TWI908113B - Anti-TIGIT antibodies and their uses - Google Patents

Abstract

This disclosure provides anti-TIGIT antibodies, nucleic acid molecules encoding anti-TIGIT antibodies, expression vectors for expressing anti-TIGIT antibodies, and host cells. This disclosure further provides methods for the prevention and treatment of cancer and immune disorders by administering anti-TIGIT antibodies.

Description

Anti-TIGIT antibodies and their uses

This application generally relates to antibodies. More specifically, this application relates to monoclonal antibodies against TIGIT, methods for preparing the same, and uses of said antibodies.

In the tumor microenvironment, continuous antigen stimulation can lead to T cell exhaustion (a state of T cell dysfunction) and overexpression of co-inhibitory receptors, including PD-1, LAG-3, TIM3, and TIGIT. Currently, various strategies are being explored to activate/restore exhausted T cells through the single or combined use of small molecule or therapeutic antibody approaches.

TIGIT (a T-cell immune receptor with Ig and ITIM domains) is a co-suppressive receptor, also known as Vstm3 and WUCAM, expressed on NK and CD8+ T cells, as well as CD4+ T cell subsets (including immunosuppressive regulatory T cells (Tregs)). Four ligands for TIGIT are known: poliovirus receptor (PVR), PVRL2, PVRL3, and PVRL4, all of which are overexpressed by tumor cells and antigen-presenting cells, leading to immunosuppression. These ligands also bind to the co-stimulatory molecule CD226 and the co-suppressive molecules PVRIG and CD96 (the latter sometimes considered a co-stimulator). Antibodies against TIGIT disrupt the binding of TIGIT to its ligands and block its inhibitory signaling, shifting the balance in favor of CD226-mediated activation, thereby inducing a strong antitumor immune response. TIGIT is upregulated in cancer and inflammatory diseases and has been identified as an exhaustion marker, like other exhaustion markers such as PD-1, LAG3, and TIM3. Furthermore, TIGIT has been identified as a key inhibitory receptor for a novel T cell population, stem cell-like memory T cells, which may be a preferred target for anti-PD-(L)1 therapy.

TIGIT could be a promising therapeutic target in tumor immunotherapy, either as a single agent or in combination with other immunomodulators.

In a broader sense, this disclosure relates to compounds, methods, compositions, and articles of antibodies that have improved efficacy. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapy and diagnosis, and can be used in combination with other antibodies that can respond to a variety of targets.

This disclosure provides chimeric and humanized monoclonal antibodies against TIGIT. It further provides methods for validating antibody function in vitro and in vivo, and methods for treating subjects with cancer or immune disorders by administering the disclosed anti-TIGIT antibodies alone or in combination with a PD-1/PD-L1 antagonist.

In some aspects, this disclosure provides isolated antibodies against TIGIT or antigen-binding portions thereof. In some embodiments, the isolated antibody or antigen-binding portion thereof comprises:

The heavy chain CDR (HCDR)1 containing the amino acid sequence of SEQ ID NO: 1; the HCDR2 containing the amino acid sequence of SEQ ID NO: 2; the HCDR3 containing the amino acid sequence of SEQ ID NO: 3; the light chain CDR (LCDR)1 containing the amino acid sequence of SEQ ID NO: 4; the LCDR2 containing the amino acid sequence of any one of SEQ ID NO: 7, 5, 8 and 9; and the LCDR3 containing the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the isolated antibody or its antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

The VH includes or consists of the following:

(i) The amino acid sequence shown in any one of SEQ ID NO: 10-11;

(ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of SEQ ID NO: 10-11; or

(iii) An amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) added, deleted, and/or substituted amino acids compared to any one of SEQ ID NO: 10-11; and/or

The VL includes or consists of the following:

(i) The amino acid sequence shown in any one of SEQ ID NO: 12-18;

(ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of SEQ ID NO: 12-18; or

(iii) An amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) added, deleted and/or substituted amino acids compared to any of SEQ ID NO: 12-18.

In some embodiments, the isolated antibody or its antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the VH region shown in any of SEQ ID NO:10-11, and LCDR1, LCDR2 and LCDR3 of the VL region shown in any of SEQ ID NO:12-18.

In some embodiments, the isolated antibody or its antigen-binding portion includes: HCDR1 shown in SEQ ID NO:1; HCDR2 shown in SEQ ID NO:2; HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4; LCDR2 shown in SEQ ID NO:7; and LCDR3 shown in SEQ ID NO:6.

In some embodiments, the isolated antibody or its antigen-binding portion comprises a VH region containing the amino acid sequence of SEQ ID NO:11 and a VL region containing the amino acid sequence of SEQ ID NO:15.

In some embodiments, the isolated antibody or its antigen-binding portion further comprises a human IgG constant region, such as a human IgG1, IgG4, IgG2, or IgG3 constant region, which may be natural or a variant thereof. Specifically, the antibody may comprise a human IgG1 Fc region or a human IgG4 Fc region (with S228P substitution).

In some embodiments, the anti-TIGIT antibodies disclosed herein are mouse antibodies, chimeric antibodies, or humanized antibodies. In some embodiments, the antibodies described herein are anti-TIGIT antagonist antibodies.

In some aspects, this disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region encoding an isolated antibody or its antigen-binding moiety disclosed herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence as shown in SEQ ID NO:21, and/or a nucleic acid sequence as shown in SEQ ID NO:22.

In some respects, this disclosure provides expression vectors that contain one or more nucleic acid molecules as disclosed herein.

In some respects, this disclosure provides host cells comprising expression vectors as disclosed herein.

In some respects, this disclosure provides pharmaceutical compositions comprising an antibody or its antigen-binding moiety as disclosed herein, and a pharmaceutically acceptable delivery vehicle.

In some aspects, this disclosure provides a method for preparing the antibody or its antigen-binding moiety, the method comprising expressing the antibody or its antigen-binding moiety in a host cell and isolating the antibody or antigen-binding moiety from the host cell. In some embodiments, the host cell has been transfected or transformed with one or more expression vectors encoding the heavy chain and light chain of the antibody described herein. The nucleic acid sequence encoding the heavy chain and the nucleic acid sequence encoding the light chain may be in the same vector or in separate vectors.

In some respects, this disclosure provides methods for modulating TIGIT-related immune responses in subjects, comprising administering to subjects an antibody or an antigen-binding portion thereof as disclosed herein.

In some respects, this disclosure provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject alone or in combination with another anticancer agent such as an antiPD-1 antibody an effective amount of the disclosed antibody or its antigen-binding portion or drug combination.

In some respects, this disclosure provides a method for treating or preventing cancer or immune-related conditions in a subject, the method comprising administering an effective amount of an antibody or its antigen-binding portion, as disclosed herein, alone or in combination with another anticancer agent, to the subject.

The anticancer agent may be a chemotherapy agent, a monoclonal antibody, an antibody-drug conjugate, etc. In some implementation schemes, the anticancer agent is an antiPD-1 antibody, an antiPD-L1 antibody, or an antiCTLA-4 antibody.

The cancers mentioned can be selected from colon cancer, lung cancer (such as NSCLC), breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, colorectal cancer, kidney cancer, clear cell renal cancer, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, central nervous system tumors, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma, myeloma, soft tissue carcinoma, and sarcoma. The immune-related conditions mentioned can be T-cell dysfunction disorders, infectious or inflammatory diseases.

In some implementation schemes, antibodies or their antigen-binding portions, as disclosed herein, are administered in combination with anti-PD-1 antibodies.

In some respects, this disclosure provides combinations of isolated antibodies or antigen-binding portions thereof as disclosed herein with anti-PD-1 antibodies.

In some aspects, this disclosure provides the use of antibodies or antigen-binding portions thereof, as disclosed herein, alone or in combination with another anticancer agent, in the preparation of medicaments for the treatment or prevention of diseases such as cancer and immune disorders. In some embodiments, the anticancer agent is an antiPD-1 antibody, an antiPD-L1 antibody, or an antiCTLA-4 antibody.

In some respects, this disclosure provides the use of antibodies or antigen-binding portions thereof as disclosed herein in the preparation of diagnostic agents for the diagnosis of diseases associated with TIGIT overexpression.

In some respects, this disclosure provides antibodies or antigen-binding portions thereof as disclosed herein for the treatment or prevention of cancer and immune disorders. In some embodiments, the antibodies disclosed herein are used in combination with PD-1/PD-L1 antagonists such as PD-1 antibodies.

In some respects, this disclosure provides a method for detecting the presence of TIGIT antigen in a sample or measuring the amount of TIGIT antigen, comprising contacting the sample with an anti-TIGIT antibody or its antigen-binding portion disclosed herein.

In some respects, this disclosure provides a kit or device in which an antibody disclosed herein or its antigen-binding moiety is contained in one or more containers.

The above is an overview and therefore includes simplifications, generalizations, and omissions of details where necessary; thus, those skilled in the art will recognize that this overview is merely illustrative and not intended to be limiting in any way. This overview is not intended to identify the key or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

While the present invention can be implemented in many different forms, what is disclosed herein are specific illustrative embodiments that verify the principles of the present invention. It should be emphasized that the present invention is not limited to the specific embodiments exemplified herein. Furthermore, any chapter headings used herein are for organizational purposes only and are not to be construed as limiting the subjects described.

Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure will have the meanings commonly understood in the art by one of ordinary skill. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise expressly indicates, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and/or.” Furthermore, the use of the term “comprising” and other forms (such as “including” and “containing”) is not restrictive. Additionally, the scope provided in the specification and the appended claims includes all points between endpoints.

Generally, the terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics and proteology, and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques disclosed herein are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual , 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology , Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science , Wiley, John & Sons, Inc. (2003). The terminology, laboratory procedures, and techniques related to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used in this field.

Definition

To better understand this invention, the definitions and explanations of relevant terms are provided below.

As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as κ and λ light chains. The heavy chains can be classified as μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, variable regions are linked to constant regions via "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three structural domains (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementary determining regions (CDRs)) separated by relatively conserved regions (called frame regions (FRs)). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy chain/light chain pair form antigen-binding sites. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

The terms "antigen-binding portion" and "antigen-binding fragment" for antibody may be used interchangeably in the context of this application, referring to a polypeptide that contains a fragment of a full-length antibody that retains the ability to specifically bind to an antigen that binds to the full-length antibody, and/or to the same antigen that competes with the full-length antibody. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y.). (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Under certain conditions, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementary determinant region (CDR) fragments, single-stranded antibodies (e.g., scFv), chimeric antibodies, bispecific antibodies, and such polypeptides comprising at least a portion sufficient to endow the polypeptide with antigen-binding specificity. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., the monoclonal anti-human TIGIT antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and can be screened for specificity in the same manner as intact antibodies.

As used herein, the term "monoantibody" or "mAb" refers to an antibody molecule preparation having a single molecular composition. Monoantibodies exhibit single-binding specificity and affinity for a specific epitope.

As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. An exemplary chimeric antibody disclosed herein is W3642-1.433.11-xIgG4.SP, which comprises a heavy chain having a rat VH fused to a human IgG4 constant region and a light chain having a rat VL fused to a human Igλ.

As used herein, the term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammalian species, such as rat or mouse, has been grafted onto a human frame sequence. Additional frame region modifications may be made within the human frame sequence. Optionally, the humanized antibody also includes at least a portion of an immunoglobulin constant region (e.g., Fc), typically a constant region of a human immunoglobulin. Exemplary humanized antibodies disclosed herein are W3642-1.433.11-z10-p1-IgG4.SP and W3642-1.433.11-z11-p1-IgG4.SP, which contain a heavy chain having a human lineage VH fused to a human IgG4 constant region and a light chain having a human lineage VL fused to human Igλ.

As used herein, the term "PTM" or "post-translational modification" refers to a process that occurs on one or more amino acids of a protein (such as an antibody) after it has been translated. Proteins are typically produced by ribosomes, which translate mRNA into polypeptide chains, which then undergo PTM to form the mature protein product. PTM processes include phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, and lipidation. Preferably, potential PTM sites are removed during antibody optimization to avoid structural and functional heterogeneity introduced by the PTM process.

As used herein, the term "TIGIT" or "T-cell immune receptor with Ig and ITIM domains" encompasses any naturally occurring TIGIT from any vertebrate source, including mammalian TIGIT such as primates (e.g., humans) and rodent TIGIT such as mice and rats, unless otherwise stated. TIGIT is also referred to in this field as DKFZp667A205, FLJ39873, protein 9 containing group V and immunoglobulin domains, protein 3 containing group V and transmembrane domains, VSIG9, VSTM3, and WUCAM. The term includes full-length unprocessed TIGIT, extracellular domains of TIGIT, and any form of TIGIT produced through in-cell processing. The term also includes naturally occurring variants of TIGIT, such as splice variants or allelic variants. An exemplary amino acid sequence of full-length human TIGIT is shown in SEQ ID NO: 23 (MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG).

As used herein, the term "PD-1/PD-L1 antagonist" includes PD-L1 antagonists (such as anti-PD-L1 antibodies) that reduce, block, inhibit, eliminate, or interfere with signal transduction caused by the interaction of PD-L1 with one or more of its binding partners (such as PD-1 or B7-1), as well as PD-1 antagonists (such as anti-PD-1 antibodies) that reduce, block, inhibit, eliminate, or interfere with signal transduction caused by the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 antagonist is selected from, but not limited to, the following anti-PD-1 antagonist antibodies: nivolumab (MDX-1106) or pembrolizumab (formerly known as lambolizumab (MK-3475), MED1-0680, PDR001 (spartalizumab), REGN2810 (cemiplimab), BGB-108, prolgolimab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, spartalizumab, sasanlimab, penpulimab, CS1003, HLX10, SCT-I10A, SHR-1316, CS1001, envafolimab, TQB2450, ZKAB001, LP- 002, zimberelimab, balstilimab, genolimzumab, BI 754091, cetrelimab, YBL-006, BAT1306, HX008, CX-072, IMC-001, KL-A167, budigalimab, AMG 404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, APL-502, cosibelimab, lodapolimab, GS-4224, INCB086550, FA Z053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, MAX-10181, RC98, BION-004, AM0001, CB201, ENUM 244C8、ENUM 388D4, AUNP-012, STI-1110, ADG104, AK-103, LBL-006, hAb21, AVA-004, PDL-GEX, INCB090244, KD036, KY1003, LYN192, MT-6035, VXM10, YBL-007, ABSK041, GB7003, JS-003, and HS-636. In some embodiments, the PD-L1 antagonist is selected from, but not limited to, the following anti-PD-L1 antagonist antibodies: MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). PD-1/PD-L1 antagonists include known antibodies and internally developed antibodies.

The term "binding affinity" is used in this paper as a measure of the strength of the nonvalent interaction between two molecules (e.g., an antibody or its antigenic moiety) and an antigen. The binding affinity between two molecules can be quantified by measuring the equilibrium dissociation constant (KD). Correspondingly, KD can be determined by measuring the kinetics of complex formation and dissociation using methods such as surface plasma resonance (SPR) (Biacore™). The rate constants corresponding to the binding and dissociation of monovalent complexes are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. The term ka (or kon) refers to the binding rate of a specific antibody-antigen interaction, while the term kd (or koff) refers to the dissociation rate of a specific antibody-antigen interaction. KD is related to ka and Kd through the equation KD = Kd/ka or koff/kon. The binding kinetics and binding affinity of antibodies can be assessed using standard methods known in the art or as described in the following embodiments section.

As used in this article, the term "high affinity" refers to an antibody with a KD of 1× ^10⁻⁹ M or lower, preferably 5× ^10⁻¹⁰ M or lower, or even more preferably 1× ^10⁻¹⁰ M or lower against the target antigen.

As used herein, the term "EC ₅₀ ," also known as "half-maximum effective concentration," refers to the concentration of a drug, antibody, or agent that induces a 50% response between a baseline and a maximum after a specific exposure time. In the context of this application, EC50 may be expressed in "nM" or "M."

As used herein, the term "isolated" refers to a state obtained artificially from the natural state. If a substance or component is naturally present, it may be due to a change in its natural environment, or the substance being isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide may be naturally present in a living animal; the same polynucleotide or polypeptide isolated from this natural state in high purity is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of mixed artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the isolated substance.

As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to the TIGIT protein is substantially free of antibodies that specifically bind to antigens other than the TIGIT protein). However, isolated antibodies that specifically bind to the human TIGIT protein may exhibit cross-reactivity with other antigens, such as TIGIT proteins from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and/or chemicals.

As used herein, the term "vector" refers to a nucleic acid medium in which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be transformed, transduced, or transfected into host cells to enable the expression of genetic material elements they carry in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). Vectors may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication origin.

As used herein, the term "host cell" refers to a cellular system that can be engineered to produce proteins, protein fragments, or peptides of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term encompasses not only the specific test cells but also the progeny of those cells. Because mutations or environmental influences may cause some modifications in offspring, such offspring may differ from the parent cells, but are still included in the term "host cell".

As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment and comparison. "Percentage identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment, if any, are preferably addressed using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in Computational Molecular Biology, (edited by Lesk, A. M.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (edited by Smith, D. W.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (edited by Griffin, A. M. and Griffin, H. G.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (edited by Gribskov, M. and Devereux, J.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAMJ. Applied Math. 48:1073.

As used in this article, the term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately produce immune response substances such as antibodies and sensitized lymphocytes, but also to the specific immune response that antibodies or sensitized T lymphocytes can form in the organism's immune system after stimulation with an antigen. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the mode of immunization.

As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the field and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In one specific embodiment of this invention, the human TIGIT gene is transfected into 293F cells.

As used herein, the term "SPR" or "surface plasma resonance" refers to and includes optical phenomena that allow analysis of real-time biospecific interactions by detecting changes in protein concentrations within the biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, NJ). For a detailed description, see the examples and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit . 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem . 198:268-277.

As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting heterogeneous mixtures of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). The instruments used to perform FACS are known to those of ordinary skill in the art and are commercially available to the public. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epis C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).

As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-carrying target cells and subsequently kill them with cytotoxins. Antibodies "arm" cytotoxic cells, and this is absolutely necessary for such killers. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of molecules of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patents 5,500,362 or 5,821,337. Effector cells available for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in animal models such as those published by Clynes et al. in PNAS (USA) 95:652-656 (1998).

The terms "subject" and "patient" are used interchangeably and include mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammals. The term does not necessarily indicate that the subject has been diagnosed with a specific disease, but usually refers to an individual under medical supervision.

As used in this article, the term "prevention" or "avoidance" in relation to a certain disease condition in mammals refers to the prevention or delay of the onset of the disease, or the prevention of the appearance of its clinical or subclinical symptoms.

As used in this article in the context of treating a disease, the terms "treatment," "curing," or "management" generally refer to the treatment and therapy of humans or animals in which some desired therapeutic effect is achieved, such as inhibiting disease progression, including slowing the rate of progression, halting the rate of progression, remission, improvement, and cure. In the context of cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or combinations thereof.

As used herein, the term "effective amount" refers to the amount of an active compound or a material, composition, or dosage form containing the active compound, which, when administered according to the desired treatment regimen, is effective in producing some desired therapeutic effect commensurate with a reasonable benefit/risk ratio. For example, when used in combination with the treatment of a disease or condition, "effective amount" refers to the amount or concentration of an antibody or its antigen-binding portion that is effective in treating said disease or condition.

As used herein, the term "pharmaceutically acceptable" means that the medium, diluent, excipient and/or its salt are chemically and/or physically compatible with the other components of the formulation and are physiologically compatible with the recipient.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active agent, and is well known in the art (see, for example, Gennaro AR, ed., Remington's Pharmaceutical Sciences, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

As used in this article, the term "adjuvant" refers to a nonspecific immunostimulant that, when delivered to an organism along with or before an antigen, enhances the organism's immune response to the antigen or alters the type of immune response. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium parvm, lipopolysaccharides, and cytokines. Freund's adjuvants are currently the most commonly used adjuvants in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

Anti-TIGIT antibody

In some respects, this disclosure provides antibodies or antigen-binding portions or variants thereof capable of binding TIGIT (such as human, mouse or cynomolgus monkey TIGIT) with sufficient affinity, thereby substantially or completely inhibiting the biological activity of TIGIT.

In some embodiments, such as those disclosed herein, the anti-TIGIT antibody is an antibody generated in rats immunized with the TIGIT antigen protein. In some embodiments, the anti-TIGIT antibody disclosed herein is a chimeric antibody. In some embodiments, the anti-TIGIT antibody disclosed herein is a humanized antibody. The antigen-binding moiety of the antibody may be Fab, Fab’, F(ab’)2, a single-stranded variable fragment (scFv), or a bispecific antibody.

In some embodiments, this document provides humanized anti-TIGIT antibodies in which the HCDR and LCDR sequences of human immunoglobulins are replaced with HCDR or LCDR sequences obtained from non-human species (such as rats) that possess the desired specificity, affinity, and/or capability. In some further embodiments, certain framework ("FR") residues of human immunoglobulins are reverted to the corresponding non-human residues. In some other embodiments, the humanized antibody may contain residues not found in the parental rat antibody or human immunoglobulin. Further modifications to the CDR and framework residues can be made to eliminate the possibility of post-translational modifications, thereby improving antibody properties such as binding affinity.

Various methods for humanizing nonhuman antibodies are known in the art. For example, humanized antibodies may have one or more amino acid residues introduced from nonhuman sources. These nonhuman amino acid residues are often referred to as "introduced" residues and are typically derived from "introduced" variable structural domains. Humanized antibodies incorporating TIGIT can be produced using techniques known to those of ordinary skill in the art (e.g., Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall’Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Patent Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

The antibodies disclosed herein are capable of binding with high affinity to at least one of human, mouse, and cynomolgus monkey TIGIT. The binding of the disclosed antibodies to TIGIT can be assessed using one or more techniques well known in the art, such as ELISA. The binding specificity of the disclosed antibodies can also be determined by monitoring the binding of the antibodies to cells expressing the TIGIT protein, for example by flow cytometry. In some embodiments, the antibodies are tested by flow cytometry assays, wherein the antibodies react with cell lines expressing human TIGIT, such as HEK293 cells that have been transfected to express TIGIT on their cell surface. Alternatively or additionally, the binding of the antibodies, including binding kinetics (e.g., KD value), can be tested in a BIAcore binding assay.

In some embodiments, the disclosed antibody or its antigen-binding moiety binds to human TIGIT at a KD of 1 x ^10⁻⁹ M or less, 5 x ^10⁻¹⁰ M or less, 1 x ^10⁻¹⁰ M or less, 5 x ^10⁻¹¹ M or less, 4 x ^10⁻¹¹ M or less, 3 x ^10⁻¹¹ M or less, 2.5 x ^10⁻¹¹ M or less, or 2 x ^10⁻¹¹ M or less, as measured by SPR. In some embodiments, the antibody or its antigen-binding moiety specifically binds to human TIGIT as well as crab-eating TIGIT and mouse TIGIT. For example, the antibody or its antigen-binding moiety can bind to cells expressing human TIGIT with an EC50 not exceeding 0.5 nM, 0.4 nM, or 0.3 nM, for example, not exceeding 0.23 nM; bind to cells expressing cynomolgus monkey TIGIT with an EC50 not exceeding 0.5 nM, 0.4 nM, 0.3 nM, or 0.2 nM, for example, not exceeding 0.18 nM; bind to cells expressing mouse TIGIT with an EC50 not exceeding 0.5 nM, 0.4 nM, or 0.3 nM, for example, not exceeding 0.33 nM, as measured by FACS.

The anti-TIGIT antibodies disclosed herein inhibit the interaction between TIGIT and one or more of its ligands PVR (CD155), PVRL2 (CD112), and PVRL3 (CD113). For example, anti-TIGIT antibodies can block signal transduction through PVR, PVRL2, and/or PVRL3, thereby restoring T cells from a dysfunctional state to a functional response to antigen stimulation (e.g., proliferation, cytokine production, target cell killing).

In some embodiments, the anti-TIGIT antibody provided herein inhibits the interaction between TIGIT and its ligand CD155. In some embodiments, the anti-TIGIT antibody provided herein inhibits the interaction between TIGIT and its ligand CD112. In some embodiments, the anti-TIGIT antibody provided herein inhibits the interaction between TIGIT and its ligand CD113. In some embodiments, the anti-TIGIT antibody provided herein inhibits the interaction between TIGIT and one or more of the ligands CD155, CD112, and CD113.

In some embodiments, the ability of anti-TIGIT antibodies to inhibit the interaction between TIGIT and CD155, CD112, or CD113 is assessed by measuring whether the physical interaction between TIGIT and CD155, CD112, or CD113 is reduced in a binding assay. In some embodiments, this binding assay is a competitive binding assay. The assay can be performed in various forms, such as, but not limited to, ELISA, flow cytometry, surface plasma resonance (SPR) assays (e.g., Biacore™), or BioLayer interferometry (e.g., ForteBio Octet™).

Include CDR of resistance TIGIT antibody

In some embodiments, this disclosure provides isolated antibodies or antigen-binding portions thereof, comprising:

A) One or more heavy chain CDRs (HCDRs) selected from the following group:

HCDR1, which contains an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that differs from SEQ ID NO: 1 by no more than 2 amino acids by the addition, deletion or substitution of amino acids;

HCDR2, comprising an amino acid sequence consisting of SEQ ID NO: 2 or an amino acid sequence differing from SEQ ID NO: 2 by no more than two amino acids by addition, deletion, or substitution; and

HCDR3, which contains an amino acid sequence of SEQ ID NO:3 or an amino acid sequence that differs from SEQ ID NO:3 by no more than 2 amino acids by the addition, deletion or substitution of amino acids;

B) One or more light chain CDRs (LCDRs) selected from the following group:

LCDR1, which contains an amino acid sequence of SEQ ID NO:4 or an amino acid sequence that differs from SEQ ID NO:4 by no more than 2 amino acids by adding, deleting or substituting amino acids;

LCDR2, comprising any one of SEQ ID NO: 5, 7, 8 and 9 or an amino acid sequence that differs from any one of SEQ ID NO: 5, 7, 8 and 9 by no more than two amino acids, including amino acid additions, deletions, or substitutions; and

LCDR3, comprising an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence differing from SEQ ID NO: 6 by no more than two amino acids by addition, deletion, or substitution; or

C) One or more HCDRs of A) and one or more LCDRs of B)

In some implementation schemes, CDR delineation is based on the Contact definition method introduced by Dr. Andrew C.R. Martin's group (http://www.bioinf.org.uk/abs/).

In some embodiments, the isolated antibody or its antigen-binding portion provided in this disclosure comprises: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in any one of SEQ ID NO:5, 7, 8 and 9, and LCDR3 shown in SEQ ID NO:6.

The extent of the frame region and CDR can be accurately identified using methods known in the art, such as the Kabat definition, Chothia definition, AbM definition, Contact definition, IMGT definition (all of which are well known in the art) and any combination thereof. See, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. 1969 May, 63(1):78-85; and Martin and Allen, in Handbook of Therapeutic Antibodies , chapter 5, 2007. Also see hgmp.mrc.ac.uk and bioinf.org.uk/abs. Correspondences or comparisons between different definitions of the numbers can be found, for example, at www.imgt.org/ (see also Giudicelli V et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206–11; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55–77).

As will be understood by those skilled in the art, the exact numbering and location of CDRs can differ between different numbering systems. However, it should be understood that disclosing a variable heavy chain sequence and/or a variable light chain sequence includes disclosing the associated (intrinsic) CDR, regardless of the numbering method used. Therefore, disclosing each variable region constitutes disclosing the CDR (e.g., HCDR1, HCDR2, and HCDR3). Two antibodies with the same VH and VL mean that when determined using the same method (e.g., the Kabat, AbM, Chothia, Contact, and IMGT numbering methods known in the art), their CDRs are identical. When determined using different numbering methods, the same antibody as disclosed herein may have a different set of CDRs.

Variable regions and CDRs in antibody sequences can be identified according to general rules already developed in the field (e.g., the Kabat, AbM, Chothia, Contact, and IMGT numbering systems) or by comparing the sequence with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel (eds.), Antibody Engineering, Springer, New York, NY, 2001, and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in and available from the Abysis website (maintained by AC Martin of the Department of Biochemistry & Molecular Biology, University College London, London, England) at www.bioinf.org.uk/abs and the VBASE2 website www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Sequences can be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. See *Protein Sequence and Structure Analysis of Antibody Variable Domains * by Dr. Andrew CR. Martin, in: * Antibody Engineering Lab Manual * (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at bioinforg.uk/abs). The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used according to the teachings in this document.

In some embodiments, the anti-TIGIT antibody disclosed herein comprises a VH region and a VL region, wherein the VH region comprises FRW1-HCDR1-FRW2-HCDR2-FRW3-HCDR3-FRW4, and wherein HCDR1 has the amino acid sequence shown in SEQ ID NO:1, HCDR2 has the amino acid sequence shown in SEQ ID NO:2, and HCDR3 has the amino acid sequence shown in SEQ ID NO:3, and/or wherein the VL region comprises FRW1-LCDR1-FRW2-LCDR2-FRW3-LCDR3-FRW4, and wherein LCDR1 has the amino acid sequence shown in SEQ ID NO:4, LCDR2 has the amino acid sequence shown in SEQ ID NO:5, 7, 8 or 9, and LCDR3 has the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the framework region is derived from a human lineage (i.e., humanized), for example, from human immunoglobulins. In some embodiments, certain residues in the framework region of a human lineage are reverted to the corresponding residues in the parental nonhuman antibody. In some embodiments, the FR region may include one or more individual FR residue substitutions that improve antibody properties, such as binding affinity, isomerization, immunogenicity, etc. In some specific embodiments, FRW3 in the VH region of a human lineage includes Val and Trp at positions 78 and 94 (according to Kabat designations), respectively. In some specific embodiments, FRW1 in the VL region of a human lineage includes one or more of Gln at position 1, Ala at position 2, and Val at position 3 (according to Kabat designations). When referring to residues in the variable region (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In some specific implementations, FRW1 and FRW4 at the N and C ends of the VH and/or VL regions can be truncated so that they only include portions of FRW1 and/or FRW4. In some implementations, optimizations to remove PTMs are performed on the CDR and FR regions.

In some embodiments, the anti-TIGIT antibody provided herein comprises one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO:10, and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO:12. In some embodiments, the anti-TIGIT antibody provided herein comprises one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO:11, and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO:13 or 16. In some embodiments, the anti-TIGIT antibody provided herein comprises one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO:10, and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO:14, 17, or 18. In some embodiments, the anti-TIGIT antibody provided herein comprises one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO:11, and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO:15.

In some embodiments, the anti-TIGIT antibody provided herein comprises at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:10, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:12. In some embodiments, the anti-TIGIT antibody provided herein comprises at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:11, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:13. In some embodiments, the anti-TIGIT antibody provided herein comprises at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:10, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:14. In some embodiments, the anti-TIGIT antibody provided herein comprises at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:11, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:15. In some embodiments, the anti-TIGIT antibody provided herein comprises at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:11, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:16. In some embodiments, the anti-TIGIT antibody provided herein comprises at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:10, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO:17. In some embodiments, the anti-TIGIT antibody provided herein comprises at least one of FRW1, FRW2, FRW3 and FRW4 of the amino acid sequence shown in SEQ ID NO:10, and at least one of FRW1, FRW2, FRW3 and FRW4 of the amino acid sequence shown in SEQ ID NO:18.

In some specific implementations, the VH region contains the amino acid Val (V) at position 78 and Trp (W) at position 94. Alternatively, the VL region contains the amino acid "QAV" (Gln-Ala-Val) at positions 1-3.

Anti-variable regions including heavy chain and light chain TIGIT antibody

In some embodiments, the isolated antibody or its antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

VH comprises (i) an amino acid sequence of one of SEQ ID NO: 10-11; (ii) an amino acid sequence having the same CDR group as one of SEQ ID NO: 10-11 and having at least 85%, 90%, or 95% identity in the frame region; or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acids added, deleted, and/or substituted in the frame region compared to an amino acid sequence of one of SEQ ID NO: 10-11; and/or

VL comprises (i) an amino acid sequence of one of SEQ ID NO: 12-18; (ii) an amino acid sequence having the same CDR group as one of SEQ ID NO: 12-18 and having at least 85%, 90%, or 95% identity in the frame region; or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acid additions, deletions, and/or substitutions in the frame region compared to an amino acid sequence of one of SEQ ID NO: 12-18.

The percentage identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0) using the PAM120 weighted residual base table with a vacancy length penalty of 12 and a vacancy penalty of 4. In addition, the percentage identity between two amino acid sequences can be determined using Needleman and Wunsch's algorithm (J. Mol. Biol. 48:444-453 (1970)), which has been incorporated into the GAP procedure in the GCG software package (available at http://www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

Alternatively or additionally, the disclosed protein sequences can be further used as "query sequences" to perform searches against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. MoI. Biol. 215:403-10. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecule of this invention. For obtaining vacancy alignments for comparative purposes, vacancy BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. When using the BLAST and vacancy BLAST procedures, the default parameters for each procedure (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

In some further embodiments, the isolated antibody or its antigen-binding portion may contain conserved substitutions or modifications of amino acids in the variable regions of the heavy and/or light chains. It should be understood in the art that certain conserved sequence modifications can be performed without removing the antigen binding. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O’ Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

As used herein, "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein/peptide containing an amino acid sequence. For example, conservative substitution can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution includes the substitution of an amino acid residue with another amino acid residue having a similar sidechain, for example, by a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar sidechains have been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), and amino acids with uncharged polar side chains (such as glycine, aspartic acid, glutamic acid, serine, threonine, tyrosine, cysteine, and tryptophan). The amino acids have nonpolar side chains (e.g., alanine, voline, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, voline, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histamine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the field (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

In one particular embodiment, the isolated antibody or its antigen-binding portion comprises an amino acid sequence containing SEQ ID NO:11 or a heavy chain variable region thereof, and an amino acid sequence containing SEQ ID NO:15 or a light chain variable region thereof.

In other embodiments, the amino acid sequences of the heavy chain variable region and/or the light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding sequences as described above.

Anti-specific properties TIGIT antibody

The antibodies disclosed herein are characterized by specific functional features or properties. The in vitro functional properties and pharmacological activities of the antibodies have been fully evaluated at both the molecular and cellular levels, based on their mechanisms of action against the target. In some embodiments, the isolated antibody or its antigen-binding moiety possesses one or more of the following properties:

(a) Specifically binds to at least one of human TIGIT protein, cynomolgus monkey TIGIT protein and mouse TIGIT protein, for example, able to bind human TIGIT with less than 1 nM of KD, as determined by SPR;

(b) It does not cross-bind with TIGIT homologous proteins;

(c) Blocking the binding between TIGIT and its ligands CD155, CD112 and CD113;

(d) Activation of immune cells such as T cells and NK cells;

(e) Induction of ADCC effect in CHOK1 cells expressing TIGIT; and

(f) As shown in in vivo mouse models, when used in combination with anti-PD-1 drugs, the efficacy in treating cancer is significantly superior to that of reference antibodies.

Dual blocking of TIGIT and PD-1 can reverse immunosuppression. As shown herein, the disclosed anti-TIGIT antibody exhibits synergistic effects with anti-PD-1 agents (e.g., anti-PD-1 antibodies) or anti-PD-L1 agents (e.g., anti-PD-L1 antibodies).

In some preferred embodiments, the disclosed antibody may be combined with other therapeutic agents, such as anticancer agents including anticancer antibodies and chemotherapy agents. The other therapeutic agents may also be antagonists or inhibitors of T-cell co-inhibitors, agonists of T-cell co-activating factors, or immunostimulatory cytokines.

In some embodiments, the additional treatment is an antibody selected from the following proteins: CD25, PD-1, PD-L1, Tim3, Lag3, CTLA4, 41BB, OX40, CD3, CD40, CD47M, GM-CSF, CSF1R, TLR, STING, RIGI, TAM receptor kinase, NKG2A, NKG2D, GD2, TIGIT, EGFR, PDGFRa, SLAMF7, VEGF, CTLA-4, CD20, cCLB8, KIR, and CD52. In some embodiments, the additional treatment agent is selected from anti-CD25 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-Tim3 antibody, anti-Lag3 antibody, anti-CTLA4 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-CD3 antibody, anti-CD40 antibody, anti-CD47M antibody, anti-CSF1R antibody, anti-TLR antibody, anti-STING antibody, anti-RIGI antibody, anti-TAM receptor kinase antibody, anti-NKG2A antibody, anti-NKG2D antibody, anti-GD2 antibody, anti-EGFR antibody, anti-PDGFR-a antibody, anti-SLAMF7 antibody, anti-VEGF antibody, anti-CTLA-4 antibody, anti-CD20 antibody, anti-cCLB8 antibody, anti-KIR antibody, and anti-CD52 antibody. In some implementations, the additional treatment is selected from SEA-CD40, avelumab, durvalumab, nivolumab, pembrolizumab, pidilizumab, atezolizumab, Hul4.18K322A, Hu3F8, dinituximab, trastuzumab, cetuximab, olaratumab, necitumumab, elotuzumab, ramucirumab, pertuzumab, ipilimumab, bevacizumab, rituximab, obinutuzumab, siltuximab, ofatumumab, lirilumab, and alemtuzumab.

Fc district

The anti-TIGIT antibody and antigen-binding portion provided herein further include an immunoglobulin-constant region comprising an Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region (natural or a variant thereof), and optionally also comprising a hinged region. In some embodiments, the Fc region is a human IgG1 Fc region, such as a wild-type Fc region or an Fc variant. The Fc variant may have at least about 80% or at least about 90% homology with the natural sequence Fc region, for example, at least about 95% homology. In some embodiments, the Fc region is a human IgG4 Fc region, such as a wild-type Fc region or an Fc variant containing an S228P substitution. In some embodiments, the anti-TIGIT antibody disclosed herein comprises a wild-type human IgG1 Fc region. The variant Fc region may contain one or more amino acid modifications (e.g., Leu234Ala/Leu235Ala or LALA) that alter antibody-dependent cytotoxicity (ADCC) or other effector function. In some embodiments, the Fc region may contain one or more amino acid changes (e.g., insertion, deletion, or substitution) that result in a modified binding interaction between Fc and FcRn or FcγR.

In some embodiments, the Fc region is the IgG4 Fc region containing the S228P mutation (according to the EU number in Kabat et al.), which prevents Fab arm crossing and stabilizes the IgG4 molecule. In some embodiments, the Fc region is the IgG1 Fc region and includes LALA mutations, namely the L234A and L235A mutations. LALA mutations are perhaps the most commonly used mutations to disrupt antibody effector function, such as eliminating the binding of Fc to specific FcγRs, reducing PBMC and monocyte-mediated ADCC activity. The "EU numbering system" or "EU index" is commonly used to refer to the residues in the immunoglobulin heavy chain stationary region (e.g., the EU index reported by Kabat et al., as above). The "EU number in Kabat" or "EU index in Kabat" refers to the residue number of the human IgG1 EU antibody. Unless otherwise stated herein, the residual number mentioned in the constant domain of an antibody refers to the residual number obtained through the EU numbering system.

Monoclonal antibodies can be prepared using a variety of techniques known in the field, including hybridoma technology, recombination technology, phage display technology, transgenic animals (such as XenoMouse®), or certain combinations thereof. For example, monoclonal antibodies can be produced using hybridomas and biochemical and genetic engineering techniques recognized in the field, such as those described in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic , John Wiley and Sons, 1st ed. 2009; Shire et al. (ed.) Current Trends in Monoclonal Antibody Development and Manufacturing , Springer Science + Business Media LLC, 1st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. It should be understood that the selected binding sequence can be further modified, for example, to increase affinity for the target, humanize the target binding sequence, increase its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies including those with modified target binding sequences are also antibodies of the present invention. In some embodiments, anti-human TIGIT monoclonal antibodies are prepared using hybridoma technology. The generation of hybridomas is well known in the field. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

Nucleic acid molecules of antibodies that are publicly known from the code

In some respects, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and/or light chain variable regions of isolated antibodies as disclosed herein.

The nucleic acids of this invention can be obtained using standard molecular biology techniques. For antibodies expressed in hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), cDNA encoding the light and heavy chains of the antibodies prepared from hybridomas can be obtained via standard PCR amplification or cDNA strain techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be recovered from the gene library.

By operatively linking the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), isolated nucleic acids encoding the VH region can be converted into full-length heavy chain genes. The sequences of human heavy chain constant region genes are known in the field (see, for example, Kabat et al. (1991), ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, preferably IgG1 or IgG4 constant regions.

By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into full-length light chain genes (and Fab light chain genes). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a κ or λ constant region.

Once the DNA fragments encoding the VH and VL regions are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible connector. The term "operatively linked" as used in this context refers to the ligation of two DNA fragments such that the amino acid sequences encoded by both fragments remain within the reading frame.

In some embodiments, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions of isolated antibodies as disclosed herein.

In some specific embodiments, the isolated nucleic acid molecule encodes the variable region of the isolated antibody's heavy chain and includes a nucleic acid sequence selected from the following group:

(A) A nucleic acid sequence that encodes a heavy chain variable region as shown in any of SEQ ID NO: 10-11;

(B) A nucleic acid sequence as shown in SEQ ID NO: 21; or

(C) Nucleic acid sequences that have been hybridized with complementary chains of (A) or (B) nucleic acid sequences under highly stringent conditions.

In some embodiments, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding a light chain variable region of an isolated antibody as disclosed herein.

In some specific embodiments, the isolated nucleic acid molecule encodes the variable light chain region of the isolated antibody and includes a nucleic acid sequence selected from the following group:

(A) A nucleic acid sequence encoding a light chain variable region as shown in any of SEQ ID NO: 12-18;

(B) A nucleic acid sequence as shown in SEQ ID NO: 22; or

(C) Nucleic acid sequences that have been hybridized with complementary chains of (A) or (B) nucleic acid sequences under highly stringent conditions.

For example, the nucleic acid molecule comprises SEQ ID NO: 21 and 22. In some other embodiments, the nucleic acid molecule has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 21 or 22. In some specific embodiments, the percentage of identity derives from the degeneracy of the genetic codon, and the encoded protein sequence remains unchanged.

Exemplary highly stringent conditions include hybridization at 45°C in 5X SSPE and 45% formamide, followed by a final wash at 65°C in 0.1X SSC. It should be understood in the art that equivalent stringent conditions can be achieved by variations in temperature and buffer or salt concentration, as described in Ausubel et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications to hybridization conditions can be determined empirically or precisely calculated based on probe length and the percentage of guanine/cytosine (GC) base pairing. Hybridization conditions can be calculated as described in Sambrook et al. (Eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47-9.51.

host cells

The host cells used in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as bacteria, yeast, fungi, plant or animal cells, preferably mammalian cells. Mammalian host cells used for expressing the antibodies of this disclosure include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77: 4216-4220, used in conjunction with DHFR selection markers, such as those described in R. J. Kaufman and P. A. Sharp (1982) J. MoI. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. This also includes monkey kidney CV1 cell lines transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells, secondary selected for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216); mouse seteloli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); and monkey kidney cells (CV1 ATCC CCL). 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NS0 (e.g., RCB0213, 1992, Bio/Technology 10:169) and SP2/0 cells (e.g., SP2/0-Ag14 cells, ATCC CRL 1581); rat myeloma cells, such as YB2/0 cells (e.g., YB2/3HL.P2.G11.16Ag.20 cells, ATCC CRL1662); PER.C6 cells; and human hepatocellular carcinoma cell line (Hep G2). CHO cells are one of the cell lines available in this paper, and CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)) and Lec13 are exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44, or CHO-DP12 host cells, these cells can be modified to lack the ability to fucosylate proteins expressed therein. In some implementations, the host cells used in this paper are selected from CHO cells, CHO-S cells, HEK cells, HEK293 cells, HEK-293F cells, Expi293F cells, PER.C6 cells, NSO cells, and lymphocytes.

Suitable prokaryotes for this purpose include fungi, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (e.g., Escherichia coli, Enterobacter), Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), Shigella, and spore-forming bacteria such as B. subtilis and B. licheniformis, and Pseudomonas (e.g., Pseudomonas aeruginosa). aeruginosa and Streptomyces.

Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable strains or expression hosts for antibody-encoding vectors. Brewing yeast or common baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and useful in this paper, such as *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces* such as *K. lactis*, *K. fragilis* (ATCC 12,424), *K. bulgaricus* (ATCC 16,045), *K. wickeramii* (ATCC 24,178), *K. waltii* (ATCC 56,500), *K. drosophilarum* (ATCC 36,906), *K. thermotolerans*, and *K. marx*. *Marxianus*; *Yarrowia* (EP 402, 226); *Pichia pastoris* (EP 183, 070); *Candida*; *Trichoderma reesia* (EP 244, 234); *Neurospora crassa*; *Schwanniomyces*, such as *Schwanniomyces occidentalis*; and filamentous fungi such as *Neurospora*, *Penicillium*, *Tolypocladium*, and *Aspergillus*, with hosts such as *A. nidulans* and *A. niger*.

When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is generated by culturing the host cells for a period of time sufficient for the antibody to be expressed in the host cells or by secreting the antibody into the culture medium in which the host cells are cultured. The antibody can be recovered from the culture medium using standard protein purification methods.

Drug Composition

In some aspects, the present invention relates to a pharmaceutical composition comprising at least one antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable delivery vehicle. In some aspects, the present invention provides a pharmaceutical composition comprising a nucleic acid encoding an antibody or antigen-binding moiety thereof disclosed herein and a pharmaceutically acceptable delivery vehicle. In some aspects, the present invention provides a pharmaceutical composition comprising cells expressing an antibody or antigen-binding moiety thereof disclosed herein and a pharmaceutically acceptable delivery vehicle.

Components of the composition

The pharmaceutical composition may optionally contain one or more other pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, other components known in the art, and combinations thereof.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, hydrogen peroxide, citric acid, cysteine, glycerol, acetic acid, sorbitol, butyl anisole, butylated hydroxytoluene, and/or propyl gallate. For example, compositions containing antibodies or their antigen-binding fragments may contain one or more antioxidants such as methionine to reduce oxidation of the antibody or its antigen-binding fragments. Reduction of oxidation can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or antigen-binding fragment thereof, extending its shelf life, and/or increasing its activity.

To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution, isotonic dextran injection, sterile aqueous injection, or dextran and lactated Ringer's solution; non-aqueous media such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents with antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or glucose; and buffers such as phosphates or lemon. Acid buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), barrier or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethylene glycol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions contained in multi-dose containers. These antimicrobial agents include phenol or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline solution, dextrose, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or reagents such as sodium acetate, dehydrated sorbitol monolaurate, triethanolamine oleate, or cyclodextrin.

Application, formulation and dosage

The pharmaceutical composition of the present invention can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or implantation or inhalation. The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous dosage forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. The appropriate formulation and application method can be selected based on the intended application and treatment plan.

Suitable formulations for intravenous administration include hard or soft gelatin capsules, pellets, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers and their controlled-release formulations.

Preparations suitable for parenteral administration (e.g., via injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the preparation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic loadings suitable for this type of preparation include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosage regimens (including dosage, timing, and repetition) will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance).

The frequency of administration can be determined and adjusted during treatment, and based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction in tumor cells, decreasing tumor cell proliferation, or delaying the development of metastasis. In some administration regimens, the dosage can be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, a continuously releasing formulation of the therapeutic combination of the invention may be suitable.

As will be understood by those with ordinary knowledge in the art, appropriate dosage can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risk or adverse side effects. The chosen dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, the method of administration, the timing of administration, the rate of compound clearance, the duration of treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the patient's type, sex, age, weight, condition, general health, and medical history. The amount of compound and the method of administration are ultimately determined by a physician, veterinarian, or clinician, but are generally chosen to achieve a local concentration at the site of action sufficient to achieve the desired effect without causing substantial harmful or adverse side effects.

Generally, the antibody or its antigen-binding moiety of the present invention can be administered in a variety of ranges. These include doses of about 5 μg/kg body weight to about 40 mg/kg body weight; doses of about 50 μg/kg body weight to about 5 mg/kg body weight; and doses of about 100 μg/kg body weight to about 10 mg/kg body weight. Other ranges include doses of about 100 μg/kg body weight to about 20 mg/kg body weight and doses of about 0.5 mg/kg body weight to about 20 mg/kg body weight. In some embodiments, the dose is at least about 100 μg/kg body weight, at least about 250 μg/kg body weight, at least about 750 μg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, or at least about 10 mg/kg body weight.

In any case, the antibody or its antigen-binding portion of the present invention is preferably administered to subjects in need as required. Those skilled in the art can determine the frequency of administration, for example, based on considerations by the attending physician, such as the condition being treated, the age of the subject, the severity of the condition being treated, and the general health condition of the subject.

In certain preferred embodiments, the treatment involving the antibody of the invention or its antigen-binding portion will comprise multiple doses of the selected pharmaceutical product administered over several weeks or months. More specifically, the antibody of the invention or its antigen-binding portion may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

The dosage and regimen of a disclosed therapeutic combination can also be determined empirically in individuals given a single or multiple doses. For example, an incremental dose of the therapeutic combination as described herein can be given to an individual. In the selected regimen, the dosage may be gradually increased, decreased, or mitigated based on empirically determined or observed side effects or toxicities. To evaluate the efficacy of the selected combination, markers of a specific disease, condition, or disease can be tracked as previously described. For cancer, these include direct measurement of tumor size through palpation or visual observation, indirect measurement of tumor size through X-rays or other imaging techniques; improvement assessed through direct tumor biopsy and microscopic examination of tumor samples; reduction of pain or numbness in measurements of indirect tumor markers (such as PSA for prostate cancer) or tumorigenic antigens; improvement in tumor-related speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life or extended survival as measured by tests performed.

Compatible formulations intended for parenteral administration (e.g., intravenous injection) may contain an antibody or its antigen-binding moiety disclosed herein at a concentration of about 10 μg/ml to about 100 mg/ml. Those skilled in the art will understand that dosage will vary depending on the individual, the type of tumor, the stage of the tumor, whether the tumor has begun to metastasize to other sites in the individual, and the dosage of previous treatments and treatments used in combination with the antibody disclosed herein.

Applications of this invention

The antibodies, antibody compositions, and methods of this invention have numerous in vitro and in vivo applications, including, for example, the detection of TIGIT or the enhancement of immune responses. For instance, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects to enhance immunity in various situations. Immune responses can be modulated, such as enhanced, stimulated, or upregulated.

For example, subjects include patients who require enhanced immune responses. This method is particularly suitable for treating patients with conditions that can be treated by enhancing immune responses, such as T-cell-mediated immune responses. In one specific implementation, this method is particularly suitable for in vivo cancer treatment. When the anti-TIGIT antibody is administered together with another agent, such as an anti-PD-1 agent, the two can be administered in any order or simultaneously.

This invention further provides a method for detecting the presence of TIGIT antigen in a sample or measuring the amount of TIGIT antigen, comprising contacting a sample or control sample with an anti-TIGIT antibody or its antigen-binding portion under conditions that allow the formation of a complex between the antibody or a portion thereof and TIGIT. The formation of the complex is then detected, wherein a difference in complex formation between the sample and the control sample indicates the presence of TIGIT antigen in the sample. Furthermore, the anti-TIGIT antibody of this invention can be used to purify TIGIT through immunoaffinity purification.

Treatment of conditions including cancer

In some aspects, the present invention provides a method for treating a symptom or disease in mammals, comprising administering to a patient (e.g., a human) in need of treatment a therapeutically effective amount of an anti-TIGIT antibody or its antigen-binding moiety, as disclosed herein, preferably in combination with a PD-1/PD-L1 antagonist. The symptom or disease includes, but is not limited to, proliferative conditions (such as cancer), immune conditions, inflammatory diseases, or infectious diseases. For example, the symptom could be cancer.

In some embodiments, the cancer is a cancer enriched by CD112, CD113, or CD155 expression. In some embodiments, the cancer is a cancer enriched by T cells or natural killer (NK) cells expressing TIGIT.

Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoma. More specific examples of this type of cancer include, but are not limited to, lung cancer, such as non-small cell lung cancer (NSCLC), which includes squamous NSCLC or non-squamous NSCLC, including locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC) or recurrent or metastatic NSCLC (e.g., stage IV NSCLC), lung adenocarcinoma or squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer, including gastrointestinal cancers and gastrointestinal stromal cancers; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer. Cancer; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-refractory non-muscle-invasive bladder cancer (NMIBC)); urethral cancer; hepatocellular carcinoma; breast cancer (e.g., TIGIT+ breast cancer and triple-negative breast cancer (TNBC), i.e., estrogen receptor (ER-), progesterone receptor (PR-), and TIGIT (TIGIT-) negative); colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer (e.g., renal cell carcinoma (RCC)); anterior Prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penis cancer; melanoma, including superficial diffuse melanoma, punctate malignant melanoma, acral punctate melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade/follicular non-Hodgkin lymphoma (NHL)); small lymphocyte (SL) NHL; intermediate/follicular NHL; intermediate diffuse NHL; advanced immunoblastic NHL; advanced lymphocytic NHL; advanced small uncut cell NHL; massive disease NHL; mantle cell lymphoma. Lymphoma; AIDS-related lymphoma; and Waldenstrom macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic promyelocytic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndrome (MDS), as well as dysvascularization, edema (such as edema associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and related metastases associated with macular degeneration.

As a co-inhibitory receptor on various immune cells, TIGIT is associated with a wide range of cancers, both malignant and benign, primary and secondary, which can be treated or prevented using the methods disclosed herein. Preferably, the anti-TIGIT antibody disclosed herein is administered in combination with another anticancer agent, preferably a PD-1/PD-L1 antagonist such as an anti-PD-1 antibody. The cancer can be a solid tumor or a hematologic malignancy. Examples of these cancers include lung cancers such as bronchial cancers (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromalacia (non-cancerous) and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and striated fibroma; and bone cancers such as osteochondroma, chondroma, chondroblastoma, chondromycinoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, and malignant fibrosarcoma. Tissue tumors, Ewing's tumor (Ewing's sarcoma), and reticulum sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pineal tumor, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermoid cyst, and hemangioma; cancers of the digestive system such as colon cancer, leiomyomas, epidermal cysts, and leiomyomas. Dermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colonic polyps, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, lamellar fibrosis, bile duct carcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, adrenal adenoid adenoma, and transitional cell carcinoma of the renal pelvis; bladder cancer; skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancers; and eye-related cancers, such as... Retinoma and intraocular melanoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal); noncancerous growth of the parathyroid glands; pancreatic cancer. In some embodiments, the cancer is colon cancer.

In some other embodiments, the condition or disease to be treated or prevented is an immune-related disease. This immune-related disease may be related to T cell dysfunction. In some embodiments, T cell dysfunction is characterized by reduced responsiveness to antigen stimulation. In some embodiments, T cell dysfunction is characterized by T cell ineffectiveness, or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments, T cell dysfunction is characterized by T cell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the immune-related disease is selected from unresolved acute infections, chronic infections, and tumor-related immunodeficiency.

Stimulation of the immune response

In some aspects, the present invention also provides methods for enhancing (e.g., stimulating) the immune response of a subject, comprising administering to the subject an antibody of the present invention or an antigen-binding portion thereof to enhance the subject's immune response. For example, the subject is a mammal. In a particular implementation, the subject is a human.

The term "enhanced immune response" or its grammatical variations mean any response that stimulates, induces, increases, improves, or enhances the immune system of a mammal. An immune response can be a cellular response (i.e., cell-mediated, such as that mediated by cytotoxic T lymphocytes) or a humoral response (i.e., an antibody-mediated response), and can be primary or secondary. Examples of enhanced immune responses include increased CD4+ helper T cell activity and the production of cytolytic T cells. Enhancement of the immune response can be assessed using a variety of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, the methods of this disclosure enhance the immune response in mammals compared to the immune response in untreated mammals or mammals not treated with the methods of this disclosure.

In one embodiment, the antibody or its antigen-binding portion is used to enhance a human immune response to a microbial pathogen (such as a virus). In another embodiment, the antibody or its antigen-binding portion is used to enhance a human immune response to a vaccine. In one embodiment, the method enhances a cellular immune response, particularly a cytotoxic T-cell response. In another embodiment, the cellular immune response is a T helper cell response. In yet another embodiment, the immune response is the production of cytokines, particularly IFN-γ or IL-2. The antibody or its antigen-binding portion can be used to enhance a human immune response to a microbial pathogen (such as a virus) or a vaccine.

The antibody or its antigen-binding portion can be used alone as a monotherapy or in combination with chemotherapy, radiotherapy, targeted therapy or cell immunotherapy.

Used in combination with chemotherapy

The disclosed antibody or its antigen-binding portion can be used in combination with anticancer agents, cytotoxic agents, or chemotherapy agents.

The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat cellular proliferative disorders such as cancer, and include, but are not limited to, cytotoxic agents, cell inhibitors, antiangiogenic agents, suppressants, chemotherapy agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, antimetastatic agents, and immunotherapeutic agents. It should be understood that such anticancer agents may contain conjugates and may be conjugated to the disclosed antibody prior to administration. More specifically, in some embodiments, the selected anticancer agent is linked to the unpaired cysteine of an engineered antibody to provide an engineered conjugate. Therefore, such engineered conjugates are explicitly considered within the scope of this invention. In some other embodiments, the anticancer agent is administered in combination with an antibody-drug conjugate containing different therapeutic agents.

As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and/or causes cell damage. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-octacocetin, localized cytotoxicin), plants (abrusatin, ricin, sphaerocarpus toxin, quercetin, American phytoalexin, saponins, white tree toxin, momoridin, pollen protein, barley toxin, tung oil (Aleurites fordii) protein, caryophyllin protein, Phytolacca Small molecule toxins or enzymatically active toxins of mericana proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitors, methaqualone toxins, croton toxins, haloxyfop inhibitors, white tree toxins, mitegellin, phenolmycins, neomycins, and trichomycin compounds) or animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNases; DNase I, including its fragments and/or variants).

For the purposes of this invention, "chemotherapeutic agents" include chemical compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and/or survival of cancer cells. These chemical agents typically target intracellular processes required for cell growth or division, and are therefore particularly effective against cancer cells that typically grow and divide rapidly. For example, vincristine dispolymerizes microtubules, thereby inhibiting cell entry into mitosis. Generally, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or produce tumorigenic progeny (e.g., TIC). These agents are often used in combination, and combinations are often the most effective, for example, in regimens such as CHOP or FOLFIRI.

Examples of anticancer agents (as components of site-specific conjugates or in their unconjugated state) that can be used in combination with the antibodies of the present invention include, but are not limited to, alkyl sulfonates, aziridine, ethyleneimine and methyl melamine, acetogenins, camptothecin, sclerotin, callystatin, CC-1065, cryptophycins, dolasstatin, etc. Elutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, espoietin, chromophores of enediyne antibiotics, aclacinomysins Actinomycin, Ammonium chloride, Azoserine, Bleomycin, Actinomycin C, Carabicin, Oxalide, Carcinomalacin, Chromones, Regeneristin, Daunorubicin, Detoxine, 6-Diazono-5-oxo-L-leucine, ADRIAMYCIN® Doxorubicin, Epirubicin, Isorubicin, Idarubicin, Mesorcinol, Mycophenolic acid, Nogamycin, Oleucine, Peropromycin, Bleomycin Potfiromycin, purinemycin, quelamycin, rhodopsin, streptozotocin, tebufenozide, ubenimex, gentamicin, zolarubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide; folic acid analogs, purine analogs, androgens, anti-adrenergics, folic acid supplements such as frolinic acid. acid), glucuronolactone, aldoxyphosphatidylglycoside, aminoacetylpropionic acid, emuramicin, acridine, bestrabucil, bisacodyl, edatrazine, defofamine, colchicine, diazinon, elfornithine, ammonium ethyl acetate, epoxigerulone, epoxigerulone, gallium nitrate, hydroxyurea, lentinan, chlordamine, maytansinoids, mitoxanthraquinone, mitoxanthraquinone, mopidanmol, nitraerine, pentostatin, methamidophos, pirarubicin, loxoanthraquinone, podophyllic acid, 2-ethylhydrazine, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, OR), razoride; rhizomycin; cizonan; geranoylamine; tinuzonic acid; triazoquinone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculosporin A and anguidine); urethane; vincristine; dacarbazine; mannomustine; dibromomannitol; dibromoeugenol; guarpobromo; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chloranbucil; GEMZAR® gemcitabine ; 6-Thioguanine; guanine; methotrexate; platinum analogs; vincristine; platinum; etoposide (VP-16); isocyclophosphamide; mitoxantrone; vincristine, NAVELBINE® vinorelbine; norsulphine; teniposide; edatrazine; daunorubicin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT-11); toprolactone isomerase inhibitor RFS 2000; difluoromethylguanine; visual pigments; capecitabine; cobustatin; methyltetrahydrofolate; oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A (which reduce cell proliferation); and pharmaceutically acceptable salts, acids or derivatives of any of the above. The definition also includes anti-hormonal agents used to regulate or inhibit the hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as trisatabin (a 1,3-dioxanecyclopentane cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and TIGIT expression inhibitors; vaccines, PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; vinorelbine and espomethasone; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

Used in combination with radiotherapy

This invention also provides combinations of antibodies or their antigen-binding portions with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combination therapies using the targeted delivery of radioisotopes to tumor cells are also considered, and the disclosed antibodies can be used in combination with targeted anticancer agents or other targeted approaches. Typically, radiotherapy is administered intravenously over a period of approximately one to two weeks. Radiotherapy can be administered to subjects with head and neck cancer for approximately six to seven weeks. Alternatively, radiotherapy can be administered as a single dose or as multiple sequential doses.

Drug packaging and reagent kits

Pharmaceutical packages and reagent kits are also provided, comprising one or more containers containing one or more doses of an antibody or its antigen-binding moiety. In some embodiments, unit doses are provided, wherein the unit dose contains a pre-quantitative composition comprising, for example, an antibody or its antigen-binding moiety, with or without one or more other reagents. For other embodiments, such unit doses may be supplied in single-use, pre-filled syringes. In still other embodiments, the composition contained in the unit dose may comprise brine, sucrose, or similar substances; buffers, such as phosphates; and/or formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., sterile water or a salt solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the packaged antibody is intended for the treatment of a selected oncological condition.

This invention also provides a kit comprising a single- or multi-dose administration unit of an antibody and optionally one or more anticancer agents. The kit includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, vials, syringes, etc. The container can be formed of various materials, such as glass or plastic, and contains a pharmaceutically effective amount of the antibody. In some embodiments, the container includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the antibody in a suitable container and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combination therapy. For example, in addition to the antibody or its antigen-binding moiety of the present invention, such a kit may contain any one or more anticancer agents, such as chemotherapy or radiotherapy agents; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and/or other anticancer agents. In some embodiments, the kit may contain an anti-PD-1 antibody.

More specifically, the kits may have a single container containing the antibody or its antigen-binding moiety, with or without additional components, or they may have different containers for each desired reagent. In the case of combination therapies, single solutions may be premixed in molar equivalents or in a manner where one component is more than another. Alternatively, the antibody and any optional anticancer agent in the kit may be stored separately in different containers before administration to the patient. The kits may also include a second/third container for containing sterile, pharmaceutically acceptable buffers or other diluents such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent.

As briefly described above, the reagent kit may also contain a tool for administering an antibody or its antigen-conjugated portion and any optional components to a patient, such as one or more needles, intravenous injection bags or syringes, or even eye drops, pipettes or other similar devices through which the preparation can be injected or introduced into an animal or applied to a diseased area of the body. The reagent kit of the present invention typically also includes a device for receiving vials or similar items and other tightly sealed components for commercial sale, such as injection or blow-molded plastic containers, in which the desired vials and other devices are placed and held.

Sequence List Summary

This application includes a sequence list comprising multiple nucleic acid and amino acid sequences. Tables A, B, and C below provide a summary of the included sequences.

W3642-1.433.11 is a parental hybrid tumor strain, which was humanized to obtain strain W3642-1.433.11-z11, and strain W3642-1.433.11-p1 was obtained by removing PTM. W3642-1.433.11-xIgG4.SP is a chimeric antibody; W3642-1.433.11-z10-p1-IgG4.SP and W3642-1.433.11-z11-p1-IgG4.SP are humanized antibodies containing the same CDR set; W3642-1.433.11-z11-p1-IgG4.SP and W3642-1.433.11-z11-p1-uIgG1L differ only in the constant region; W3642-1.433.11-p1-xIgG4.SP, W3642-1.433.11-p2-xIgG4.SP and W3642-1.433.11-p3-xIgG4.SP are antibodies containing different substitutions of PTM removed in LCDR2.

Table A: CDR sequences of antibody/strain HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 W3642-1.433.11-xIgG4.SP;W3642-1.433.11; W3642-1.433.11-z11 GFTFSVAWIH (SEQ ID NO: 1) LIKAGSNYATDYAESVKG (SEQ ID NO: 2) TLSHGSLNWFAS (SEQ ID NO: 3) TLSSGNIENKYVH (SEQ ID NO: 4) NDDKRPD (SEQ ID NO: 5) HSYVSSINV (SEQ ID NO: 6) W3642-1.433.11-p1-xIgG4.SP; W3642-1.433.11-z10-p1-IgG4.SP; W3642-1.433.11-z11-p1-IgG4.SP; W3642-1.433.11-z11-p1-uIgG1L SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 NDDKRP E (SEQ ID NO: 7, with D56E) SEQ ID NO: 6 W3642-1.433.11-p2-xIgG4.SP SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 NDDKRP Q (SEQ ID NO: 8, with D56Q) SEQ ID NO: 6 W3642-1.433.11-p3-xIgG4.SP SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 NDDKRP S (SEQ ID NO: 9, with D56S) SEQ ID NO: 6

Table B: Amino acid sequences in the variable regions strain/antibody amino acid sequence W3642-1.433.11-xIgG4.SP VH EVQLVETGGSLVQPGKSLKLTCVSS GFTFSVAWIH WVRQSPDKQLEWIG LIKAGSNNYATDYAESVKG RFTMSRDDSKSSVYLQMNSLREEDTAIYYCAW TLSHGSLNWFAS WGRGTLVTVSS (SEQ ID NO: 10) VL QAVLTQPDSVSTSLGGTVRLSC TLSSGNIENKYVH WYQQHEGRSPTTMIY NDDKRPD GVPDRFSGSIDSSSNSAFLTITNVEIEDEAIYFC HSYVSSINV FGGGTKLTVL (SEQ ID NO: 12) W3642-1.433.11-z11 VH EVQLVESGGGLVQPGGSLRLSCAAS GFTFSVAWIH WVRQAPGKGLEWVG LIKAGSNNYATDYAESVKG RFTISRDDDSKNSVYLQMNSLKTEDTAVYYCAW TLSHGSLNWFAS WGRGTLVTVSS (SEQ ID NO: 11) VL QAVLTQPHSVSESPGKTVTISC TLSSGNIENKYVH WYQQRPGSSPTTVIY NDDKRPD GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC HSYVSSINV FGGGTKLTVL (SEQ ID NO: 13) W3642-1.433.11-p1-xIgG4.SP VH EVQLVETGGSLVQPGKSLKLTCVSS GFTFSVAWIH WVRQSPDKQLEWIG LIKAGSNNYATDYAESVKG RFTMSRDDSKSSVYLQMNSLREEDTAIYYCAW TLSHGSLNWFAS WGRGTLVTVSS (SEQ ID NO: 10) VL QAVLTQPDSVSTSLGGTVRLSC TLSSGNIENKYVH WYQQHEGRSPTTMIY NDDKRPE GVPDRFSGSIDSSSNSAFLTITNVEIEDEAIYFC HSYVSSINV FGGGTKLTVL (SEQ ID NO: 14) W3642-1.433.11-z11-p1-uIgG1L; W3642-1.433.11-z11-p1-IgG4.SP VH EVQLVESGGGLVQPGGSLRLSCAAS GFTFSVAWIH WVRQAPGKGLEWVG LIKAGSNNYATDYAESVKG RFTISRDDDSKNSVYLQMNSLKTEDTAVYYCAW TLSHGSLNWFAS WGRGTLVTVSS(SEQ ID NO: 11) GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAGCCCGGTGGATCTTTAAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCGTGGCTTGGATCCACTGGGTGAGGCAAGCTCCCGGTAAGGGTTTAGAGTGGGTGGGTTTAATCAAGGCCGGCAGCAACAACTACGCCACAGACT ACGCCGAGAGCGTGAAGGGTCGTTTCACCATTCTCGTGACGACAGCAAGAACTCTGTGTATTTACAGATGAACTCTTTAAAGACCGAGGACACCGCCGTGTACTACTGCGCTTGGACTTTAAGCCATGGCTCTTTAAACTGGTTCGCCAGCTGGGGCAGAGGCACTTTAGTGACCGTGAGCAGC (SEQ ID NO: 21, coding nucleic acid sequence) VL QAVLTQPHSVSESPGKTVTISC TLSSGNIENKYVH WYQQRPGSSPTTVIY NDDKRPE GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC HSYVSSINV FGGGTKLTVL(SEQ ID NO: 15) CAAGCTGTGCTGACCCAGCCTCACAGCGTGAGCGAGAGCCCCGGCAAGACCGTGACCATCAGCTGCACTTTAAGCAGCGGCAACATCGAGAACAAGTACGTGCACTGGTACCAGCAGAGGCCCGGTAGCAGCCCCACCACAGTGATCTACAACGACGACAAGAGG CCCGAGGGCGTGCCCGATCGTTTCAGCGGCAGCATCGACAGCAGCAGCAACAGCGCCTCTTTAACCATCAGCGGTTTAAAGACCGAGGACGAGGCCGACTACTGCCACAGCTACGTGAGCAGCATCAACGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTG (SEQ ID NO: 22, encoding nucleic acid sequence) W3642-1.433.11-z10-p1-IgG4.SP VH EVQLVESGGGLVQPGGSLRLSCAAS GFTFSVAWIH WVRQAPGKGLEWVG LIKAGSNNYATDYAESVKG RFTISRDDDSKNSVYLQMNSLKTEDTAVYYCAW TLSHGSLNWFAS WGRGTLVTVSS(SEQ ID NO: 11) VL NFMLTQPHSVSESPGKTVTISC TLSSGNIENKYVH WYQQRPGSSPTTVIY NDDKRPE GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC HSYVSSINV FGGGTKLTVL (SEQ ID NO: 16) W3642-1.433.11-p2-xIgG4.SP VH EVQLVETGGSLVQPGKSLKLTCVSS GFTFSVAWIH WVRQSPDKQLEWIG LIKAGSNNYATDYAESVKG RFTMSRDDSKSSVYLQMNSLREEDTAIYYCAW TLSHGSLNWFAS WGRGTLVTVSS (SEQ ID NO: 10) VL QAVLTQPDSVSTSLGGTVRLSC TLSSGNIENKYVH WYQQHEGRSPTTMIY NDDKRPQ GVPDRFSGSIDSSSNSAFLTITNVEIEDEAIYFC HSYVSSINV FGGGTKLTVL (SEQ ID NO: 17) W3642-1.433.11-p3-xIgG4.SP VH EVQLVETGGSLVQPGKSLKLTCVSS GFTFSVAWIH WVRQSPDKQLEWIG LIKAGSNNYATDYAESVKG RFTMSRDDSKSSVYLQMNSLREEDTAIYYCAW TLSHGSLNWFAS WGRGTLVTVSS (SEQ ID NO: 10) VL QAVLTQPDSVSTSLGGTVRLSC TLSSGNIENKYVH WYQQHEGRSPTTMIY NDDKRPS GVPDRFSGSIDSSSNSAFLTITNVEIEDEAIYFC HSYVSSINV FGGGTKLTVL (SEQ ID NO: 18)

Table C: Sequences of heavy and light chains antibody amino acid sequence W3642-1.433.11-z11-p1-uIgG1L heavy chain amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSVAWIHWVRQAPGKGLEWVGLIKAGSNNYATDYAESVKGRFTISRDDSKNSVYLQMNSLKTEDTAVYYCAWTLSHGSLNWFASW GRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:19) Light chain amino acid sequence QAVLTQPHSVSESPGKTVTISCTLSSGNIENKYVHWYQQRPGSSPTTVIYNDDKRPEGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCHSYVSSINVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 20)

Implementation Examples

The disclosure generally described herein will be more readily understood by referring to the following embodiments, which are provided by way of example and are not intended to limit the invention. These embodiments are not intended to represent all or only the experiments conducted.

Example 1: Preparation of antigens, reference antibodies, and cell lines

1.1 Antigen generation

W364-hPro1.ECD.His is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal polyhistamine tag; W364-hPro1.ECD.hFc is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal Fc region containing human IgG1; W364-mPro1.ECD.His is a mouse TIGIT domain with a C-terminal polyhistamine tag. The extracellular domains of GIT (NP_001139797.1); W364-mPro1.ECD.hFc is the extracellular domain of mouse TIGIT (NP_001139797.1) with a C-terminal Fc region containing human IgG1; W364-hPro1L1.ECD.mFc is the extracellular domain of human CD155 (NP_006496.3) with a C-terminal Fc region containing human IgG1. W364-hPro1L1.ECD.mFc is the extracellular domain of human CD155 (NP_006496.3) with a C-terminal Fc region containing mouse IgG1. These antigens were purchased from suppliers or prepared in-house.

1.2 Preparation of benchmark antibody (BMK)

Based on the sequences disclosed in their respective patents, the amino acid sequences encoding the variable domains of the anti-TIGIT reference antibodies WBP364-BMK1, WBP364-BMK4, and WBP364-BMK6 were synthesized, and the sequence information is summarized in Table 1.

Table 1: Reference Antibody Information Antibody code company Patent number Serial ID WBP364-BMK1 Roche (Genentech) US20170088613 4.1D3 WBP364-BMK4 BMS (Ono) US20160176963 22G2 WBP364-BMK6 Astellas (Potenza) WO2017059095 MAB10

1.3 Generation of cell pools/cell lines

The following cell lines were used to generate human TIGIT-expressing cells: W364-293F.hPro1.pool (293F cells transfected with full-length human TIGIT (NP_776160.2)), W364-CHOK1.hPro1.2A11 (CHOK1 cells transfected with full-length human TIGIT (NP_776160.2)), and W364-FlpinCHO.cynoPro1.pool (FlpinCHO cells transfected with full-length cynomolgus monkey TIGIT (XP_015300911.1)). The W364-FlpinCHO.mPro1.pool of cells expressing mouse TIGIT was generated using FlpinCHO cells transfected with full-length mouse TIGIT (NP_001139797.1).

Example 2: Generation of chimeric and humanized antibodies

2.1 Immunity

Two 6- to 8-week-old female SD rats were purchased from Shanghai SLAC Experiment Animal Co., Ltd. and housed in an IACUC-approved animal facility. The two animals were alternately immunized with W364-hPro1.ECD.His and W364-mPro1.ECD.His.

2.2 Serum titer detection

The titer of anti-human/mouse TIGIT antibodies in serum samples was determined by ELISA. Microplates were coated with W364- _{hPro1.ECD.hFc} or W364-mPro1.ECD.hFc at a concentration of 1 μg/mL in 100 μL of coating buffer ( _Na₂CO₃ / _NaHCO₃ , pH 9.2) per well and incubated overnight at 4°C. On the day of assay, diluted rat serum samples (first 1:100, then 3-fold diluted in 1×PBS/2% BSA) and a negative control were added to plates blocked with 1×PBS/2% BSA for 1 hour, and the plates were then incubated at ambient temperature for 1 hour. After washing three times with 1×PBST (PBS containing 0.05% Tween-20), HRP-labeled goat anti-rat IgG Fc (Bethyl, cat#A110-236P) was added and incubated at ambient temperature for 1 hour. After removing unbound material, TMB (3,3',5,5'-tetramethylbenzidine) substrate was added, and the reaction was stopped with 2M HCl. The absorbance at 450 nm was measured using a microplate spectrophotometer.

Serum titers of immunized SD rats are shown in Table 2. Both animals were euthanized after final booster immunization with W364-hPro1.ECD.His and W364-mPro1.ECD.His, and lymph nodes were collected for fusion.

Table 2. Serum titers of anti-TIGIT antibodies Animal ID # antigen Titration information Before blood collection First blood draw Second blood draw 1 W364-hPro1.ECD.hFc 100 1968300 656100 2 100 1968300 656100 1 W364-mPro1.ECD.hFc / / 1968300 2 / / 1968300

2.3 Heteroma formation

Lymph nodes were collected from immunized rats under sterile conditions and dissociated into single-cell suspensions. B cells were isolated from the lymph nodes and then mixed with myeloma cells SP2/0 at a ratio of 1:1.2. Electrofusion was performed using a BTX 2001 Electro cell manipulator according to an optimized electrofusion procedure. After fusion, the cells were transferred to 96-well plates (1.2 × ^10⁴ cells/well) in DMEM medium supplemented with 20% FBS and 1% HAT select agent. The plates were incubated at 37°C, 5% _CO₂ , and monitored periodically. When the strains reached approximately 80% confluence in the wells, 100 μL of supernatant was transferred from the tissue culture plate to a 96-well assay plate for antibody screening.

2.4 Antibody Screening and Secondary Selection

The high-throughput screening process includes preliminary screening of cynomolgus monkey TIGIT conjugates using cell-based ELISA, secondary screening of human/cynomolgus monkey/mouse TIGIT conjugates using cell-based FACS, and functional screening of TIGIT/PVR blockers using cell-based FACS.

Dilute the logarithmically growing positive cell lines to 200–300 cells per 1.5 mL semi-solid HAT medium. Gently mix the cell suspension on a vortex mixer for 5–10 seconds, then seed in 6-well plates. Incubate the plates at 37°C and 5% _CO2 for 7–8 days. When cell clusters have grown, pick each visible single colony and seed it into a 96-well plate in DMEM medium supplemented with 10% fetal bovine serum. After 2–3 days, collect the supernatant from each strain and screen again to obtain positive hybridoma single cells.

2.5 Sequencing of Heteroma

RNA was isolated from monoclonal hybridoma cells using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa Bio Inc.) according to the manufacturer's instructions. cDNA was amplified using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Inc.) according to the manufacturer's instructions. The resulting cDNA was used as a template for subsequent PCR amplification using primers specific to the genes of interest. The PCR product was inserted into the pMD18-T vector, and the ligation product was sent to GENEBIX for sequencing.

Fifty positive cell lines were selected for secondary selection through primary and secondary binding screening, as well as TIGIT/PVR blocking screening. After confirming the monoclonal antibody, 39 positive hits were selected for sequencing, of which 3 were subsequently transformed into human IgG.

2.6 Sequence Optimization

2.6.1 Chimeric Antibody Production

The amino acid sequences of the VH and VL domains were codon-optimized for mammalian expression. Codon-optimized DNA sequences were synthesized using GENEWIZ and then selectively implanted into pcDNA expression vectors containing human IgG1 or IgG4 constant regions. Plasmids containing the VH and VL genes were co-transfected into Expi293 cells, and the cells were cultured for approximately 5 days until the supernatant was harvested. Antibodies were purified from the supernatant using a Protein A column.

The W3642-1.433.11 strain was identified, and its variable domain sequence is shown in Table B. The chimeric antibody of W3642-1.433.11 was named W3642-1.433.11-xIgG4.SP, which contains a heavy chain with rat VH fused to the human IgG4 constant region and a light chain with rat VL fused to human Igλ.

2.6.2 PTM Removal

For the parent antibody W3642-1.433.11, the amino acid residue "DG" at the VL CDR2 boundary was identified as a potentially unstable residue with isomerization risk. Mutagenesis was performed to eliminate the risk of post-translational modification (PTM), and SPR analysis was used to measure the binding kinetics of the antibody binding to human TIGIT. Mutagenesis of the "DG" residue to EG, QG, or SG did not significantly alter the binding affinity. The results are shown in Table 3.

Table 3. Koff rate of PTM-removed variants binding with human TIGIT antibody VH structural domain VL structure domain k _off (1/s) W3642-1.433.11-xIgG4.SP rVH rVL 5.95E-05 W3642-1.433.11-p1-xIgG4.SP rVH rVL+D056E 5.27E-05 W3642-1.433.11-p2-xIgG4.SP rVH rVL+D056Q 5.30E-05 W3642-1.433.11-p3-xIgG4.SP rVH rVL+D056S 5.31E-05

2.6.3 Humanization of rat antibodies

Humanization of W3642-1.433.11 was performed via CDR transplantation. The CDRs were identified according to the Contact definition introduced by Dr. Andrew C.R. Martin's team (http://www.bioinf.org.uk/abs/). The VH and VL sequences of W3642-1.433.11 were compared with the human germline V gene database, and the human IGVH and IGVL sequences with the highest homology to W3642-1.433.11 were selected as humanization templates. The CDRs of the VH and VL domains of W3642-1.433.11 were transplanted into the framework of the humanization templates to construct the germline VH and VL domain sequences.

Several sites within the frame region underwent "reversal mutations" to convert amino acids in the germline sequence to their corresponding amino acids in the original rat sequence. This also produced a hybrid chimeric antibody (W3642-1.433.11-hyAbL) consisting of a rat VH fused to the human IgG4 constant region and a germline VL fused to human Igλ.

To determine whether the humanized variant retained antigen-binding activity, surface plasma resonance (SPR) was performed using a Biacore 8K instrument (Cytiva). In short, goat anti-human IgG Fc antibody (Jackson ImmunoResearch, catalog number 109-005-098) was immobilized on a CM5 biosensor chip (GE, catalog number 29-1496-03). The goat anti-human IgG Fc antibody was used to capture the antibody from the transfection supernatant at a pilot scale. The antigen W364-hPro1.ECD.His was injected into a running buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at a flow rate of 100 μL/min. The binding period was 120 s, followed by a dissociation period of 900 s. The dissociation rate ( koff ) was calculated using a simple one-to-one Langmuir binding model. The results are shown in Table 4.

Table 4. Koff rate of antibody variants binding to human TIGIT antibody VH structural domain VL structure domain k _off (1/s) W3642-1.433.11-xIgG4.SP rVH rVL 5.95E-05 W3642-1.433.11-hyAbL rVH GL 9.83E-05

Note: rVH = rat hybridoma sequence of the heavy chain variable region, rVL = rat hybridoma sequence of the light chain variable region, GH = human phylogenetic sequence of the heavy chain variable region, GL = human phylogenetic sequence of the light chain variable region.

Two variants, W3642-1.433.11-z10-p1-IgG4.SP and W3642-1.433.11-z11-p1-IgG4.SP, combining humanization and PTM removal, showed high affinity for human TIGIT, as measured by Biacore. The affinity of W3642-1.433.11-z11-p1-IgG4.SP was approximately 2 times higher than that of W3642-1.433.11-z10-p1-IgG4.SP. The results are shown in Table 5.

Table 5. Affinity of humanized and PTM-removed variants to human TIGIT antibody VH structural domain VL structure domain kon (1/Ms) koff (1/s) KD (M) W3642-1.433.11-xIgG4.SP rVH rVL 1.68E+06 2.58E-05 1.54E-11 W3642-1.433.11-z10-p1-IgG4.SP GH+L078V+R094W GL+D056E 1.55E+06 6.15E-05 3.96E-11 W3642-1.433.11-z11-p1-IgG4.SP GH+L078V+R094W GL+N001Q+F002A+M003V+D056E 1.72E+06 4.11E-05 2.39E-11

The corresponding IgG1 antibody for W3642-1.433.11-z11-p1-IgG4.SP was generated, and it is called W3642-1.433.11-z11-p1-uIgG1L, or simply W3642.

Implementation Examples 3 External signs

3.1 Human TIGIT Combination Assay

W364-293F.hPro1.pool cells (1× ^10⁵ cells/well) were incubated with different concentrations of anti-TIGIT antibodies at 4°C for 1 hour. After washing with 1×PBS/1% BSA, a second antibody, Alexa Fluor 647-labeled goat anti-human IgG (Jackson Immuno Research cat#109-605-098), was added and incubated with the cells in the dark at 4°C for 1 hour. Anti-human TIGIT antibodies WBP364-BMK1 and WBP364-BMK4 were used as positive controls. Human IgG1 isotype antibodies were used as isotype controls. The cells were then washed and resuspended in 1×PBS/1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

The binding results of W3642-1.433.11-z11-p1-uIgG1L on W364-293F.hPro1.pool cells are shown in Figure 1, indicating that W3642-1.433.11-z11-p1-uIgG1L can strongly bind to cells expressing human TIGIT, with binding potency comparable to the reference antibody. A summary of antibody binding is shown in Table 7.

3.2 Measurement of TIGIT binding in cynomolgus monkeys

W364-FlpinCHO.cynoPro1.pool cells (1× ^10⁵ cells/well) were incubated with different concentrations of anti-TIGIT antibodies at 4°C for 1 hour. After washing with 1×PBS/1%BSA, a second antibody, Alexa Fluor647-labeled goat anti-human IgG (Jackson ImmunoResearch CAT #109-605-098), was added and incubated with the cells in the dark at 4°C for 1 hour. Anti-human TIGIT antibodies WBP364-BMK1 and WBP364-BMK4 were used as positive controls. Human IgG1 isotype antibodies were used as isotype controls. The cells were then washed and resuspended in 1×PBS/1%BSA. MFI of cells was measured by flow cytometry (BD) and analyzed by FlowJo.

The binding results of W3642-1.433.11-z11-p1-uIgG1L on W364-FlpinCHO.cynoPro1.pool cells are shown in Figure 2, indicating that W3642-1.433.11-z11-p1-uIgG1L can strongly bind to TIGIT-expressing cells in cynomolgus monkeys, with binding potency comparable to the reference antibody. A summary of antibody binding is shown in Table 7.

3.3 TIGIT binding assay in mice

W364-FlpinCHO.mPro1.pool cells (1× ^10⁵ cells/well) were incubated with different concentrations of anti-TIGIT antibody at 4°C for 1 hour. After washing with 1×PBS/1%BSA, a second antibody, Alexa Fluor647-labeled goat anti-human IgG (Jackson ImmunoResearch cat#109-605-098), was added and incubated with the cells in the dark at 4°C for 1 hour. The anti-human TIGIT antibody WBP364-BMK6, which is known to cross-react with mouse TIGIT, was used as a positive control. A human IgG1 isotype antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS/1%BSA. MFI of cells was measured by flow cytometry (BD) and analyzed by FlowJo.

The binding results of W3642-1.433.11-z11-p1-uIgG1L to the extracellular domain of mouse TIGIT are shown in Figure 3. This indicates that W3642-1.433.11-z11-p1-uIgG1L strongly binds to mouse TIGIT-expressing cells, with a higher binding potency than the reference antibody WBP364-BMK6. WBP364-BMK1 and WBP364-BMK4 did not bind to mouse TIGIT-expressing cells. A summary of antibody binding is shown in Table 7.

3.4 Human TIGIT Affinity Determination

The affinity of anti-TIGIT antibodies for recombinant human TIGIT was determined using a Biacore 8K instrument (Cytiva) via surface plasma resonance (SPR). Goat anti-human IgG Fc antibody (Jackson ImmunoResearch, catalog number 109-005-098) was immobilized on a CM5 biosensor chip (GE catalog number 29-1496-03), and anti-TIGIT antibodies were captured using the goat anti-human IgG Fc antibody. For kinetic measurements, a series of concentrations of W364-hPro1.ECD.His were injected into a running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) at a flow rate of 30 μL/min at 25 °C, initially for a binding period followed by a dissociation period. The binding rate ( kon ) and dissociation rate ( koff ) were calculated using a simple one-to-one Languir binding model. The equilibrium dissociation constant (kD) was calculated as the ratio of koff/kon .

The binding affinity of W3642-1.433.11-z11-p1-uIgG1L to the extracellular domain of human TIGIT is shown in Table 6. W3642-1.433.11-z11-p1-uIgG1L binds to human TIGIT with an affinity of 2.39E-11 M.

Table 6. Affinity constants between anti-TIGIT antibodies and human TIGIT antibody ka (1/Ms) kd (1/s) KD (M) WBP364-BMK1 5.95E+05 2.14E-05 3.59E-11 WBP364-BMK4 1.46E+06 7.19E-05 4.92E-11 W3642-1.433.11-z11-p1-uIgG1L 1.72E+06 4.11E-05 2.39E-11

3.5 Assay for TIGIT paraprotein binding

Pre-coat the plates with 1 μg/mL of W364-hPro1.ECD.His, recombinant human CD28, CTLA-4, PD-1, ICOS, or PVRIG extracellular domain in 50 μL of coating buffer per well and incubate overnight at 4°C. After blocking with 200 μL of 1×PBS/2% BSA, add 50 μL of test antibody at a concentration of 1 nM, 0.1 nM, or 0.01 nM to the plates and incubate at ambient temperature for 1 hour. After incubation, wash the plates three times with 1×PBST. Add HRP-labeled goat anti-human IgG antibody (Bethyl cat#A80-304P) diluted in 1×PBS/2% BSA and incubate at ambient temperature for 1 hour. Anti-CD28 antibody (US7585960, TGN1412), anti-CTLA-4 antibody (US8784815, 10D1), anti-PD-1 antibody (US9084776, 5C4), anti-ICOS antibody (US10023635, 37A10), and anti-PVRIG antibody (US20180244774, CHA.7.518.1) were used as positive controls for CD28, CTLA-4, PD-1, ICOS, and PVRIG, respectively. Human IgG1 isotype antibody was used as an isotype control. After washing six times with 1×PBST, color development was performed by adding 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. The absorbance was read at 450 nm using an M5e microplate reader (Molecule Devices).

The binding results of W3642-1.433.11-z11-p1-uIgG1L to TIGIT paraprotein are shown in Figure 4. This indicates that W3642-1.433.11-z11-p1-uIgG1L specifically binds to TIGIT, but has no cross-reactivity with human CD28, ICOS, PVRIG, PD-1, or CTLA-4.

3.6 Human TIGIT/ligand blocking assay

The ability of anti-TIGIT antibodies to block human TIGIT/PVR interaction was tested using FACS. W364-CHOK1.hPro1.2A11 cells were washed with 1×PBS/1% BSA and seeded at 1× ^10⁵ cells per well in 96-well round-bottom plates. Excess buffer was removed from the wells by centrifugation. Serially diluted anti-TIGIT antibody (2-fold concentration) was premixed with 4 µg/mL (2-fold concentration) of W364-hPro1L1.ECD.mFc at a 1:1 volume ratio, and 100 μL of the antibody/ligand mixture was added to each well. The plates were incubated at 4°C for 1 hour. After washing with 1×PBS/1%BSA, the cells were incubated with the second antibody, PE-labeled goat anti-mouse IgG (Bethyl cat#A90-239PE), in the dark at 4°C for 1 hour. Anti-human TIGIT antibodies WBP364-BMK1 and WBP364-BMK4 were used as positive controls. Human IgG1 isotype antibodies were used as isotype controls. The cells were then washed and resuspended in 1×PBS/1%BSA. MFI of the cells was measured by flow cytometry (BD) and analyzed using FlowJo.

The ability of anti-TIGIT antibodies to block the interaction between human TIGIT/CD112 and TIGIT/CD113 was also tested using FACS.

The results of blocking human TIGIT binding to its ligands (CD155, CD112, and CD113) are shown in Figures 5-7. The results indicate that W3642-1.433.11-z11-p1-uIgG1L effectively blocks the binding of human CD155, CD112, and CD113 to TIGIT. A summary of antibody blocking activity is shown in Table 7. Ligand binding rate was calculated as binding % = MFI _sample / MFI _max × 100%, where MFI _max is defined as the MFI in the absence of an antibody.

3.7 Jurkat TIGIT/NFAT-luciferase reporter gene assay (RGA)

Jurkat cells, co-expressing human TIGIT and NFAT-luciferase reporter genes, were stimulated via co-culturing with CHOK1 cells co-expressing human PVR and TCR activators, through the participation of T cell receptors. CHOK1/PVR/TCR activator cells were seeded in 96-well plates at a density of 2 × ^10⁴ cells/well and incubated _overnight at 37°C and 5% CO₂. The next day, after removing the supernatant and non-adherent cells, serially diluted anti-TIGIT antibody and Jurkat/TIGIT/NFAT-luciferase cells (2 × ^10⁴ cells/well) were added to the plates and co-cultured at 37°C and 5% _CO₂ for 5–6 hours. After incubation, the reconstituted luciferase substrate (Promega cat#E6130) was added to each well and mixed thoroughly. The luciferase intensity was read using an EnVision microplate reader (PerkinElmer).

Figure 8 shows the results of reversing the inhibition of TIGIT/PVR interaction-induced NFAT signaling by W3642-1.433.11-z11-p1-uIgG1L in RGA, indicating that W3642-1.433.11-z11-p1-uIgG1L can enhance TCR/NFAT activation by inhibiting the TIGIT/PVR pathway. Table 7 shows the antibody activity results in RGA assays.

3.8 Jurkat Function Measurement

HT1080 is a human fibrosarcoma cell line expressing human PVR, PVRL2, and PVRL3. Jurkat cells overexpressing human TIGIT were stimulated via co-culturing with HT1080 cells overexpressing human TCR activator factor, with the participation of T cell receptors. 1 × ^10⁴ Jurkat/TIGIT cells and 5 × ^10³ HT1080/TCR activator factor cells were co-cultured for 2 days at 37°C and 5% _CO₂ in the presence of continuously diluted anti-TIGIT antibody. After incubation, the supernatant was collected for IL-2 measurement by ELISA (capture antibody R&D catalog number MAB602, detection antibody R&D catalog number BAF202). Absorbance was measured using an M5e microplate reader (Molecule Devices).

Jurkat cells expressing TIGIT were co-cultured with HT1080/TCR activator cells in the presence of antibodies, and IL-2 in the supernatant was quantified by ELISA. The results showed that W3642-1.433.11-z11-p1-uIgG1L dose-dependently enhanced Jurkat cell activation. Data are shown in Figure 9. The results of antibody-induced IL-2 secretion are shown in Table 7.

Table 7. Summary of Antibody Characterization Results Determination W3642-1.433.11-z11-p1-uIgG1L WBP364-BMK1 WBP364-BMK4 Binding to human TIGIT-engineered cells, EC50 (nM) 0.23 0.50 0.32 Binding to TIGIT-engineered cynomolgus monkey cells, EC50 (nM) 0.18 0.47 0.20 Binding to mouse TIGIT-engineered cells, EC50 (nM) 0.33 Unbound Unbound Blocking CD155 combined with TIGIT, IC50 (nM) 0.13 0.28 0.21 Blocking CD112 binding to TIGIT, IC50 (nM) 0.52 0.31 0.34 Blocking CD113 binding to TIGIT, IC50 (nM) 0.42 0.28 0.28 NFAT reporting gene assay, EC50 (nM) 2.5 3.0 2.1 IL-2 secretion by Jurkat cells, EC50 (nM) 0.50 ~2.58 ~0.77

3.9 Assay of human primary NK cell activation

HT1080 cells expressing human PVR, PVRL2, and PVRL3 were used as target cells, and primary human NK cells isolated from human PBMCs were used as effector cells. 1 × ^10⁴ HT1080 cells pre-loaded with EuTDA (PerkinElmer cat#AD0116) and 1 × ^10⁴ primary human NK cells were co-cultured at 37°C and 5% _CO₂ for 2 hours in the presence of continuously diluted anti-TIGIT antibody. Target cell lysis in the culture supernatant was then tested by time-resolved fluorescence according to the manufacturer's instructions (PerkinElmer cat#AD0116). Fluorescence was detected using an EnVision microplate reader (PerkinElmer).

Human NK cells were co-cultured with HT1080 cells in the presence of antibodies. The results showed that W3642-1.433.11-z11-p1-uIgG1L could enhance NK cell killer activity in a dose-dependent manner. The data are shown in Figure 10.

3.10 Antibody-dependent cytotoxicity assay

To evaluate the ADCC effect of anti-TIGIT antibody on TIGIT-expressing cells, human TIGIT-engineered W364-CHOK1.hPro1.2A11 cells were used as target cells, and primary human NK cells isolated from human PBMCs were used as effector cells. 1 × ^10⁴ target cells and different concentrations of anti-TIGIT antibody were pre-incubated in 96-well plates at 37°C and 5% _CO₂ for half an hour, followed by adding 5 × ^10⁴ freshly isolated human NK cells to each well. The plates were incubated at 37°C and 5% _CO₂ for 6 hours. Target cell lysis was measured using an LDH-based cytotoxicity assay kit (Roche CAT#0744926001) according to the manufacturer's instructions. Absorbance was measured using an M5e microplate reader (Molecule Devices).

The ADCC assay results are shown in Figure 11. The results indicate that W3642-1.433.11-z11-p1-uIgG1L can induce ADCC in TIGIT-expressing CHOK1 cells in a dose-dependent manner, with efficacy similar to that of the reference antibody.

3.11 Antibody serum stability assay

Human serum was freshly isolated from a healthy donor. Anti-TIGIT antibody was diluted in the serum. Samples were aliquoted into five tubes and incubated at 37°C. Samples were then collected on days 0, 1, 4, 7, and 14, rapidly frozen, and stored at -70°C until analysis. Binding activity of the samples was assessed by FACS according to the method described in Section 3.1.

The binding of serum-incubated W3642-1.433.11-z11-p1-uIgG1L to W364-293F.hPro1.pool cells is shown in Figure 12. Antibodies incubated with serum for up to two weeks maintained similar binding activity and very similar EC50 as fresh antibodies. These results indicate that W3642-1.433.11-z11-p1-uIgG1L is stable in human serum at 37°C for at least two weeks.

3.12 Determination of antibody thermal stability

Conformational stability is a crucial characteristic of successful antibodies. Conformational stability can be assessed by measuring thermal stability using differential scanning fluorometry (DSF), which is sensitive to changes in protein folding. DSF measures the protein disfolding transition temperature (Tm) based on changes in the fluorescence intensity of the environmentally sensitive dye SYPRO Orange.

DSF was performed in a Quant Studio 7 Flex Real-Time PCR System (Applied Biosystems) with appropriate reagent buffer. SYPRO orange dye (Invitrogen cat#S6651) was added to the antibody, and the mixture was transferred to 96-well plates. The plates were placed in the Quant Studio® 7 Flex Real-Time PCR system, with the temperature range set to 26°C to 95°C and a heating rate of 0.9°C/min. The first two temperatures of protein unfolding transition were recorded as Tm1 and Tm2. These two values were calculated from the melting curve using QuantStudio® Real-Time PCR software (v1.3).

The DSF thermal analysis plot of the W3642-1.433.11-z11-p1-uIgG1L antibody showed two transition temperatures: a lower melting temperature (Tm1) and a higher melting temperature (Tm2), at 69.0℃ and 79.0℃, respectively. The results are shown in Table 8.

Table 8. Tm values of antibodies antibody Tm1 (°C) Tm2 (°C) W3642-1.433.11-z11-p1-uIgG1L 69.0 79.0

Implementation Examples 4 Internal signs

4.1 In vivo efficacy of combined therapy with anti-TIGIT and anti-PD-1 antibodies

To investigate the antitumor activity of anti-TIGIT antibodies in an MC38 syngeneic mouse model, human TIGIT transgenic h-TIGIT C57BL/6 mice (Jiangsu GemPharmatech Co., Ltd) were inoculated with tumor cells. Wild-type MC38 tumor cells (1 × ^10⁶ ) suspended in 0.1 mL DPBS were subcutaneously injected into the right forearm axilla of h-TIGIT C57BL/6 mice to induce tumor formation. When the average tumor size reached approximately 73 ^mm³ , the tumor-bearing animals were randomly divided into six study groups, with 8 mice in each group. The study design is shown in Table 9. Monoclonal anti-PD-1 antibodies (“anti-mPD-1”, strain ID 2E5 disclosed in WO2018053709) were combined with anti-TIGIT antibodies in several groups.

All antibodies were administered intraperitoneally to tumor-bearing mice twice weekly. Body weight and tumor volume were measured twice weekly. All procedures related to animal handling, care, and treatment in this study were performed in accordance with guidelines approved by the International Association for the Care and Use of Biological Models (IACUC) and the Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). Mice were euthanized according to predefined health criteria, and the study was terminated 24 days after the first administration of antibodies.

Table 9. Research Design of the MC38 Model Group handle N/group Dosage (mg/kg) Drug delivery route Frequency of drug administration Number of doses G1 PBS 8 / IP BIW 6 times G2 Anti-mPD-1 8 0.3 IP BIW 6 times G3 WBP364-BMK1 8 10 IP BIW 6 times G4 W3642-1.433.11-z11-p1-uIgG1L 8 10 IP BIW 6 times G5 Anti-mPD-1 + WBP364-BMK1 8 0.3 + 10 IP BIW 6 times G6 Anti-mPD-1 + W3642-1.433.11-z11-p1-uIgG1L 8 0.3 + 10 IP BIW 6 times

This study aimed to determine the antitumor activity of W3642-1.433.11-z11-p1-uIgG1L in h-TIGIT mice inoculated with MC38 tumor cells. Throughout the experiment, tumor growth and body weight were closely monitored in all mice, with tumor size measured and recorded twice weekly. Tumor growth inhibition (TGI) was calculated and analyzed at the optimal treatment time (24 days after grouping, with the first administration on the same day as grouping). Tumor volume results are shown in Figure 13 and summarized in Tables 10 and 11. Body weight changes are shown in Figure 14, indicating no abnormal changes between groups.

The results showed that W3642-1.433.11-z11-p1-uIgG1L alone exhibited weak tumor-suppressive activity, but it enhanced the effect of the combination with the anti-mPD-1 antibody. Compared with the combination of WBP364-BMK1 and the anti-mPD-1 antibody, the combination of W3642 and the anti-mPD-1 antibody showed significantly better tumor-suppressive efficacy from the start to the end of treatment.

Table 10. Summary of Tumor Volume Days after grouping Tumor volume ( ^mm³ ) ^a G1 G2 G3 G4 G5 G6 0 73.08±6.68 72.65±6.22 73.98±6.09 73.09±4.64 73.80±5.39 73.51±5.98 3 138.65±21.51 128.98±8.86 137.49±14.18 125.63±12.12 132.34±19.30 112.16±9.37 7 228.22±46.78 165.75±18.54 221.80±39.92 227.21±29.54 141.45±44.44 94.99±19.15 10 394.86±106.40 194.46±26.00 372.44±75.42 330.66±48.02 216.14±81.90 100.59±31.80 14 645.15±160.86 308.91±49.83 611.54±110.48 533.45±80.47 288.89±119.78 121.24±44.96 17 1069.40±253.48 435.83±74.73 959.99±213.11 825.23±147.30 450.50±182.32 207.56±84.20 twenty one 1814.22±403.06 655.85±146.33 1527.09±310.10 1293.32±229.96 383.82±122.20 286.65±114.30 twenty four 2176.32±532.13 907.09±215.22 2404.98±530.73 1834.10±326.10 723.05±251.46 432.77±181.64

Note: a, mean ± SEM.

Table 11. Summary of tumor growth inhibition Group Test sample Dosage (mg/kg) Tumor volume ( ^mm³ ) ^a TGI (%) P ^b Before treatment 24 days after treatment G1 PBS / 73.08±6.68 2176.32±532.13 / / G2 Anti-mPD-1 0.3 72.65±6.22 907.09±215.22 60.33 0.037 G3 WBP364-BMK1 10 73.98±6.09 2404.98±530.73 -10.83 0.768 G4 W3642-1.433.11-z11-p1-uIgG1L 10 73.09±4.64 1834.10±326.10 16.27 0.582 G5 Anti-mPD-1 + WBP364-BMK1 0.3 + 10 73.80±5.39 723.05±251.46 69.13 0.030 G6 Anti-mPD-1 + W3642-1.433.11-z11-p1-uIgG1L 0.3 + 10 73.51±5.98 432.77±181.64 82.92 0.006

Note:

a, mean ± SEM.

b. The mean tumor volume on day 24 after division into the treatment group and the PBS group was statistically analyzed using an independent samples t-test.

Those skilled in the art will further recognize that the invention can be implemented in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the invention discloses only exemplary embodiments, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims to indicate the scope and content of the invention.

without.

Figures 1 through 3 show the binding of antibodies to human TIGIT (Figure 1), cynomolgus monkey TIGIT (Figure 2), and mouse TIGIT (Figure 3), as determined by FACS.

Figure 4 shows the binding of the antibody to the TIGIT paraprotein, as determined by ELISA.

Figure 5 shows the results of antibody blocking CD155 binding to TIGIT, as determined by FACS.

Figure 6 shows the results of antibody blocking CD112 binding to TIGIT, as determined by FACS.

Figure 7 shows the results of antibody blocking of CD113 binding to TIGIT, as determined by FACS.

Figure 8 shows the results of antibody assay in the NFAT reporter gene assay.

Figure 9 shows the role of antibodies in stimulating IL-2 release in Jurkat cells.

Figures 10 and 11 show the roles of antibodies in the NK cell activation assay and ADCC assay, respectively.

Figure 12 shows the stability of the antibody in human serum.

Figure 13 shows the results of tumor volume changes after antibody treatment in the MC38 xenograft study.

Figure 14 shows the results of body weight changes after antibody treatment in the MC38 xenograft study.

A0101_OR_PSEQ.xml

without.

Claims (17)

An isolated antibody or its antigen-binding moiety that specifically binds to the TIGIT protein, comprising: a heavy chain CDR (HCDR)1 as shown in the amino acid sequence of SEQ ID NO: 1; an HCDR2 as shown in the amino acid sequence of SEQ ID NO: 2; an HCDR3 as shown in the amino acid sequence of SEQ ID NO: 3; a light chain CDR (LCDR)1 as shown in the amino acid sequence of SEQ ID NO: 4; an LCDR2 as shown in the amino acid sequence of SEQ ID NO: 7; and an LCDR3 as shown in the amino acid sequence of SEQ ID NO: 6. The isolated antibody or its antigen-binding portion as described in claim 1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH consists of an amino acid sequence shown in any one of SEQ ID NO: 10-11; and the VL consists of an amino acid sequence shown in any one of SEQ ID NO: 14-16. The isolated antibody or its antigen-binding portion as described in claim 1 comprises a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 11 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 15. An isolated antibody or its antigen-binding portion as described in any of claims 1 to 3, wherein the isolated antibody further comprises a human IgG constant region. The isolated antibody or its antigen-binding portion as described in claim 4, wherein the human IgG constant region is a human IgG1, IgG4, IgG2 or IgG3 constant region or a variant thereof. An isolated antibody or its antigen-binding portion as described in any of claims 1 to 3, wherein the antibody comprises a human IgG1 Fc region or a human IgG4 Fc region having an S228P substitution. The isolated antibody or its antigen-binding portion as described in any of claims 1 to 3, wherein the antibody is a chimeric antibody or a humanized antibody. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region and a light chain variable region of an isolated antibody or its antigen-binding moiety as described in any of claims 1 to 7. A carrier comprising isolated nucleic acid molecules as described in claim 8. A host cell comprising a vector as described in claim 9. A pharmaceutical composition comprising an isolated antibody or antigen-binding moiety thereof as described in any one of claims 1 to 7, and a pharmaceutically acceptable carrier. A method for producing an isolated antibody or antigen-binding portion thereof as described in any one of claims 1 to 7, comprising the steps of: - culturing host cells containing one or more expression vectors encoding the antibody or antigen-binding portion thereof under suitable conditions; and - harvesting the antibody or antigen-binding portion thereof from the cell culture. Use of an isolated antibody or an antigen-binding moiety thereof as described in any one of claims 1 to 7 in combination with an anti-PD-1 antibody in the preparation of a medicament for treating cancer in a subject. For the purposes described in claim 13, the cancers are selected from lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, leukemia, lymphoma, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, colorectal cancer, kidney cancer, clear cell renal cancer, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, central nervous system tumors, mesothelioma, myeloma, soft tissue carcinoma, and sarcoma. For the purpose described in claim 14, wherein the cancer is colon cancer or melanoma. A combination of an isolated antibody or its antigen-binding portion as described in any one of claims 1 to 7 with an anti-PD-1 antibody. A reagent kit comprising a container containing an isolated antibody or antigen-binding portion thereof as described in any one of claims 1 to 7.