scaffolds, such as affinity variants based on the Z domain of staphylococcal protein A, engineered Kunitz domains, monomers based on the 10th extracellular domain of human fibronectin III, or adenettin, anticarrier proteins derived from lipid carriers, DARPin (designed anchorin repetitive domain), human ubiquitin molecules, polymerized LDLR-A modules, high-affinity polymers, or cysteine-rich knotting peptides. See, for example, Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference.

In a preferred embodiment, the antigen-binding region used in the antigen-binding protein conjugated with the 4-1BBL ECD mutant protein as described herein comprises or is composed of an immunoglobulin variable region. Such immunoglobulin variable regions may comprise or be composed of variable domains typically derived from antibodies (immunoglobulin chains), such as the associated _VL and _VH domains found on two polypeptide chains, as present in Fab. Alternatively, the immunoglobulin variable domain may comprise or be composed of a single-stranded antigen-binding domain, such as scFv, _VH , _VL , or immunoglobulin single variable domains (ISVDs), such as dAb, V-NAR, or _VHH domains. The immunoglobulin variable region to be used in the antigen-binding protein of the conjugate of the 4-1BBL ECD mutant protein as described herein can be a human or humanized immunoglobulin variable region or an immunoglobulin monovariable domain as defined above herein. It specifically binds to at least the TROP2 antigen-binding region.

Therefore, in one embodiment, the antigen-binding protein in the conjugate of the 4-1BBL ECD mutant protein as described herein comprises at least one of the following: i) a first antigen-binding region specifically binding to a TROP2-bound TAA; and ii) a second antigen-binding region specifically binding to TROP2 or another TAA. In one embodiment, the TAA-binding antigen-binding region is an antigen-binding region derived from an immunoglobulin or non-immunoglobulin scaffold as defined above. Preferably, the TAA-bound antigen-binding region comprises or is composed of at least one immunoglobulin variable domain. More preferably, the TAA-bound antigen-binding region comprises or is composed of a TAA-bound Fab or a TAA-bound immunoglobulin single variable domain (ISVD). In one embodiment, the antigen-binding region that specifically binds to the TAA is an antigen-binding region that binds to the TAA with a _KD value not exceeding ^10⁻³ M or ^10⁻⁴ M, which can be determined as described above.

In one embodiment, the antigen-binding region of a specific TAA includes or is composed of a human or humanized immunoglobulin variable region or an immunoglobulin monovariable region as defined above herein.

In one embodiment, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein comprises two antigen-binding regions that specifically bind to a TAA, namely, a first and a second antigen-binding region. In an antigen-binding protein comprising two antigen-binding regions that specifically bind to a TAA, the two antigen-binding regions may bind to one and the same TAA, may bind to at least two different TAAs, or may bind to at least two different antigenic determinants on the same TAA. In one embodiment of an antigen-binding protein comprising two antigen-binding regions that specifically bind to a TAA, the two antigen-binding regions are identical. Therefore, with respect to the two antigen-binding regions that specifically bind to a TAA, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein may be a homodimer or a heterodimer antigen-binding protein.

As used herein, the term tumor-associated antigen (TAA) refers to an antigen that is differentially expressed by cancer/tumor cells compared to normal cells (i.e., non-tumor cells). Alternatively, a TAA can be an antigen expressed by non-tumor cells (e.g., immune cells) that have pro-tumor effects (e.g., immunosuppressive effects) and can thus be used to target cancer cells. Therefore, a TAA can be any antigen that may stimulate a tumor-specific immune response. Some of these antigens are encoded by normal cells, although not necessarily expressed by normal cells, or expressed by normal cells at a lower level or lower frequency. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are temporarily expressed (such as embryonic antigens and fetal antigens). Other TAAs are encoded by mutated cellular genes, such as oncogenes (e.g., activated ras oncogenes), repressor genes (e.g., mutated p53), and fusion proteins resulting from internal deletions or chromosomal translocations, including neoantigens. Other TAA antigens can also be encoded by viral genes, such as those carried by RNA and DNA oncoviruses. Other TAAs can be expressed on immune cells that can promote or mediate tumorigenesis, such as immune evasion cells, monocytes or macrophages, and, depending on the situation, suppress T cells, regulatory T cells or myeloid-derived suppressor cells.

TAAs are typically normal cell surface antigens that are overexpressed, expressed at abnormal times, or expressed by target cell populations. Ideally, target TAAs would be expressed only on proliferating cells (e.g., tumor cells) or tumor-promoting cells (e.g., immunosuppressive immune cells), but this is rarely observed in practice. Therefore, in many cases, target antigens are selected based on the differential expression between proliferating/disease tissues and healthy tissues.

In one embodiment, the antigen-binding protein in a conjugate with the 4-1BBL ECD mutant protein as described herein includes at least one antigen-binding region that specifically binds to TAA:TROP2.

TROP2 is a transmembrane glycoprotein encoded by the human Tacstd2 gene. The 323-amino acid sequence of human TROP2 is described in NCBI accession number NP_002344, the contents of which are incorporated herein by reference. The human TROP2 mRNA sequence is described in NCBI accession number NM_002353, the contents of which are incorporated herein by reference. TROP2 is an intracellular calcium signaling transducer that is differentially expressed in many cancers. TROP2 plays a role in tumor progression by actively interacting with several key molecular signaling pathways traditionally associated with cancer development and progression. Aberrant overexpression of TROP2 has been described in several solid cancers. TROP2 influences cancer cell growth, proliferation, invasion, metastasis, and survival, thus being associated with tumor invasiveness and poor prognosis. These facts make TROP2 a potential prognostic biomarker for identifying high-risk patients and an attractive therapeutic target for advanced disease.

Therefore, in one embodiment, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein comprises at least one antigen-binding region specifically binding to TROP2, the antigen-binding region comprising a combination of complementary determinant regions (CDRs) selected from the group consisting of: a) the CDR-H1, CDR-H2, and CDR-H3 sequences as defined in SEQ ID NO: 93, and the CDR-L1, CDR-L2, and CDR-L3 sequences as defined in SEQ ID NO: 94 (sacituzumab); b) the CDR-H1, CDR-H2, and CDR-H3 sequences as defined in SEQ ID NO: 338, and the CDR-L1, CDR-L2, and CDR-L3 sequences as defined in SEQ ID NO: 94. The sequences included in SEQ ID NO: 339 (datopotamab); c) the sequences included in SEQ ID NO: 540 (DDR-L1, CDR-L2, and CDR-L3), and the sequences included in SEQ ID NO: 541 (AR46A6); d) the sequences included in SEQ ID NO: 542 (CDR-H1, CDR-H2, and CDR-H3), and the sequences included in SEQ ID NO: 543 (KM4097); and e) the sequences included in SEQ ID NO: 544 (CDR-H1, CDR-H2, and CDR-H3), and the sequences included in SEQ ID NO: 545 (KM4097). The CDR-L1, CDR-L2 and CDR-L3 sequences (K5-70) included in 545.

Therefore, in one embodiment, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein includes at least one antigen-binding region specifically binding to TROP2, the antigen-binding region including a combination of complementary determinant regions (CDRs) selected from the group consisting of: a) CDR-H1 containing the sequence of SEQ ID NO: 552, CDR-H2 containing the sequence of SEQ ID NO: 553, CDR-H3 containing the sequence of SEQ ID NO: 554, CDR-L1 containing the sequence of SEQ ID NO: 555, CDR-L2 containing the sequence of SEQ ID NO: 556, and CDR-L3 containing the sequence of SEQ ID NO: 557 (goxatocilizumab); b) containing the sequence of SEQ ID NO: CDR-H1 containing the sequence of SEQ ID NO: 558, CDR-H2 containing the sequence of SEQ ID NO: 559, CDR-H3 containing the sequence of SEQ ID NO: 560, CDR-L1 containing the sequence of SEQ ID NO: 561, CDR-L2 containing the sequence of SEQ ID NO: 562, and CDR-L3 containing the sequence of SEQ ID NO: 563 (dadabutumab); c) CDR-H1 containing the sequence of SEQ ID NO: 564, CDR-H2 containing the sequence of SEQ ID NO: 565, CDR-H3 containing the sequence of SEQ ID NO: 566, CDR-L1 containing the sequence of SEQ ID NO: 567, CDR-L2 containing the sequence of SEQ ID NO: 568, and CDR-L3 containing the sequence of SEQ ID NO: 569 (KM4097); d) containing the sequence of SEQ ID NO: CDR-H1 of sequence 570, CDR-H2 of sequence SEQ ID NO: 571, CDR-H3 of sequence SEQ ID NO: 572, CDR-L1 of sequence SEQ ID NO: 573, CDR-L2 of sequence SEQ ID NO: 574, and CDR-L3 of sequence SEQ ID NO: 575 (AR47A6.4.2); and e) CDR-H1 of sequence SEQ ID NO: 576, CDR-H2 of sequence SEQ ID NO: 577, CDR-H3 of sequence SEQ ID NO: 578, CDR-L1 of sequence SEQ ID NO: 579, CDR-L2 of sequence SEQ ID NO: 580, and CDR-L3 of sequence SEQ ID NO: 581 (K5-70).

In one embodiment, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein includes at least one antigen-binding region specifically binding to TROP2, the antigen-binding region comprising a combination of variable heavy ( _VH ) and variable light ( _VL ) domains selected from the following groups: a) the VH sequence as contained in SEQ ID NO: 138 and the _VL sequence as contained in SEQ ID NO: 139 (goxatocilizumab); b) the _VH sequence as contained in SEQ ID NO: 338 and _{the VL} sequence as contained in SEQ ID NO: 339 (dadabutumab); c) the _VH sequence as contained in SEQ ID NO: 540 and the _VL sequence as contained in SEQ ID NO: 541 (AR46A6); d) the VH sequence as contained in SEQ ID NO: 542 and the _VL sequence as contained in SEQ ID NO: 543. _L sequence (KM4097); and e) V _H sequence as contained in SEQ ID NO: 544 and V _L sequence (K5-70) as contained in SEQ ID NO: 545.

In one embodiment, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein comprises two antigen-binding regions that specifically bind to TAAs, namely a first and a second antigen-binding region. The first antigen-binding region is primarily a region that specifically binds to the TROP2 antigen-binding region as defined above, and the second antigen-binding region binds to TAAs selected from the group consisting of: 5T4, ADAM9, ADAM10, ADAM12, AFP, ALK, ALPP, ALPP2, and ALPPL2. AXL, angiopoietin-2, apelin receptor, B7-H3, B7-H4, B7-H6, B7.1, B7.2, BCMA, BTLA, CA125, CAIX, CCR4, CCR6, CCR7, CD123, CD133, CD138, CD142, CD147, CD166, CD171, CD19, CD2, CD20, CD205, CD22, CD228, CD2 4. CD25, CD27, CD276, CD3, CD30, CD317, CD33, CD38, CD3E, CD4, CD40, CD44v6, CD45, CD46, CD47, CD 52. CD56, CD70, CD71, CD73, CD74, CD79, CD79B, CD80, CD80/CD86, CDCP1, CDH3, CDK4, CEA, CEACAM5 CLDN18, CLEC14A, CLEC4, CSF1R, CSPG4, CT-7, CTLA4, Calcinin 17, Calcinin 6, CanAg, Necrolin 18.2, Necrolin 6, cMet, Necrolin 37, Cripto-1, Crypto, DC3, DLK1, DLL3, DLL4, DR5, E-Calcinin, E-Selectin, EBV-encoded nuclear antigen (EBNA)-I, EDA, EDB, EDNRB, EGF, EGFR, EGFRvIII, EPCAM, EPHA4, EphA10, EphA2, EphA3, EphB2, EphB4, Extraterrestrial B (Extradomain B, EDB) Fibronectin, F3, FAP, FGFR2, FGFR2b, FGFR4, FOLH1, FOLR1, FRα, FSHR, FcRL5/FcRH5, Fibronectin extradomain B, Flt3, GFRa4, GM3, GPCR5D, GPRC5D, GRP78, GUCY2C, Glycoprotein NMB, Phosphatidylinositol proteoglycan 1, Phosphatidylinositol protein Glycan 2, phosphatidylinositol proteoglycan 3, GnT-V, HAVCR2, HER-2/ERBB2, HER-3/ERBB3, HER-4/ERBB4, HER2, HER3, HER4, HLA-G, HSP70, ICAM-1, IFNG, IGF-1R, IL-1 co-protein, IL-6 receptor, IL-8 receptor, IL13Ra2, IL3RA, Ig-unique, integrin β 6. KAAG-1, KDR, KLK2, KLRC1, Killer lg-like receptor, Killer lg-like receptor 3DL2 (KIR3DL2), L1-CAM, L1CAM, LAG3, LAGE-1, LGR5, LIV-1, Lewis-Y, MART-1/Melan-A, MET, MIC-A/B, MICB, MISIIR, MMP2, MS4A1, MSLN, MUC1, MUC1-C, MUC16, MUM-1, melanocyte transferrin, mesothelin, Mud 6, NAG, NKG2D, NT5E, NTRKR1 (EC 2.7.10.1), NaPi2b, ligand-4, OLR1, OX40, P-calcium mucin, P1A, PD-L1, PD1, PDGF, PDGF α-receptor, PDGF β-receptor, PDGFR, PDGFRA, PLAUR, PRAME, PSCA, PSMA, PTK7, PTPRC, PVRL4, plexiform-A1, RAGE, ROBO1, ROR1, ROR2, SCP-1, SEZ6, SLAMF7, SLC3A2, SSTR2, SSX-1, SSX-2 (HOM-MEL-40), SSX-4, SSX-5, STEAP1, STEAP2, T-cell receptor/CD3-ζ chain, TACSTD2, TGF-α, TIGIT, tissue factor/TF, TM4SF1, TMEFF2, TNFRSF10B, TNFRSF17, TNFRSF4, TNFRSF8, TRAILR1, TRAILR2, TROP2, TSHR, TYRP1, VEGF, VEGFA, VEGFR1, VEGFR2, VH1/VL1, VH2/VL2, VH3/VL3, GAGE tumor antigen, GD2 ganglioside, GM2 ganglioside, RAET1 protein, UL16-binding protein Proteins (ULBP), heterodimeric receptors containing at least one HER subunit, human papillomavirus protein, avβ1 integrin, avβ3 integrin, avβ6 integrin, adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (ADAbp), anti-Müllerian hormone type II receptor, brain glycogen phosphorylase, c-erbB-2, colon-rectal related antigen (CRC)-C017-1A/GA733, gastrin-releasing peptide receptor antigen, gp100, gp75, gpA33, hCG, human papillomavirus protein, integrin receptor, MMP9, muc17, p15, prostate-specific antigen (PSA), protein tyrosine kinase 7 (protein tyrosine kinase 7). The list includes kinase 7 (PTK7), receptor protein tyrosine kinase 3 (TYRO-3), sVE-calcium mucin, scattering factor receptor kinase, α-catenin, α-fetalin, α11bβ3-integrin, β-catenin, and γ-catenin, although this is not exhaustive. Antigen-binding regions with affinity for surface antigens expressed on NK cells .

In one embodiment, the antigen-binding protein in a conjugate with the 4-1BBL ECD mutant protein as described herein may further include a third antigen-binding region, which is an antigen-binding region having or potentially having an affinity for surface antigens expressed on NK cells.

In one embodiment of an antigen-binding protein conjugated with the 4-1BBL ECD mutant protein as described herein, a third antigen-binding region having or potentially having affinity for surface antigens expressed on NK cells comprises or is composed of an immunoglobulin Fc region or at least a portion thereof, which binds to a type III Fcγ receptor (FcγHIIIA) expressed on (human) NK cells, also referred to herein as CD16A. CD16A is an immunoglobulin gamma Fc region receptor (FcγRIIIa) expressed on NK cells, and NK cells recognize IgG bound to the surface of target cells infected with pathogens or expressing TAAs via CD16A. Any naturally occurring isotype, allele, ortholog, or variant is encompassed by the term CD16A polypeptide (e.g., a CD16A polypeptide that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the continuous sequence of at least 20, at least 30, at least 50, at least 100, or at least 200 amino acid residues thereof). The 254 amino acid residue sequence of human CD16A is shown in SEQ ID NO: 55, which corresponds to UniProt accession number P08637, the disclosure of which is incorporated herein by reference.

In one embodiment, the immunoglobulin Fc region includes at least one of the CH2 and CH3 domains. In one embodiment, the immunoglobulin Fc region includes at least one of the CH2 and CH3 domains, and a hinged region. In one embodiment, the immunoglobulin Fc region includes or is composed of a hinged region and the CH2 and CH3 domains. In one embodiment, the immunoglobulin Fc region is a CD16A-binding dimer Fc region or at least a portion thereof.

In one embodiment, the Fc region of CD16A or a portion thereof is a wild-type region or a portion thereof.

In one embodiment, the Fc region or a portion thereof that binds to CD16A can be modified to enhance or reduce its binding affinity to CD16A. Within the Fc region, CD16A binding is mediated by a hinge region and a CH2 domain. For example, in human IgG1, the interaction with CD16 is primarily concentrated on the amino acid residues D265-E269, N297-T299, A327-I332, L234-S239 and the carbohydrate residue N-acetylglucosamine in the CH2 domain (see Sondermann et al., 2000 Nature, 406(6793):267-273). Based on known domains, mutations can be selectively performed to enhance or reduce binding affinity to CD16A, such as by using phage display libraries or yeast surface-displayed cDNA libraries, or by designing based on known three-dimensional structures of interactions. In one embodiment, the Fc region or part thereof is IgG2.

Therefore, in one embodiment, when the antigen-binding protein is intended to have increased affinity for CD16A, the Fc region of CD16A, or a portion thereof, may contain modifications that increase affinity for CD16A. Thus, the Fc region of CD16A, or a portion thereof, may contain one or more amino acid modifications (e.g., amino acid substitution, deletion, insertion) that increase binding to (human) CD16A and, where appropriate, another receptor such as FcRn. Typical modifications include modified human IgG1-derived constant regions containing at least one amino acid modification (e.g., substitution, deletion, insertion) and/or altered glycosylation type, such as hypofucosylation. For example, modifications may increase the binding of the Fc region to FcγRIIIa (CD16A) on NK cells. Examples of modifications are provided in US10,577,419, the disclosure of which is incorporated herein by reference. Specific mutations (in the IgG1 Fc region) that enhance FcyRIIIa (CD16A) binding include E333A, S239D/I332E, and S239D/A330L/I332E.

In one embodiment, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein includes an Fc region or a portion thereof that binds to CD16A, the Fc region or a portion thereof containing at least one amino acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) relative to the wild-type Fc region, such that the molecule has an enhanced binding affinity for (human) CD16A relative to molecules containing the wild-type Fc region, as appropriate. The intermediate variant Fc region contains substitutions at any one or more of locations 239, 298, 330, 332, 333 and/or 334 (e.g., S239D, S298A, A330L, I332E, E333A and/or K334A substitutions), whereby the variant Fc region contains substitutions at the residue S239 and I332, such as S239D and I332E substitutions (Kabat EU designation).

In one embodiment, the antigen-binding protein conjugate with the 4-1BBL ECD mutant protein as described herein includes an Fc region or a portion thereof that binds to CD16A, the Fc region or a portion thereof containing a glycosylation pattern that increases the binding affinity for (human) CD16A. Such carbohydrate modification can be achieved, for example, by expressing nucleic acids encoding antigen-binding proteins in host cells having an altered glycosylation mechanism. Cells having an altered glycosylation mechanism are known in the art and can be used as host cells in which recombinant antibodies are expressed, thereby generating antibodies with altered glycosylation. See, for example, Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, and European Patents: EP 1,176,195; WO 06/133148; WO 03/035835; WO 99/54342, each of which is incorporated herein by reference in its entirety. In one embodiment, the antigen-binding protein comprises one or more hypofucosylated constant regions. Such an antigen-binding protein may or may not contain amino acid modifications and/or may be expressed, synthesized, or treated under conditions leading to hypofucosylation. In one embodiment, in an assembly comprising an antigen-binding protein conjugated with the 4-1BBL ECD mutant protein as described herein, at least 20, at least 30, at least 40, at least 50, at least 60, at least 75, at least 85, at least 90, at least 95%, or substantially all of the antigen-binding protein has a constant region comprising a fucoidan-deficient core carbohydrate structure (e.g., complex, mixed, and high-mannose structure). In one embodiment, an antigen-binding protein is provided that does not contain N-linked glycans and comprises a fucoidan-containing core carbohydrate structure. The core carbohydrate will preferably be a glycochain at Asn297.

In one embodiment, the antigen-binding protein in a conjugate with the 4-1BBL ECD mutant protein as described herein includes an Fc region or a portion thereof that binds to CD16A, the Fc region or a portion thereof being modified to have increased binding affinity for CD16A, having a binding affinity for human CD16A at least 1, 2 or 3 log orders higher than that of known or wild-type human IgG1 antibodies, for example, as assessed by surface plasma resonance.

In another embodiment, when the antigen-binding protein is intended to have reduced affinity for CD16A, the CH2 and/or CH3 domains, the CD16A-binding Fc region, or a portion thereof may contain modifications that reduce affinity for CD16A. For example, a CH2 mutation in the dimer Fc region protein at N297 (Kabat code) can eliminate CD16A binding. Other modifications in the Fc region reduce or eliminate binding to CD16A, including L234A/L235A modification (also known as "LALA"), L235A/G237A modification (also known as "LAGA", and described in Liu et al., Antibodies. 2020; 9(4): 64; Szapacs et al., Bioanalysis. 2023; 15(16): 955–6) and L234A/L235A/P329G modification (also known as LALAPG), and L234A/L235A/P329G modification eliminates CD16 binding more completely than LALA modification. In embodiments where the antigen-binding protein in a conjugate with the 4-1BBL ECD mutant protein as described herein includes an antigen-binding region that specifically binds to the antigenic determinant of γδ TCR, preferably the antigen-binding protein includes an Fc region modified to reduce or eliminate binding to CD16A.

Modifying the Fc region to reduce or eliminate its binding to CD16A can prevent or at least reduce the "sink effect," in which at least a portion of the amount of multispecific antigen-binding protein administered is lost due to binding to NK cells or monocytes. Modifying the Fc region to reduce or eliminate its binding to CD16A can also be used on antigen-binding proteins to further weaken their significant affinity for NK cells, thereby reducing activity in the absence of target cells. Modifying the Fc region to reduce or eliminate its binding to CD16A can also be used on antigen-binding proteins to reduce or avoid NK cell self-killing. The absence of NK cell self-killing is a favorable feature of the antigen-binding proteins described herein. Crossover between NK cells and other NK cells is expected to reduce the therapeutic efficacy of NK cell involvement. Most importantly, NK cells can induce immune cell activation by crossover with one or more NK cells or other immune cells through bivalent or multivalent interactions with FcRy or by combining with secondary immune cell antigens (such as NKp46, NKG2D, NKp30, SLAMF7, or CD38). This can lead to target cell-induced autocytic or immune cell killing (e.g., NK-NK cell lysis), ultimately resulting in depletion of viable NK cells in vivo, as previously described with CD16-guided murine IgG antibodies (3G8), CD38-guided daratumumab, and other methods (Choi et al. 2008 Immunology 124(2) 215-22; DOI: 10.111 l/j.l365-2567.2007.02757.x; Yoshida 2010 Front.Microbiol 1 : 128 DOI: 10.3389/fmicb.2010.00128; Wang et al. 2018 Clin Cancer Res, 24(16): 4006-4017; DOI: 10.1158/1078-0432.CCR-17-3117；His et al. 2008; Nakamura 2013 PNAS; 110(23) 9421-9426; DOI: 10.1073/pnas.1300140110；Breman et al. 2018 Front Immunol, 12(9)2940; DOI: 10.3389/fimmu.2018.02940).

Those skilled in this art will understand that other configurations of Fc region modification can be implemented. For example, substitution of human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 showed a significant reduction in binding to Fcy receptors, thereby reducing ADCC and CDC. Furthermore, Idusogie et al. (2000) J. Immunol. 164(8):4178-84 demonstrated that alanine substitution at different positions (including K322) significantly reduced complement activation.

In one embodiment, the antigen-binding protein in a conjugate with the 4-1BBL ECD mutant protein as described herein includes an Fc region or a portion thereof that binds to CD16A, the Fc region or a portion thereof being modified to have a reduced binding affinity for CD16A, having a binding affinity for human CD16A that is at least 1, 2 or 3 log-orders lower than that of known or wild-type human IgG1 antibodies, for example, as assessed by surface plasma resonance.

In one embodiment, the antigen-binding protein in a conjugate of the 4-1BBL ECD mutant protein as described herein includes an Fc region having an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% amino acid identical to the Fc region of at least one of SEQ ID NOs: 1, 3, 5, 7, 9, 11-19 and 23, and preferably has one or more of the above structural and/or functional features.

In one embodiment, in an antigen-binding protein conjugated with the 4-1BBL ECD mutant protein as described herein, the third antigen-binding region may be an antigen-binding region that specifically binds to NK cell activation receptors, such as the antigen-binding regions described above regarding the second antigen-binding region. In one embodiment, at least one of the second or third antigen-binding regions activates an NK cell activation receptor. Additional activators that activate NK cells.

In one embodiment, the antigen-binding protein in the conjugate of the 4-1BBL ECD mutant protein as described herein contains at least one additional agonist, which may be at least one other NK cell activating cytokine besides the 4-1BBL ECD mutant protein.

In one embodiment of the conjugate, the additional activator is a heterologous portion. In another embodiment of the conjugate, the additional activator is directly attached to the heterologous portion, or the additional activator is attached to the heterologous portion via a linker, preferably a (flexible) peptide linker as described above, such as (GGGGS) _n , wherein the replication number "n" can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In one embodiment of the conjugate, the additional NK cell activating cytokines may be selected from the group consisting of: IL-21 receptor agonists, IL-15 receptor agonists, IL-2 receptor agonists, type I interferon (IFN-1) receptor agonists, IL-12 receptor agonists, and IL-18 receptor agonists, as further described below.

In one embodiment, the conjugate of the 4-1BBL ECD mutant protein as described herein therefore contains at least an IL-21 receptor (IL-21R) agonist.

Interleukin-21 (IL-21) is a protein encoded by the IL-21 gene in humans (Entrez Gene ID: 59067). IL-21 is a cytokine that plays a powerful regulatory role in immune system cells (including natural killer (NK) cells) and induces cell division/proliferation in its target cells. The amino acid sequences (including their signaling sequences) of the human IL-21 precursor are described in NCBI accessions NP_001193935 and NP_068575, the disclosures of which are incorporated herein by reference. IL-21 (matured/processed) comprises amino acids 30–153 of NP_001193935 or amino acids 30–162 of NP_068575 (i.e., SEQ ID NO: 38). IL-21 exerts its effects on target cells through the IL-21 receptor (IL-21R), which is expressed on the surface of T cells, B cells, and NK cells. IL-21R is structurally similar to the receptors of other type I cytokines (such as IL-2R or IL-15) and requires dimerization with a common gamma chain (γc) to bind to IL-21. In humans, IL-21R is encoded by the IL-21R gene (Entrez Gene ID: 50615). The amino acid sequence of human IL-21R is described in NCBI accessions NP_068570, NP_851564, and NP_851565, the contents of which are incorporated herein by reference.

As used herein, an "IL-21R agonist" is an agent with "agonist" activity toward the IL-21 receptor, meaning that the agent can induce or increase "IL-21R signaling." "IL-21R signaling" refers to the ability of IL-21R to activate or transduce intracellular signaling pathways, for example, when expressed on the surface of T cells, B cells, and NK cells and triggered by its natural ligand IL-21. "Natural ligand IL-21" in this context should be understood as human wild-type IL-21 containing or composed of the aforementioned amino acid sequence. When bound to IL-21, the IL-21 receptor acts via the Jak/STAT pathway, utilizing Jak1 and Jak3, as well as the STAT3 homodimer, to activate its target genes. IL-21R agonist activity, i.e. changes in IL-21R signaling activity, can be measured, for example, by tests designed to measure changes in IL-21R signaling pathways. These tests may include monitoring phosphorylation of signal transduction components, measuring the binding of certain signal transduction components to other proteins or intracellular structures, measuring the biochemical activity of components such as kinases, measuring the expression of reporter genes under the control of IL-21R-sensitive promoters and enhancers, or indirectly through downstream effects mediated by IL-21R (activation of specific cell lysis mechanisms in NK cells or γδ T cells). Suitable cell-based assays for the bioactivity of the IL-21R agonist are described, for example, in Maurer et al. (mAbs. 2012; 4(1): 69–83.), in which a mouse preB cell line was transfected with the human IL-21R and STAT-responsive luciferase reporter gene. The activity of the IL-21R agonist was determined using this cell line by measuring the level of STAT3 phosphorylation using anti-pSTAT3 antibody-coupled beads and/or by detecting luciferase luminescence after the cell line was exposed to the IL-21R agonist. The natural ligand IL-21 can be used as a positive control in the assay of IL-21R agonist activity, and can also be used as a reference for determining the IL-21R agonist activity of non-natural IL-21R agonists (such as conjugates containing IL-21R agonists as described herein).

In one embodiment, the conjugate as described herein comprises an IL-21R agonist having reduced IL-21R agonist activity compared to human wild-type IL-21. In one embodiment, the IL-21R agonist has IL-21R agonist activity that is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 10000, or 100000 times lower than that of human wild-type IL-21.

In one embodiment, the conjugate as described herein comprises an IL-21R agonist that has enhanced IL-21R agonist activity compared to human wild-type IL-21. In one embodiment, the IL-21R agonist has IL-21R agonist activity that is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 10000, or 100000 times higher than human wild-type IL-21.

In one embodiment, the conjugate as described herein comprises an IL-21R activator, which is an IL-21 polypeptide comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 38, and preferably having the IL-21R activator activity as defined above, and/or preferably having an affinity for IL-21R as defined below.

In one embodiment, the conjugate as described herein contains an IL-21R activator that has a decreased or increased affinity for IL-21R compared to human wild-type IL-21. The affinity of the IL-21R activator for IL-21R can be determined using methods commonly known in this art, such as surface plasma resonance.

In one embodiment, the conjugate as described herein comprises an IL-21R agonist having a reduced affinity for IL-21R compared to human wild-type IL-21. In one embodiment, the IL-21R agonist has an affinity for IL-21R that is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times lower than that for human wild-type IL-21.

In one embodiment, the conjugate as described herein comprises an IL-21R agonist having an enhanced affinity for IL-21R compared to human wild-type IL-21. In one embodiment, the IL-21R agonist has an affinity for IL-21R that is 2, 5, 10, 20, 50, 100, 200, 500, or 1000 times greater than that for human wild-type IL-21.

In one embodiment, the conjugate as described herein comprises an IL-21R agonist, which is IL-21 or a fragment thereof having IL-21R agonist activity. Preferably, the IL-21R agonist is human IL-21 or a fragment thereof having IL-21R agonist activity. In one embodiment, the IL-21R agonist is an IL-21 mutant protein having a reduced affinity for IL-21R compared to human wild-type IL-21. The IL-21 mutant protein with reduced affinity for IL-21R compared to human wild-type IL-21 is described in one of the following: Shen et al. (Front Immunol. 2020; 11: 832), WO2019/028316, WO2006/111524, WO2008/049920 and a concurrent application by the same applicant, reference number P62009936EP. Therefore, in one embodiment, the IL-21R agonist is an IL-21 mutant protein having a mutation (i.e., amino acid substitution, deletion, or insertion) of one or more of the following amino acids: D4, R5, I8, R9, R11, Q12, L13, I14, D15, I16, D18, Q19, L20, K21, Y23, R65, I66, I67, N68, V69, S70, K72, K73, L74, K75, R76, K77, P78, P79, S80, E100, E109, R110, K112, S113, Q116, K117, I119, H120, and L123 (amino acid positions refer to SEQ ID NO: (The position in 38 or the corresponding position in the IL-21 allele variant).

In one embodiment, the IL-21R agonist is an IL-21 mutant protein comprising at least one amino acid substitution, deletion, or insertion selected from the group consisting of: (N82-A83-G84-R85-R86-Q87-K88-), L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65-, and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11E, R11Q, R11N, R 11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, K21H, K21N, K21Q, K21E, K21D , I67T, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74E, L74K, L74R, L7 4N, L74Q, L74S, L74P, K75E, K75Q, K75N, K75S, K75-, K76-, K112H, K112N, K112 Q, K112E, K112D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N59-, T60-, G61-, N62-, N63-, E64-, R65-, and I66-), (K75- and R76-), (K75-, R76-, and R77-), G84 insGGGG and K75 insX (where X is one, two, or three amino acids selected from the group consisting of G, S, and D). In one embodiment, the IL-21 mutant protein does not contain any amino acid sequence modifications other than at least one amino acid substitution, deletion, or insertion selected from the group consisting of: (N82- A83- G84- R85- R86- Q87- K88-), L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L 74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11E, R11Q, R11N, R 11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, K21H, K21N, K21Q, K21E, K21D , I67T, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74E, L74K, L74R, L7 4N, L74Q, L74S, L74P, K75E, K75Q, K75N, K75S, K75-, K76-, K112H, K112N, K112 Q, K112E, K112D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG and K75 insX (where X is one, two or three amino acids selected from the group consisting of G, S and D).

In one embodiment, the IL-21R agonist is an IL-21 mutant protein having at least one, two, three, four, five, six, seven, or eight amino acids missing in the regions N59 to I66, or having at least one, two, three, four, five, six, seven, or eight amino acids missing in the regions N82 to R90. In one embodiment, the IL-21R agonist is an IL-21 mutant protein with deletions of (N59-, T60-, G61-, N62-, N63-, E64-, R65-, and I66-), (N63-, E64-, R65-, and I66-), (N82-, A83-, G84-, R85-, R86-, Q87-, and K88-), and (N82-, A83-, G84-, R85-, R86-, Q87-, K88-, H89-, and R90-).

In one embodiment, the IL-21R agonist is an IL-21 mutant protein having an insertion of at least two, three, or four amino acids immediately adjacent to the C-terminus to G84. In another embodiment, the IL-21 mutant protein comprises an insertion of at least one, two, three, or four glycine acids immediately adjacent to the C-terminus to G84 (with increasing preference).

In one embodiment, the IL-21R agonist is an IL-21 mutant protein comprising at least one amino acid substitution, deletion, or insertion selected from those listed in Table C. Table C. Additional Substitutions, Deletions, and Insertions of IL-21 Mutants Location WT Additional mutation preferences (preference gradually decreases from left to right) 4 D R, K > Q, N > all others 8 I F, W, Y, L > All others 11 R Q, N, Y > All others 12 Q R > All others 13 L S, V, T, G > All others 19 Q E, K, R, H > G, S, T > all others 20 L D, E, R, K > Q, H > G > all others twenty one K N, Q, E, D > All others 59 N G, P, S, D > All others 60 T G, P, S, D > All others 61 G P, S, D > All others 62 N G, P, S, D > All others 63 N G, P, S, D > All others 64 E G, P, S > All others 65 R All the rest 66 I R, K, E, H > Q, N, Y > all others 67 I D, E, K, R > N, Q > S, G > All others 74 L E, K, R > N, Q > S, P > all others 75 K Q, N, S, missing > all others 84 G Insertion of any combination of G, S, P, D 112 K N, Q, E, D > All others

In one embodiment, the IL-21 mutant protein binds to the human IL-21 receptor with a reduced affinity relative to wild-type IL-21, wherein preferably the IL-21 mutant protein binds to the human IL-21 receptor having the amino acid sequence of SEQ ID NO: 173 with the reduced affinity, or the IL-21 mutant protein binds to the IL-21 receptor γ chain having the amino acid sequence of SEQ ID NO: 174 with the reduced affinity. Therefore, in one embodiment, the IL-21 mutant protein comprises at least one amino acid substitution or deletion selected from the group consisting of: (N82-A83-G84-R85-R86-Q87-K88-); L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63-E64-R65-I66-); and L74F. In one embodiment, the IL-21 mutant protein does not contain any modifications other than substitution or deletion of at least one amino acid selected from the following group: (N82-A83-G84-R85-R86-Q87-K88-), L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63-E64-R65-I66-); and L74F.

In one embodiment, the IL-21 mutant protein provided herein exhibits a binding affinity for human IL-21R, expressed as _pKD , which is at least 0.5 lower than the _pKD of wild-type IL-21 for IL-21R. In one embodiment, the IL-21 mutant protein has a _pKD of at least 0.5 lower than the _pKD of wild-type IL-21 for IL-21R, and the IL-21 mutant protein is an IL-21 mutant protein comprising at least one amino acid substitution or deletion selected from the group consisting of: (N82-A83-G84-R85-R86-Q87-K88-); L20W; R5H; I8V; I8Q; Q12K; K73Y; L13E; D4H; I8H; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutant protein does not contain any amino acid sequence modifications other than substitutions or deletions of at least one amino acid selected from the following group: (N82- A83- G84- R85- R86- Q87- K88-); L20W; R5H; I8V; I8Q; Q12K; K73Y; L13E; D4H; I8H; I67N; L74D; L20S; and L20N.

In one embodiment, the IL-21 mutant protein provided herein exhibits a binding affinity for human IL-21R, expressed as _pKD , which is at least 1.0 lower than the _pKD of wild-type IL-21 for IL-21R. In one embodiment, the IL-21 mutant protein has a _pKD of at least 1.0 lower than the _pKD of wild-type IL-21 for IL-21R, and the IL-21 mutant protein is an IL-21 mutant protein comprising at least one amino acid substitution or deletion selected from the group consisting of: (N82-A83-G84-R85-R86-Q87-K88-); L20W; R5H; I8Q; Q12K; K73Y; L13E; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutant protein does not contain any amino acid sequence modifications other than substitutions or deletions of at least one amino acid selected from the following group: (N82- A83- G84- R85- R86- Q87- K88-); L20W; R5H; I8Q; Q12K; K73Y; L13E; I67N; L74D; L20S; and L20N.

In one embodiment, the IL-21 mutant protein provided herein exhibits a binding affinity for human IL-21R, expressed as pKD, which is at least 1.6 lower than the _pKD of wild-type IL-21 for IL-21R. In one embodiment, the IL-21 mutant protein with a _pKD at least 1.6 lower than the _pKD of wild-type IL-21 for IL-21R is an IL-21 mutant protein comprising at least one amino acid substitution or deletion selected from the group consisting of: (N82-A83-G84-R85-R86-Q87-K88-); L20W; R5H; Q12K; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutant protein does not contain any amino acid sequence modifications other than substitutions or deletions of at least one amino acid selected from the following group: (N82- A83- G84- R85- R86- Q87- K88-); L20W; R5H; Q12K; I67N; L74D; L20S; and L20N.

In one embodiment, an antigen-binding protein in a conjugate as described herein is provided, the antigen-binding protein comprising a combination of the 4-1BBL ECD mutant protein as described herein and the IL-21 mutant protein as described herein, as shown in Table D.

Table D. Combinations of 4-1BBL mutant proteins and IL-21 mutant proteins coupled with antigen-binding proteins. AVC-ID 4-1BBL ECD mutant protein IL-21 mutant protein AVC72 V100T R9E AVC73 Q227R R9E AVC74 Q230S R9E AVC75 V100T R76A AVC76 Q227R R76A AVC77 Q230S R76A AVC78 V100T I8Q AVC79 Q227R I8Q AVC80 Q230S I8Q AVC85 Q227E R9E R76A AVC86 Q227E L13E AVC87 V153Q L13E I8H K21H D4H AVC88 V153Q D4H I8H K21H AVC89 V153Q L13E I8H K21H AVC90 V153Q L13E D4H I8H AVC91 V153Q I8H K21H AVC92 V153Q L13E D4H K21H AVC93 V153Q D4H I8H AVC94 V153Q L13E I8H AVC95 V153Q D4H K21H AVC96 V153Q L13E K21H AVC97 V153Q L13E D4H AVC98 Y110Q V153Q Q227E I8H AVC99 L101N Y110Q V153Q I8H AVC100 V100Q Y110Q V153Q I8H AVC101 L101N V153Q Q227E I8H AVC102 Y110Q A154D Q227E I8H AVC103 Y110Q V153Q I8H AVC104 V153Q Q227E I8H AVC105 Y110Q Q227E I8H AVC106 L101N Q227E I8H AVC107 V153Q I8H AVC176 A154D L74D AVC187 A154D L20N AVC188 A154D L20W AVC189 A154D L20S AVC190 A154D N82- A83- G84- R85- R86- Q87- K88- AVC285 A154E N82- A83- G84- R85- R86- Q87- K88- AVC286 A154D + G155Q L20W AVC287 A154E L20W AVC288 A154D +G155Q N82- A83- G84- R85- R86- Q87- K88-

In one embodiment, the conjugate as described herein comprises a combination of 4-1BBL ECD mutant protein and IL-21 mutant protein, wherein the 4-1BBL ECD mutant protein is selected from the group consisting of: 4-1BBL mutant protein A154D; 4-1BBL mutant protein A154E; 4-1BBL mutant protein V153Q; 4-1BBL mutant protein Q227E; 4-1BBL mutant protein Q227R; 4-1BBL mutant protein L101N; 4-1BBL mutant protein Y110Q; 4-1BBL mutant protein Q230K; and 4-1BBL mutant protein V100Q; and wherein the IL-21 mutant protein is selected from the group consisting of: IL-21 mutant protein (N82-A83-G84-R85-R86-Q87- K88-); IL-21 mutant L20W; IL-21 mutant L74D; IL-21 mutant L20N; IL-21 mutant I67N; IL-21 mutant L20S; IL-21 mutant L13E; IL-21 mutant I8H; IL-21 mutant (N63- E64- R65- I66-); and IL-21 mutant L74F. 4-1BBL and IL-21 mutants should be understood herein to contain at least the substitutions or deletions shown, or not to contain any other amino acid sequence modifications besides the substitutions or deletions shown. In one embodiment of the symbiont, the 4-1BBL ECD mutant protein and the IL-21 mutant protein are each coupled to an antigen-binding protein, wherein preferably the 4-1BBL ECD mutant protein exists as a heterotrimer or homotrimer of 4-1BBL ECD linked by a polypeptide linker.

In a specific embodiment, the conjugate comprises a combination of 4-1BBL ECD mutant proteins and IL-21 mutant proteins selected from the following groups: 4-1BBL mutant protein A154D and IL-21 mutant protein (N82-A83-G84-R85-R86-Q87- K88-); 4-1BBL mutant protein A154D and IL-21 mutant protein L20W; 4-1BBL mutant protein A154D and IL-21 mutant protein L74D; 4-1BBL mutant protein A154D and IL-21 mutant protein L20N; 4-1BBL mutant protein A154D and IL-21 mutant protein I67N; 4-1BBL mutant protein A154D and IL-21 mutant protein L20S; 4-1BBL mutant protein A154D and IL-21 mutant protein L13E; 4-1BBL mutant protein A154D and IL-21 mutant protein I8H; 4-1BBL mutant protein A154D and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein A154D and IL-21 mutant protein L74F; 4-1BBL mutant protein A154E and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein A154E and IL-21 mutant protein L20W; 4-1BBL mutant protein A154E and IL-21 mutant protein L74D; 4-1BBL mutant protein A154E and IL-21 mutant protein L20N; 4-1BBL mutant protein A154E and IL-21 mutant protein I67N; 4-1BBL mutant protein A154E and IL-21 mutant protein L20S; 4-1BBL mutant protein A154E and IL-21 mutant protein L13E; 4-1BBL mutant protein A154E and IL-21 mutant protein I8H; 4-1BBL mutant protein A154E and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein A154E and IL-21 mutant protein L74F; 4-1BBL mutant protein V153Q and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein V153Q and IL-21 mutant protein L20W; 4-1BBL mutant protein V153Q and IL-21 mutant protein L74D; 4-1BBL mutant protein V153Q and IL-21 mutant protein L20N; 4-1BBL mutant protein V153Q and IL-21 mutant protein I67N; 4-1BBL mutant protein V153Q and IL-21 mutant protein L20S; 4-1BBL mutant protein V153Q and IL-21 mutant protein L13E; 4-1BBL mutant protein V153Q and IL-21 mutant protein I8H; 4-1BBL mutant protein V153Q and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein V153Q and IL-21 mutant protein L74F; 4-1BBL mutant protein Q227E and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein Q227E and IL-21 mutant protein L20W; 4-1BBL mutant protein Q227E and IL-21 mutant protein L74D; 4-1BBL mutant protein Q227E and IL-21 mutant protein L20N; 4-1BBL mutant protein Q227E and IL-21 mutant protein I67N; 4-1BBL mutant protein Q227E and IL-21 mutant protein L20S; 4-1BBL mutant protein Q227E and IL-21 mutant protein L13E; 4-1BBL mutant protein Q227E and IL-21 mutant protein I8H; 4-1BBL mutant protein Q227E and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein Q227E and IL-21 mutant protein L74F; 4-1BBL mutant protein Q227R and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein Q227R and IL-21 mutant protein L20W; 4-1BBL mutant protein Q227R and IL-21 mutant protein L74D; 4-1BBL mutant protein Q227R and IL-21 mutant protein L20N; 4-1BBL mutant protein Q227R and IL-21 mutant protein I67N; 4-1BBL mutant protein Q227R and IL-21 mutant protein L20S; 4-1BBL mutant protein Q227R and IL-21 mutant protein L13E; 4-1BBL mutant protein Q227R and IL-21 mutant protein I8H; 4-1BBL mutant protein Q227R and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein Q227R and IL-21 mutant protein L74F; 4-1BBL mutant protein L101N and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein L101N and IL-21 mutant protein L20W; 4-1BBL mutant protein L101N and IL-21 mutant protein L74D; 4-1BBL mutant protein L101N and IL-21 mutant protein L20N; 4-1BBL mutant protein L101N and IL-21 mutant protein I67N; 4-1BBL mutant protein L101N and IL-21 mutant protein L20S; 4-1BBL mutant protein L101N and IL-21 mutant protein L13E; 4-1BBL mutant protein L101N and IL-21 mutant protein I8H; 4-1BBL mutant protein L101N and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein L101N and IL-21 mutant protein L74F; 4-1BBL mutant protein Y110Q and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein Y110Q and IL-21 mutant protein L20W; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L74D; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L20N; 4-1BBL mutant protein Y110Q and IL-21 mutant protein I67N; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L20S; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L13E; 4-1BBL mutant protein Y110Q and IL-21 mutant protein I8H; 4-1BBL mutant protein Y110Q and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein Y110Q and IL-21 mutant protein L74F; 4-1BBL mutant protein Q230K and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein Q230K and IL-21 mutant protein L20W; 4-1BBL mutant protein Q230K and IL-21 mutant protein L74D; 4-1BBL mutant protein Q230K and IL-21 mutant protein L20N; 4-1BBL mutant protein Q230K and IL-21 mutant protein I67N; 4-1BBL mutant protein Q230K and IL-21 mutant protein L20S; 4-1BBL mutant protein Q230K and IL-21 mutant protein L13E; 4-1BBL mutant protein Q230K and IL-21 mutant protein I8H; 4-1BBL mutant protein Q230K and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein Q230K and IL-21 mutant protein L74F; 4-1BBL mutant protein V100Q and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein V100Q and IL-21 mutant protein L20W; 4-1BBL mutant protein V100Q and IL-21 mutant protein L74D; 4-1BBL mutant protein V100Q and IL-21 mutant protein L20N; 4-1BBL mutant protein V100Q and IL-21 mutant protein I67N; 4-1BBL mutant protein V100Q and IL-21 mutant protein L20S; 4-1BBL mutant protein V100Q and IL-21 mutant protein L13E; 4-1BBL mutant protein V100Q and IL-21 mutant protein I8H; 4-1BBL mutant protein V100Q and IL-21 mutant protein (N63- E64- R65- I66-); as well as the 4-1BBL mutant protein V100Q and the IL-21 mutant protein L74F.

In one embodiment, a conjugate comprising the combination of 4-1BBL ECD mutant protein and IL-21 mutant protein is a conjugate that, when composed of trastuzumab conjugated with IL-21 mutant protein and 4-1BBL ECD mutant protein, induces maximum NK cell proliferation at a saturation concentration of 25 nM in the presence of SKOV3 tumor cells in a normalized 5-day NK cell proliferation assay, with the proliferation being at least 60% of that induced by a corresponding control conjugate comprising wild-type IL-21 and wild-type 4-1BBL in the same assay, wherein the 4-1BBL ECD mutant protein exists as a homotrimer, wherein the ECD monomers are linked via (GGGGS) ₄ linkers. Therefore, in one embodiment, the conjugate comprises a combination of 4-1BBL ECD mutant proteins and IL-21 mutant proteins selected from the group consisting of: 4-1BBL mutant protein A154D and IL-21 mutant protein (N82-A83-G84-R85-R86-Q87-K88-); 4-1BBL mutant protein A154D and IL-21 mutant protein L20W; 4-1BBL mutant protein A154D and IL-21 mutant protein L20S; 4-1BBL mutant protein A154E and IL-21 mutant protein (N82-A83-G84-R85-R86-Q87- K88-); 4-1BBL mutant protein A154E and IL-21 mutant protein L20W; and 4-1BBL mutant protein A154E and IL-21 mutant protein L20S.

In one embodiment, a conjugate comprising the combination of 4-1BBL ECD mutant protein and IL-21 mutant protein is a conjugate that, when composed of trastuzumab conjugated with IL-21 mutant protein and 4-1BBL ECD mutant protein, induces maximum NK cell proliferation at a saturation concentration of 25 nM in the presence of SKOV3 tumor cells in a normalized 5-day NK cell proliferation assay, with the proliferation being at least 89% of that induced by a corresponding control conjugate comprising wild-type IL-21 and wild-type 4-1BBL in the same assay, wherein the 4-1BBL ECD mutant protein exists as a homotrimer, wherein the ECD monomers are linked via (GGGGS) ₄ linkers. Therefore, in one embodiment, the conjugate comprises a combination of 4-1BBL ECD mutants and IL-21 mutants selected from the group consisting of: 4-1BBL mutant A154D and IL-21 mutant (N82-A83-G84-R85-R86-Q87-K88-); 4-1BBL mutant A154D and IL-21 mutant L20W; 4-1BBL mutant A154E and IL-21 mutant (N82-A83-G84-R85-R86-Q87-K88-); and 4-1BBL mutant A154E and IL-21 mutant L20W.

In one embodiment, a conjugate comprising the combination of 4-1BBL ECD mutant protein and IL-21 mutant protein is a conjugate that, when composed of trastuzumab conjugated with IL-21 mutant protein and 4-1BBL ECD mutant protein, induces maximum NK cell proliferation at a saturation concentration of 25 nM in the presence of SKOV3 tumor cells in a normalized 5-day NK cell proliferation assay, with the proliferation being at least 105% of that induced in the same assay by a corresponding control conjugate comprising wild-type IL-21 and wild-type 4-1BBL, wherein the 4-1BBL ECD mutant protein exists as a homotrimer, wherein the ECD monomers are linked via (GGGGS) ₄ linkers. Therefore, in one embodiment, the conjugate comprises the following combination: 4-1BBL mutant protein A154D and IL-21 mutant protein (N82-A83-G84-R85-R86-Q87-K88-).

In one embodiment, the conjugate as described herein is an IL-21R agonist comprising an antigen-binding region that specifically binds to IL-21R and has IL-21R agonist activity. The antigen-binding region may be an antigen-binding region as described above herein.

In one embodiment, the conyzosome as described herein contains more than one IL-21R agonist as described above. Therefore, in one embodiment, the conyzosome has an IL-21R agonist valence higher than one. The IL-21R agonist valence of the conyzosome can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or higher. The structure of the conyzosome containing the 4-1BBL ECD mutant protein and the antigen-binding protein is described.

In one embodiment, in a conjugate comprising the 4-1BBL ECD mutant protein and an antigen-binding protein as described herein, at least one of the first and second antigen-binding regions (specifically binding TAAs or NK cell activation receptors) is coupled to a third antigen-binding region that has or may have affinity for surface antigens expressed on NK cells. Preferably, at least one of the first and second antigen-binding regions that specifically binds TAAs or NK cell activation receptors is coupled to at least one polypeptide chain of the third antigen-binding region. The coupling of the two domains/regions should be understood to mean that they are covalently linked to each other. These two domains/regions can be chemically crosslinked to each other using crosslinking agents to link the two protein molecules, as is well known in the art. Many commercially available crosslinking reagents are available for the preparation of protein or peptide bioconjugates. Many of these crosslinking reagents allow biomolecules to undergo dimeric homo- or hetero-coupling via free amines or hydroxyl groups in the protein side chains. Other crosslinking methods involve coupling via carbohydrate groups to acehydrazine moieties. For crosslinking the first and/or second binding regions to the third binding region, crosslinking agents with heterofunctional specificity are preferably used. In one embodiment, the crosslinking agent includes flexible spacers to provide flexibility or freedom of movement between the two regions relative to each other.

However, in one embodiment, at least one of the first and second antigen-binding regions is coupled to the third antigen-binding region by being contained in a single polypeptide chain. Since the antigen-binding region may contain two polypeptide chains, such as _VH and _VL chains, in one embodiment, at least one polypeptide chain of the antigen-binding region specifically binding to a TAA or NK cell activation receptor forms a single polypeptide chain with at least one polypeptide chain of the second antigen-binding region. Similarly, since the third antigen-binding region, having or potentially having affinity for surface antigens expressed on NK cells, may also contain two polypeptide chains, such as the dimer Fc region of an antibody, in one embodiment, at least one polypeptide chain of the third antigen-binding region forms a single polypeptide chain with at least one polypeptide chain of at least one of the first and second antigen-binding regions.

Therefore, in one embodiment, the conjugate comprising the 4-1BBL ECD mutant protein and antigen-binding protein as described herein comprises a single polypeptide chain comprising, in order from N-terminus to C-terminus: i) at least one polypeptide chain of at least one of the first and second antigen-binding regions; ii) a flexible linker, if applicable; and iii) at least one polypeptide chain of a third antigen-binding region. The flexible linker may be an immunoglobulin hinge region or a linker as described herein.

In one embodiment, the third antigen-binding region, i.e., the domain having or potentially having affinity for surface antigens expressed on NK cells, is a dimer immunoglobulin Fc region, wherein each of the two polypeptide chains of the Fc region is linked to a CH1 domain, and each CH1 domain is linked to at least one of the immunoglobulin variable regions (specifically binding to TAAs or NK cell activation receptors) of the first and second antigen-binding regions. The dimer immunoglobulin Fc region is preferably a dimer of the Fc region as described above herein. The immunoglobulin variable region may be scFv, _VH domain, _VL domain, or immunoglobulin single variable domain (ISVD), such as dAb, V-NAR domain, or _VHH domain. In one embodiment, the immunoglobulin variable region linked to the CH1 domain is a _VH domain paired with the _VL domain, which is linked to a Cκ or _Cλ domain. In this embodiment, preferably, the _VH and _VL domains together specifically bind to TAA or NK cell activation receptors.

In one embodiment, both immunoglobulin variable regions bind to the same TAA. In another embodiment, each of the two immunoglobulin variable regions binds to a different TAA. In one embodiment, the first immunoglobulin variable region binds to a TAA and the second immunoglobulin variable region binds to an NK cell activation receptor. In one embodiment, both the first and second immunoglobulin variable regions bind to NK cell activation receptors. The two immunoglobulin variable regions may each bind to a different NK cell activation receptor, or they may both bind to the same NK cell activation receptor.

In terms of their specificity, conjugates comprising the 4-1BBL ECD mutant protein and antigen-binding protein as described herein can therefore be homodimers having two identical immunoglobulin variable regions, both of which bind the same TAA. Alternatively, conjugates comprising the 4-1BBL ECD mutant protein and antigen-binding protein as described herein can therefore be heterodimers in terms of specificity to TAAs and/or NK cell activation receptors, wherein each of the two immunoglobulin variable regions binds a different TAA or a TAA and NK cell activation receptor. In embodiments where the antigen-binding protein is bispecific, it is preferable that one of the two immunoglobulin variable regions is an immunoglobulin monovariable domain, while the other immunoglobulin variable region is not. Secondly, the assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which may lead to the assembly of homodimers and heterodimers of each antibody heavy chain. The preferential assembly of heterodimers can be promoted by incorporating different mutations into the CH3 domain of the constant region of each antibody heavy chain, as shown in US13/494,870, US16/028850, US11/533,709, US12/875,015, US13/289,934, US14/773,418, US12/811,207, US13/866,756, US14/647,480, US14/830,336, and WO2019/195409. For example, mutations can be made in the CH3 domain of human IgG1, and different amino acid substitution pairs can be incorporated into the first and second polypeptides, causing the two chains to selectively heterodimerize with each other. For example, a CH3 domain containing amino acid substitutions, wherein a mutation occurs at the CH3 domain interface of the antibody Fc region to produce a changed charge polarity at the Fc dimer interface, such that the co-expression of electrostatically matched Fc chains supports favorable attractive interactions, thereby promoting the formation of desired Fc heterodimers, while unfavorable repulsive charge interactions inhibit the formation of unwanted Fc homodimers.

In one embodiment, a "mortar and pestle" approach is used, wherein a mutation occurs at the CH3 domain interface of the antibody's Fc region, causing the antibody to preferentially form a heterodimer (further including attached light chains). This mutation produces altered charge polarity at the Fc dimer interface, resulting in electrostatically matched Fc chains co-expressing favorable attractive interactions that promote the formation of the desired Fc heterodimer, while unfavorable repulsive charge interactions inhibit the formation of the unwanted Fc homodimer. For example, one heavy chain contains a T366W substitution, while the second heavy chain contains T366S, L368A, and Y407V substitutions, see, for example, Ridgway et al. (1996) Protein Eng., 9, pp. 617-621; Atwell (1997) J. Mol. Biol., 270, pp. 26-35; and WO2009/089004, the disclosure of which is incorporated herein by reference. In another approach, one heavy chain contains an F405L substitution, while the second heavy chain contains a K409R substitution, see, for example, Labrijn et al. (2013) Proc. Natl. Acad. Sci. USA, 110, pp. 5145-5150. In another approach, one heavy chain comprises substitutions of T350V, L351Y, F405A, and Y407V, while the second heavy chain comprises substitutions of T350V, T366S, K392L, and T394W, see, for example, Von Kreudenstein et al., (2013) mAbs 5:646-654. In yet another approach, one heavy chain comprises substitutions of K409D and K392D, while the second heavy chain comprises substitutions of D399K and E356K, see, for example, Gunasekaran et al., (2010) J. Biol. Chem. 285:19637-19646. In another approach, one heavy chain contains D221E, P228E, and L368E substitutions, while the second heavy chain contains D221R, P228R, and K409R substitutions, see, for example, Strop et al., (2012) J. Mol. Biol. 420: 204-219. In another approach, one heavy chain contains S364H and F405A substitutions, while the second heavy chain contains Y349T and T394F substitutions, see, for example, Moore et al., (2011) mAbs 3: 546-557. In yet another approach, one heavy chain contains H435R substitution, while the second heavy chain may or may not contain substitutions, see, for example, U.S. Patent No. 8,586,713. When such heteropolymeric antibodies possess an Fc region derived from human IgG2 or IgG4, the Fc region of these antibodies can be engineered to contain amino acid modifications that allow or prevent CD16 binding. In some embodiments, the antibody may include glycosylation of the mammalian antibody type N-link at residue N297 (Kabat EU number).

In a preferred embodiment, the conjugate comprising the 4-1BBL ECD mutant protein and the antigen-binding protein as described herein includes a dimer immunoglobulin Fc region, which is a (homo- or hetero-)dimer of at least one Fc region as described above herein, wherein each of the two Fc polypeptide chains is operatively linked to a Fab that specifically binds to a TAA or NK cell activation receptor. In addition to the presence of the 4-1BBL ECD mutant protein (and, where applicable, other agonists), the antigen-binding protein in this conjugate comprising dimer Fc linked to the two Fabs can thus form an immunoglobulin structure, such as the known IgG immunoglobulin.

In one embodiment, in a conjugate comprising the 4-1BBL ECD mutant protein and an antigen-binding protein as described herein, at least one of the 4-1BBL ECD mutant protein and the additional agonist is coupled to at least one of the first and second antigen-binding regions of a specific TAA or NK cell activation receptor, or to a third antigen-binding region. As mentioned above, the coupling of two protein entities should be understood to mean that they are covalently linked to each other, which can be achieved by chemical crosslinking using a crosslinking agent to link the two protein molecules, such that, as is well known in the art, the crosslinking agent may comprise a flexible spacer.

However, in one embodiment, at least one of the 4-1BBL ECD mutant protein and the other activator forms a single polypeptide chain with at least one of the following: i) at least one polypeptide chain of at least one of the first and second antigen-binding regions that specifically bind to TAA or NK cell activation receptors; and ii) at least one polypeptide chain of a second antigen-binding region having or potentially having affinity for surface antigens expressed on NK cells. In one embodiment, a flexible linker (described below) exists between the activator and the region defined in i) or ii).

In one embodiment, at least one of the 4-1BBL ECD mutant protein and another agonist forms a single polypeptide chain with at least one of: i) a light chain in at least one of two Fabs that specifically bind to TAA or NK cell activation receptors; and ii) at least one of two Fc chains in the Fc region of a dimer immunoglobulin. In one embodiment, a flexible linker (described below) exists between at least one of the 4-1BBL ECD mutant protein and another agonist and the light chain defined in i) or the Fc chain defined in ii).

In one embodiment, at least one of the 4-1BBL ECD mutant protein and another agonist is fused with at least one of the following: i) the N-terminus of the light chain of at least one of the two Fabs that specifically bind to TAA or NK cell activation receptors, via a flexible linker, if applicable; ii) the C-terminus of the light chain of at least one of the two Fabs that specifically bind to TAA or NK cell activation receptors, via a flexible linker, if applicable; iii) the N-terminus of the heavy chain of at least one of the two Fabs that specifically bind to TAA or NK cell activation receptors; and iv) the C-terminus of the heavy chain of at least one of the two Fc chains in the Fc region of the dimer immunoglobulin, via a flexible linker, whereby the flexible linker may be as described below.

In one embodiment, the conjugate comprises a (homo- or hetero-)dimeric antigen-binding protein as described above, the dimer possibly comprising a 4-1BBL ECD mutant protein on only one of the two monomers of the dimer and at least one of a further activator, or the dimer possibly comprising a 4-1BBL ECD mutant protein on each (i.e., both) of the two monomers of the dimer and at least one of a further activator. Thus, in one embodiment, the conjugate comprises an immunoglobulin structure, and at least one of the 4-1BBL ECD mutant protein and a further activator may be present on at least one or both sides of the immunoglobulin structure. In embodiments where at least one of the 4-1BBL ECD mutant protein and an additional activator is present on each of the two monomers in the dimer or immunoglobulin structure, the antigen-binding protein in the conjugate may, for example, comprise the 4-1BBL ECD mutant protein on both monomers, an additional activator (e.g., an IL-21R activator) on both monomers, or the 4-1BBL ECD mutant protein on the first monomer and an additional activator (e.g., an IL-21R activator) on the second monomer. It should be understood that when the antigen-binding protein in the conjugate comprises a heterodimeric heavy chain, the "mortar and pestle structure" technique described above can be applied, wherein the CH3 domain of the first chain is modified to have a "protrusion" ("mortar") and the second chain is modified to have a corresponding "cavity" ("mortar").

Therefore, in one embodiment, the conjugate comprising the antigen-binding protein as described herein is a heterodimer for at least one of: i) first and second antigen-binding regions; and ii) at least one of a fused 4-1BBL ECD mutant protein and a fused additional agonist, and the dimer Fc region comprises different first and second polypeptide chains, the first and second polypeptide chains comprising a club-and-mortar structure modification that promotes the bonding of the first and second polypeptide chains in the Fc region.

The amino acid sequences of suitable peptide linkers for linking the various functional domains and regions in a conjugate containing the 4-1BBL ECD mutant protein and antigen-binding protein as described herein are known in the art, as described above herein.

In one embodiment, the conjugate comprising the 4-1BBL ECD mutant protein and antigen-binding protein as described herein is a conjugate as exemplified herein, such as, for example, AVC323, AVC421, AVC245 or AVC267 (see Table 1.1.10.2) or derivatives of AVC72 – AVC107, AVC176, AVC187 – AVC190, AVC285 or AVC287, wherein the trastuzumab variable weight ( _VH ) and variable light ( _VL ) domains are replaced by the variable weight ( _VH ) and variable light ( _VL ) domains of a monoclonal antibody against TROP2, such as goxatozumab, daboteurumab, AR46A6, KM4097 or K5-70 as described above.

In one embodiment, the conjugate comprises a TROP2-targeting antigen-binding protein, the HER2-targeting antigen-binding protein comprising: a) a first heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with any one of SEQ ID NO: 585, 591, and 588; b) a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with any one of SEQ ID NO: 586, 674, 592, and 589; and c) a light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with any one of SEQ ID NO: 584, 593, and 590.

In a preferred embodiment, the conjugate comprises: a) a first heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 585; b) a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 586 or 674; and c) a light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 584. In a preferred embodiment, the conjugate comprises: a) a first heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 585; b) a second heavy chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 586; and c) a light chain comprising an amino acid sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 584. Bioactivity of the conjugate comprising the 4-1BBL ECD mutant protein and the antigen-binding protein.

Conjugates containing the 4-1BBL ECD mutant protein or antigen-binding protein as described herein may have one or more biological activities, including, for example, antigen (TAA, preferably TROP2) binding, binding to NK cells, the ability to direct NK cells to target cells expressing TAA, the ability to activate NK cells, including inducing superfunctional NK cells, and/or the ability to induce (activated/superfunctional) NK cells to lyse target cells.

In one embodiment, a conjugate containing the 4-1BBL ECD mutant protein or antigen-binding protein as described herein induces an increase in at least one of the following NK cell activities: degranulation of CD107a, CD69 expression, IFNγ production, NK cell proliferation, and NK cell cytotoxicity. Preferably, this increase is at least 1.0, 1.1, 1.2, 1.5, 2.0, 5.0, 10, 20, 50, 100, 110, 120, 150, 200, 210, 220, 250, or 300 times greater than that achieved using the same effector: target cell ratio, or using the same NK cells and target cells not in contact with the conjugate containing the 4-1BBL ECD mutant protein or antigen-binding protein.

In one embodiment, a conjugate comprising the 4-1BBL ECD mutant protein or antigen-binding protein as described herein induces an increase in at least one of the following NK cell activities: degranulation of CD107a, CD69 expression, IFNγ production, NK cell proliferation, and NK cell cytotoxicity. Preferably, this increase is at least 1.0, 1.1, 1.2, 1.5, 2.0, 5.0, 10, 20, 50, 100, 110, 120, 150, 200, 210, 220, 250, or 300-fold higher than that achieved using the same effector: target cell ratio, and using the same NK cells and target cells in contact with the reference antigen-binding protein (all other things being equal).

In one embodiment, the reference antigen-binding protein is a known human IgG1 monoclonal antibody that binds the same TAA to a conjugate comprising the 4-1BBL ECD mutant protein and the antigen-binding protein, preferably to the same antigenic determinant, and more preferably to the same TAA-specific antigen-binding region. For example, compared to the monoclonal antibody goxatuzumab, the conjugate comprising the 4-1BBL ECD mutant protein and the antigen-binding protein (having a TROP2 binding region, preferably having a goxatuzumab variable domain) is superior in inducing an increase in NK cell activity.

In one embodiment, the reference antigen-binding protein is a (multispecific) antigen-binding protein comprising at least one antigen-binding region that binds to the same TAA as a conjugate containing a 4-1BBL ECD mutant protein or an antigen-binding protein, preferably binding to the same antigenic determinant, more preferably binding to an antigen-binding region having the same TAA-specific antigen-binding region, and comprising at least one antigen-binding region that specifically binds to NK cell activation receptors (such as NKp46, NKp44, NKp30, NKG2D, DNAM1, and CD16A). In one embodiment, the reference antigen-binding protein is a (multispecific) antigen-binding protein that includes at least one conjugate that binds the same TAA to a 4-1BBL ECD mutant protein or an antigen-binding protein, preferably to the same antigenic determinant, more preferably to an antigen-binding region having the same TAA-specific antigen-binding region, and includes at least one NK cell-activating cytokine other than the 4-1BBL ECD mutant protein. NK cell activating cytokines other than the 4-1BBL ECD mutant protein can be selected from the group consisting of IL-21R agonists, IL-15 receptor agonists, type I interferon (IFN-1) agonists, IL-2 receptor agonists, IL-12 receptor agonists, and IL-18 receptor agonists, as described above. For example, the IL21 mutant protein as described above, or IL-15 receptor agonists, such as IL15 (such as human modified IL-15 crosslinkers described in US2018282386 and Vallera et al. (2016, Clin Cancer Res.; 22(14): 3440–3450)).

In one embodiment, the reference antigen-binding protein is an NK cell binder, such as those described, for example, in WO2016/207278, WO 2018/148445, WO2018/152518, WO2019195409, US2018282386, Vallera et al. (2016, ibid .) and Demaria et al. (2021, ibid .). One example of a (multispecific) reference antigen-binding protein is, for example, AVC-006 as described in WO2024/056862, which includes a HER2-binding region and an NKG2D-binding region.

The detection of NK activation markers or assays for NK cell cytotoxicity, or assays for detecting NK cell activation and cytotoxicity (e.g., short-term and long-term cytotoxicity assays), are described in examples in this paper, and in, for example, in Pessino et al., J. Exp. Med, 1998, 188 (5): 953-960; Sivori et al., Eur J Immunol, 1999.29:1656-1666; Brando et al., (2005) J. Leukoc. Biol.78:359-371; El-Sherbiny et al., (2007) Cancer Research 67(18):8444-9; Nolte-'t Hoen et al., (2007) Blood 109:670-673); WO As described in 2016/207278 and WO 2018/148445.

In one embodiment, a conjugate containing the 4-1BBL ECD mutant protein or antigen-binding protein as described herein has the ability to induce superfunctionality (or superfunctional phenotype) in NK cells or NK cell populations. The superfunctional NK cell phenotype should be understood herein as a phenotype possessing one or more characteristics of a phenotype acquired by amplifying NK cells obtained from a donor, wherein in vitro this is achieved by co-culturing NK cells with irradiated K562 cultured cells modified to express membrane-bound IL-21 (mbIL-21) and 4-1BB ligands (FC21 cultured cells), as described by Denman et al. (2012, ibid .). Thus, in one embodiment, by co-culturing with NK cells containing 4-1BB ligands as described herein... Co-culturing donor NK cells with a conjugate of ECD mutant protein or antigen-binding protein (e.g., for 7, 14, or 21 days) in vitro amplification produces NK cell populations having one or more (or preferably all) of the following characteristics: a) fold expansion of amplified NK cells by at least 0.5, 1.0, or 2 times the fold expansion of amplified NK cells obtained by co-culturing with irradiated FC21 cultured cells in vitro. a) 0 or 5.0 times; b) the telomere length of amplified NK cells increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55% compared to the telomere length of fresh NK cells, preferably the percentage increase in telomere length compared to the telomere length of fresh NK cells obtained by in vitro amplification through co-culturing with irradiated FC21 cells. The telomere length increased by at least 0.5, 1.0, 2.0, or 5.0 times; c) the expression level of at least one NK cell activation receptor selected from NKG2D, NKp30, NKp44, NKp46, and CD16 on the amplified NK cells was at least 0.5, 1.0, 2.0, or 5.0 times that of NK cells obtained after in vitro amplification in the presence of FC21 cultured cells; d) the amplified NK cells e) The amount of at least one of the cytokines TNF-α, IFN-γ, and IL-6 secreted by the cells is 0.5, 1.0, 2.0, or 5.0 times that of the cytokines secreted by NK cells obtained after in vitro amplification in the presence of FC21 cultured cells; e) The cytotoxicity of the amplified NK cells is at least 0.5, 1.0, 2.0, or 5.0 times that of the NK cells obtained after in vitro amplification in the presence of FC21 cultured cells. In a preferred embodiment, the in vitro amplification of donor NK cells further includes co-culturing the NK cells with tumor cells expressing TAAs that specifically bind to conjugates containing 4-1BBL ECD mutant proteins or antigen-binding proteins. The formulation of in vitro amplification of donor NK cells and the assays used to determine fold expansion, telomere length increase, NK cell activation receptor expression levels, cytokine secretion, and cytotoxicity (e.g., short-term or long-term cytotoxicity assays) are described in Denman et al. (2012, ibid .) and in the examples herein. Pharmaceutical Composition

In another embodiment, the invention relates to a pharmaceutical composition comprising the 4-1BBL ECD mutant protein as described herein, or a conjugate comprising the 4-1BBL ECD mutant protein as described herein and an antigen-binding protein, and a pharmaceutically acceptable carrier (adjuvant). The pharmaceutically acceptable carrier (such as an adjuvant or mediator) is used to administer the protein to a subject. The pharmaceutical composition can be used in the treatment methods described below by administering an effective amount of the composition to a subject in need. As used herein, the term "subject" refers to all animals classified as mammals, including but not limited to primates and humans. Subjects are preferably males or females of any age or race.

As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonics and absorption delayers, and the like (see, for example, "Handbook of Pharmaceutical Excipients," Rowe et al., eds., 7th ed., 2012, www.pharmpress.com). The use of such media and agents in pharmaceutically active substances is well known in the art. Unless any known media or agent is incompatible with the active compound, its use in the composition is covered. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosage and concentration used, and include buffers such as phosphates, citrates, and other organic acid antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexadecyl quaternary ammonium chloride; benzyl alkyl ammonium chloride; benzothorium chloride; phenol, butyl or benzyl alcohol; p-hydroxybenzoic acid esters, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); Low molecular weight (less than about 10 residues) proteins; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannitol, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions such as sodium; metal complexes (e.g., ^Zn²⁺- protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

Complementary active compounds may also be incorporated into the pharmaceutical composition of the present invention. Therefore, in certain embodiments, the pharmaceutical composition of the present invention may contain more than one active compound required for a specific indication to be treated, preferably compounds that have complementary activities and do not adversely affect each other. For example, it may be desirable to further provide chemotherapy agents, cytokines, analgesics, thrombolytics, or immunomodulators, such as immunosuppressants or immunostimulants. The effective amount of such other active agents depends particularly on the amount of the protein of the present invention present in the pharmaceutical composition, the type of disease or condition, or the treatment.

In one embodiment, the protein of the invention is prepared using a carrier that protects the compound from rapid autologous elimination, such as controlled-release formulations, including implants and microencapsulated delivery systems, such as liposomes. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be readily apparent to those skilled in the art. Liposome suspensions, including targeted liposomes, can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art (e.g., as described in US 4,522,811 or WO2010/095940).

The protein of this invention can be administered extragastrically. As used herein, "extragastric" includes intravenous, intraarterial, intralymphatic, intraperitoneal, intramuscular, or subcutaneous administration. Intravenous or intramuscular forms of parenteral administration are preferred. "Systemic administration" means intravenous, intraperitoneal, and intramuscular administration. Of course, the amount of protein required for therapeutic or preventative effects will vary depending on the protein selected, the nature and severity of the condition being treated, and the patient. In addition, the protein can be appropriately administered via pulse infusion, for example, using a decreasing dose of protein. Preferably, the drug is administered by injection, either intravenously, intramuscularly, or subcutaneously, depending in part on whether the administration is short-term or long-term.

Therefore, in certain embodiments, the pharmaceutical composition of the present invention may be in a form suitable for parenteral administration, such as a sterile solution, suspension, or lyophilized product in an appropriate unit dosage form. Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (in the case of water-soluble solutions) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial aqueous solutions, CremophorEM (BASF, Parsippany, N.J.), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and its flowability should be sufficient for easy injection. It should be stable under manufacturing and storage conditions and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, pharmaceutically acceptable polyols such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings (such as lecithin), maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial activity can be achieved using various antibacterial and antifungal agents, such as para-hydroxybenzoic acid, chlorobutanol, phenol, ascorbic acid, thimerosal, and analogues. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride.

The absorption of injectable compounds can be prolonged by including agents that delay absorption (such as aluminum monostearate and gelatin) in the composition.

A sterile injectable solution can be prepared by incorporating an active compound (such as the protein of this invention) in the required amount, if necessary, with one or a combination of the components listed above into a suitable solvent, followed by filtration and sterilization. Generally, dispersions are prepared by incorporating an active compound into a sterile medium containing an alkaline dispersion medium and other desired components from the components listed above. In the case of sterile powders used to prepare sterile injectable solutions, a preferred preparation method is vacuum drying and freeze-drying, which produces a powder containing the active component plus any other desired components from its previously sterile filtered solution.

In certain embodiments, the pharmaceutical composition is administered via intravenous (IV), intramuscular (IM), or subcutaneous (SC) routes. Appropriate excipients, such as bulking agents, buffers, or surfactants, may be used. The formulations mentioned will be prepared using standard methods well known in the art for preparing parenteral-applied compositions, and are described in more detail from various sources, including, for example, "Remington: The Science and Practice of Pharmacy" (Ed. Allen, L. V. 22nd ed., 2012, www.pharmpress.com).

Formulating pharmaceutical compounds (i.e., parenteral compounds) into dosage units is particularly advantageous for ease of administration and dosage uniformity. As used herein, dosage units refer to physical discrete units suitable as unit doses for the intended treatment; each unit contains a pre-quantitative active compound (the protein of this invention) bound to the desired therapeutic effect and the required pharmaceutical carrier. The specifications of the dosage units of this invention depend on and are directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of techniques for mixing such active compounds for treating individuals.

Generally, for the prevention and/or treatment of the diseases and conditions mentioned herein, and depending on the specific disease or condition to be treated and its severity, the potency of the specific protein of the invention to be used, the specific route of administration, and the specific pharmaceutical formulation or composition used, the protein of the invention will generally be administered in the range of 0.001 to 1,000 mg/kg body weight/day, preferably about 0.01 to about 100 mg/kg body weight/day, most preferably about 0.05 to 10 mg/kg body weight/day, such as about 1, 10, 100, or 1,000 mg/kg body weight/day, continuously (e.g., by infusion), as a single daily dose, or as multiple doses throughout the day. Clinicians will generally be able to determine the appropriate daily dose based on the factors mentioned herein. It is also evident that, in certain circumstances, clinicians may choose to deviate from this dosage, for example, based on the factors mentioned above and their expert judgment. These pharmaceutical compositions may be included in containers, packaging, or dispensers along with instructions for use. Therapeutic Use

In another embodiment, a 4-1BBL ECD mutant protein as described herein, or a conjugate comprising the 4-1BBL ECD mutant protein as described herein and an antigen-binding protein, is provided for use as a pharmaceutical. In one embodiment, the 4-1BBL ECD mutant protein as described herein or the conjugate as described herein is used as an active ingredient, component, or substance in a pharmaceutical.

In one embodiment, the present invention relates to the use of a 4-1BBL ECD mutant protein as described herein or a conjugate comprising the 4-1BBL ECD mutant protein as described herein and an antigen-binding protein for the manufacture of a pharmaceutical preparation, for example, a pharmaceutical preparation comprising the 4-1BBL ECD mutant protein, a fusion protein or a conjugate as an active ingredient, for the treatment, prevention or diagnosis of a disease in a subject of need.

In one embodiment, the present invention relates to a pharmaceutical preparation containing the 4-1BBL ECD mutant protein as described herein, or a conjugate comprising the 4-1BBL ECD mutant protein and an antigen-binding protein as described herein, or comprising the 4-1BBL ECD mutant protein, a fusion protein, or a conjugate as an active ingredient, for the treatment, prevention, or diagnosis of diseases in a subject in need.

In one embodiment, the present invention relates to a method for treating a disease in a subject of need, wherein the method comprises administering to the subject (an effective amount) the 4-1BBL ECD mutant protein as described herein, or a conjugate comprising the 4-1BBL ECD mutant protein and an antigen-binding protein as described herein, or a pharmaceutical preparation comprising the 4-1BBL ECD mutant protein, a fusion protein, or a conjugate as an active ingredient.

Diseases treated, prevented, or diagnosed using the 4-1BBL ECD mutant protein, fusion protein, or conjugate can include cancer, infectious diseases, inflammatory diseases, or autoimmune diseases.

In one embodiment, the 4-1BBL ECD mutant protein, fusion protein, or conjugate is used to treat, prevent, or diagnose a disease such as cancer, as described below. The cancer is preferably a cancer exhibiting the TAA described above, and more preferably, the cancer exhibits a TAA bound to an antigen-binding protein in the conjugate. In a preferred embodiment, the TAA is TROP2.

In one embodiment, treatment may include the following steps: a) identifying the TAA expressed by (tumor) cells in cancer; b) selecting a conjugate containing an antigen-binding protein that specifically binds to the TAA as described herein; c) treating the cancer with the conjugate selected in b). The cancer may be any cancer as described below. In a preferred embodiment, the TAA is TROP2.

In one embodiment, the invention relates to a method for enhancing the antitumor activity of NK cells in a subject, the method comprising administering to the subject a 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein, or a pharmaceutical preparation containing a 4-1BBL ECD mutant protein, fusion protein, or conjugate as an active ingredient. In one embodiment, the subject suffers from cancer, such as the cancer described below. Preferably, the cancer comprises tumor cells expressing a TAA, more preferably, the tumor cells express a TAA that binds to an antigen-binding protein in the conjugate. In a preferred embodiment, the TAA is TROP2.

In one embodiment, the invention relates to a method for amplifying and/or inducing hyperfunctional NK cells in a subject, the method comprising administering to the subject a 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein, or a pharmaceutical preparation containing a 4-1BBL ECD mutant protein, fusion protein, or conjugate as an active ingredient. The amplification and hyperfunctionality are preferably as described above. In one embodiment, the subject has cancer, preferably cancer comprising tumor cells expressing a TAA, more preferably tumor cells expressing a TAA that binds to an antigen-binding protein in the conjugate. In a preferred embodiment, the TAA is TROP2.

Cancer patients typically exhibit low NK cell counts and/or NK cell depletion. Therefore, the 4-1BBL ECD mutant protein or conjugate of this invention can be advantageously used to increase the number of NK cells and/or induce NK cell superfunction in cancer patients. Another advantage of NK cell superfunction induced by the 4-1BBL ECD mutant protein, fusion protein, or conjugate of this invention includes increased secretion of cytokines (such as TNF-α, IFN-γ, and IL-6), which contributes to the formation of adaptive immune responses involving dendritic cells (DCs) and T cells. In fact, it has been reported that NK cells promote the recruitment of DC subsets to the tumor microenvironment, and that DC subsets specifically cross-present tumor antigens to CD8 ⁺ T cells, indicating that NK cells play a key role in enhancing the anti-tumor CD8 ⁺ T cell response (Bottcher et al., Cell, 2018.172: 1022–1037; and Barry et al., Nat. Med.2018.24: 1178–1191). The contribution of NK cells to coordinating antitumor T cell responses has also been experimentally demonstrated in mice, indicating that in addition to direct effector functions, NK cells can also promote T cell responses and durable immune control against tumors (Bonavita et al., Immunity 2020.53: 1215–1229).

In various embodiments, the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein is provided for use in the manufacture of drugs, for example, for application to subjects suffering from cancer, infectious diseases, or inflammatory diseases.

In one embodiment, TAA is an antigen expressed on the surface of malignant cells of cancer types as defined above herein and/or as described below. In one embodiment, TAA is TROP2 as defined above and/or TROP2 antigen expressed on the surface of malignant cells of cancer types as described below. The treatment methods described herein are suitable for patients with cancers selected from the following groups: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, tailbone carcinoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, osteosarcoma, malignant fibrous histiocytoma, brainstem glioma, brain tumor, brain tumor, brainstem glioma, atypical teratoma of the central nervous system, striated leiomyoma, central nervous system... Embryogenic tumors, cerebellar astrocytomas, cerebral astrocytomas/malignant gliomas, craniopharyngiomas, ependymoblastomas, ependymomas, medulloblastomas, medullary epitheliomas, moderately differentiated pineal solid tumors, supratentorial primitive neuroectodermal tumors and pinealoblastomas, visual pathway and hypothalamic neurogliomas, brain and spinal cord tumors, breast cancer, bronchial tumors, Burkitt lymphoma. Lymphoma, carcinoid tumors, gastrointestinal carcinoid tumors, atypical teratomas of the central nervous system (striated leiomyomas), embryonal tumors of the central nervous system, central nervous system lymphomas, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colorectal cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, esophageal cancer, Ewing family tumors. Tumors, including: extragonadal genital cell tumors, extrahepatic bile duct carcinoma, intraocular melanoma, retinal blastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (gist), germ cell tumors, gestational trophoblastomas, gliomas, brainstem gliomas, cerebral astrocytomas, visual pathway and hypothalamic gliomas, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Langerhans cell histiocytosis, and Hodgkin's lymphoma. Lymphoma, hypothalamic cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumor, renal (renal cell) cancer, Langerhans cell histiocytosis, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia. Macroglobulinemia, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, melanoma, Merkel cell carcinoma Carcinoma, mesothelioma, occult primary metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasmoma, fungal infections, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative disorders, myeloid leukemia, myeloid leukemia, acute myeloid leukemia, multiple myeloma, myeloproliferative disorders, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumors. Low-grade malignant ovarian tumors, pancreatic cancer, papilloma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal gland tumors, pineal-blastoma and supratentorial primitive ectodermal tumors, pituitary adenoma, plasmacytoma/multiple myeloma, pleural-pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter, respiratory tract cancers involving the nut gene on chromosome 15, retinoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing's tumor, Kaposi's sarcoma. Sarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cervical cancer, gastric (stomach) cancer, supratentorial primitive ectodermal tumor, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor. In various embodiments, cancers treated by the disclosed methods and combinations can be treated while healthy cells are preserved.

In one embodiment, the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein can be used as a monotherapy (i.e., without the need for other treatments). In another embodiment, the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein can be used for combination therapy.

In one embodiment, the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein is used in combination with another immunotherapy, such as cell immunotherapy. Therefore, the 4-1BBL ECD mutant protein, fusion protein, or conjugate can be used in combination with the redistribution of immune cells, including T cells, such as CAR T cells, or the redistribution of NK cells. NK cells can be enriched or amplified, for example, by methods known in this art, or can be ex vivo amplified NK cells, as described below.

In one embodiment, the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein may be used in combination therapy with one or more other therapeutic agents. Additional therapeutic agents are typically used for the specific therapeutic purpose of administering an antibody against a TAA (e.g., TROP2). The additional therapeutic agent will usually be administered in the dosage and regimen of a single therapy commonly used for the specific disease or symptom being treated. When used to treat cancer, such therapeutic agents include, but are not limited to, anticancer agents and chemotherapy agents. Exemplary treatments that can be used as part of a combination therapy for cancer treatment include, for example, radiation, mitomycin, retinoic acid, ribomustin, gemcitabine, vincristine, etoposide, cladrolidine, dibromomannitol, methotrexate, doxorubicin, carboquinone, pentostatin, diammoniazide, netsistatin, cetrexate, letrozole, raltitrexed, daunorubicin, faldazole, formustin, thymosin, solbufos, nedaplatin, cytidine, bicalutamide, vinorelbine, vesnarinone, ampicillin, acridine, and proglutamine. Eritrethroid acetate, Ketocerin, Deoxyfluorouridine, Etratiate, Isotretinoin, Strazotocin, Nimustine, Vindysin Flutamide, Drolone, Butosin, Carmofol, Razosen, Cizofir-Lan, Carboplatin, Mitolprol, Tegafur, Isocyclophosphamide, Pnimos, Pisipanib, Levamisole, Teniposide, Inprofen, Enoxapane, Ergothione, Hydroxymethylolone, Tamoxifen, Progesterone, Meandran, Cyclothotherapeutic acid, Formexan, Interferon-Alfa, Interferon-2-Alfa, Interferon-Beta, Interferon-Gamma, Colony-Stimulating Factor-1, Colony-Stimulating Factor-2, Denileukin (diftitox), interleukin-2, and luteinizing hormone-releasing factor.

Another class of agents that can be used as part of combination therapy (including the use of 4-1BBL ECD mutants, fusion proteins, or conjugates as described herein) for the treatment of cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include those that inhibit one or more of the following: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (Pd1), (iii) PD-L1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. Further agents that can be used as part of combination therapy for the treatment of cancer are monoclonal antibodies against TAAs as described above. In a preferred embodiment, the TAA is TROP2.

In some embodiments, administration of 4-1BBL ECD mutant proteins, fusion proteins or conjugates, and other therapeutic agents as described herein may produce additional or synergistic effects on immunity and/or therapeutic efficacy.

In one embodiment, in addition to primary therapies including, for example, surgery, chemotherapy, and/or radiation therapy, the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein is also used as at least one of novel adjuvant and adjuvant therapies. As a novel adjuvant therapy, the 4-1BBL ECD mutant protein, fusion protein, or conjugate is administered prior to primary treatment, for example, to help shrink tumor size (so that less extensive surgery and/or radiation therapy is required), kill already spread cancer cells (e.g., micrometastatic disease), and/or reduce the risk of tumor cell spread after surgery. As an adjunctive therapy, the 4-1BBL ECD mutant protein, fusion protein, or conjugate is administered after primary treatment, for example, to treat minimal residual disease (eliminating remaining cancer cells). The 4-1BBL ECD mutant protein, fusion protein, or conjugate can also be used as maintenance therapy, i.e., long-term adjunctive therapy, such as repeated administration over a period of at least one month or one year. Using the 4-1BBL ECD mutant protein or conjugate as a novel adjunctive therapy and/or adjunctive therapy reduces recurrence rates. In novel adjunctive therapies and/or adjunctive therapies, the 4-1BBL ECD mutant protein, fusion protein, or conjugate can be used as a monotherapy or in combination with the treatments described above. Ex vivo methods

In another embodiment, the invention relates to a method for treating NK cells or NK cell populations in vitro ( in vivo ) using the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein. The method may be for amplification, preactivation, activation, enhancement of cytotoxicity and/or cytokine production, and induction of at least one of the superfunctional phenotypes defined above. The method includes at least the step of contacting NK cells or populations thereof with the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein, or an assembly containing the 4-1BBL ECD mutant protein, fusion protein, or conjugate. In a preferred embodiment, the method includes the further step of co-culturing NK cells with tumor cells expressing TAA that specifically binds to antigen-binding proteins in the conjugate. Preferably, the NK cells are co-cultured with the tumor cells expressing TAA that specifically binds to antigen-binding proteins in the conjugate in the presence of the conjugate. In a preferred embodiment, the TAA is TROP2.

NK cells or NK cell populations for in vitro processing can be enriched from peripheral blood mononuclear cells (PBMCs). Methods for enriching NK cells from PBMCs and for in vitro processing of NK cells are described, for example, in Denman et al. (pLoS One.2012;7(1):e30264) and US2020/0061115. For example, NK cells enriched from PBMCs can be seeded at a rate of 0.1 x ^10⁶ NK cells/mL in SCGM (CellGenix, Portsmouth, NH) supplemented with 10% FBS, 2 mM Glutamax, 100 U/mL IL-2 (Peprotech, Rocky Hill, NJ), and one or more of the 4-1BBL ECD mutant proteins or conjugates described herein at concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 μg/mL. The supplemented medium should be refreshed every 2–3 days.

It should be understood that the duration of contact between NK cells and the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein (i.e., the duration of preactivation or activation (i.e., the duration of amplification, preactivation, activation, enhancement of cytotoxicity and/or cytokine production and induction of a superfunctional phenotype) can be any length of time required to achieve the desired phenotype of NK cells. For example, contact can be as short as 1 minute or as long as 7 days (e.g., NK cells cultured for 7 days in the presence of the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein). In one embodiment of the method, NK cells are subjected to contact with 4-1BBL... ECD mutant protein, fusion protein, or conjugate is exposed to the protein for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours. In one embodiment of the method, NK cells are exposed to 4-1BBL... Exposure to ECD mutant proteins, fusion proteins, or conjugates for 5, 6, 7, 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 days.

In one embodiment, the in vitro treated (expanded) NK cells have one or more of the following characteristics: a) the expanded NK cells have increased by at least 0.5, 1.0, 2.0, or 5.0 times compared to the expanded NK cells obtained by co-culturing with irradiated FC21 cells; b) the expanded NK cells have increased telomere length by at least 10, 1, or 10 times compared to fresh NK cells. 5, 20, 25, 30, 35, 40, 45, 50, or 55%, preferably, the percentage increase in telomere length of amplified NK cells is at least 0.5, 1.0, 2.0, or 5.0 times that of amplified NK cells obtained by co-culturing with irradiated FC21 cells in vitro, compared to the telomere length of fresh NK cells; c) Amplification The expression level of at least one NK cell activation receptor selected from NKG2D, NKp30, NKp44, NKp46, and CD16 on NK cells was at least 0.5, 1.0, 2.0, or 5.0 times that of NK cells obtained after in vitro amplification in the presence of FC21 cultured cells; d) the expression level of TNF-α and IFN-γ secreted by amplified NK cells. e) The cytotoxicity of the expanded NK cells is at least 0.5, 1.0, 2.0, or 5.0 times that of the NK cells obtained after in vitro amplification in the presence of FC21 cultured cells;

In another embodiment, the invention relates to a method for treating a disease in a recipient, wherein the method comprises the step of administering (effective amounts) of NK cells to the recipient, which are obtained in the aforementioned method of in vitro treatment of NK cells or NK cell populations. Once a sufficient number of NK cells with the desired (superfunctional) phenotype have been amplified by in vitro treatment, the NK cells can be administered to the recipient.

In one embodiment, the treatment method comprises the administration of ex vivo treated NK cells with a combination of a 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein, or a pharmaceutical preparation comprising a 4-1BBL ECD mutant protein, fusion protein, or conjugate as an active ingredient.

In one embodiment, the treatment method comprises administering ex vivo treated NK cells in combination with another NK cell binding agent (such as those described, for example, in WO2016/207278, WO 2018/148445, WO2018/152518, WO2019195409, US2018282386, Vallera et al. (2016, ibid. ) and Demaria et al. (2021, ibid .)), or with a multispecific antigen-binding protein (such as those described in concurrent pending applications WO2024/056862 and WO2024/056861 by the same applicant). Another example of an NK cell binding agent is, for example, AVC-006 as described in WO2024/056862, which includes a HER2 binding region and an NKG2D binding region. In other embodiments, ex vivo treated NK cells may be used in combination with other binding agents and 4-1BBL ECD mutant proteins, fusion proteins, or conjugates as described herein.

As described above, the disease to be treated can be cancer, infectious disease, inflammatory disease, or autoimmune disease. Preferably, the disease to be treated is cancer, as described above. The cancer is preferably a cancer expressing a TAA that specifically binds to the antigen-binding protein in the conjugate, which is administered in combination with ex vivo treated NK cells. The combined administration of ex vivo treated NK cells and the conjugate will facilitate the targeting of the ex vivo treated NK cells to tumor cells expressing a TAA that specifically binds to the antigen-binding protein in the conjugate.

In one embodiment, the ex vivo treated NK cells are autologous to the subject. In another embodiment, the ex vivo treated NK cells are allogeneic, for example, derived from donor PBMCs. Methods for producing 4-1BBL ECD mutant protein, fusion protein, or conjugate containing such mutant protein include nucleic acids, host cells, and methods for this purpose.

In one embodiment, the present invention relates to a nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of a 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein. The nucleotide sequence encoding such a polypeptide chain preferably encodes a signal peptide operatively linked to the polypeptide chain. The nucleic acid molecule comprising one or more nucleotide sequences encoding the polypeptide chain further preferably comprises regulatory elements for (or facilitating) the expression of the polypeptide chain in a suitable host cell, such regulatory elements being operatively linked to the nucleotide sequence.

In one embodiment, the invention relates to a host cell comprising a nucleic acid molecule containing one or more nucleotide sequences of a polypeptide chain encoding a 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein. In one embodiment, the host cell is an isolated cell or a cultured cell. Suitable host cells are, in particular, prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as * Escherichia coli* or bacilli. Suitable yeast cells include * Saccharomyces cerevisiae* and * Pichia pastoris* . Higher eukaryotic cells include insect cells and well-defined cell lines of mammalian origin. Examples of suitable mammalian host cell lines include monkey kidney cells such as COS-1 and COS-7 (Gluzman et al., 1981, Cell 23:175), L cells, HEK 293 cells (e.g., Expi293, HEK293-F, and HEK293-E cells), C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells (e.g., ExpiCHO cells), HeLa cells, BHK cell lines such as BHK21, BSC-1, Hep G2, 653, SP2/0, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI, such as those described by McMahan et al. (1991, EMBO J. 10: As described in 2821). The host cell can be any suitable species or organism capable of producing N-linked glycosylated polypeptides, such as mammalian host cells capable of producing human or rodent IgG-type N-linked glycosylated polypeptides. Suitable selection and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985). Host cells containing the nucleic acid molecules of this invention can be cultured under conditions that promote polypeptide expression.

Therefore, another aspect of the invention relates to a method for producing the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein. The method preferably comprises culturing host cells as described above, such that one or more nucleotide sequences are expressed and the 4-1BBL ECD mutant protein, fusion protein, or conjugate is produced. The method preferably comprises culturing host cells containing one or more nucleotide sequences of a polypeptide chain encoding the 4-1BBL ECD mutant protein, fusion protein, or conjugate. Preferably, the host cells are cultured under conditions conducive to the expression of one or more polypeptide chains. The method may further comprise a step of recovering the 4-1BBL ECD mutant protein, fusion protein, or conjugate. 4-1BBL ECD mutant proteins, fusion proteins, or conjugates can be recovered using familiar protein purification procedures, including, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, size exclusion chromatography, or affinity chromatography, using, for example, streptavidin/biotin (see, for example, Low et al., 2007, J. Chromatography B, 848:48-63; Shukla et al., 2007, J. Chromatography B, 848:28-39).

In another embodiment, the present invention relates to a method for producing a pharmaceutical composition comprising the 4-1BBL ECD mutant protein, fusion protein, or conjugate as described herein, the method comprising the steps of: a) producing the 4-1BBL ECD mutant protein, fusion protein, or conjugate as defined above; and b) co-formulating the 4-1BBL ECD mutant protein, fusion protein, or conjugate with a pharmaceutically acceptable carrier as defined above to obtain the pharmaceutical composition.

The present invention has been described above with reference to several illustrative embodiments shown in the accompanying figures. Modifications and substitutions to some parts or elements are possible and are included within the scope of protection defined by the appended patent applications. Example 1 1.1 Methods and Materials 1.1.1 Reagents NK Cell Amplification Reagent List Reagents Suppliers and product codes Skin color hematopoietic layer N/A CD56 Freeze-dried Microbeads Miltenyi 130-097-042 IL-2 R&D Systems, 202-IL-050 FITC anti-human CD56 (REA196) Miltenyi, 130-114-549 Hu CD3 BUV395 UCHT1 BD, 563546 PE anti-human CD360 (IL-21R) Biolegend 359506 APC is anti-human CD137 (4-1BB) Biolegend 309810 It can fix the reactive dye eFluor 780 eBioscience, 65-0865-18 UltraComp beads Invitrogen, 01-2222-42 LS tubing Miltenyi, 130-042-401 Pre-separation filter, 30 μM Miltenyi, 130-041-407 Fc blocking Miltenyi, 130-059-901 Histopaque Sigma Aldrich, 10771-500ML HEPES Pan Biotech, P05-01100 HBSS Fisher Sci, 11500476 ACK lysis buffer Fisher Sci, 11509876 SK-OV-3 cell line culture medium Components Volume (mL) Login Number McCoy's 5a culture medium 418.5 Gibco, 16600082 1% Penicillin-Streptomycin 5.0 Hyclone, SV30010 FCS 75.0 Pan BioTech, P30-193602 L-glutamic acid 1.5 Gibco, 25030-081 Complete culture medium for effector cells (NK cells) Components Volume (mL) Login Number RPMI 1640 425.0 Pan BioTech, P04-17500 1% Penicillin-Streptomycin 5.0 Hyclone, SV30010 10% AB 50.0 Access Cell Culture, 515-HI 20 mM HEPES 10.0 Pan BioTech, PO5-01100 L-glutamic acid 10.0 Gibco, 25030-081 1.1.2 Maintenance and isolation sub-combination culture (70-80%) of SKOV3 WT cell line SK-OV-3

1. Remove and discard the culture medium.

2. Briefly rinse the cell layer with 0.25% (w/v) trypsin-0.53 mM EDTA solution to remove any traces of serum containing trypsin inhibitor.

3. Add 2 to 3 mL of trypsin-EDTA solution to the flask and observe the cells under an inverted microscope until the cell layer disperses (usually within 5 to 15 minutes).

Note: To avoid clumping, do not stir the cells by hitting or shaking the flask while waiting for cell separation. Cells that are difficult to separate can be placed at 37°C to promote dispersion.

4. Add 6 to 8 mL of complete growth medium and gently pipette and aspirate the cells.

5. Add appropriate aliquots of the cell suspension to new culture containers.

6. Incubate the culture at 37°C. Subculture ratio: A subculture ratio of 1:2 to 1:3 is recommended, i.e., 3-6 x 10,000 cells/cm². Medium renewal: Every 2-3 days. 1.1.3 Culture Medium: Growth and testing medium: McCoy's 5a modified medium + 2mM glutamic acid + 15% FBS; 1% penicillin/streptomycin frozen medium FCS + 10% DMSO. 1.1.4 PBMC sorting and NK cell separation.

Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using density gradient centrifugation. In short, skin-colored hemoglobin fractions were placed on a histopaque in falcon tubes and density centrifuged. After centrifugation at 500g for 30 min at room temperature, the PBMC fractions were harvested from the interface into new falcon tubes. The cells underwent several washing and lysis steps to remove red blood cell contamination. Subsequently, CD56 ⁺ cells were obtained by magnetic cell sorting using a system from Miltenyi Biotec, according to the manufacturer's instructions (Miltenyi-130-097-042). After separation, aliquots of the sorted NK cells were taken for post-sorting purity testing. 1.1.5 SK-OV-3 radiation E:T ratio = 1:2 (NK: SK-OV-3) • 8 x ^10⁶ cells/flask x (X condition) x (Y donor)

1. Irradiate additional cells to address potential cell loss following irradiation. Retain sufficient cells for proliferation for restimulation on day 7.

2. Resuspend the required number of cells, 5 x ^10⁶ cells/mL.

3. Irradiate with 100 Gy.

4. Count the irradiated cells and adjust the count to 5 x ^10⁶ cells/mL. 1.1.6 Cognitive Effects of SK-OV-3 Cells

1. Prepare test compounds.

2. Spike the required reserve volume into the irradiated SK-OV-3 to reach the desired concentration (25 nM), and place it in a flask in a 37°C incubator for 30 minutes.

3. Wash the cells with 300 x g for 5 minutes.

4. Count the cells and adjust the density to 3.2 x ^10⁶ /mL. 1.1.7 Inoculate with NK cells for 14- day expansion.

1. Adjust NK cells to the desired density - 1.6 x ^10⁶ /mL in NK cell culture medium containing IL-2 (50 IU/mL).

2. Inoculate 2.5 mL of NK cells per bottle (4 x ^10⁶ total cells per bottle).

3. Inoculate 2.5 mL of SK-OV-3 cells conditioned with 3.2 x ^10⁶ /mL of compound or mediator according to the appropriate flask diagram.

4. Add 17.5 mL of 2 x IL-2 complete medium and 17.5 mL of IL-2-free complete medium to all flasks. This is to achieve an IL-2 activity of 50 IU/mL.

5. The final total volume of each T75 flask will be 40 mL. 1.1.8 Cytotoxicity Assay

NK cells and NucLight-Red (NLR)-transduced SKOV3 tumor cells were co-cultured at a 2:1 ratio for 5 days using different concentrations of NK enhancer in a 7-step, 4-fold dilution series, starting at 25 nM and covering a range from 25 nM to 0.01 nM. Throughout the culture period, the number of NLR-positive cells was quantified every 4 hours using an Incucyte automated microscope and compared to a control condition where NK cells were co-cultured with NLR SKOV3 cells in the absence of NK enhancer. Cytotoxicity was calculated as follows: 1.1.9 Proliferation Measurement: NK cells were labeled with amine-reactive cell proliferation dyes (CPDs), such as CellTrace Violet (Thermo Scientific, accession number C34557). The labeled NK cells were then used to co-culture with SKOV3 cells or alone with different concentrations of NK enhancers. When co-culturing with SKOV3 cells, an alternative 7-step 4-fold dilution series was used, starting at 25 nM and covering the range from 25 nM to 0.01 nM. When culturing NK cells alone, an 8-step 3-fold dilution series was used, covering the range from 100 nM to 0.01 nM. After 5 days of culture, the levels of cell proliferation dyes in NK cells were analyzed by flow cytometry, and the percentage of CPD-hypoactive cells was used as the proliferation reading. 1.1.10 Production of NK enhancers

The NK cell enhancers (also referred to herein as conjugates) listed in Tables 1.1.10.1 and 1.1.10.2 have amino acid sequences as shown in the sequence listing and are prepared, purified and characterized substantially as described in WO2024/056862.

Table 1.1.10.1 shows HER2-targeting NK cell enhancers containing the 4-1BBL and/or IL-21 mutant proteins, along with an overview of their corresponding amino acid sequences in the sequence listing. AVC1 is a control NK cell enhancer containing wild-type 4-1BBL and IL-21. AVC-ID HC1 mutation in code 4-1BBL HC1 SEQ ID NO HC2 mutation in IL-21 HC2 SEQ ID NO LC SEQ ID NO AVC1 - 11 - 12 2 AVC22 - 11 R76K 177 2 AVC23 - 11 R5Q 178 2 AVC24 - 11 R9E 179 2 AVC25 - 11 R76A 180 2 AVC26 - 11 R5H 181 2 AVC27 - 11 I8V 182 2 AVC28 - 11 I8Q 183 2 AVC29 - 11 R11E 184 2 AVC30 - 11 Q12K 185 2 AVC31 - 11 Q19S 186 2 AVC32 - 11 K73Y 187 2 AVC33 - 11 K75E 188 2 AVC34 - 11 L13E 189 2 AVC35 - 11 R76H 190 2 AVC36 - 11 D4H 191 2 AVC37 - 11 I8H 192 2 AVC38 - 11 K21H K112H 193 2 AVC39 - 11 K21H 194 2 AVC40 - 11 K112H 195 2 AVC46 V100T 196 - 12 2 AVC47 V100Q 197 - 12 2 AVC48 L101N 198 - 12 2 AVC49 Y110Q 199 - 12 2 AVC50 G114K 200 - 12 2 AVC51 L115R 201 - 12 2 AVC52 V153Q 202 - 12 2 AVC53 R171D 203 - 12 2 AVC54 Q227E 204 - 12 2 AVC55 Q227R 205 - 12 2 AVC56 Q230S 206 - 12 2 AVC57 Q230K 207 - 12 2 AVC58 A116D 208 - 12 2 AVC59 A154D 209 - 12 2 AVC72 V100T 196 R9E 179 2 AVC73 Q227R 205 R9E 179 2 AVC74 Q230S 206 R9E 179 2 AVC75 V100T 196 R76A 180 2 AVC76 Q227R 205 R76A 180 2 AVC77 Q230S 206 R76A 180 2 AVC78 V100T 196 I8Q 183 2 AVC79 Q227R 205 I8Q 183 2 AVC80 Q230S 206 I8Q 183 2 AVC85 Q227E 204 R9E+R76A 2 AVC86 Q227E 204 L13E 189 2 AVC87 V153Q 202 L13E I8H K21H D4H 2 AVC88 V153Q 202 D4H I8H K21H 2 AVC89 V153Q 202 L13E I8H K21H 2 AVC90 V153Q 202 L13E D4H I8H 2 AVC91 V153Q 202 I8H K21H 2 AVC92 V153Q 202 L13E D4H K21H 2 AVC93 V153Q 202 D4H I8H 2 AVC94 V153Q 202 L13E I8H 2 AVC95 V153Q 202 D4H K21H 2 AVC96 V153Q 202 L13E K21H 2 AVC97 V153Q 202 L13E D4H 2 AVC98 Y110Q V153Q Q227E 210 I8H 192 2 AVC99 L101N Y110Q V153Q 211 I8H 192 2 AVC100 V100Q Y110Q V153Q 212 I8H 192 2 AVC101 L101N V153Q Q227E 213 I8H 192 2 AVC102 Y110Q A154D Q227E 214 I8H 192 2 AVC103 Y110Q V153Q 215 I8H 192 2 AVC104 V153Q Q227E 216 I8H 192 2 AVC105 Y110Q Q227E 217 I8H 192 2 AVC106 L101N Q227E 218 I8H 192 2 AVC107 V153Q 202 I8H 192 2 AVC108 - 11 I14D 219 2 AVC109 - 11 D18A 220 2 AVC110 - 11 E100R 221 2 AVC111 - 11 E109R 222 2 AVC112 - 11 I119D 223 2 AVC113 - 11 E100R E109R 224 2 AVC114 - 11 N82- A83- G84- R85- R86- Q87- K88- 225 2 AVC115 - 11 N82- A83- G84- R85- R86- Q87- K88- H89- R90- 226 2 AVC116 - 11 N63- E64- R65- I66- 227 2 AVC117 - 11 N59- T60- G61- N62- N63- E64- R65- I66- 228 2 AVC118 - 11 I67T 229 2 AVC119 - 11 I67N 230 2 AVC120 - 11 L74G 231 2 AVC121 - 11 L74D 232 2 AVC122 - 11 L74F 233 2 AVC123 - 11 L20S 234 2 AVC124 - 11 L20N 235 2 AVC125 - 11 L20W 236 2 AVC126 - 11 G84 insGGGG 237 2 AVC176 A154D 209 L74D 232 2 AVC187 A154D 209 L20N 235 2 AVC188 A154D 209 L20W 236 2 AVC189 A154D 209 L20S 234 2 AVC190 A154D 209 N82- A83- G84- R85- R86- Q87- K88- 225 2 AVC285 A154E 675 N82- A83- G84- R85- R86- Q87- K88- 225 2 AVC286 A154D G155Q 697 L20W 236 2 AVC287 A154E 675 L20W 236 2 AVC288 A154D G155Q 697 N82- A83- G84- R85- R86- Q87- K88- 225 2

Table 1.1.10.2 shows TROP2-targeting NK cell enhancers containing the 4-1BBL and/or IL-21 mutant protein and the control protein AVC137 (excluding 4-1BBL and IL-21), along with an overview of their corresponding amino acid sequences in the sequence listing. AVC-ID HC1 mutation in code 4-1BBL HC1 SEQ ID NO HC2 mutation in IL-21 HC2 SEQ ID NO LC SEQ ID NO Based on moAb AVC16 - 582 - 583 584 Gosatozumab AVC221 - 547 - 548 546 AR47A6.4.2 AVC227 - 550 - 551 549 KM4097 AVC245 A154D 585 N82- A83- G84- R85- R86- Q87- K88- 586 587 Gosatozumab AVC323 A154D 591 N82- A83- G84- R85- R86- Q87- K88- 592 593 KM4097 AVC421 A154D 588 N82- A83- G84- R85- R86- Q87- K88- 589 590 AR47A6.4.2 AVC267 A154D 585 L20W 674 584 Gosatozumab AVC137 No 4-1BBL 672 No IL-21 673 584 Gosatozumab 1.1.11 Affinity of NK enhancers to 4-1BB or IL-21R

The affinity of the enhancers for 4-1BB or IL-21R was analyzed using a Biacore T200 instrument at 25 °C and a flow rate of 50 μl/min. The C1 sensor chip was functionalized for reversible biotin capture assays in all flow cells (fc1–fc4). The analytical buffer consisted of 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Tween 20, and 3 mM EDTA. Each assay cycle is performed as follows: Biotin capture reagent is introduced into all surfaces (fc1–fc4), 4-1BB or IL-21R is specifically captured in flow cell 2 (fc2), and the enhancer is injected at five consecutive dilutions (starting from different concentrations), followed by a 60-second binding phase and a 600/1200-second dissociation phase (longer dissociation is applied after the highest concentration). The ligand-receptor complex is then completely removed from all surfaces using a standard regeneration protocol, and the wafer is restored to baseline before the next cycle. 1.1.12 4-1BB Reporting Gene Assay

NK cell enhancers were incubated with 4-1BB effector cells (Promega accession number JA2351) for 6 hours, followed by the addition of Bio-Glo reagent and measurement of luciferase activity using a spectrophotometer. 1.1.13 Long-term repeated NK cell cytotoxicity assay

A repetitive co-culture system (similar to the setup used by Thakur et al. (J Cancer Res Clin Oncol. 2020 Aug; 146(8): 2007–2016.)) was established to investigate the repetitive cytotoxicity of CAR-T cells containing a HER2-EGFR bispecific binder. Since understanding the general cell culture status is desirable for such long-term measurements, the ^Incucyte® live cell analysis system was used. SKOV-3 target cells and NK cells (purified from the aforementioned normal donor skin-colored hematopoietic layer using RosetteSep via negative selection) were used at a 1:1 E:T ratio. SKOV-3 target cells were transduced with lentivirus using Nuclight Red (Sartorius accession number 4625) to allow for the counting of fluorescently labeled target cells. Three copies were prepared for testing.

A fixed amount of 10,000 fluorescently labeled target cells were used in 200 μl of medium per well to ensure adequate nutrition for at least 3 days of culture. Co-culture was performed in the presence of 50 IU/mL IL-2. After 3 days, NK cells were harvested and used to establish a new round of co-culture with fresh target cells at a 1:1 E:T ratio. At the beginning of each 3-day round, 25 nM of NK enhancer was added to expose the NK cells to the new enhancer protein at the start of each round. Control wells contained only NK cells and SKOV-3 target cells, or only SKOV-3 target cells. Cell monitoring was performed every 3 hours, and fluorescently labeled target cells were counted. The experiment lasted for 6 co-culture cycles, totaling 18 days. 1.1.14 In vivo pharmacokinetic data

Male C57BI16/J mice (n = 3) were administered intravenously at a dose of 1 mg test product/kg body weight. The test products were the NK cell enhancers AVC16 and AVC245, and the control antibodies AVC137 and trastuzumab. Blood samples were collected at 15 min, 2 h, and 1, 3, and 7 days post-injection for analysis. Serum levels of enhancer antibodies and control antibodies were detected and quantified using a sandwich ELISA with TROP2 or HER2-coated plates (with or without trastuzumab). Serum levels were converted from absorbance to ng/mL using the corresponding standard curves for each test product, and plotted over time. 1.1.15 In vivo efficacy data of NK cell enhancers

The efficacy of AVC245 in in vivo inducing NK cell proliferation and reducing tumor burden was evaluated in SKOV-3 xenograft mice relative to an IgG TROP2-targeted control. Age-matched female NRG mice received a non-lethal low-dose whole-body irradiation of 230 cGy on day -1. On day 0, each mouse received an intraperitoneal injection of 400 μL of 1 x ^10⁶ human donor NK cells. On day 7, mice were implanted with ⁵ x 10⁵ luciferase-expressing SKOV-3 cells (SKOV-3-Luc) via intraperitoneal injection. For AVC137 and AVC245 treatment, 10 mice were used per group, and for tumor-only and NK+ tumor-only controls, 5 mice were used per group. Starting from day 0, administer intraperitoneal injections of AVC137 (5.09 mg/kg) or isomer of AVC245 (8 mg/kg) or a mediator. Three times a week for the first week (i.e., Monday, Wednesday, and Friday), and twice a week thereafter (i.e., Monday and Friday). Starting from day 0, administer intraperitoneal injections of 5000 IU/mouse of recombinant human IL-2. Three times a week (i.e., Monday, Wednesday, and Friday).

On days 7 and 18, 100 μL of peripheral blood was collected from each mouse and stained to determine the absolute NK cell count per mL of blood by flow cytometry. Blood was collected prior to any treatment scheduled for that day.

Tumor burden was quantified using bioluminescence imaging (BLI) via automatic exposure on the first day of each week (days 7, 14, 21, etc.). The first BLI was performed on day -1 instead of day 0, and the last BLI was performed the day before the experimental endpoint. BLI was performed before any injections (i.e., NK cells, AVC, IL-2) on the day of the experiment. 1.2 Results

Figures 1 and 2 show that NK enhancers containing IL-21 and 4-1BBL ECD mutant proteins exhibit impaired ability to induce proliferation, but this only occurs in the absence of tumor cells. Figure 1 shows the dose-response curves for NK enhancers AVC37, AVC52, and the control NK enhancer AVC1. Figure 2 shows the dose-response curves for NK enhancer AVC54 and the control NK enhancer AVC1. NK enhancers AVC37, AVC52, and AVC54 show significantly impaired ability to induce NK cell proliferation, but this only occurs in the absence of HER2-expressing SKOV3 tumor cells. In the presence of SKOV3 tumor cells, the ability of NK enhancers AVC37, AVC52, and AVC54 to induce NK cell proliferation was comparable to that of the control NK enhancer AVC1.

Figure 3 shows the dose-response curves of various other NK enhancers containing different 4-1BBL ECD mutant proteins, as shown by their ability to induce NK cell proliferation in the absence of tumor cells. It can be seen that, compared to the control NK enhancer AVC1 containing wild-type 4-1BBL ECD, the various NK enhancers with different 4-1BBL ECD mutant proteins exhibit varying degrees of reduced ability to induce NK cell proliferation in the absence of tumor cells.

Figure 4 shows that NK cell cytotoxicity against SKOV3 tumor cells induced by NK enhancers containing IL-21 and 4-1BBL ECD mutant proteins was not affected by IL-21 or 4-1BBL ECD mutations in AVC37 and AVC54, respectively. The dose-response curves for NK cell cytotoxicity against SKOV3 tumor cells induced by AVC37 and AVC54 were not significantly different from the dose-response curve of the corresponding control NK enhancer AVC1 containing wild-type IL-21 and 4-1BBL ECD.

Figure 5 shows that the ability of NK enhancers AVC37 or AVC52 (containing IL-21 or 4-1BBL ECD mutant proteins, respectively) to induce and support long-term NK cell proliferation is almost unaffected.

Table 1.2.1 shows that the binding affinity of the NK enhancer containing the 4-1BBL ECD mutant protein shown to 4-BB is reduced compared to the corresponding control NK enhancer AVC1 containing wild-type 4-1BBL ECD.

Figures 6A, 6B, and 6C show that NK enhancers with reduced affinity for the 4-1BBL ECD mutant protein also have reduced potency in the detection of 4-1BB reporter cells. In fact, Figure 7 shows a strong correlation between affinity measured by SPR and functionality in the detection of the 4-1BB reporter gene.

Table 1.2.1 summarizes the reduced binding affinity of the enhancer to 4-1BB and its ability to induce NK cell proliferation in the presence of SKOV3 tumor cells at a saturated concentration (25 nM) containing a combination of the trimer of the indicated 4-1BBL ECD mutant protein and wild-type IL-21, expressed as a percentage of the control enhancer AVC1 (containing the trimer of wild-type 4-1BBL ECD and wild-type IL-21). AVC ID 4-1BBL mutation K _D in nM Logarithmic reduction of pK _D %AVC1 proliferation at 25 nM AVC1 wild type 39.81 0.00 - AVC46 V100T 218.78 0.74 - AVC47 V100Q 776.25 1.29 75% AVC48 L101N 812.83 1.31 84% AVC49 Y110Q 1202.26 1.48 81% AVC50 G114K 57.54 0.16 - AVC51 L115R ND - AVC52 V153Q 588.84 1.17 92% AVC53 R171D 61.66 0.19 - AVC54 Q227E 1819.70 1.66 93% AVC55 Q227R >4400 >2.05 71% AVC56 Q230S 53.70 0.13 - AVC57 Q230K 1621.81 1.61 80% AVC58 A116D 107.15 0.43 - AVC59 A154D 776.25 1.29 97% AVC285 A154E 830 0.83 - AVC286 A154D G155Q 2810 5.55 - AVC287 A154E 732 0.77 - AVC288 A154D G155Q 3390 5.47 -

Table 1.2.1 presents the binding affinity of NK enhancers containing the trimer of the 4-1BBL ECD mutant protein to 4-1BB as reduced compared to the corresponding control NK enhancer AVC1 containing the trimer of wild-type 4-1BBL ECD. Table 1.2.1 further presents the ability of the enhancers to induce NK cell proliferation in the presence of SKOV3 tumor cells at saturation concentration (25 nM), expressed as a percentage of the control enhancer AVC1. As can be seen from Table 1.2.1, most NK enhancers containing the 4-1BBL mutant protein exhibit at least an order of magnitude reduction in affinity for 4-1BB compared to the AVC1 enhancer containing wild-type 4-1BBL. Surprisingly, given the strong correlation between affinity and functionality, some of these enhancers retained most of their ability to induce NK cell proliferation in the presence of SKOV3 tumor cells, to a degree similar to the control enhancer AVC1 with wild-type IL-21, as their ability to induce proliferation was reduced by less than 30%, or even less than 3%, relative to the control enhancer.

Most NK enhancers with the 4-1BBL mutant protein retained their potency in inducing NK cell proliferation compared to AVC1 control enhancers with wild-type 4-1BBL (Figure 8). NK cells were seeded in the presence of the enhancer or mediator (conditioned SK-OV-3 cells), and NK cell proliferation was tracked over 14 days as described in Example 1.1.7. As shown in Figure 8, the reduced affinity of the enhancer for 4-1BB compared to AVC1 did not significantly affect proliferation, except for AVC55, which showed lower proliferation compared to AVC1 and other enhancers.

Figure 9 shows the results of a long-term repeated NK cell cytotoxicity assay as described in Example 1.1.13. The NK enhancer AVC59, containing the A154D 4-1BBL ECD mutant protein, was compared to the control enhancer AVC1, containing wild-type 4-1BBL ECD. Other controls included the mediator, NK cells only, and SKOV-3 target cells, or SKOV-3 target cells only. As can be seen from Figure 9, the AVC59 enhancer exhibited similar efficacy to the wild-type enhancer AVC1 in the long-term cytotoxicity assay.

Table 1.2.2. Overview of the reduced binding affinity of selected enhancers containing the IL-21 mutant protein and the trimer of wild-type 4-1BBL ECD to IL-21R compared to the control enhancer AVC1 (containing the trimer of wild-type 4-1BBL ECD and wild-type IL-21). AVC ID mutation K _D [nM] pK _D Logarithmic reduction pK _D AVC1 - 0.040 10.39 0.00 AVC34 L13E 0.838 9.08 1.32 AVC36 D4H 0.305 9.52 0.88 AVC37 I8H 0.251 9.60 0.79 AVC114 N82- A83- G84- R85- R86- Q87- K88- 15.85 7.80 2.59 AVC116 N63- E64- R65- I66- 0.1 10.02 0.37 AVC118 I67T 0.04 10.41 -0.02 AVC119 I67N 0.45 9.35 1.05 AVC120 L74G ND ND AVC121 L74D >500 <6.3 >4 AVC122 L74F 0.12 9.91 0.48 AVC123 L20S 3.24 8.49 1.91 AVC124 L20N 9.12 8.04 2.35 AVC125 L20W 1.82 8.74 1.65 AVC126 G84GGGGG 0.04 10.36 0.03

In Table 1.2.3, the ability of enhancers to induce maximum NK cell proliferation in the presence of SKOV3 tumor cells was determined at a saturation concentration of 25 nM. The ability of enhancers to induce maximum NK cell proliferation was determined by enhancers containing a combination of IL-21 mutant protein and wild-type 4-1BBL ECD or 4-1BBL A154D mutant protein, wherein the affinity of wild-type 4-1BBL ECD or 4-1BBL A154D mutant protein for its homologous receptor 4-1BB was reduced compared to the control enhancer AVC1. Table 1.2.3 also presents the pEC ₅₀ values for NK cell proliferation induced by each enhancer in the presence of SKOV3 tumor cells.

Table 1.2.3. An overview of the ability of enhancers containing a combination of IL-21 mutant protein and the trimer of wild-type 4-1BBL ECD or the 4-1BBL A154D mutant protein to induce NK cell proliferation in the presence of SKOV3 tumor cells at a saturation concentration (25 nM), as shown. The control enhancer AVC1 (containing the trimer of wild-type 4-1BBL ECD and wild-type IL-21) is also presented. pEC ₅₀ values for enhancer-induced NK cell proliferation in the presence of SKOV3 tumor cells are also shown. AVC ID IL-21 4-1BBL pEC ₅₀ %AVC1 proliferation at 25 nM AVC1 WT WT 9.66 100% AVC114 N82- A83- G84- R85- R86- Q87- K88- WT 10.42 102% AVC121 L74D WT 9.16 95% AVC123 L20S WT 10.18 105% AVC124 L20N WT 10.25 92% AVC125 L20W WT 9.66 95% AVC187 L20N A154D 9.20 49% AVC188 L20W A154D 9.14 89% AVC189 L20S A154D 9.28 61% AVC190 N82- A83- G84- R85- R86- Q87- K88- A154D 9.32 108%

As shown in Table 1.2.3, compared with the corresponding enhancer containing wild-type IL-21, the ability of the NK cell enhancer containing wild-type 4-1BBL ECD and a certain number of selected IL-21 mutant proteins with reduced affinity for IL-21R to induce NK cell proliferation in the presence of SKOV3 tumor cells was almost unaffected. pEC ₅₀ was reduced by more than log 0.5 compared with the control enhancer, while the maximum induced proliferation at saturated enhancer concentrations was reduced by no more than 10% compared with the control enhancer.

However, when the wild-type 4-1BBL ECD in these enhancers was replaced by the reduced-affinity 4-1BBL ECD A154D mutant protein, the pEC ₅₀ value was still reduced by more than log 0.5 compared to the control enhancer. However, compared to the control group, the maximum induced proliferation at saturated enhancer concentrations was significantly reduced by more than 50% for at least two IL-21 mutant proteins. In contrast, the combination of the IL-21 mutant protein L20W and the 4-1BBL mutant protein retained most of the ability to maximally induce NK cell proliferation, with a reduction in maximum induced proliferation of no more than about 10% compared to the corresponding wild-type control enhancer AVC1. The AVC190 enhancer, which contains the combination of 4-1BBL ECD mutant protein A154D and IL-21 mutant protein DEL7 (N82-A83-G84-R85-R86-Q87-K88-), even outperformed the wild-type control enhancer AVC1 in terms of maximum induced proliferation at saturated enhancer concentrations.

Similar results to those obtained above using an enhancer targeting HER2 as a TAA were also obtained using an enhancer targeting TROP2 as a TAA. The ability of the enhancers AVC1 and AVC16 (targeting HER2 and TROP2, respectively) to induce NK cell cytotoxicity was compared. The long-term NK cell cytotoxicity induced by the conjugates AVC1 and AVC16 was determined substantially as described in Example 1.1.13, except that only a single round of co-culturing of NK cells and target cells was performed for 96 hours. In summary, the cytotoxicity of NK cells against tumor cell lines exhibiting specific binding to the conjugates AVC1 and AVC16 was determined, as shown in Table 1.2.4. NK cells and target cells were co-cultured in a single round at a 2:1 E:T ratio (20,000 NK cells: 10,000 tumor cells) for 96 hours in the presence or absence of 25 nM of multispecific antigen-binding proteins. Therefore, the control wells contained only NK cells and the corresponding target tumor cells. Triples were used for testing. Interferon-gamma levels in the supernatant were determined using the MACSplex cytotoxic IFN-γ kit (catalog number 130-125-800).

Table 1.2.4 Target tumor cell lines used to identify NK cell cytotoxicity induced by multispecific antigen-binding proteins AVC1 and AVC16. Multispecific antigen binding protein target antigen UNIPROT Login Target tumor cell lines AVC1 HER2 P04626 SKOV-3 AVC16, AVC221, AVC227, AVC245, AVC267 TROP2 P09758 BxPC3

The results for AVC1 and AVC16 are shown in Figures 10A and 10B, respectively. Compared to NK cells alone, both multispecific antigen-binding proteins AVC1 and AVC16 significantly induced increased cytotoxicity against tumor cells expressing the corresponding antigens. Similarly, Figures 11A and 11B show that the same group of multispecific antigen-binding proteins responded to increased interferon-gamma production by co-culturing with tumor cells expressing the corresponding antigens. Therefore, when targeting HER2 or TROP2, which are tumor-associated antigens, similar stimulatory effects of multispecific antigen-binding proteins were achieved in inducing TAA-targeted NK cell cytotoxicity or inducing interferon-gamma production.

Figure 12 shows the NK enhancers AVC16 (based on the Fab fragment from goxatozumab), AVC221 (based on the Fab fragment from AR47A6.4.2, described in US20120237518A1), and AVC227 (based on the Fab fragment from AR47A6.4.2, described in US2008131428A1) in long-term repeated NK cell cytotoxicity assays, as described in Example 1.1.13. The additional controls were NK cells and BxPC3 target cells only (i.e., without enhancers) or BxPC3 target cells only. As can be seen from Figure 12, the NK enhancers AVC16, AVC221 and AVC227, each based on different TROP2 antigen-binding proteins, have the same efficacy in inducing NK cell cytotoxicity against TROP2-expressing BxPC3 tumor cells.

Figure 13 shows a comparison of the NK enhancers AVC16 (based on the Fab fragment of gosatuzumab, containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD), AVC267 (based on the Fab fragment of gosatuzumab, containing a trimer of IL-21 L20W mutant protein and wild-type 4-1BBL ECD), and the control protein AVC 137 (a gosatuzumab analogue, without IL-21 or 4-1BBL cytokines) in long-term repeated NK cell cytotoxicity assays, as described in Example 1.1.13. Additional controls included NK cells only and BxPC3 target cells (i.e., without enhancers) or BxPC3 target cells only. As can be seen from Figure 13, the NK enhancer AVC267, which contains the IL-21 mutant protein L20W, exhibits similar long-term in vivo tumor control as the AVC16 enhancer containing wild-type IL-21.

Figure 14 shows a comparison of the NK enhancers AVC16 (based on the Fab fragment of gosatuzumab, containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD), AVC245 (based on the Fab fragment of gosatuzumab, containing a trimer of the IL-21 DEL7 (N82-A83-G84-R85-R86-Q87-K88-) mutant and the 4-1BBL ECD A154D mutant) and the control protein AVC 137 (a gosatuzumab analogue without IL-21 or 4-1BBL cytokines) in long-term repeated NK cell cytotoxicity assays, as described in Example 1.1.13. The other controls consisted of NK cells and BxPC3 target cells only (i.e., without the enhancer) or BxPC3 target cells only. As shown in Figure 14, the NK enhancer AVC245, containing both the IL-21 mutant and the 4-1BBL ECD mutant, exhibited even better long-term in vivo tumor control than the AVC16 enhancer, which contains both wild-type IL-21 and 4-1BBL.

Figure 15 shows the in vivo pharmacokinetics of the NK enhancers AVC16 and AVC245 compared to the control antibodies AVC137 and trastuzumab. Mice were intravenously injected with 1 mg/kg body weight of the enhancer or antibody. Blood samples were collected at 15 min, 2 h, and 1, 3, and 7 days post-injection for analysis, and the serum levels of the enhancer antibody and control antibody were determined by ELISA. As shown in Figure 15, after a decrease in the first 15 minutes, serum levels stabilized for the remainder of the 7-day testing period. Within this time frame, there were no significant differences between the enhancers and antibodies tested.

Figure 16 shows that the in vivo efficacy of the AVC245 enhancer in reducing tumor burden in a mouse xenograft model (see Example 1.1.15) was significantly higher than that of the control antibody AVC137, which lacked the 4-1BBL and IL-21 mutant proteins. Table 1.2.5 confirms the statistical significance of the tumor burden-reducing efficacy of AVC245 relative to the control antibody AVC137 and the tumor-only and NK cell + tumor controls.

Table 1.2.5. Two-way ANOVA + Tukey multiple comparison test. Tumor only NK NK + AVC245 NK ns NK + AVC245 **** (<0.0001) **** (<0.0001) NK + AVC137 *** (0.0006) **** (<0.0001) ** (0.0083)

Figure 17 shows that the in vivo efficacy of the AVC245 enhancer in inducing donor NK cell proliferation in a mouse xenograft model (see Example 1.1.15) was significantly higher than that of the control antibody AVC137, which lacks the 4-1BBL and IL-21 mutant proteins. Therefore, Figures 16 and 17 convincingly demonstrate the therapeutic efficacy of conjugates containing an antigen-binding protein targeting TAA, along with the 4-1BBL and IL-21 mutant proteins as described herein, in treating tumors exhibiting TAA.

without

Figure 1. NK enhancers containing IL-21 or 4-1BBL ECD mutant proteins only exhibit impaired ability to induce proliferation in the absence of tumor cells. NK enhancers AVC37 (trastuzumab and a conjugate of I8H IL-21 mutant protein and wild-type 4-1BBL ECD) and AVC52 (trastuzumab and a conjugate of wild-type IL-21 and V153Q 4-1BBL ECD mutant protein) showed severely impaired ability to induce NK cell proliferation, but this only occurred in the absence of HER2-expressing SKOV3 tumor cells. In the presence of SKOV3 tumor cells, the NK enhancers AVC37 and AVC52 were comparable to the control NK enhancer AVC1 (a conjugate of trastuzumab and wild-type IL-21 and wild-type 4-1BBL ECD mutant proteins). NK cells were cultured for 5 days with or without tumor cells (dashed lines) and the indicated concentrations of NK enhancers were shown. Data presented are from measurements taken at the end of co-culture. Dark gray and light gray areas indicate the historical mean and standard deviation obtained using AVC1 in the presence and absence of tumor cells, respectively.

Figure 2. The NK enhancer AVC54 (a conjugate of trastuzumab, wild-type IL-21, and the Q227E 4-1BBL ECD mutant protein) also showed severely impaired ability to induce NK cell proliferation, but this only occurred in the absence of HER2-expressing SKOV3 tumor cells. In the presence of SKOV3 tumor cells, the NK enhancers AVC37 and AVC52 were comparable to the control group NK enhancer AVC1 (a conjugate of trastuzumab, wild-type IL-21, and the wild-type 4-1BBL ECD mutant protein). NK cells were cultured for 5 days with or without tumor cells (dashed lines), and the concentrations of NK enhancers shown are illustrated. The data presented are from measurements taken at the end of co-culture.

Figure 3. As shown, the ability of NK enhancers containing various 4-1BBL ECD mutant proteins to induce proliferation was impaired compared to the corresponding control NK enhancer AVC1 containing wild-type 4-1BBL ECD. NK cells were cultured for 5 days without tumor cells and with the NK enhancers shown; the data shown are from measurements taken at the end of co-culture.

Figure 4. NK cell cytotoxicity against SKOV3 tumor cells, induced by NK enhancers containing IL-21 or 4-1BBL ECD mutant proteins, is unaffected by IL-21 or 4-1BBL mutations. NK cells were co-cultured with tumor cells for 5 days in the presence of either NK enhancer AVC37 (a conjugate of trastuzumab, I8H IL-21 mutant protein, and wild-type 4-1BBL ECD), NK enhancer AVC52 (a conjugate of trastuzumab, wild-type IL-21, and V153Q 4-1BBL ECD mutant protein), or control NK enhancer AVC1 (a conjugate of trastuzumab, wild-type IL-21, and wild-type 4-1BBL ECD mutant protein), at concentrations as indicated. Data shown are from measurements performed at the end of co-culture.

Figure 5. The ability of NK enhancers containing IL-21 or 4-1BBL ECD mutant proteins to induce and support long-term NK cell proliferation is unaffected. NK cell proliferation was achieved for 14 days using SKOV3 tumor cells conditioned with an indicator NK enhancer. The fold increase in NK cells after 14 days is indicated.

Figure 6. The left panel shows the dose-response curve of the NK enhancer and the 4-1BBL ECD mutant protein in the 4-1BB reporting cell assay, compared to the dose-response curves of the control enhancer AVC1 and wild-type 4-1BBL. The right panel shows the reaction at 100 nM enhancer, with the _EC50 value in nM given on the far right. A) Comparison of reinforcing agents AVC50, AVC51, AVC52, AVC53 and AVC54 with the control reinforcing agent AVC1; B) Comparison of reinforcing agents AVC55, AVC56, AVC57, AVC58 and AVC59 with the control reinforcing agent AVC1; and C) Comparison of reinforcing agents AVC46, AVC47, AVC48 and AVC49 with the control reinforcing agent AVC1.

Figure 7. Plotting the pEC ₅₀ values (x-axis) of NK enhancer and the indicated 4-1BBL ECD mutant protein, and the control enhancer AVC1 and wild-type 4-1BBL for 4-1BB reporter cell assays, as determined by surface plasma resonance (y-axis).

Figure 8. NK enhancer and the NK cell proliferation induced by the 4-1BBL ECD mutant protein shown, compared with the NK cell proliferation induced by the control enhancer AVC1 and wild-type 4-1BBL.

Figure 9. Long-term cytotoxicity of NK cells stimulated with the NK enhancer AVC59 and the 4-1BBL ECD A154D mutant protein or the wild-type control enhancer AVC1 against SKOV-3 tumor cells after repeated co-culture. NK cells were harvested after each 3-day cycle and used to establish new co-culture cycles with fresh target cells at a 1:1 E:T ratio. At the beginning of each 3-day cycle, enhancer AVC1 or AVC59 or a control protein (trastuzumab analogue) was added to expose NK cells to the new enhancer protein at the start of each cycle. The control wells contained only NK cells and SKOV-3 target cells (NK + SKOV3), or only SKOV-3 target cells (SKOV3 only).

Figure 10. Long-term NK cell cytotoxicity induced by multispecific antigen-binding proteins AVC-001 and AVC-016 against tumor cell lines expressing their respective tumor-associated antigens: (A) AVC-001 (AVC1) and SKOV-3 tumor cells expressing HER2; (B) AVC-016 (AVC16) and BxPC3 tumor cells expressing TROP2. Changes in NK cell cytotoxicity (hollow squares) induced by multispecific antigen-binding proteins against tumor cells expressing corresponding antigens over time (hours), compared with NK cell cytotoxicity alone (solid circles).

Figure 11. Interferon-gamma levels after co-culturing with tumor cells expressing the corresponding antigen. Data are shown as fold changes with mediator (NK cells + tumor cells). (A) AVC-001 (AVC1) and SKOV-3 tumor cells expressing HER2; (B) AVC-016 (AVC16) and BxPC3 tumor cells expressing TROP2.

Figure 12. Long-term cytotoxicity of NK cells stimulated with the NK enhancers AVC16 (based on goxatozumab), AVC221 (based on AR47A6.4.2, described in US2008131428A1), and AVC227 (based on KM4097, described in US20120237518A1) against BxPC3 tumor cells (expressing TROP2) after repeated co-culture. NK cells were harvested after each 3-day cycle and used to establish new co-culture cycles with fresh target cells at a 1:1 E:T ratio. An NK enhancer was added at the start of each 3-day cycle to expose NK cells to the new enhancer protein at the beginning of each cycle. Control wells contained only NK cells and BxPC3 target cells (NK + BxPC3), or only BxPC3 target cells (BxPC3 only).

Figure 13. Long-term cytotoxicity of NK cells against TROP2-expressing BxPC3 tumor cells using the following stimuli: NK enhancers AVC16 (based on the Fab fragment of gosatuzumab, containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD), AVC267 (based on the Fab fragment of gosatuzumab, containing a trimer of IL-21 L20W mutant protein and wild-type 4-1BBL ECD), and control protein AVC 137 (a gosatuzumab analogue without IL-21 or 4-1BBL cytokines) (after repeated co-culture with BxPC3 tumor cells). After each 3-day cycle, NK cells were harvested and used to establish new co-culture cycles with fresh target cells at a 1:1 E:T ratio. An NK enhancer was added at the start of each 3-day cycle to expose the NK cells to the new enhancer protein at the beginning of each cycle. Control wells contained only NK cells and BxPC3 target cells (NK + BxPC3), or only BxPC3 target cells (BxPC3 only).

Figure 14. Long-term cytotoxicity of NK cells against (TROP2-expressing) BxPC3 tumor cells stimulated with the following: NK enhancers AVC16 (based on the Fab fragment of gosatuzumab, containing a trimer of wild-type IL-21 and wild-type 4-1BBL ECD), AVC245 (based on the Fab fragment of gosatuzumab, containing a trimer of IL-21 DEL7 (N82-A83-G84-R85-R86-Q87-K88-) mutant protein and 4-1BBL ECD A154D mutant protein), and control protein AVC 137 (a gosatuzumab analogue without IL-21 or 4-1BBL cytokines) (after repeated co-culture with BxPC3 tumor cells). After each 4-5 day cycle, NK cells are harvested and used to establish new co-culture cycles with fresh target cells at a 1:1 E:T ratio. An NK enhancer is added at the beginning of each cycle to expose the NK cells to the new enhancer protein at the start of each cycle. Control wells contain only NK cells and BxPC3 target cells (NK + BxPC3), NK cells and BxPC3 target cells and PBS (medium), or only BxPC3 target cells (BxPC3 only).

Figure 15. In vivo pharmacokinetics of NK enhancers AVC16 and AVC245 compared to control antibodies AVC137 and trastuzumab. Mice were intravenously injected with 1 mg/kg body weight of the enhancer or antibody. Blood samples were collected at specified time points after injection for analysis, and the levels of enhancer antibody and control antibody in mouse serum were determined by ELISA.

Figure 16. The in vivo efficacy of the AVC245 enhancer in reducing tumor burden in a mouse xenograft model was significantly higher than that of the control antibody AVC137, which was used only for tumors, NK cells + tumors, or without the 4-1BBL and IL-21 mutant proteins.

Figure 17. The in vivo efficacy of AVC245 enhancer in inducing donor NK cell proliferation in a mouse xenograft model was significantly higher than that of the control antibody AVC137, which was used for tumor only, NK cells + tumor, or without 4-1BBL and IL-21 mutant proteins.

TW202600601A_114109842_SEQL.xml

Claims (15)

A conjugate comprising: a) an antigen-binding protein including at least one antigen-binding region specifically binding to TROP2; b) a mutant protein of the extracellular domain (ECD) of a 4-1BB ligand (4-1BBL), wherein the 4-1BBL ECD mutant protein exhibits a binding affinity for human 4-1BB, expressed as _pKD , which is at least 1.0 lower than _{the pKD} of wild-type 4-1BBL ECD for human 4-1BB, wherein the 4-1BBL ECD mutant protein is used as a conjugate of an antibody specifically binding to a tumor-associated antigen (TAA). In the presence of a portion of the homotrimer of the ECD mutant protein, in the presence of tumor cells expressing the TAA, the conjugate induced maximum NK cell proliferation at a saturation concentration of 25 nM for 5 days in a normalized NK cell proliferation assay, the proliferation being not less than 50% of that induced by a corresponding control conjugate containing the trimer of wild-type 4-1BBL ECD in the same assay, the conjugate further comprising human IL-21; and c) The IL-21 mutant protein binds to human IL-21R with a reduced affinity relative to wild-type IL-21 for the human IL-21 receptor (IL-21R), wherein the IL-21 mutant protein, when present in a conjugate of an antibody that specifically binds to a tumor-associated antigen (TAA), has an EC50 that induces NK cell proliferation in the presence of tumor cells expressing the TAA in a normalized 5-day NK cell proliferation _assay , the _EC50 being no more than 5-fold higher than the _EC50 of a corresponding control conjugate containing wild-type IL-21 in the same assay, the conjugate further comprising a trimer of the wild-type 4-1BB ligand extracellular domain (4-1BBL ECD). The conjugate as described in claim 1, wherein the antigen-binding region specifically binding to TROP2 includes a combination of complementary determinant regions (CDRs) selected from the following groups: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3: a) CDR-H1 containing the sequence of SEQ ID NO: 552, CDR-H2 containing the sequence of SEQ ID NO: 553, CDR-H3 containing the sequence of SEQ ID NO: 554, CDR-L1 containing the sequence of SEQ ID NO: 555, CDR-L2 containing the sequence of SEQ ID NO: 556, and CDR-L3 containing the sequence of SEQ ID NO: 557 (goxatozumab); b) CDR-H1 containing the sequence of SEQ ID NO: 558, CDR-L2 containing the sequence of SEQ ID NO: 559, CDR-L2 containing the sequence of SEQ ID NO: 559, and CDR-L3 containing the sequence of SEQ ID NO: 557. c) CDR-H2 of the sequence containing SEQ ID NO: 559, CDR-H3 of the sequence containing SEQ ID NO: 560, CDR-L1 of the sequence containing SEQ ID NO: 561, CDR-L2 of the sequence containing SEQ ID NO: 562, and CDR-L3 of the sequence containing SEQ ID NO: 563 (dadabutumab); c) CDR-H1 of the sequence containing SEQ ID NO: 564, CDR-H2 of the sequence containing SEQ ID NO: 565, CDR-H3 of the sequence containing SEQ ID NO: 566, CDR-L1 of the sequence containing SEQ ID NO: 567, CDR-L2 of the sequence containing SEQ ID NO: 568, and CDR-L3 of the sequence containing SEQ ID NO: 569 (KM4097); d) CDR-L3 of the sequence containing SEQ ID NO: CDR-H1 of sequence 570, CDR-H2 of sequence SEQ ID NO: 571, CDR-H3 of sequence SEQ ID NO: 572, CDR-L1 of sequence SEQ ID NO: 573, CDR-L2 of sequence SEQ ID NO: 574, and CDR-L3 of sequence SEQ ID NO: 575 (AR47A6.4.2); and e) CDR-H1 of sequence SEQ ID NO: 576, CDR-H2 of sequence SEQ ID NO: 577, CDR-H3 of sequence SEQ ID NO: 578, CDR-L1 of sequence SEQ ID NO: 579, CDR-L2 of sequence SEQ ID NO: 580, and CDR-L3 of sequence SEQ ID NO: 581 (K5-70). The conjugate as described in claim 1 or 2, wherein the antigen-binding region specifically binding to TROP2 comprises a combination of variable heavy ( _VH ) and variable light ( _VL ) domains selected from the following groups: a) the _VH sequence as contained in SEQ ID NO: 138 and the _VL sequence as contained in SEQ ID NO: 139 (goxatocilizumab); b) the VH sequence as contained in SEQ ID NO: 338 and the _VL sequence as contained in SEQ ID NO: 339 (dadabutumab); c) the _VH sequence as contained in SEQ ID NO: 540 and the _VL sequence as contained in SEQ ID NO: 541 (AR46A6); d) the _VH sequence as contained in SEQ ID NO: 542 and the _VL sequence as contained in SEQ ID NO: 543 (KM4097); and e) as contained in SEQ _ID NO: 138. The V _H sequence contained in ID NO: 544, and the V _L sequence (K5-70) contained in SEQ ID NO: 545. The conytonic as described in any of the preceding claims, wherein at least one of the following is true: a) the amino acid sequence of the 4-1BBL ECD mutant protein differs from the wild-type human 4-1BBL ECD amino acid sequence of SEQ ID NO: 37 in that the 4-1BBL ECD mutant protein contains at least one substitution selected from the group consisting of: A154D, A154E, a combination of A154D and G155Q, V153Q, Q227E, L101N, Y110Q, Q230K and V100Q; and b) the amino acid sequence of the IL-21 mutant protein differs from the wild-type human IL-21 amino acid sequence of SEQ ID NO: 38 in that the IL-21 mutant protein contains at least one amino acid substitution or deletion selected from the group consisting of: (N82- A83- G84- R85- R86- Q87- K88-), L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63- E64- R65- I66-); and L74F. The conjugate as described in any of the preceding claims, wherein the 4-1BBL ECD mutant protein exists as part of a fusion protein comprising three 4-1BBL ECD monomers, wherein one, two, or three of the monomers are the 4-1BBL ECD mutant protein as described in claim 1 or 4, wherein the three 4-1BBL ECD monomers are fused together in a single polypeptide chain, and wherein, where appropriate, the three 4-1BBL ECD monomers are linked by a polypeptide linker. The conjugate as described in claim 5, wherein the fusion protein comprises three identical 4-1BBL ECD monomers as described in claims 1 or 4, and wherein preferably the three 4-1BBL ECD monomers are linked by a (GGGGS) ₄ polypeptide linker. The conytomer as described in any of the preceding claims, wherein the conytomer comprises a combination of 4-1BBL ECD mutant proteins and IL-21 mutant proteins selected from the group consisting of: 4-1BBL mutant protein A154D and IL-21 mutant protein (N82-A83-G84-R85-R86-Q87- K88-); 4-1BBL mutant protein A154D and IL-21 mutant protein L20W; 4-1BBL mutant protein A154D and IL-21 mutant protein L74D; 4-1BBL mutant protein A154D and IL-21 mutant protein L20N; 4-1BBL mutant protein A154D and IL-21 mutant protein I67N; 4-1BBL mutant protein A154D and IL-21 mutant protein L20S; 4-1BBL mutant protein A154D and IL-21 mutant protein L13E; 4-1BBL mutant protein A154D and IL-21 mutant protein I8H; 4-1BBL mutant protein A154D and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein A154D and IL-21 mutant protein L74F; 4-1BBL mutant protein A154E and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein A154E and IL-21 mutant protein L20W; 4-1BBL mutant protein A154E and IL-21 mutant protein L74D; 4-1BBL mutant protein A154E and IL-21 mutant protein L20N; 4-1BBL mutant protein A154E and IL-21 mutant protein I67N; 4-1BBL mutant protein A154E and IL-21 mutant protein L20S; 4-1BBL mutant protein A154E and IL-21 mutant protein L13E; 4-1BBL mutant protein A154E and IL-21 mutant protein I8H; 4-1BBL mutant protein A154E and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein A154E and IL-21 mutant protein L74F; 4-1BBL mutant protein A154D+ G155Q and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein A154D+ G155Q and IL-21 mutant protein L20W; 4-1BBL mutant protein A154D+ G155Q and IL-21 mutant protein L74D; 4-1BBL mutant protein A154D+ G155Q and IL-21 mutant protein L20N; 4-1BBL mutant protein A154D+ G155Q and IL-21 mutant protein I67N; 4-1BBL mutant protein A154D+ G155Q and IL-21 mutant protein L20S; 4-1BBL mutant protein A154D + G155Q and IL-21 mutant protein L13E; 4-1BBL mutant protein A154D + G155Q and IL-21 mutant protein I8H; 4-1BBL mutant protein A154D + G155Q and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein A154D + G155Q and IL-21 mutant protein L74F; 4-1BBL mutant protein V153Q and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87-) K88-); 4-1BBL mutant protein V153Q and IL-21 mutant protein L20W; 4-1BBL mutant protein V153Q and IL-21 mutant protein L74D; 4-1BBL mutant protein V153Q and IL-21 mutant protein L20N; 4-1BBL mutant protein V153Q and IL-21 mutant protein I67N; 4-1BBL mutant protein V153Q and IL-21 mutant protein L20S; 4-1BBL mutant protein V153Q and IL-21 mutant protein L13E; 4-1BBL mutant protein V153Q and IL-21 mutant protein I8H; 4-1BBL mutant protein V153Q and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein V153Q and IL-21 mutant protein L74F; 4-1BBL mutant protein Q227E and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein Q227E and IL-21 mutant protein L20W; 4-1BBL mutant protein Q227E and IL-21 mutant protein L74D; 4-1BBL mutant protein Q227E and IL-21 mutant protein L20N; 4-1BBL mutant protein Q227E and IL-21 mutant protein I67N; 4-1BBL mutant protein Q227E and IL-21 mutant protein L20S; 4-1BBL mutant protein Q227E and IL-21 mutant protein L13E; 4-1BBL mutant protein Q227E and IL-21 mutant protein I8H; 4-1BBL mutant protein Q227E and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein Q227E and IL-21 mutant protein L74F; 4-1BBL mutant protein L101N and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein L101N and IL-21 mutant protein L20W; 4-1BBL mutant protein L101N and IL-21 mutant protein L74D; 4-1BBL mutant protein L101N and IL-21 mutant protein L20N; 4-1BBL mutant protein L101N and IL-21 mutant protein I67N; 4-1BBL mutant protein L101N and IL-21 mutant protein L20S; 4-1BBL mutant protein L101N and IL-21 mutant protein L13E; 4-1BBL mutant protein L101N and IL-21 mutant protein I8H; 4-1BBL mutant protein L101N and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein L101N and IL-21 mutant protein L74F; 4-1BBL mutant protein Y110Q and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein Y110Q and IL-21 mutant protein L20W; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L74D; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L20N; 4-1BBL mutant protein Y110Q and IL-21 mutant protein I67N; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L20S; 4-1BBL mutant protein Y110Q and IL-21 mutant protein L13E; 4-1BBL mutant protein Y110Q and IL-21 mutant protein I8H; 4-1BBL mutant protein Y110Q and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein Y110Q and IL-21 mutant protein L74F; 4-1BBL mutant protein Q230K and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein Q230K and IL-21 mutant protein L20W; 4-1BBL mutant protein Q230K and IL-21 mutant protein L74D; 4-1BBL mutant protein Q230K and IL-21 mutant protein L20N; 4-1BBL mutant protein Q230K and IL-21 mutant protein I67N; 4-1BBL mutant protein Q230K and IL-21 mutant protein L20S; 4-1BBL mutant protein Q230K and IL-21 mutant protein L13E; 4-1BBL mutant protein Q230K and IL-21 mutant protein I8H; 4-1BBL mutant protein Q230K and IL-21 mutant protein (N63- E64- R65- I66-); 4-1BBL mutant protein Q230K and IL-21 mutant protein L74F; 4-1BBL mutant protein V100Q and IL-21 mutant protein (N82- A83- G84- R85- R86- Q87- K88-); 4-1BBL mutant protein V100Q and IL-21 mutant protein L20W; 4-1BBL mutant protein V100Q and IL-21 mutant protein L74D; 4-1BBL mutant protein V100Q and IL-21 mutant protein L20N; 4-1BBL mutant protein V100Q and IL-21 mutant protein I67N; 4-1BBL mutant protein V100Q and IL-21 mutant protein L20S; 4-1BBL mutant protein V100Q and IL-21 mutant protein L13E; 4-1BBL mutant protein V100Q and IL-21 mutant protein I8H; 4-1BBL mutant protein V100Q and IL-21 mutant protein (N63- E64- R65- I66-); as well as the 4-1BBL mutant protein V100Q and the IL-21 mutant protein L74F. The conyzosome as described in claim 7, wherein the conyzosome comprises the 4-1BBL mutant protein A154D and the IL-21 mutant protein (N82-A83-G84-R85-R86-Q87-K88-); or a combination of the 4-1BBL mutant protein A154D and the IL-21 mutant protein L20W. The conjugate as described in any of the preceding claims, wherein the conjugate comprises: a) an antigen-binding protein comprising at least one antigen-binding region specifically binding to TROP2, wherein the antigen-binding region comprises: CDR-H1 comprising the sequence of SEQ ID NO: 552, CDR-H2 comprising the sequence of SEQ ID NO: 553, CDR-H3 comprising the sequence of SEQ ID NO: 554, CDR-L1 comprising the sequence of SEQ ID NO: 555, CDR-L2 comprising the sequence of SEQ ID NO: 556, and CDR-L3 comprising the sequence of SEQ ID NO: 557 (goxatocilizumab); b) a 4-1BBL ECD mutant protein, wherein the amino acid sequence of the 4-1BBL ECD mutant protein is identical to that of wild-type human 4-1BBL in SEQ ID NO: 37. The difference in the ECD amino acid sequence is that the 4-1BBL ECD mutant protein contains the substitution A154D; and c) the IL-21 mutant protein, the difference in the amino acid sequence of which is different from the wild-type human IL-21 amino acid sequence of SEQ ID NO: 38, the difference being that the IL-21 mutant protein contains the deletion (N82- A83- G84- R85- R86- Q87- K88-). The conytomer as described in any of the preceding claims, wherein the conytomer comprises a dimer Fc region bound to CD16A. The conjugate as described in any one of claims 1 to 10, wherein at least one antigen-binding region of TROP2 specifically binds to the dimer Fc region to form a dimer immunoglobulin structure, and wherein at least one of the 4-1BBL ECD mutant protein and the IL-21 mutant protein is present on at least one or both sides of the dimer immunoglobulin structure. A pharmaceutical composition comprising a conjugate as described in any one of claims 1 to 11 and a pharmaceutically acceptable delivery agent. The conjugate as described in any one of claims 1 to 11 or the combination as described in claim 12 is used for the treatment of cancer, wherein, as appropriate, the conjugate or the combination is used in combination with the transfer of immune cells, wherein preferably the immune cells are selected from T cells and NK cells. The conytomer described in any of claims 1 to 11 or the composition described in claim 12, used for the purpose described in claim 13, wherein the cancer is a cancer comprising tumor cells expressing TROP2. The conjugate or combination thereof described in any one of claims 1 to 11 or as described in claim 12 is used for the purposes described in claims 13 or 14, wherein at least one of the following is employed: a) the conjugate or combination thereof is applied as a novel adjunctive therapy prior to at least one of the primary therapies comprising surgery and radiotherapy for the cancer; and b) the conjugate or combination thereof is applied as an adjunctive therapy after at least one of the primary therapies comprising surgery and radiotherapy for the cancer.