Anti-TIGIT antibodies and uses thereof

Abstract

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

Description

[0001] Cross-references

[0002] This application claims the benefit of international application PCT / CN2023 / 098574, filed on June 6, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list, which is incorporated herein by reference in its entirety. Technical Field

[0005] This application generally relates to antibodies. More specifically, this application relates to monoclonal antibodies against TIGIT, methods for their preparation, and uses of the antibodies. Background Technology

[0006] In the tumor microenvironment, persistent antigen stimulation can lead to T cell exhaustion, T cell dysfunction, and high expression of co-inhibitory receptors including PD-1, LAG-3, TIM3, and TIGIT. Currently, various strategies are being explored to revitalize exhausted T cells using small molecule or therapeutic antibody approaches, either alone or in combination.

[0007] TIGIT (a T-cell immune receptor with Ig and ITIM domains), also known as Vstm3 and WUCAM, is a co-inhibitory receptor expressed on NK and CD8+ T cells, as well as CD4+ T cell subsets, including immunosuppressive regulatory T cells (Tregs). There are four known ligands for TIGIT: the poliovirus receptor (PVR), PVRL2, PVRL3, and PVRL4, all of which are overexpressed by tumor and antigen-presenting cells, leading to immunosuppression. These ligands also bind to the co-stimulatory molecule CD226 and the co-inhibitory molecules PVRIG and CD96 (the latter sometimes considered a co-stimulatory agent). Antibodies against TIGIT disrupt the binding of TIGIT to its ligands and block its inhibitory signaling, shifting the balance to favor CD226-mediated activation, which induces a strong anti-tumor immune response. Like other exhaustion markers such as PD-1, LAG3, and TIM3, TIGIT is upregulated in cancer and inflammatory diseases and has been identified as an exhaustion marker. In addition, TIGIT has been identified as a key inhibitory receptor for a new population of T cells (stem cell-like memory T cells), which may be a preferred target for anti-PD-(L)1 efficacy.

[0008] TIGIT may be a promising therapeutic target for tumor immunotherapy, either as a single agent or in combination with other immunomodulators. Summary of the Invention

[0009] This disclosure provides for these and other purposes. In a broader sense, this disclosure relates to compounds, methods, compositions, and articles of antibody with improved efficacy. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapeutics and diagnostics, and can be used in combination with other antibodies that respond to a variety of targets.

[0010] This disclosure provides chimeric and humanized monoclonal antibodies against TIGIT. Further methods are provided for validating antibody function in vitro and in vivo, as well as methods for treating subjects with cancer or immune disorders by administering anti-TIGIT antibodies, as disclosed herein, alone or in combination with a PD-1 / PD-L1 antagonist.

[0011] 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:

[0012] Heavy chain CDR (HCDR)1 containing the amino acid sequence of SEQ ID NO: 1;

[0013] HCDR2 containing the amino acid sequence of SEQ ID NO: 2;

[0014] HCDR3 containing the amino acid sequence of SEQ ID NO: 3;

[0015] The light chain CDR(LCDR)1 containing the amino acid sequence of SEQ ID NO: 4;

[0016] LCDR2 containing the amino acid sequence of any one of SEQ ID NO: 7, 5, 8, and 9; and

[0017] LCDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0018] 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).

[0019] VH contains or is composed of the following:

[0020] (i) An amino acid sequence as shown in any one of SEQ ID NO: 10-11;

[0021] (ii) An amino acid sequence that is at least 85%, 90%, or 95% identical to any one of SEQ ID NO: 10-11; or

[0022] (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

[0023] VL includes or consists of the following:

[0024] (i) An amino acid sequence as shown in any one of SEQ ID NO: 12-18;

[0025] (ii) An amino acid that is at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NO: 12-18; or

[0026] (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: 12-18.

[0027] In some embodiments, the isolated antibody or its antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the VH region as shown in any one of SEQ ID NO: 10-11, and LCDR1, LCDR2 and LCDR3 of the VL region as shown in any one of SEQ ID NO: 12-18.

[0028] In some embodiments, the isolated antibody or its antigen-binding portion comprises: HCDR1 as shown in SEQ ID NO: 1; HCDR2 as shown in SEQ ID NO: 2; HCDR3 as shown in SEQ ID NO: 3; LCDR1 as shown in SEQ ID NO: 4; LCDR2 as shown in SEQ ID NO: 7; and LCDR3 as shown in SEQ ID NO: 6.

[0029] 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.

[0030] 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.

[0031] In some embodiments, the anti-TIGIT antibodies disclosed herein are murine antibodies, chimeric antibodies, or humanized antibodies. In some embodiments, the antibodies described herein are anti-TIGIT antagonist antibodies.

[0032] In some aspects, this disclosure provides isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of isolated antibodies or their antigen-binding moieties as disclosed herein. In some embodiments, the nucleic acid molecule comprises nucleic acid sequences as shown in SEQ ID NO: 21 and / or as shown in SEQ ID NO: 22.

[0033] In some respects, this disclosure provides expression vectors that contain nucleic acid molecules as disclosed herein.

[0034] In some respects, this disclosure provides a host cell containing expression vectors as disclosed herein.

[0035] In some respects, this disclosure provides pharmaceutical compositions comprising an antibody or its antigen-binding portion as disclosed herein, and a pharmaceutically acceptable carrier.

[0036] In some aspects, this disclosure provides methods for preparing antibodies or antigen-binding moieties thereof, comprising expressing the antibody or antigen-binding moieties thereof in host cells and isolating the antibody or antigen-binding moieties thereof from the host cells. In some embodiments, the host cells have been transfected or transformed with expression vectors encoding the heavy and light chains of the antibodies disclosed herein. The heavy chain encoding nucleic acid sequences and the light chain encoding nucleic acid sequences may be in the same vector or in separate vectors.

[0037] In some respects, this disclosure provides methods for modulating TIGIT-related immune responses in subjects, comprising administering to subjects an antibody or its antigen-binding portion as disclosed herein.

[0038] In some aspects, 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 an antibody or antigen-binding portion thereof disclosed herein, or a pharmaceutical composition thereof.

[0039] In some aspects, this disclosure provides methods for treating or preventing cancer or immune-related conditions in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof, as disclosed herein, alone or in combination with another anticancer agent.

[0040] Anticancer agents can be chemotherapeutic agents, monoclonal antibodies, antibody-drug conjugates, etc. In some implementations, the anticancer agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0041] The cancers mentioned can be selected from colon cancer, lung cancer (e.g., 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 cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, central nervous system vegetations, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma, myeloma, soft tissue cancer, and sarcoma. Immune-related conditions can be T-cell dysfunction disorders, infections, or inflammatory diseases.

[0042] In some implementations, antibodies or their antigen-binding portions, as disclosed herein, are administered in combination with anti-PD-1 antibodies.

[0043] In some respects, this disclosure provides a combination of isolated antibodies or their antigen-binding portions as disclosed herein with anti-PD-1 antibodies.

[0044] 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.

[0045] 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 diagnosing diseases associated with TIGIT overexpression.

[0046] In some aspects, 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, antibodies as disclosed herein are used in combination with PD-1 / PD-L1 antagonists such as PD-1 antibodies.

[0047] In some aspects, this disclosure provides methods 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 as disclosed herein.

[0048] In some respects, this disclosure provides kits and devices comprising antibodies or antigen-binding portions thereof as disclosed herein in one or more containers.

[0049] The foregoing is an overview and therefore necessarily contains simplifications, generalizations, and omissions of details; thus, those skilled in the art will understand that this overview is merely illustrative and not intended to be limiting in any way. This overview is not intended to identify 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. Attached Figure Description

[0050] Figure 1-3 The results showed that the antibody and human TIGIT (as determined by FACS) Figure 1 ), crab-eating macaques (TIGIT) Figure 2 ) and mouse TIGIT ( Figure 3 The combination of ).

[0051] Figure 4 The binding of the antibody to the TIGIT paralog protein, as measured by ELISA, is shown.

[0052] Figure 5 The results show the antibody blocking the binding of CD155 to TIGIT, as determined by FACS.

[0053] Figure 6 The results show the antibody blocking the binding of CD112 to TIGIT, as determined by FACS.

[0054] Figure 7 The results show the antibody blocking the binding of CD113 to TIGIT, as determined by FACS.

[0055] Figure 8 The results of the antibody in the NFAT reporter gene assay are shown.

[0056] Figure 9 The effect of the antibody in stimulating the release of IL-2 through Jurkat cells was demonstrated.

[0057] Figure 10 and 11 The effects of the antibody in NK cell activation assay and ADCC assay are shown respectively.

[0058] Figure 12 The stability of the antibody in human serum was demonstrated.

[0059] Figure 13 The results show the changes in tumor volume after antibody treatment in the MC38 xenograft study.

[0060] Figure 14 The results show the changes in body weight after antibody treatment in the MC38 xenotransplantation study. Detailed Implementation

[0061] While the invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that illustrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments shown. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0062] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly specifies otherwise. 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 (e.g., “comprises” and “comprised”) is not restrictive. Moreover, the scope provided in the specification and appended claims includes both endpoints and all points in between.

[0063] Generally, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of this disclosure are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout the specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6 thed., 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); Ausubelet 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 nomenclature, laboratory procedures, and techniques described herein for use in conjunction with analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry are those well-known and commonly used in the art.

[0064] definition

[0065] To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0066] As used herein, the term "antibody" or "Ab" generally refers to a Y-tetrameric protein, which comprises two heavy (H) and two light (L) polypeptide chains 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 antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of 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 complementarity-determining regions (CDRs)), which are separated by relatively conserved regions (called frame regions (FRs)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / 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.

[0067] In the context of this application, the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, which are used interchangeably, refer to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to the antigen to which the full-length antibody specifically binds, and / or competes with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology 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 complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and such polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., the monoclonal anti-human TIGIT antibody provided herein) by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) and specificity can be screened in the same manner as for intact antibodies.

[0068] As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of an antibody molecule consisting of a single molecule. Monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope.

[0069] 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 murine 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 the constant region of human IgG4 and a light chain having a rat VL fused to human Igλ.

[0070] As used herein, the term "humanized antibody" refers to an antibody in which a germline CDR sequence derived from another mammalian species (e.g., rat or mouse) has been grafted onto a human frame sequence. Further frame region modifications may be made within the human frame sequence. Humanized antibodies may optionally also contain at least a portion (e.g., Fc) of an immunoglobulin constant region, typically a constant region of human immunoglobulins. Exemplary chimeric 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 humanized VH fused to a human IgG4 constant region and a light chain having a humanized VL fused to human Igλ.

[0071] As used herein, the term "PTM" or "post-translational modification" refers to the process that occurs on one or more amino acids of a protein (e.g., an antibody) after it has been translated. Proteins are typically produced by ribosomes, which translate mRNA into polypeptide chains, which are then converted into mature protein products via PTM. PTM processes include phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, and esterification. Preferably, potential PTM sites are removed during antibody optimization to avoid structural and functional heterogeneity resulting from the PTM process.

[0072] 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 mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise stated. TIGIT is also known in the art as DKFZp667A205, FLJ39873, protein 9 containing the V-set and the immunoglobulin domain, protein 3 containing the V-set and the transmembrane domain, VSIG9, VSTM3, and WUCAM. The term encompasses full-length unprocessed TIGIT, the extracellular domains of TIGIT, and any form of TIGIT produced by processing in the cell. The term also encompasses 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).

[0073] As used herein, the term "PD-1 / PD-L1 antagonist" includes PD-L1 antagonists (e.g., anti-PD-L1 antibodies) and PD-1 antagonists (e.g., anti-PD-1 antibodies). PD-L1 antagonists reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (e.g., PD-1 or B7-1).In some implementations, the PD-1 antagonist is an anti-PD-1 antagonist antibody selected from, but not limited to, the following: nivolumab (MDX-1106) or pembrolizumab (formerly 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, Budugralimab, AMG 404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, APL-502, cosibelimab, lodapolimab, GS-4224, INCB086550, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, MAX-10181, RC98, BION-004, AM0001, CB201, ENUM 244C8, ENUM388D4, 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 implementations, the PD-L1 antagonist is an anti-PD-L1 antagonist antibody selected from, but not limited to, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). PD-1 / PD-L1 antagonists include known antibodies and internally developed antibodies.

[0074] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or its antigenic moiety and an antigen). Binding affinity between two molecules can be quantified by determining the equilibrium dissociation constant (KD). In turn, KD can be determined by measuring the kinetics of complex formation and dissociation using a surface plasmon resonance (SPR) method (Biacore™) (as a non-limiting example). The rate constants corresponding to the binding and dissociation of a monovalent complex are referred to as 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 via the equation KD = kd / ka or koff / kon. Antibody binding kinetics and binding affinity can be assessed using standard assays known in the art or as described in the Examples section below.

[0075] As used in this article, the term "high affinity" refers to a 1x10 bond strength against the target antigen. -9 M or smaller, preferably 5x10 -10 M or smaller, or even better, 1x10 -10 M or smaller, or even better 5x10 -11 M or smaller K D Antibodies.

[0076] As used in this article, the term "EC" 50 The term "half-maximal effective concentration" (EC50), also known as "half-maximal effective concentration," refers to the concentration of a drug, antibody, or toxin that induces half the response between baseline and maximum response after a specific exposure time. In the context of this application, EC50... 50 It is expressed in units of “nM” or “M”.

[0077] As used herein, the term "separated" refers to a state obtained artificially from a natural state. If a "separated" substance or component exists in nature, it may be due to changes in its natural environment, separation of the substance from its natural environment, or both. For example, an unseparated polynucleotide or polypeptide may naturally exist in a living animal, and a highly pure copy of the same polynucleotide or polypeptide separated from such a natural state is called a separated polynucleotide or polypeptide. The term "separated" 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 separated substance.

[0078] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen 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, an isolated antibody that specifically binds to the human TIGIT protein may be cross-reactive with other antigens (e.g., TIGIT proteins from other species). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0079] As used herein, the term "vector" refers to a nucleic acid medium that may have an intercalated polynucleotide. When a vector allows expression of a protein encoded by the intercalated polynucleotide, it is called an expression vector. Vectors may have elements of carried genetic material that are expressed in a host cell through transformation, transduction, or transfection. 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 chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (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 (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Vectors may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0080] 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 covers not only the specific subject cells but also the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may differ from the parent cells but are still included within the scope of the term "host cell."

[0081] 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. "Identity percentage" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably resolved 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, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48:1073.

[0082] As used herein, 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 antigens to stimulate the activation, proliferation, and differentiation of specific immune cells in order to ultimately generate immune effector 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 a host depends on three factors: the characteristics of the antigen, the host's reactivity, and the immunization method.

[0083] As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, lipid transfection and chemical and physical methods, such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In a specific embodiment of the invention, the human TIGIT gene is transfected into 293F cells.

[0084] As used herein, the term “SPR” or “surface plasmon resonance” refers to and includes an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Example 5 and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0085] 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 a heterogeneous mixture of biological cells into two or more containers (one cell at a time) based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence-Activated Cell Sorting". Retrieved 2017-11-09.). Instruments used to perform FACS are known to those skilled 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, Calif.), the Epics C from Coulter Epics Division (Hialeah, Fla.), and the MoFlo from Cytomation (Colorado Springs, Colo.).

[0086] 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 target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely necessary for this type of killing. Primary cells used to mediate 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 the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, for example, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells 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 assessed in vivo, for example, in animal models disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0087] 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 mammal species. The term does not necessarily mean that a subject has been diagnosed with a specific disease, but generally refers to an individual under medical supervision.

[0088] As used in this article, the term “prevent, prevention, or preventing” in relation to a disease condition in mammals refers to preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms.

[0089] As used herein, in the context of treating a condition, the term "treatment" (treating or treated) generally refers to the treatment and therapy, whether in humans or animals, in which some desired therapeutic effect is achieved, such as inhibiting the progression of the condition, and includes a reduction in the rate of progression, cessation of the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. In the case of cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof.

[0090] As used herein, the term "effective amount" refers to an amount of an active compound or a material, composition, or dosage form containing an active compound that, when administered according to a desired treatment regimen, effectively produces some of the desired therapeutic efficacy in proportion to a reasonable benefit / risk ratio. For example, when used in conjunction with the treatment of a disease or condition, "effective amount" refers to an amount or concentration of an antibody or its antigen-binding portion that is effective in treating said disease or condition.

[0091] As used herein, the term "pharmaceuticalally acceptable" means that the medium, diluent, excipient and / or salt thereof is chemically and / or physically compatible with other components in the formulation and is physiologically compatible with the recipient.

[0092] 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, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 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.

[0093] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the organism's immune response to the antigen or alter 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 breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0094] Anti-TIGIT antibody

[0095] In some respects, this disclosure provides antibodies or antigen-binding portions or variants thereof that are capable of binding TIGIT (e.g., human, mouse, or cynomolgus monkey TIGIT) with sufficient affinity such that they substantially or completely inhibit the biological activity of TIGIT.

[0096] In some embodiments, the anti-TIGIT antibody disclosed herein 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-chain variable fragment (scFv), or a biantibody.

[0097] In some embodiments, this document provides humanized anti-TIGIT antibodies in which the HCDR and LCDR sequences of human immunoglobulins are replaced with HCDR and LCDR sequences obtained from a non-human species (such as rats) having the desired specificity, affinity, and / or ability. In some other embodiments, certain framework (“FR”) residues of the human immunoglobulin 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 residues and framework residues may be made to remove potential post-translational modifications to improve antibody performance, such as binding affinity.

[0098] Various methods for humanizing nonhuman antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a nonhuman source. These nonhuman amino acid residues are generally referred to as “input” residues, which are typically derived from an “input” variable domain. Humanized antibodies that bind to TIGIT can be generated using techniques known to those skilled 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).

[0099] The antibodies disclosed herein can bind with high affinity to at least one of human, mouse, and cynomolgus monkey TIGIT. Binding of the antibodies of this disclosure to TIGIT can be assessed using one or more well-established techniques in the art (e.g., ELISA). The binding specificity of the antibodies of this disclosure can also be determined by monitoring the binding of the antibody to cells expressing the TIGIT protein, for example, flow cytometry. In some embodiments, the antibody is tested by flow cytometry assay in which the antibody reacts with a cell line expressing human TIGIT, such as HEK293 cells that have been transfected to express TIGIT on their cell surface. Additionally or alternatively, antibody binding includes binding kinetics (e.g., K... D The value can be tested in conjunction with BIAcore assays.

[0100] In some embodiments, the antibody or its antigen-binding portion of this disclosure is in the form of 1x10 -9 M or smaller, 5x10 -10 M or smaller, 1x10 -10 M or smaller, 5x10 -11 M or smaller, 4x10 -11 M or smaller, 3x10 -11 M or smaller, 2.5x10 -11 M or smaller, or 2x10 -11 M or smaller K DBinding to human TIGIT, as measured by SPR. In some embodiments, the antibody or its antigen-binding portion is capable of specifically binding to human TIGIT, as well as cynomolgus monkey TIGIT and mouse TIGIT. For example, the antibody or its antigen-binding portion can bind to human TIGIT-expressing cells at an EC50 of no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, such as no more than 0.23 nM; to cynomolgus monkey TIGIT-expressing cells at an EC50 of no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, such as no more than 0.18 nM; and to mouse TIGIT-expressing cells at an EC50 of no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, such as no more than 0.33 nM, as measured by FACS.

[0101] The anti-TIGIT antibodies disclosed herein can 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 signaling via PVR, PVRL2, and / or PVRL3 to restore functional T cell responses (e.g., proliferation, cytokine production, target cell killing) from a dysregulated state of response to antigen stimulation.

[0102] 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.

[0103] In some embodiments, the ability of the anti-TIGIT antibody 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, the binding assay is a competitive binding assay. This assay can be performed in various forms, such as, but not limited to, ELISA assays, flow cytometry, surface plasmon resonance (SPR) assays (e.g., Biacore™), or biolayer interferometry (e.g., ForteBio Octet™).

[0104] Anti-TIGIT antibody containing CDR

[0105] In some embodiments, this disclosure provides isolated antibodies or antigen-binding portions thereof, comprising:

[0106] A) One or more heavy chain CDRs (HCDRs) selected from the following group:

[0107] HCDR1, comprising an amino acid sequence that is different from SEQ ID NO: 1 in that it consists of no more than two amino acid additions, deletions, or substitutions; HCDR2, comprising an amino acid sequence that is different from SEQ ID NO: 2 in that it consists of no more than two amino acid additions, deletions, or substitutions; and HCDR3, comprising an amino acid sequence that is different from SEQ ID NO: 3 in that it consists of no more than two amino acid additions, deletions, or substitutions.

[0108] B) One or more light chain CDRs (LCDRs) selected from the following group:

[0109] LCDR1, comprising an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence differing from SEQ ID NO: 4 in that it consists of no more than two amino acid additions, deletions, or substitutions; LCDR2, comprising any one of SEQ ID NO: 5, 7, 8, and 9 or an amino acid sequence differing from any one of SEQ ID NO: 5, 7, 8, and 9 in that it consists of no more than two 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 in that it consists of no more than two amino acid additions, deletions, or substitutions; or

[0110] C) One or more HCDRs of A) and one or more LCDRs of B).

[0111] In some implementations, CDR identification is based on the Contact definition introduced by Dr. Andrew CR Martin's team (http: / / www.bioinf.org.uk / abs / ).

[0112] In some embodiments, this disclosure provides isolated antibodies or antigen-binding portions thereof comprising: HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 3, LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in any one of SEQ ID NO: 5, 7, 8 and 9, and LCDR3 as shown in SEQ ID NO: 6.

[0113] The scope of the frame area and CDR can be precisely identified using methodologies known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, the contact definition, the IMGT definition (all of which are well known in the art) and any combination thereof. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins ofImmunological 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. 1969May, 63(1):78-85; and Martin and Allen, in “Handbook of Therapeutic Antibodies”, chapter 5, 2007. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Correspondences or comparisons between the numberings according to different definitions 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 CompImmunol. (2003) 27:55–77).

[0114] As those skilled in the art will understand, the exact numbering and location of CDRs can differ in different numbering systems. However, it should be understood that the disclosure of variable heavy and / or variable light sequences includes the disclosure of the associated (inherent) CDR, regardless of the numbering method used. Therefore, the disclosure of each variable region is the disclosure of the CDR (e.g., HCDR1, HCDR2, and HCDR3). Two antibodies having the same VH and VL mean that when measured using the same method (e.g., numbering methods such as Kabat, AbM, Chothia, Contact, and IMGT known in the art), their CDRs are identical. When measured using different numbering methods, the same antibodies disclosed herein may have different CDR groups.

[0115] Variable regions and CDRs in antibody sequences can be identified according to general rules already developed in the art (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering systems) or by aligning the sequence against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., AntibodyEngineering, 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 on the “Abysis” website at www.bioinf.org.uk / abs (maintained by AC. Martin in the Department of Biochemistry and Molecular Biology, University College London, UK) and the VBASE2 website at www.vbase2.org, and can be evaluated through these websites, as described in Retter et al., Nucl. Acids Res., 33 (Databaseissue): 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 the chapter "Protein Sequence and Structure Analysis of Antibody Variable Domains" in Dr. Andrew C. Martin's book, available 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 further includes general rules that have been developed for identifying CDRs that can be used in accordance with the teachings herein.

[0116] In some embodiments, such as the anti-TIGIT antibody disclosed herein, a VH region and a VL region are included, wherein the VH region includes 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 includes 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.

[0117] In some embodiments, the framework region is derived from a human lineage (i.e., humanized), such as human immunoglobulin. In some embodiments, certain residues in the humanized framework region are reverted to 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 humanized VH region comprises Val and Trp at positions 78 and 94 (according to Kabat numbering), respectively. In some specific embodiments, FRW1 in the humanized VL region comprises one or more of Gln at position 1, Ala at position 2, and Val at position 3 (according to Kabat numbering). When referring to residues in the variable region (approximately residues 1-107 of the light chain and residues 1-113 of 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 embodiments, the N-terminal and C-terminal FRW1 and FRW4 of the VH and / or VL regions may be truncated so that they contain only a portion of FRW1 and / or FRW4. In some embodiments, the CDR and FR regions have undergone PTM removal optimization.

[0118] In some embodiments, this document provides an anti-TIGIT antibody comprising 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, this document provides an anti-TIGIT antibody comprising 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, this document provides an anti-TIGIT antibody comprising 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, this document provides an anti-TIGIT antibody comprising 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.

[0119] In some embodiments, this document provides an anti-TIGIT antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 10, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 12. In some embodiments, this document provides an anti-TIGIT antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 11, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 13. In some embodiments, this document provides an anti-TIGIT antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 10, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 14. In some embodiments, this document provides an anti-TIGIT antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 11, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 15. In some embodiments, this document provides an anti-TIGIT antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 11, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 16. In some embodiments, this document provides an anti-TIGIT antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 10, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 17. In some embodiments, this document provides an anti-TIGIT antibody comprising at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 10, and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 as shown in SEQ ID NO: 18.

[0120] In some specific embodiments, the VH region contains amino acid Val (V) at position 78 and amino acid Trp (W) at position 94. Alternatively, the VL region contains amino acids “QAV” (Gln-Ala-Val) at positions 1-3.

[0121] Anti-TIGIT antibodies containing heavy chain variable regions and light chain variable regions

[0122] 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:

[0123] VH includes:

[0124] (i) The amino acid sequence of one of SEQ ID NO: 10-11;

[0125] (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

[0126] (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 the amino acid sequence of one of SEQ ID NO: 10-11; and / or

[0127] VL includes:

[0128] (i) The amino acid sequence of one of SEQ ID NO: 12-18;

[0129] (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

[0130] (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 the amino acid sequence of one of SEQ ID NO: 12-18.

[0131] The percentage of 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 a PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been integrated into the GAP program 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.

[0132] Alternatively or additionally, the protein sequences disclosed herein can be further used as “query sequences” to search public databases for, for example, to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J.MoI. Biol. 215:403-10. BLAST protein searches 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 molecules of this disclosure. For obtaining vacancy alignments for comparative purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res.25(17):3389-3402, can be used. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0133] In some further embodiments, the isolated antibody or its antigen-binding moiety may contain conserved substitutions or modifications of amino acids in the variable regions of the heavy and / or light chains. It will be understood in the art that certain conserved sequence modifications that do not remove antigen binding can be made. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildtet 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.

[0134] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide containing that amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, for example, 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, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). 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 art (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).

[0135] In a specific implementation, the isolated antibody or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 or composed of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or composed of the amino acid sequence of SEQ ID NO: 15.

[0136] 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 shown above.

[0137] Anti-TIGIT antibodies with certain properties

[0138] The antibodies disclosed herein are characterized by specific functional features or properties. Based on their target-target mechanism of action, the in vitro functional properties and pharmacological activities of the antibodies have been fully evaluated at the molecular and cellular levels. In some embodiments, the isolated antibody or its antigen-binding moiety possesses one or more of the following properties:

[0139] (a) Specific binding to at least one of human TIGIT protein, cynomolgus monkey TIGIT protein, and mouse TIGIT protein, for example, at a concentration of less than 1 nM K. D It binds to human TIGIT, as measured by SPR;

[0140] (b) It does not cross-bind with TIGIT homologous proteins;

[0141] (c) Blocking the binding between TIGIT and its ligands CD155, CD112 and CD113;

[0142] (d) Activation of immune cells such as T cells and NK cells;

[0143] (e) Induction of ADCC effect on TIGIT-expressing CHOK1 cells; and

[0144] (f) When combined with anti-PD-1 agents, it has significantly better efficacy than the baseline antibody in treating cancer, as demonstrated in in vivo mouse models.

[0145] Dual blockade of TIGIT and PD-1 can reverse immunosuppression. As shown herein, the anti-TIGIT antibody of this disclosure exhibits a synergistic effect with anti-PD-1 agents (e.g., anti-PD-1 antibodies) or anti-PD-L1 agents (e.g., anti-PD-L1 antibodies).

[0146] In some preferred embodiments, the antibodies disclosed herein can be combined with other therapeutic agents, such as anticancer agents, including anticancer antibodies and chemotherapeutic agents. The other therapeutic agents may also be antagonists or inhibitors of T-cell co-inhibitors, agonists of T-cell co-activators, or immunostimulatory cytokines.

[0147] In some implementations, additional therapeutic agents are antibodies that bind to proteins selected from 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 implementations, additional therapeutic agents are selected from anti-CD25 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-Tim3 antibodies, anti-Lag3 antibodies, anti-CTLA4 antibodies, anti-4-1BB antibodies, anti-OX40 antibodies, anti-CD3 antibodies, anti-CD40 antibodies, anti-CD47M antibodies, anti-CSF1R antibodies, anti-TLR antibodies, anti-STING antibodies, anti-RIGI antibodies, anti-TAM receptor kinase antibodies, anti-NKG2A antibodies, anti-NKG2D antibodies, anti-GD2 antibodies, anti-EGFR antibodies, anti-PDGFR-a antibodies, anti-SLAMF7 antibodies, anti-VEGF antibodies, anti-CTLA-4 antibodies, anti-CD20 antibodies, anti-cCLB8 antibodies, anti-KIR antibodies, and anti-CD52 antibodies. In some implementations, additional therapeutic agents are selected from SEA-CD40, avelumab, durvalumab, nivolumab, pembrolizumab, pidilizumab, atezolizumab, Hul4.18K322A, Hu3F8, dinituximab, trastuzumab, cetuximab, olaratumab, necitumumab, and elotuzumab. The following are listed: lotuzumab, ramucirumab, pertuzumab, ipilimumab, bevacizumab, rituximab, obinutuzumab, siltuximab, ofatumumab, lirilumab, and alemtuzumab.

[0148] Fc area

[0149] The anti-TIGIT antibody and antigen-binding portion provided herein further includes an immunoglobulin constant region containing an Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region (natural or a variant thereof), and optionally a hinge 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% homology with the natural sequence Fc region, or at least about 90% homology with it, for example, at least about 95% homology with it. 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 contains a wild-type human IgG1 Fc region. The variant Fc region may contain one or more amino acid modifications that alter antibody-dependent cytotoxicity (ADCC) or other effector function (e.g., Leu234Ala / Leu235Ala or LALA). In some embodiments, the Fc region may contain one or more amino acid variations (e.g., insertions, deletions, or substitutions) that produce a modified Fc region having a modified binding interaction between Fc and FcRn or FcγR.

[0150] In some embodiments, the Fc region is the IgG4 Fc region containing the S228P mutation (according to the EU numbering shown in Kabat et al.) that prevents Fab arm exchange and stabilizes the IgG4 molecule. In some embodiments, the Fc region is the IgG1 Fc region and contains the LALA mutation, namely the mutations in L234A and L235A. The LALA mutation is likely the most commonly used mutation to disrupt antibody effector function, such as eliminating Fc binding to specific FcγR and reducing ADCC activity mediated by PBMCs and monocytes. When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., the EU index reported in Kabat et al., ibid.). “EU numbering as shown in Kabat” or “EU indexing as shown in Kabat” refers to the residue number of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the antibody constant domain mean residue numbers obtained through the EU numbering system.

[0151] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, and transgenic animals (e.g., XenoMouse). ®(or some combination thereof.) For example, monoclonal antibodies can be produced using hybridomas and biochemical and genetic engineering techniques recognized in the art, such as those described in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1 st ed. 2009; Shire et. al. (Eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science + Business Media LLC, 1 st 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 improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention. In some embodiments, anti-human TIGIT monoclonal antibodies are prepared using hybridoma technology. Hybridoma generation is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0152] Nucleic acid molecules encoding the antibodies disclosed herein

[0153] In some respects, this disclosure 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.

[0154] The nucleic acids disclosed herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), the cDNA encoding the light and heavy chains of the antibody prepared from the hybridoma can be obtained by standard PCR amplification or cDNA cloning 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.

[0155] The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the VH-encoding nucleic acid to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), ibid.) and DNA fragments encompassing 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, but more preferably IgG1 or IgG4 constant regions.

[0156] Isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ constant region.

[0157] 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 linker. As used herein, the term "operatively linked" is intended to mean the joining of two DNA fragments such that the amino acid sequences encoded by both fragments remain within the frame.

[0158] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions of isolated antibodies as disclosed herein.

[0159] In some specific implementations, the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated antibody and contains a nucleic acid sequence selected from the group consisting of:

[0160] (A) A nucleic acid sequence encoding the heavy chain variable region as shown in any one of SEQ ID NO: 10-11;

[0161] (B) The nucleic acid sequence shown in SEQ ID NO: 21; or

[0162] (C) Nucleic acid sequences that hybridize with the complementary strand of (A) or (B) nucleic acid sequences under highly stringent conditions.

[0163] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding a light chain variable region of an isolated antibody as disclosed herein.

[0164] In some specific implementations, the isolated nucleic acid molecule encodes the light chain variable region of the isolated antibody, comprising a nucleic acid sequence selected from the group consisting of:

[0165] (A) A nucleic acid sequence encoding the variable region of the light chain as shown in any one of SEQ ID NO: 12-18;

[0166] (B) The nucleic acid sequence shown in SEQ ID NO: 22; or

[0167] (C) Nucleic acid sequences that hybridize with the complementary strand of (A) or (B) nucleic acid sequences under highly stringent conditions.

[0168] For example, the nucleic acid molecule contains SEQ ID NO: 21 and 22. In some other embodiments, the nucleic acid molecule shares 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 identity percentage is derived from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0169] Exemplary high-strict 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 will be understood in the art that equivalently stringent conditions can be achieved by varying the temperature and buffer or salt concentration, as described in Ausubel, et al. (ed.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3–6.4.10. Modifications in the hybridization conditions can be determined empirically or precisely calculated based on the length and percentage of guanosine / cytosine (GC) base pairs on the probe. Hybridization conditions can be calculated as described in Sambrook, et al. (ed.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47–9.51.

[0170] host cells

[0171] The host cells disclosed in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as yeast, bacteria, fungi, plant and animal cells, preferably mammalian cells. Mammalian host cells for expressing the antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with DHFR selection markers, for example, as described in RJ Kaufman and PA 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 line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile 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 supporting cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); and human cervical cancer cells (HELA, ATCC). CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NSO (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 CRL 1662); PER.C6 cells; and human hepatocellular carcinoma cell line (Hep G2). CHO cells are one of the cell lines that can be used in this study, of which 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 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, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, NSO cells, and lymphocytes.

[0172] Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae including Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella including Salmonella typhimurium, Serratia including Serratia marcescens, and Shigella, as well as Bacilli including Bacillus subtilis and Bacillus licheniformis, Pseudomonas including Pseudomonas aeruginosa, and Streptomyces.

[0173] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or Bacillus baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and can be used in this paper, such as *Schizosaccharomyces pombe*; hosts of the *Kluyveromyces* genus such as, for example, *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wicken* (ATCC 24,178), *Kluyveromyces walter* (ATCC 56,500), *Kluyveromyces fruitfly* (ATCC 36,906), *Kluyveromyces thermosus*, and *Kluyveromyces marx*; *Yersinia* genus (EP 402,226); *Pichia pastoris* (EP 183,070); *Candida* genus; *Trichoderma reesei* (EP 244,234); *Neurospora crassa*; *Schwanyophytes* genus such as *Schwanyophyces serrata*; and filamentous fungi such as, for example, hosts of the *Neurospora*, *Penicillium*, *Cyclophorus*, and *Aspergillus* genera such as *Aspergillus nidus* and *Aspergillus niger*.

[0174] When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow the antibody to be expressed in the host cells or by secreting the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods.

[0175] Pharmaceutical Composition

[0176] In some aspects, this disclosure relates to pharmaceutical compositions comprising at least one antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising a nucleic acid encoding an antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising cells expressing an antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable carrier.

[0177] Components of the composition

[0178] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions disclosed herein may also be administered in combination therapy 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 various combinations thereof.

[0179] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and / or propyl gallate. For example, compositions containing antibodies or antigen-binding fragments of the present disclosure may include one or more antioxidants, such as methionine, to reduce antibodies or antigen-binding fragments of the present disclosure that may be oxidized. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or antigen-binding fragments of the present disclosure and one or more antioxidants such as methionine. This disclosure further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or its antigen-binding fragment to extend its shelf life and / or increase its activity.

[0180] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution, isotonic dextran injection, sterile water 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 at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or dextran; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers. These antimicrobial agents include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Suitable excipients may include, for example, water, saline, dextran, 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, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0181] Application, formulation and dosage

[0182] The pharmaceutical compositions disclosed herein can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal administration, or otherwise via implantation or inhalation. The subject compositions can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0183] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers, and their controlled-release forms.

[0184] Formulations suitable for parenteral administration (e.g., by 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 delivered (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation 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 suitable isotonic carriers for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosing regimen, including dose, time, and repetition, will depend on the specific individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0185] The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such proliferative cells, reducing the proliferation of proliferative cells, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, a sustained-release formulation of the subject therapeutic composition may be suitable.

[0186] Those skilled in the art will understand that appropriate dosage can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit against any risks or harmful side effects. The chosen dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, route of administration, time of administration, rate of excretion of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and the patient's species, sex, age, weight, condition, general health, and medical history. The amount of compound and route of administration will ultimately be determined by a physician, veterinarian, or clinician, although a dosage is generally chosen to achieve a local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

[0187] Typically, the antibodies or antigen-binding portions thereof disclosed herein can be administered in a variety of ranges. These include about 5 μg / kg body weight to about 40 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; and about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. 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, and at least about 10 mg / kg body weight.

[0188] In summary, the antibody or its antigen-binding portion disclosed herein can preferably be administered according to the needs of the subject in need. The frequency of administration can be determined by those skilled in the art, such as attending physicians, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health status of the subject being treated.

[0189] In some preferred embodiments, treatment involving the antibody or its antigen-binding portion of this disclosure may comprise multiple doses of the selected pharmaceutical product over a period of weeks or months. More specifically, the antibody or its antigen-binding portion of this disclosure 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 should be understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0190] Dosage and regimen of the disclosed therapeutic composition may also be determined empirically in individuals who have been given one or more administrations. For example, individuals may be given incremental doses of the therapeutic composition prepared as described herein. In selected embodiments, the dosage may be gradually increased, decreased, or weakened based on empirically determined or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, symptom, or condition may be tracked as previously described. For cancer, these include direct measurement of tumor size via palpation or visual observation, indirect measurement of tumor size via X-ray or other imaging techniques; improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein; reduction of pain or paralysis; improvement in tumor-related speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life or prolonged survival as measured by recognized tests.

[0191] Compatible formulations for parenteral administration (e.g., intravenous injection) may comprise an antibody or antigen-binding portion thereof as disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. It will be apparent to those skilled in the art that the dosage of the antibody or antigen-binding portion thereof as disclosed herein can vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other sites in the individual, past and contemporaneous treatments being used, and the dosage of therapeutic agents used in combination with the antibody as disclosed herein.

[0192] Application of this disclosure

[0193] The antibodies, antibody compositions, and methods disclosed herein have numerous in vitro and in vivo uses, relating to, for example, the detection of TIGIT or the enhancement of immune responses. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects, to enhance immunity under various conditions. Immune responses can be modulated, for example, enhanced, stimulated, or upregulated.

[0194] 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 (e.g., T-cell-mediated immune responses). In certain embodiments, this method is particularly suitable for treating cancer in vivo. When the anti-TIGIT antibody is administered together with another agent, such as an anti-PD-1 agent, the two can be administered either sequentially or simultaneously.

[0195] This disclosure 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 and a control sample with an anti-TIGIT antibody or its antigen-binding portion, under conditions allowing the formation of a complex between the antibody or a portion thereof and TIGIT. The formation of the complex is then detected, wherein the 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 disclosure can be used to purify TIGIT via immunoaffinity purification.

[0196] Treatment of conditions including cancer

[0197] In some aspects, this disclosure provides methods for treating a symptom or disease in mammals, comprising administering to a subject (e.g., a human) a therapeutically effective amount of an anti-TIGIT antibody or its antigen-binding portion as disclosed herein, preferably in combination with a PD-1 / PD-L1 antagonist. Symptoms or diseases include, but are not limited to, proliferative conditions (such as cancer), immune conditions, inflammatory diseases, or infectious diseases. For example, a symptom could be cancer.

[0198] In some implementations, the cancer is cancer rich in CD112, CD113, or CD155 expression. In some implementations, the cancer is cancer rich in T cells or natural killer (NK) cells expressing TIGIT.

[0199] Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoma. More specific examples of such cancers 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 or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal carcinoma; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-refractory non-muscle-invasive bladder cancer (NMIBC)); urinary tract cancer; hepatocellular carcinoma; breast cancer (e.g., TIGIT+ breast cancer and stage III breast cancer). The following cancers are considered to be non-negative: TNBC (estrogen receptor-negative, progesterone receptor-negative, and TIGIT-negative); colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney or renal cell carcinoma (e.g., renal cell carcinoma (RCC)); prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial diffuse melanoma, malignant lentigines melanoma, acral lentigines melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; and bulky NHL. (bulky disease NHL); mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloid leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal angiogenesis, edema (e.g., edema associated with brain tumors), Megs syndrome, brain cancer, head and neck cancer, and related metastases associated with phakomatoses.

[0200] As a co-inhibitory receptor on various immune cells, TIGIT is involved in a variety of cancers, whether malignant or benign, primary or secondary, which can be treated or prevented by the methods provided in this disclosure. Preferably, the anti-TIGIT antibody disclosed herein is combined 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 such cancers include lung cancers such as bronchogenic carcinomas (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondroma-like hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyosarcoma; and bone cancers such as osteochondroma, chondroma, chondroblastoma, chondromycinous fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, and malignant fibrous tissue. Histiocytoma, Ewing's sarcoma, and reticulum cell 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; digestive system cancers such as colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, and smooth muscle cell carcinoma. Tumors, gastric adenocarcinoma, intestinal lipoma, enterofibroma, intestinal fibroma, colorectal polyps, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, adrenal adenoid tumor, 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; eye-related cancers such as retinoblastoma and intraocular melanoma; Cancers of the male reproductive system such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system such as uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary carcinoma, follicular carcinoma, undifferentiated carcinoma, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid gland; and pancreatic cancer. In a specific implementation, the cancer is colon cancer.

[0201] In some other embodiments, the condition or disease to be treated or prevented is an immune-related disease. Immune-related diseases may be associated with T-cell dysfunction. In some embodiments, T-cell dysfunction is characterized by a reduced responsiveness to antigen stimulation. In some embodiments, T-cell dysfunction is characterized by T-cell impotence, or a 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, immune-related diseases are selected from the group consisting of unresolved acute infections, chronic infections, and reduced tumor immunity.

[0202] Stimulation of immune response

[0203] In some aspects, this disclosure also provides methods for enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject an antibody of this disclosure or an antigen-binding portion thereof, such that an immune response in the subject is enhanced. For example, the subject is a mammal. In a particular embodiment, the subject is a human.

[0204] The term “enhanced immune response” or its grammatical variations refer to 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 cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+ helper T cell activity and the generation of cytolytic T cells. Enhancement of an immune response can be assessed using many 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. Generally, the methods of this disclosure enhance the immune response of mammals when compared to the immune response of untreated mammals or mammals not treated with methods as disclosed herein. In one embodiment, an 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, an antibody or its antigen-binding portion is used to enhance a human immune response to a vaccine. In one embodiment, the method enhances cellular immune responses, particularly cytotoxic T-cell responses. In another embodiment, the cellular immune response is a helper T-cell response. In yet another embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-2 production. Antibodies or their antigen-binding portions can be used to enhance human immune responses to microbial pathogens (such as viruses) or vaccines.

[0205] Antibodies or their antigen-binding portions can be used alone as a monotherapy or in combination with chemotherapy, radiotherapy, targeted therapy or cell immunotherapy.

[0206] Used in combination with chemotherapy

[0207] Antibodies or their antigen-binding portions can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0208] The terms "anticancer agent" or "antiproliferative agent" refer to any pharmaceutical agent that can be used to treat cell-proliferating diseases such as cancer, and include, but are not limited to, cytotoxic agents, cell growth inhibitors, anti-angiogenic agents, tumor-reducing agents, chemotherapeutic agents, radiotherapy and radiation therapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and antimetastatic agents, and immunotherapy agents. It should be understood that such anticancer agents may comprise conjugates and may be associated with the disclosed antibody prior to administration. More specifically, in some embodiments, selected anticancer agents may be linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate. Therefore, such engineered conjugates are explicitly considered within the scope of this disclosure. In some other embodiments, the anticancer agent may be administered in combination with an antibody-drug conjugate comprising a different therapeutic agent.

[0209] As used herein, the term "cytotoxic agent" means 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, the following small molecule toxins or enzyme-active toxins: bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcus enterotoxin A), fungi (e.g., α-ascorbic acid, restrictive mycotoxins), plants (e.g., absinthecin, ricin, modeccin, mistletoe lectin, pokeweed antiviral protein, saporin, white tree toxin, momoridin, trichosanthin, barley toxin, tung oil protein, dianthin protein, Phytolacca mericana protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, Jatropha curcas toxin, croton toxin, soapwort inhibitor, white tree toxin, mitegellin, localized trachomatis, phenolmycin, neomycin, and trichothecene toxins) or animals (e.g., cytotoxic ribonucleases, such as extracellular pancreatic ribonuclease; DNase). I, including its fragments and / or variants).

[0210] For the purposes of this disclosure, "chemotherapeutic agents" comprise chemical compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents typically target intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering 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). Such agents are often administered in combination and are usually most effective in combination, for example, in regimens such as CHOP or FOLFIRI.

[0211] Examples of anticancer agents that can be used in combination with the antibodies disclosed herein (either as components of site-specific conjugates or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, camptothecin, bryostatin, carrithione, CC-1065, cryptophycin, dolastatin, duocarmycin, eleutherobin, pancratistatin, and sphagnum moss. Sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromogens of enediyne antibiotics, aclacinomysins, actinomycins, atrazomycin, azaserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinomamycin, chromomycinis, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN ®Doxorubicin, Epirubicin, Isorubicin, Idarubicin, Marcellomycin, Mitomycin, Mycophenolic acid, Nogamycin, Oligomycin derivatives, Pepromycin, Potfiromycin, Puromycin, Quelamycin, Rodobicin, Streptozocin, Streptozocin, Tuberculin, Ubenimex, Zinostatin, Zorobacterium; Antimetabolites, Erlotinib, Vimurafenib, Crizotinib, Sorafenib, Ibrutinib, Enzalutamide, Folic acid analogs, Purine analogs, Androgens, Antiadrenergic agents, Folic acid supplements such as folinic acid, Acetaldehyde lactone, Aldophosphamide glycoside, Aminolevulinic acid acid), uracil, acridine, amustine, bisulfite, edatraxate, defofamine, colchicine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine compounds, mitoxantrone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, loxoantrone, podophyllin, 2-ethylhydrazide, procarbazine, PSK ® Polysaccharide complexes (JHS Natural Products, Eugene, OR), razorcinol; radicin; cizonan; spirogermanium; Alternaria alternifolia ketoacid; triaminoquinone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and serpentin); urethan; vindesin; dacarbazine; mannomustine; dibromomannitol; dibromoeusin; pipebroman; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chlorambucil; GEMZAR ®Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, vincristine; Platinum compounds; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Navelbine ® Vinorelbine; novantrone; teniposide; edaraxacum; donomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; compressoritine; leucovorin; oxaliplatin; PKC-α, Raf, H-Ras, EGFR and VEGF-A inhibitors that reduce cell proliferation; and any pharmaceutically acceptable salts, acids or derivatives thereof. This definition also includes anti-hormonal agents used to regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors (aromatase regulates estrogen production in the adrenal glands), and anti-androgens; as well as troxatabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and TIGIT expression inhibitors; vaccines, such as PROLEUKIN® rIL-2; and LURTOTECAN. ® Topoisomerase 1 inhibitor; ABARELIX ® rmRH; vinorelbine and esporomycin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0212] Used in combination with radiotherapy

[0213] This disclosure 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 radiation, X-rays, UV radiation, microwaves, electron emission, etc.). Combination therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed antibodies can be used in combination with targeted anticancer agents or other targeted modalities. Typically, radiotherapy is administered in pulses over a period of approximately 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for approximately 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0214] Drug packaging and reagent kits

[0215] Pharmaceutical packages and kits comprising one or more containers containing one or more doses of an antibody or its antigen-binding moiety are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding moiety, with or without one or more other agents. In other embodiments, such unit doses are supplied in disposable pre-filled syringes for injection. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, etc.; buffers, such as phosphates, etc.; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline 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 encapsulated antibody is intended for the treatment of selected aplastic disease conditions.

[0216] This disclosure also provides kits comprising single-dose or multi-dose administration units of antibodies 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, bottles, vials, syringes, etc. The container can be formed from various materials such as glass or plastic and contains a pharmaceutically effective amount of the disclosed 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 penetrated by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the antibody in a suitable container and optionally contain one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the antibodies or their antigen-binding portions disclosed herein, such kits may contain any one or more of a range of anticancer agents, such as chemotherapy or radiotherapy drugs; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents. In some embodiments, the kit may contain an antiPD-1 antibody.

[0217] More specifically, the kits may have a single container containing an antibody or its antigen-binding moiety, with or without additional components, or they may have different containers for each desired agent. In cases where combination therapies are provided for conjugation, the single solutions may be premixed in molar equivalents or in an excess of one component over the other. Alternatively, the antibody and any optional anticancer agent in the kit may be maintained separately in different containers prior to 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 dextran solution.

[0218] When the reagent kit components are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, and particularly preferably sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent.

[0219] As briefly noted above, the kit may also contain a device for administering the antibody or its antigen-binding portion and any optional components to a patient, such as one or more needles, IV bags, or syringes, or even an eye dropper, pipette, or other similar device from which the formulation may be injected or introduced into an animal or applied to a diseased area of ​​the body. The kits disclosed herein may also typically include a device for tightly enclosing vials and other components for commercial sale, such as an injection or blow-molded container, in which the desired vial and other equipment are placed and held.

[0220] Summary of sequence lists

[0221] This application includes a sequence listing containing numerous nucleic acid and amino acid sequences. Tables A, B, and C below provide a summary of the included sequences.

[0222] W3642-1.433.11 is a parental hybridoma clone, which is humanized to obtain the W3642-1.433.11-z11 clone and PTM is removed to obtain the W3642-1.433.11-p1 clone. 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 group; W3642-1.433.11-z11-p1-IgG4.SP and W3642-1.433.11-z11-p1-uIgG1L differ only in constant regions; W3642-1.433.11-p1-xIgG4.SP, W3642-1.433.11-p2-xIgG4.SP and W3642-1.433.11-p3-xIgG4.SP are antibodies that contain different substitutions in LCDR2 for PTM removal.

[0223] Table A: CDR sequences of antibodies / clones

[0224] 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 IDNO: 2) TLSHGSLNWFAS(SEQ ID NO:3) TLSSGNIENKYVH (SEQ ID NO: 4) NDDKRPD(SEQ ID NO: 5) HSYVSSINV(SEQID 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 IDNO: 2 SEQ IDNO: 3 SEQ IDNO: 4 <![CDATA[NDDKRP E (SEQ ID NO: 7, with D56E) SEQ IDNO: 6 W3642-1.433.11-p2-xIgG4.SP SEQ ID NO:1 SEQ IDNO: 2 SEQ IDNO: 3 SEQ IDNO: 4 <![CDATA[NDDKRP Q (SEQ ID NO: 8, with D56Q) SEQ IDNO: 6 W3642-1.433.11-p3-xIgG4.SP SEQ ID NO:1 SEQ IDNO: 2 SEQ IDNO: 3 SEQ IDNO: 4 <![CDATA[NDDKRP S (SEQ ID NO: 9, with D56S) SEQ IDNO: 6

[0225] Table B: Amino acid sequence of the variable region

[0226]

[0227]

[0228] Table C: Sequences of heavy and light chains

[0229]

[0230] Example

[0231] The present disclosure, as generally described above, will be more readily understood by referring to the following embodiments, which are provided by way of illustration and are not intended to limit the present disclosure. The embodiments are not intended to represent all or only the experiments conducted.

[0232] Example 1

[0233] Preparation of antigens, reference antibodies and cell lines

[0234] 1.1 Antigen generation

[0235] W364-hPro1.ECD.His is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal multihistidine tag; W364-hPro1.ECD.hFc is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal Fc region of human IgG1; W364-mPro1.ECD.His is the extracellular domain of mouse TIGIT (NP_001139797.1) with a C-terminal multihistidine tag; W364-mPro1.ECD.hFc is the extracellular domain of mouse TIGIT (NP_001139797.1) with a C-terminal Fc region of human IgG1; W364-hPro1L1.ECD.hFc is the extracellular domain of human CD155 (NP_006496.3) with a C-terminal Fc region of human IgG1. W364-hPro1L1.ECD.mFc is the extracellular domain of human CD155 (NP_006496.3) with the Fc region of mouse IgG1 at the C-terminus. These antigens are purchased from suppliers or prepared in-house.

[0236] 1.2 Preparation of the reference antibody (BMK)

[0237] The amino acid sequences of the variable domains encoding the anti-TIGIT reference antibodies WBP364-BMK1, WBP364-BMK4 and WBP364-BMK6 were synthesized based on the sequences disclosed in their respective patents, and their information is summarized in Table 1.

[0238] Table 1 Reference Antibody Information

[0239] Antibody code company Patent number Sequence ID WBP364-BMK1 Roche (Genentech) US20170088613 4.1D3 WBP364-BMK4 BMS (Ono) US20160176963 22G2 WBP364-BMK6 Astellas (Potenza) WO2017059095 MAB10

[0240] 1.3 Cell Pool / Cell Line Generation

[0241] A cell pool expressing human TIGIT, W364-293F.hPro1.pool, was generated using 293F cells transfected with full-length human TIGIT (NP_776160.2). A cell line expressing human TIGIT, W364-CHOK1.hPro1.2A11, was generated using CHOK1 cells transfected with full-length human TIGIT (NP_776160.2). A cell pool expressing cynomolgus monkey TIGIT, W364-FlpinCHO.cynoPro1.pool, was generated using FlpinCHO cells transfected with full-length cynomolgus monkey TIGIT (XP_015300911.1). A cell pool expressing mouse TIGIT, W364-FlpinCHO.mPro1.pool, was generated using FlpinCHO cells transfected with full-length mouse TIGIT (NP_001139797.1).

[0242] Example 2

[0243] Generation of chimeric and humanized antibodies

[0244] 2.1 Immunization

[0245] Two female SD rats, aged 6-8 weeks, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and housed in an IACUC-approved animal facility. The two animals were immunized alternately with W364-hPro1.ECD.His and W364-mPro1.ECD.His.

[0246] 2.2 Serum titer detection

[0247] 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 in 100 μL of coating buffer (Na2CO3 / NaHCO3, pH 9.2) at 1 μg / mL per well and incubated overnight at 4°C. On the day of assay, after blocking with 1xPBS / 2% BSA for 1 hour, diluted rat serum samples (first 1:100, then 3-fold diluted in 1xPBS / 2% BSA) and negative controls were added to the plates, which were then incubated at ambient temperature for 1 hour. After washing three times with 1xPBST (PBS containing 0.05% Tween-20), HRP-labeled goat anti-rat IgG Fc (Bethyl, catalog number A110-236P) was added and incubated at ambient temperature for 1 hour. After removing unbound substances, a TMB (3,3',5,5'-tetramethylbenzidine) matrix was added, and the reaction was terminated with 2M HCl. The absorbance at 450 nm was measured using a microplate spectrophotometer.

[0248] Serum titers of immunized SD rats are shown in Table 2. After a final booster immunization with W364-hPro1.ECD.His and W364-mPro1.ECD.His, both animals were euthanized and lymph nodes were collected for fusion.

[0249] Table 2 Serum titers of anti-TIGIT antibodies

[0250]

[0251] 2.3 Hybridoma formation

[0252] 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 electrocytometer according to an optimized electrofusion program. After fusion, cells were transferred to 96-well plates (1.2 x 10⁻⁶) containing DMEM medium supplemented with 20% FBS and 1% HAT selective reagent. 4 Cells / well. The plate was incubated at 37°C and 5% CO2, and monitored periodically. When the clones 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.

[0253] 2.4 Antibody screening and subcloning

[0254] The high-throughput screening process includes primary screening of cynomolgus monkey TIGIT binders using cell-based ELISA, secondary screening of human / cynomolgus monkey / mouse binders using cell-based FACS, and functional screening of TIGIT / PVR blockers using cell-based FACS.

[0255] Dilute the logarithmically growing positive cell lines to 200–300 cells per 1.5 mL of semi-solid HAT medium. Gently mix the cell suspension on a vortex mixer for 5–10 seconds, then seed into 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 containing DMEM medium supplemented with 10% fetal bovine serum. After 2–3 days, collect the supernatant from each clone and screen again to obtain single positive hybridoma clones.

[0256] 2.5 Hybridoma Sequencing

[0257] 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 SMARTRACE 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 gene of interest. The PCR product was inserted into the pMD18-T vector, and the ligation product was sent to GENEWIZ for sequencing.

[0258] Fifty positive cell lines were selected for subcloning through a combination of primary and secondary screening, as well as TIGIT / PVR blockade. After confirming the monoclonal antibody, 39 positive results were selected for sequencing, and three of these were subsequently converted to human IgG.

[0259] 2.6 Sequence Optimization

[0260] 2.6.1 Chimeric antibody production

[0261] The amino acid sequences of the VH and VL domains were codon-optimized for mammalian expression. The codon-optimized DNA sequences were synthesized using GENEWIZ and then subcloned into pcDNA expression vectors containing constant regions of human IgG1 or IgG4. 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.

[0262] The W3642-1.433.11 clone was identified, and its variable domain sequence is shown in Table B. A chimeric antibody containing a rat VH heavy chain fused with the human IgG4 constant region and a rat VL light chain fused with the human Igλ was named W3642-1.433.11-xIgG4.SP.

[0263] 2.6.2 PTM Removal

[0264] For the parental antibody W3642-1.433.11, the amino acid residue “DG” at the VLCDR2 boundary was identified as a potentially unstable residue with isomerization risk. Mutations were performed to remove the risk of post-translational modification (PTM), and SPR analysis was used to measure the binding kinetics of the antibody to human TIGIT. Mutations of the “DG” residue to EG, QG, or SG did not significantly alter the binding affinity. The results are shown in Table 3.

[0265] Table 3. Koff rate of PTM-removed variants binding to human TIGIT

[0266] Antibody VH domain VL domain <![CDATA[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

[0267] 2.6.3 Humanization of Rat Antibodies

[0268] Humanization of W3642-1.433.11 was primarily achieved through CDR transplantation. CDRs were identified according to the contact definition introduced by Dr. Andrew CR Martin's team (http: / / www.bioinf.org.uk / abs / ). The VH and VL sequences of W3642-1.433.11 were aligned (blast) against the human germline V gene database, and human IGVH and IGVL sequences with the highest homology to W3642-1.433.11 were selected as templates for humanization. The CDRs of the VH and VL domains of W3642-1.433.11 were transplanted into the framework of the humanization template to construct the germline VH and VL domain sequences.

[0269] Several "reverse mutation" sites were performed within the framework to convert amino acids in the germline sequence to their corresponding amino acids in the original rat sequence. We also generated 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.

[0270] To determine whether the humanized variant retained antigen-binding activity, surface plasmon resonance (SPR) was performed using a Biacore 8K instrument (Cytiva). Briefly, 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 captured the antibody from the supernatant of the pilot-scale transfection. Antigen W364-hPro1.ECD.His was injected at a flow rate of 100 μL / min into running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) for a 120 s binding phase, followed by a 900 s dissociation phase. The dissociation rate (koff) was calculated using a simple one-to-one Langmuir binding model. The results are shown in Table 4.

[0271] Table 4. Koff rate of antibody variants binding to human TIGIT

[0272] Antibody VH domain VL domain <![CDATA[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

[0273] Note: rVH = rat hybridoma sequence of variable heavy chain, rVL = rat hybridoma sequence of variable light chain, GH = humanized sequence of variable heavy chain, GL = humanized sequence of variable light chain.

[0274] 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 binding to 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.

[0275] Table 5: Affinity of humanized and PTM-removed variants to human TIGIT

[0276] Antibody VH domain VL 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

[0277] The corresponding IgG1 formatted antibody for W3642-1.433.11-z11-p1-IgG4.SP was generated and named W3642-1.433.11-z11-p1-uIgG1L, or simply W3642.

[0278] Example 3

[0279] In vitro characterization

[0280] 3.1 Human TIGIT Binding Assay

[0281] W364-293F.hPro1.pool(1x10 5 Cells (number of cells / well) were incubated with different concentrations of anti-TIGIT antibody at 4°C for 1 hour. After washing with 1xPBS / 1% BSA, secondary antibody, Alexa Fluor 647-labeled goat anti-human IgG (Jackson Immuno Research CAT #109-605-098), was added, and the cells were incubated 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 antibody was used as an isotype control. Cells were then washed and resuspended in 1xPBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0282] The binding results of W3642-1.433.11-z11-p1-uIgG1L to W364-293F.hPro1.pool cells are shown in the figure. Figure 1 The study demonstrated that W3642-1.433.11-z11-p1-uIgG1L can bind strongly to human TIGIT-expressing cells, and that the binding potency is equivalent to that of the reference antibody. A summary of antibody binding is shown in Table 7.

[0283] 3.2 TIGIT binding assay in cynomolgus monkeys

[0284] W364-FlpinCHO.cynoPro1.pool (1x10 5 Cells (number of cells / well) were incubated with different concentrations of anti-TIGIT antibody at 4°C for 1 hour. After washing with 1xPBS / 1% BSA, secondary antibody, Alexa Fluor 647-labeled goat anti-human IgG (Jackson Immuno Research CAT #109-605-098), was added, and the cells were incubated 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 antibody was used as an isotype control. Cells were then washed and resuspended in 1xPBS / 1% BSA. The MFI of cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0285] The binding results of W3642-1.433.11-z11-p1-uIgG1L to W364-FlpinCHO.cynoPro1.pool cells are shown in the figure. Figure 2 The study demonstrated that W3642-1.433.11-z11-p1-uIgG1L can bind strongly to TIGIT-expressing cells in cynomolgus monkeys, and that the binding potency is equivalent to that of the reference antibody. A summary of antibody binding is shown in Table 7.

[0286] 3.3 Mouse TIGIT Binding Assay

[0287] W364-FlpinCHO.mPro1.pool (1x10 5 Cells (cells / well) were incubated with different concentrations of anti-TIGIT antibody at 4°C for 1 hour. After washing with 1xPBS / 1% BSA, secondary antibody, Alexa Fluor 647-labeled goat anti-human IgG (Jackson Immuno Research CAT #109-605-098), was added, and the cells were incubated in the dark at 4°C for 1 hour. The anti-human TIGIT antibody WBP364-BMK6, known to have cross-reactivity with mouse TIGIT, was used as a positive control. A human IgG1 isotype antibody was used as an isotype control. Cells were then washed and resuspended in 1xPBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed using FlowJo.

[0288] The binding results of W3642-1.433.11-z11-p1-uIgG1L to the extracellular domain of mouse TIGTI cells are shown in... Figure 3The study demonstrated that W3642-1.433.11-z11-p1-uIgG1L strongly binds to mouse TIGIT-expressing cells, and its binding potency is higher than that of 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.

[0289] 3.4 Human TIGIT Affinity Determination

[0290] Affinity of anti-TIGIT antibody against recombinant human TIGIT was determined using a Biacore 8K instrument (Cytiva) via surface plasmon 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 antibody was captured by the goat anti-human IgG Fc antibody. For kinetic measurements, serial concentrations of W364-hPro1.ECD.His were injected at 25°C at a flow rate of 30 μL / min into running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) for the binding phase, followed by a dissociation phase. The binding rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Languir binding model. The equilibrium dissociation constant (kD) is calculated as the ratio koff / kon.

[0291] 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 2.39 E-11 M.

[0292] Table 6. Affinity constants of anti-TIGIT antibody and human TIGIT

[0293] 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

[0294] 3.5 Binding assay of TIGIT paralogous proteins

[0295] The plates were pre-coated overnight at 4°C with 50 μL of coating buffer per well containing 1 μg / mL of W364-hPro1.ECD.His, recombinant human CD28, CTLA-4, PD-1, ICOS, or PVRIG extracellular domains. After blocking with 200 μL of 1xPBS / 2% BSA, 50 μL of test antibody was added to the plates at concentrations of 1 nM, 0.1 nM, or 0.01 nM, and the plates were incubated at ambient temperature for 1 hour. After incubation, the plates were washed three times with 1xPBST. HRP-labeled goat anti-human IgG antibody (Bethyl catalog number A80-304P) diluted in 1xPBS / 2% BSA was added, and the plates were incubated 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 1xPBST, color development was achieved by dispensing 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. The absorbance at 450 nm was read using an M5e microplate reader (Molecule Devices).

[0296] The binding results of W3642-1.433.11-z11-p1-uIgG1L to TIGIT paralogous protein are shown in... Figure 4 In this study, it was demonstrated that W3642-1.433.11-z11-p1-uIgG1L specifically binds to TIGIT and has no cross-reactivity with human CD28, ICOS, PVRIG, PD-1, or CTLA-4.

[0297] 3.6 Human TIGIT / ligand blocking assay

[0298] The ability of anti-TIGIT antibodies to block human TIGIT / PVR interaction was tested using FACS. W364-CHOK1.hPro1.2A11 cells were washed with 1xPBS / 1% BSA and then... 5Cells were seeded per well in 96-well round-bottom plates. Excess buffer was removed from the wells by centrifugation. Serially diluted anti-TIGIT antibody (2x concentration) was premixed with 4 μg / mL (2x concentration) W364-hPro1L1.ECD.mFc at a 1:1 volume ratio, and 100 μL of the antibody / ligand mixture was added to each well. The plate was incubated at 4°C for 1 hour. After washing with 1xPBS / 1% BSA, PE-labeled goat anti-human IgG (Bethyl catalog number A90-239PE) was added, and the cells were incubated with the secondary antibody 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 antibody was used as an isotype control. Cells were then washed and resuspended in 1xPBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0299] The ability of the anti-TIGIT antibody to block the interaction between human TIGIT / CD112 and TIGIT / CD113 was also tested by FACS.

[0300] The results of human TIGIT and its ligands (CD155, CD112 and CD113) binding blockade are shown in Figure 5-7 The results showed that W3642-1.433.11-z11-p1-uIgG1L effectively blocked the binding of human CD155, CD112, and CD113 to TIGIT. The antibody blocking activity is summarized in Table 7. Ligand binding rate was calculated as binding% = MFI. 样品 / MFI max x 100%, of which MFI max Defined as MFI in the absence of antibodies.

[0301] 3.7 Jurkat TIGIT / NFAT-luciferase reporter gene assay (RGA)

[0302] Jurkat cells co-expressing human TIGIT and NFAT-luciferase reporter genes were stimulated via T cell receptor conjugation by co-culturing CHOK1 cells with co-expressing human PVR and TCR activators. CHOK1 / PVR / TCR activated cells were cultured at 2 x 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and incubated overnight at 37°C with 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 x 10⁻⁶ cells / well) were seeded. 4Cells per well were added to the plate and co-cultured at 37°C and 5% CO2 for 5–6 hours. After incubation, the reconstituted luciferase substrate (Promega catalog number E6130) was added to each well and mixed thoroughly. The luciferase intensity was read using an EnVision microplate reader (PerkinElmer).

[0303] The results of W3642-1.433.11-z11-p1-uIgG1L reversing the inhibition of NFAT signaling induced by TIGIT / PVR interaction in RGA are shown in... Figure 8 The study demonstrated that W3642-1.433.11-z11-p1-uIgG1L can enhance TCR / NFAT activation by inhibiting the TIGIT / PVR pathway. A summary of antibody RGA activity is shown in Table 7.

[0304] 3.8 Jurkat Functional Measurement

[0305] HT1080 is a human fibrosarcoma cell line that expresses human PVR, PVRL2, and PVRL3. Jurkat cells overexpressing human TIGIT are stimulated via conjugation of HT1080 cells overexpressing human TCR activator through T-cell receptor conjugation. 1 x 102 4 Jurkat / TIGIT cells with 5 x 10 3 HT1080 / TCR activated cells were co-cultured for 2 days at 37°C and 5% CO2 in the presence of serially diluted anti-TIGIT antibody. After incubation, the supernatant was collected for IL-2 measurement using 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).

[0306] Jurkat cells expressing TIGIT were co-cultured with HT1080 / TCR activated 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 Table 9. A summary of antibodies that promote IL-2 secretion is shown in Table 7.

[0307] Table 7 Summary of antibody characterization

[0308] Measurement 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 No combination No combination Blocking the binding of CD155 to TIGIT, IC50 (nM) 0.13 0.28 0.21 Blocking the binding of CD112 to TIGIT, IC50 (nM) 0.52 0.31 0.34 Blocking the binding of CD113 to TIGIT, IC50 (nM) 0.42 0.28 0.28 NFAT reporter gene assay, EC50 (nM) 2.5 3.0 2.1 IL-2 secretion in Jurkat cells, EC50 (nM) 0.50 ~2.58 ~0.77

[0309] 3.9 Human primary NK cell activation assay

[0310] HT1080 cells expressing human PVR, PVRL2, and PVRL3 were used as target cells, while primary human NK cells isolated from human PBMCs were used as effector cells. 1x10⁸ cells pre-loaded with EuTDA (PerkinElmer catalog number AD0116) were used as effector cells. 4 HT1080 cells and 1x10 4 Personal primary NK cells were co-cultured at 37°C and 5% CO2 for 2 hours in the presence of serially diluted anti-TIGIT antibody. Target cell lysis in the culture supernatant was then assessed by time-resolved fluorescence according to the manufacturer's instructions (PerkinElmer catalog number AD0116). Fluorescence was detected using an EnVision microplate reader (PerkinElmer).

[0311] Human NK cells were co-cultured with HT1080 cells in the presence of an antibody. The results demonstrated that W3642-1.433.11-z11-p1-uIgG1L dose-dependently enhanced NK cell cytotoxic activity. Data are shown in Table 10.

[0312] 3.10 Antibody-dependent cytotoxicity assay

[0313] To evaluate the ADCC effect of anti-TIGIT antibody on TIGIT-expressing cells, human TIGIT-engineered W364-CHOK1.hPro1.2 A11 cells were used as target cells, and primary human NK cells isolated from human PBMCs were used as effector cells. 1 x 10-1 4 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, and then 5 x 10 4 Freshly isolated human NK cells were added to each well. The plate was incubated at 37°C with 5% CO2 for 6 hours. Target cell lysis was determined using an LDH-based cytotoxicity assay kit (Roche catalog number 04744926001) according to the manufacturer's instructions. Absorbance was measured using an M5e microplate reader (Molecule Devices).

[0314] The results of the ADCC assay are shown in Table 11. The results show that W3642-1.433.11-z11-p1-uIgG1L can induce ADCC effects on TIGIT-expressing CHOK1 cells in a dose-dependent manner, and its potency is similar to that of the reference antibody.

[0315] 3.11 Antibody Serum Stability Assay

[0316] Fresh human serum was isolated from healthy donors. 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.

[0317] The binding of serum-incubated W3642-1.433.11-z11-p1-uIgG1L to W364-293F.hPro1.pool cells was shown to be... Figure 12 The antibody, after incubation with serum for up to two weeks, maintained similar binding activity and a very similar EC50 as the fresh antibody. Results demonstrated that W3642-1.433.11-z11-p1-uIgG1L was stable in human serum at 37°C for at least 2 weeks.

[0318] 3.12 Antibody thermal stability assay

[0319] 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 temperature of protein unfolding transition (Tm) based on changes in the fluorescence intensity of the environmentally sensitive dye SYPRO Orange.

[0320] DSF was performed on a Quant Studio 7 Flex Real-Time PCR instrument (Applied Biosystems) in its respective formulation buffer. SYPRO orange dye (Invitrogen catalog number S6651) was added to the antibody, and the mixture was transferred to a 96-well plate. The plate was placed in the Quant Studio. ® A 7-flex Real-Time PCR system was used, with temperatures ranging from 26°C to 95°C and a heating rate of 0.9°C / min. The first two temperatures for protein unfolding transitions were recorded as Tm1 and Tm2. These values ​​were obtained using QuantStudio. ® The Real Time PCR software (v1.3) calculates based on the melting curve.

[0321] The DSF thermogram of the W3642-1.433.11-z11-p1-uIgG1L antibody showed two transitions: the first with a lower melting temperature (Tm1) and the second with a higher melting temperature (Tm2), at 69.0°C and 79.0°C, respectively. The results are shown in Table 8.

[0322] Table 8. Tm values ​​of antibodies

[0323] Antibody Tm1(°C) Tm2 (°C) W3642-1.433.11-z11-p1-uIgG1L 69.0 79.0

[0324] Example 4

[0325] In vivo characterization

[0326] 4.1 In vivo efficacy of combination therapy with anti-TIGIT and anti-PD-1 antibody

[0327] To investigate the anti-TIGIT antibody's anti-tumor activity in an MC38 syngeneic mouse model, human TIGIT transgenic h-TIGIT C57BL / 6 mice (Jiangsu GemPharmatech Co., Ltd) were used for tumor cell inoculation. h-TIGIT C57BL / 6 mice were subcutaneously injected with wild-type MC38 tumor cells (1 x 10⁻⁶ cells) suspended in 0.1 mL DPBS under the right forelimb axilla. 6 This is used to monitor tumor development. When the average tumor size reaches approximately 73 mm... 3 At that time, tumor-bearing animals were randomly assigned to six study groups. Each group consisted of 8 mice. The study design is shown in Table 9. In several groups, a monoclonal anti-PD-1 antibody (“anti-mPD-1”, as disclosed in WO2018053709, clone ID 2E5) was used in combination with an anti-TIGIT antibody.

[0328] 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 the study were performed in accordance with guidelines approved by the International Association for the Care and Use of Animals (IACUC) of Shanghai Bio-model Institute and followed the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice were euthanized according to predefined health criteria, and the study was terminated 24 days after the first administration.

[0329] Table 9 Research Design of the MC38 Model

[0330]

[0331] 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. Tumor growth and body weight were closely monitored in all mice throughout the experiment, with tumor size measured and recorded twice weekly. Tumor growth inhibition (TGI) was calculated and analyzed at the optimal treatment time point (24 days after grouping, with the first dose administered on the same day as grouping). Tumor volume results are shown in... Figure 13The results are summarized in Tables 10 and 11. The results of weight change are shown in... Figure 14 The study also indicated that there were no abnormal weight changes in different groups.

[0332] Based on the results, W3642-1.433.11-z11-p1-uIgG1L alone showed a weak tumor-suppressive effect, but it enhanced the efficacy of the anti-mPD-1 antibody when used in combination. 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 suppression results from the start to the end of treatment.

[0333] Table 10 Summary of Tumor Volume

[0334]

[0335] Note: a, mean ± SEM.

[0336] Table 11 Summary of tumor volume inhibition

[0337]

[0338] Note:

[0339] a, mean ± SEM.

[0340] b. The mean tumor volume of the treatment group relative to the PBS group on day 24 after grouping was statistically analyzed by independent samples t-test.

[0341] Those skilled in the art will further understand that this disclosure may be implemented in other specific forms without departing from its spirit or core attributes. Since the foregoing description of this disclosure only discloses exemplary embodiments thereunder, it should be understood that other variations are considered within the scope of this disclosure. Therefore, the invention is not limited to the specific embodiments already described in detail herein. Rather, reference should be made to the appended claims as an indication of the scope and content of the invention.

Claims

1. An isolated antibody, or an antigen binding portion thereof, comprising: a heavy chain CDR (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1 ; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain CDR (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LCDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 7, 5, 8, and 9; and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

6.

2. The isolated antibody, or antigen binding portion thereof, of claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 10-11 ; or (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of SEQ ID NOs: 10-11 ; and the VL comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 12-18; or (ii) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 12-18.

3. The isolated antibody, or antigen binding portion thereof, of claim 1 or 2, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

15.

4. The isolated antibody, or antigen binding portion thereof, of any one of the preceding claims, wherein the isolated antibody further comprises a human IgG constant region.

5. The isolated antibody, or antigen binding portion thereof, of claim 4, wherein the human IgG constant region is a human IgG1, IgG4, IgG2, or IgG3 constant region or a variant thereof.

6. The isolated antibody, or antigen binding portion thereof, of any one of the preceding claims, wherein the antibody comprises a human IgG1 Fc region or a human IgG4 Fc region with a S228P substitution.

7. The isolated antibody, or antigen binding portion thereof, of any one of the preceding claims, wherein the antibody is a chimeric antibody or a humanized antibody.

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of the isolated antibody, or antigen binding portion thereof, of any one of claims 1-7.

9. A vector comprising the isolated nucleic acid molecule of claim 8.

10. A host cell comprising the vector of claim 9.

11. A pharmaceutical composition comprising an antibody, or antigen binding portion thereof, as defined in any one of claims 1-7, and a pharmaceutically acceptable carrier.

12. A method for producing an antibody, or antigen binding portion thereof, as defined in any one of claims 1-7, the method comprising the steps of: - culturing a host cell comprising an expression vector encoding the antibody or antigen binding portion thereof under suitable conditions; and - harvesting the antibody or antigen binding portion thereof from the cell culture.

13. A method for inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen binding portion thereof as defined in any one of claims 1-7 or a pharmaceutical composition of claim 11.

14. A method for treating or preventing a cancer or an immune-related disorder in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen binding portion thereof as defined in any one of claims 1-7 or a pharmaceutical composition of claim 11.

15. The method of claim 14, wherein the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, colorectal cancer, renal cancer, clear cell renal cancer, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, central nervous system neoplasm, mesothelioma, myeloma, and sarcoma.

16. The method of claim 14, wherein the immune-related disorder is a T cell dysfunctional disorder or an infection.

17. The method of any one of claims 14-16, wherein the method further comprises administering an additional therapeutic agent, such as an anti-PD-1 antibody.

18. A combination of an isolated antibody or antigen binding portion thereof as defined in any one of claims 1-7 and an anti-PD-1 antibody.

19. Use of an antibody or antigen binding portion thereof as defined in any one of claims 1-7, alone or in combination with an anti-PD-1 antibody, for the manufacture of a medicament for the treatment or prevention of a cancer or an immune-related disorder.

20. The use of claim 19, wherein the cancer is colon cancer or lung cancer.

21. An antibody or antigen binding portion thereof as defined in any one of claims 1-7 for use in the treatment or prevention of a cancer or an immune-related disorder.

22. A kit comprising a container containing an antibody or antigen binding portion thereof as defined in any one of claims 1-7.

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