Anti-TIGIT antibodies and their use

Abstract

This invention provides an anti-TIGIT antibody, a nucleic acid molecule encoding the anti-TIGIT antibody, an expression vector used for expressing the anti-TIGIT antibody, and host cells. It also provides a method for preventing and treating cancer and immune disorders by administering the anti-TIGIT antibody.

Description

[Technical Field]

[0001] <Cross reference> This application claims the benefits of the international patent application PCT / CN2023 / 098574, filed on June 6, 2023. The entire contents of said application are incorporated herein by reference.

[0002] <Sequence Listing> This application includes a sequence listing, the entire contents of which are incorporated herein by reference.

[0003] <Technical field> This application generally relates to antibodies. More specifically, it relates to a monoclonal antibody against TIGIT, a method for preparing the same, and the use of the said antibody. [Background technology]

[0004] In the tumor microenvironment, sustained antigen stimulation can lead to T cell exhaustion (a state of T cell dysfunction), and co-inhibitory receptors such as PD-1, LAG-3, TIM3, and TIGIT may be overexpressed. Currently, numerous strategies are being investigated to reactivate exhausted T cells using small molecules or therapeutic antibodies, either alone or in combination.

[0005] TIGIT (T cell immunoreceptor with Ig and ITIM domains), also known as Vstm3 and WUCAM, is a coinhibitory receptor expressed on NK and CD8+ T cells, as well as subsets of CD4+ T cells such as immunosuppressive regulatory T cells (Tregs). Four ligands of TIGIT are known, namely poliovirus receptor (PVR), PVRL2, PVRL3, and PVRL4, all of which are overexpressed in tumors and antigen-presenting cells and cause immunosuppression. These ligands also bind to the costimulatory molecule CD226 and the coinhibitory molecules PVRIG and CD96 (the latter may also be regarded as a costimulatory molecule). Antagonistic antibodies of TIGIT prevent TIGIT from binding to its ligands, block its inhibitory signal, and induce a strong antitumor immune response by predominating the CD226-mediated activation signal. Similar to 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. Furthermore, TIGIT has been identified as an important inhibitory receptor of stem cell-like memory T cells, a new T cell population that can be a favorable target for anti-PD-(L)1 efficacy.

[0006] TIGIT may be a promising therapeutic target in tumor immunotherapy when used alone or in combination with other immunomodulatory agents.

Summary of the Invention

Means for Solving the Problems

[0007] These and other objects are provided by the present disclosure, which, in a broad sense, relates to compounds, methods, compositions, and products that provide antibodies with improved efficacy. The advantages provided by the present disclosure are widely applicable in the fields of antibody pharmaceuticals and diagnostics and can be used in combination with other antibodies that react with various targets.

[0008] The present disclosure provides chimeric and humanized monoclonal antibodies against TIGIT. Further provided are methods for verifying the function of the antibodies in vitro and in vivo, and methods for treating a subject having cancer or an immune disorder by administering the anti-TIGIT antibodies disclosed herein alone or in combination with a PD-1 / PD-L1 antagonist.

[0009] In some aspects, the present disclosure provides an isolated antibody against TIGIT or an antigen-binding portion thereof. In some embodiments, the isolated antibody or antigen-binding portion thereof: comprises a heavy-chain CDR (HCDR) 1 having the amino acid sequence of SEQ ID NO: 1; an HCDR2 having the amino acid sequence of SEQ ID NO: 2; an HCDR3 having the amino acid sequence of SEQ ID NO: 3; a light-chain CDR (LCDR) 1 having the amino acid sequence of SEQ ID NO: 4; an LCDR2 having the amino acid sequence of any one of SEQ ID NOs: 7, 5, 8, and 9; and an LCDR3 having the amino acid sequence of SEQ ID NO: 6 and comprises.

[0010] In some embodiments, the isolated antibody or antigen-binding portion thereof comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), wherein the VH: (i) has the amino acid sequence shown in any one of SEQ ID NOs: 10-11; (ii) has an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of SEQ ID NOs: 10-11; or (iii) has an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions compared to any one of SEQ ID NOs: 10-11 and comprises or consists of, and / or wherein the VL: (i) has the amino acid sequence shown in any one of SEQ ID NOs: 12-18; (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to any of sequence numbers 12-18; or (iii) Amino acid sequences having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions compared to any of SEQ ID NOs. 12-18 It includes or consists of.

[0011] In some embodiments, the isolated antibody or its antigen-binding moiety includes HCDR1, HCDR2, and HCDR3 of the VH region shown in any of SEQ ID NOs: 10 to 11, and LCDR1, LCDR2, and LCDR3 of the VL region shown in any of SEQ ID NOs: 12 to 18.

[0012] In some embodiments, the isolated antibody or its antigen-binding portion includes: HCDR1 shown in SEQ ID NO: 1; HCDR2 shown in SEQ ID NO: 2; HCDR3 shown in SEQ ID NO: 3; LCDR1 shown in SEQ ID NO: 4; LCDR2 shown in SEQ ID NO: 7; and LCDR3 shown in SEQ ID NO: 6.

[0013] In some embodiments, the isolated antibody or its antigen-binding portion includes 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.

[0014] In some embodiments, the isolated antibody or its antigen-binding moiety further comprises a human IgG constant region, such as a human IgG1, IgG4, IgG2, or IgG3 constant region. The human IgG constant region may be native or a variant thereof. Specifically, the antibody may comprise a human IgG4 Fc region or a human IgG1 Fc region having an S228P substitution.

[0015] In some embodiments, the anti-TIGIT antibody disclosed herein is a mouse antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody herein is an anti-TIGIT antagonist antibody.

[0016] In some embodiments, the Disclosure provides isolated nucleic acid molecules comprising nucleic acid sequences encoding a heavy chain variable region and / or light chain variable region of an isolated antibody or its antigen-binding moiety disclosed herein. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 21 and / or the nucleic acid sequence shown in SEQ ID NO: 22.

[0017] In some embodiments, the present disclosure provides an expression vector comprising nucleic acid molecules disclosed herein.

[0018] In some embodiments, the present disclosure provides host cells comprising the expression vectors disclosed herein.

[0019] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody or an antigen-binding moiety thereof disclosed herein and a pharmaceutically acceptable carrier.

[0020] In some embodiments, the Disclosure provides a method for preparing the antibody or its antigen-binding moiety, comprising expressing the antibody or its antigen-binding moiety in a host cell and isolating the antibody or its antigen-binding moiety from the host cell. In some embodiments, the host cell is transfected or transformed with an expression vector encoding the heavy chain and light chain of the antibody disclosed herein. The nucleic acid sequence encoding the heavy chain and the nucleic acid sequence encoding the light chain may be in the same vector or in separate vectors.

[0021] In some embodiments, the Disclosure provides a method for modulating a TIGIT-related immune response in a subject, comprising administering an antibody or an antigen-binding moiety thereof disclosed herein to the subject.

[0022] In some embodiments, the present disclosure provides a method for inhibiting the proliferation of tumor cells in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding moiety thereof or a pharmaceutical composition disclosed herein, either alone or in combination with another anticancer agent such as an anti-PD-1 antibody.

[0023] In some embodiments, the Disclosure provides a method for treating or preventing cancer or an immune-related disorder in a subject, comprising administering an effective amount of an antibody or antigen-binding moiety disclosed herein, either alone or in combination with another anticancer agent, to the subject.

[0024] The above anticancer agent may be a chemotherapeutic agent, a monoclonal antibody, or an antibody-drug conjugate. In some embodiments, the above anticancer agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0025] The cancers listed above can be selected from colon cancer, lung cancer (NSCLC, etc.), 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, stomach 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 tumors, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, soft tissue cancer, and sarcoma. The immune-related disorders listed above may be T-cell dysfunction, infections, or inflammatory diseases.

[0026] In some embodiments, the antibodies or antigen-binding moieties disclosed herein are administered in combination with an anti-PD-1 antibody.

[0027] In some embodiments, the present disclosure provides combinations of an isolated antibody or its antigen-binding moiety disclosed herein with an anti-PD-1 antibody.

[0028] In some embodiments, the Disclosure provides the use of antibodies or their antigen-binding moieties disclosed herein, alone or in combination with other anticancer agents, in the manufacture of pharmaceuticals for treating or preventing diseases such as cancer and immunodeficiencies. In some embodiments, the anticancer agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0029] In some embodiments, the disclosure provides the use of antibodies or antigen-binding moieties disclosed herein in the manufacture of diagnostic agents for diagnosing diseases associated with TIGIT overexpression.

[0030] In some embodiments, this disclosure provides antibodies or antigen-binding moieties disclosed herein for use in the treatment or prevention of cancer and immunodeficiency. In some embodiments, the antibodies disclosed herein are used in combination with PD-1 / PD-L1 antagonists, such as PD-1 antibodies.

[0031] In some embodiments, the Disclosure provides a method for detecting the presence of a TIGIT antigen in a sample or for measuring the amount of a TIGIT antigen, the method comprising contacting the sample with an anti-TIGIT antibody or its antigen-binding moiety disclosed herein.

[0032] In some embodiments, the present disclosure provides a kit or apparatus comprising an antibody or its antigen-binding portion disclosed herein in one or more containers.

[0033] As the above is a summary, it naturally includes simplifications, generalizations, and omissions of details, and therefore, those skilled in the art will understand that this summary is merely illustrative and not intended to be limiting. This summary is not intended to identify the main 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. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 shows the binding of antibodies to human TIGIT as determined by FACS. [Figure 2] Figure 2 shows the binding of antibodies to cynomolgus monkey TIGIT as determined by FACS. [Figure 3] Figure 3 shows the binding of antibodies to mouse TIGIT as determined by FACS. [Figure 4] Figure 4 shows the binding of antibodies to the TIGIT paralogus protein as determined by ELISA. [Figure 5] Figure 5 shows the results determined by FACS, indicating that the antibody blocked the binding of CD155 to TIGIT. [Figure 6] Figure 6 shows the results determined by FACS, indicating that the antibody blocked the binding of CD112 to TIGIT. [Figure 7] Figure 7 shows the results determined by FACS, indicating that the antibody blocked the binding of CD113 to TIGIT. [Figure 8] Figure 8 shows the antibody results in the NFAT reporter gene assay. [Figure 9] Figure 9 shows the effect of antibodies on stimulating IL-2 release in Jurkat cells. [Figure 10] Figure 10 shows the effect of the antibody in the NK cell activation assay. [Figure 11] Figure 11 shows the effect of the antibody in the ADCC assay. [Figure 12] Figure 12 shows the stability of antibodies in human serum. [Figure 13] Figure 13 shows the results of tumor volume changes after antibody treatment in the MC38 xenograft study. [Figure 14] Figure 14 shows the results of weight changes after antibody treatment in the MC38 xenotransplantation study. [Modes for carrying out the invention]

[0035] While the present invention can be embodied in many different forms, this specification discloses specific exemplary embodiments illustrating the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated. Furthermore, section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0036] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" shall include multiple references unless otherwise explicitly indicated in the context. Thus, for example, a reference to "a protein" includes multiple proteins, and a reference to "a cell" includes a mixture of cells. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "comprising," as well as other forms such as "comprises" and "comprised," is not limited. Also, the scopes indicated herein and in the appended claims include both endpoints and all points between those endpoints.

[0037] In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein are well known and commonly used in the art. The methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art, unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout this specification. For example, Abbas et al., Cellular and Molecular Immunology, 6 thSee also WBSaunders Company (2010), Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000), Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002), Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998), and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature, experimental procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well known and commonly used in the art.

[0038] definition To better understand this disclosure, the following definitions and explanations of relevant terms are provided.

[0039] As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified into κ light chains and λ light chains. The heavy chains can be classified into μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region consisting of about 12 or more amino acids, and the heavy chain further contains a "D" region consisting of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1, C H 2, and C H 3). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The V H region and the V L region can further be divided into hypervariable regions (called complementarity-determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each V H and V L consists of three CDRs and four FRs in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L ) of each heavy chain / light chain pair form an antigen-binding site, respectively. The antibody can be of different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0040] In the context of this application, the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, which can be used interchangeably, refer to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen to which a full-length antibody specifically binds, and / or competes with the full-length antibody for binding to the same antigen. Generally, please refer to Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be prepared 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, diabodies, and polypeptides containing at least a portion of an antibody capable of conferring specific antigen-binding ability to the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., monoclonal anti-human TIGIT antibody as shown herein) by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and their specificity can be screened in the same manner as for screening intact antibodies.

[0041] As used herein, the terms “monoclonal antibody” or “mAb” refer to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

[0042] As used herein, the term “chimeric antibody” refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody. An exemplary chimeric antibody disclosed herein is W3642-1.433.11-xIgG4.SP, which comprises a heavy chain in which rat VH is fused to the human IgG4 constant region and a light chain in which rat VL is fused to the human Ig lambda.

[0043] As used herein, the term “humanized antibody” refers to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as rat or mouse, is grafted onto a human framework sequence. Further framework region modifications can be made within the human framework sequence. The humanized antibody will also optionally include an immunoglobulin constant region (e.g., Fc), typically at least a portion of the human immunoglobulin constant region. Exemplary humanized antibodies disclosed herein are W3642-1.433.11-z10-p1-IgG4.SP and W3642-1.433.11-z11-p1-IgG4.SP, which include a heavy chain in which human germline VH is fused to the human IgG4 constant region and a light chain in which human germline VL is fused to the human Ig lambda.

[0044] As used herein, the term "PTM" or "post-translational modification" refers to a process that occurs in 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, and then undergo PTM to become mature protein products. Examples of PTM processes include phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, and lipidization. It is preferable to remove potential PTM sites during antibody optimization to avoid structural and functional heterogeneity caused by the PTM process.

[0045] As used herein, the terms “TIGIT” or “T-cell immune receptor having Ig and ITIM domains” encompass all native TIGITs derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. The term encompasses untreated full-length TIGIT, the extracellular domain of TIGIT, and all forms of TIGIT resulting from processing in cells. The term also encompasses naturally occurring variants of TIGIT, such as splice variants and allele variants. An exemplary amino acid sequence of full-length human TIGIT is shown in SEQ ID NO: 23 (MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG).

[0046] As used herein, the term "PD-1 / PD-L1 antagonist" includes PD-L1 antagonists (such as anti-PD-L1 antibodies) that reduce, block, inhibit, inactivate, or interfere with signaling resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1 or B7-1), and PD-1 antagonists (such as anti-PD-1 antibodies) that reduce, block, inhibit, inactivate, or interfere with signaling resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2, etc.). In some embodiments, the PD-1 antagonist is nivolumab (MDX-1106) or pembrolizumab (formerly lambrolizumab (MK-3475)), MED1-0680, PDR001 (spartalizumab), REGN2810 (semiprimab), BGB-108, prorugolimab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, retifanlimab, spartalizumab, sasa Nlimab, Penplimab, CS1003, HLX10, SCT-I10A, SHR-1316, CS1001, Embafolimab, TQB2450, ZKAB001, LP-002, Zimbererimab, Valstilimab, Genolimuzumab, BI754091, Cetrelimab, YBL-006, BAT1306, HX008, CX-072, IMC-001, KL-A167, Budigalimab, AMG404, CX-188, JT X-4014, 609A, Sym021, LZM009, F520, SG001, APL-502, Kosiberimab, Rhodapolimab, GS-4224, INCB086550, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, MAX-10181, RC98, BION-004, AM0001, CB201, ENUM244C8, E This refers to, but is not limited to, anti-PD-1 antagonist antibodies selected from NUM388D4, AUNP-012, STI-1110, ADG104, AK-103, LBL-006, hAb21, AVA-004, PDL-GEX, INCB090244, KD036, KY1003, LYN192, MT-6035, VXM10, YBL-007, ABSK041, GB7003, JS-003, and HS-636.In some embodiments, the PD-L1 antagonist is an anti-PD-L1 antagonist antibody selected from MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab), but is not limited to these. PD-1 / PD-L1 antagonists include known antibodies and in-house developed antibodies.

[0047] In this specification, the term “binding affinity” is used as a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or its antigenic portion and an antigen. The binding affinity between two molecules can be quantified by determining the equilibrium dissociation constant (KD). KD can also be determined, in non-limiting examples, by measuring the reaction rates of complex formation and dissociation using surface plasmon resonance (SPR) (Biacore®). The rate constants corresponding to the binding and dissociation of a monovalent complex are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. The term ka (or kon) refers to the binding rate of an individual antibody-antigen interaction, and the term kd (or koff) refers to the dissociation rate of an individual antibody-antigen interaction. KD is related to ka and kd by the formula KD = kd / ka or koff / kon. The antibody binding reaction rate and binding affinity can be evaluated by standard assays known in the art or by methods described in the Examples section below.

[0048] As used herein, the term "high affinity" refers to K for the target antigen. D is 1 x 10 -9 M or less, more convenient 5×10 -10 M or less, more preferably 1 × 10 -10 M or less, more preferably 5 × 10 -11 This refers to antibodies with a M value of 0 or less.

[0049] The term "EC" as used herein 50 The term "effective concentration," also known as "50% effective concentration," refers to the concentration of a drug, antibody, or toxin that induces an intermediate response between baseline and maximum after a specific exposure period. In the context of this application, EC50 It is expressed in units of "nM" or "M".

[0050] As used herein, the term “isolated” refers to a state obtained from its natural state by artificial means. Where a particular “isolated” substance or component exists in nature, this is made possible by alteration of its natural environment, or by isolation of the substance from its natural environment, or both. For example, certain non-isolated polynucleotides or polypeptides exist naturally within certain animal organisms, and a high-purity version of the same polynucleotide or polypeptide isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term “isolated” does not exclude any artificial or synthetic substances or other impurities that do not affect the activity of the isolated substance.

[0051] As used herein, the term “isolated antibody” is intended to refer to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the TIGIT protein substantially does not contain antibodies that specifically bind to antigens other than the TIGIT protein). However, an isolated antibody that specifically binds to the human TIGIT protein may cross-react to other antigens, such as TIGIT proteins from other species. Also, an isolated antibody may substantially not contain other cellular material and / or chemical substances.

[0052] As used herein, the term “vector” refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it is capable of expressing a protein encoded by the inserted polynucleotide. A vector can express its genetic material elements in a host cell by transformation, transduction, or transfection of the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes, e.g., yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), phages such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but is not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may contain an origin of replication.

[0053] As used herein, the term “host cell” refers to a cell line that can be manipulated to produce the protein, protein fragment, or peptide of interest. Examples of host cells include, but are not limited to, cultured mammalian cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, and YB2 / 0; human tissue or hybridoma cells; yeast cells; insect cells; and cells contained within transgenic animals or cultured tissues. This term encompasses not only individual target cells but also their offspring. Such offspring may not be identical to their parent cells due to mutations or environmental influences, but they are still included within the scope of the term “host cell.”

[0054] 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, determined by aligning and comparing their sequences. “Identity percentage” means the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the smallest size of the compared molecules. For these calculations, any gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., “algorithm”). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides 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.

[0055] 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 properties of an antigen that stimulate specific immune cells to be activated, proliferate, and differentiate to ultimately produce immunological effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response in which antibodies or sensitized T lymphocytes are formed in the organism’s immune system after the organism has been stimulated by the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the production of an immune response in a host depends on three factors: the properties of the antigen, the host’s reactivity, and the means of immunization.

[0056] As used herein, the term “transfection” refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods, such as electroporation. Several 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, Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981, Gene 13:197. In a particular embodiment of the present invention, the human TIGIT gene was transfected into 293F cells.

[0057] As used herein, the terms “SPR” or “Surface Plasmon Resonance” refer to, and include, optical phenomena that enable real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix using systems such as the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, New Jersey). For further explanation, see Example 5 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26, Jonsson, U., et al. (1991) Biotechniques 11:620-627, Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131, and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0058] As used herein, the terms “fluorescence-activated cell sorting” or “FACS” refer to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of living cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell (FlowMetric. “Sorting Out Fluorescence Activated Cell Sorting”. Retrieved 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are generally commercially available. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, California), the Epics C from Coulter Epics Division (Hyalia, Florida), and the MoFlo from Cytomation (Colorado Springs, Colorado).

[0059] As used herein, the terms “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to a cytotoxic form in which secreted immunoglobulins (Ig) bound to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-carrying target cells and subsequently kill them with cytotoxicity. Antibodies “arm” the cytotoxic cells and are absolutely necessary for such killing. NK cells, the primary cells for mediating ADCC, 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 evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. 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 may be evaluated in vivo, for example, in animal models, such as those disclosed in Clynes et al. PNAS (USA), 95:652-656 (1998).

[0060] 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 mammalian species. This term does not necessarily indicate that the subject has been diagnosed with a specific disease and typically refers to an individual under medical supervision.

[0061] As used herein with reference to specific disease conditions in mammals, the terms “prevent,” “prevent,” or “preventing” mean preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms.

[0062] As used herein in the context of treating a condition, the terms “treatment,” “treating,” or “treated” generally refer to treatments and therapies, whether in humans or animals, that achieve any desired therapeutic effect, such as inhibiting the progression of a condition, including a reduction in the rate of progression, cessation of progression, regression of the condition, improvement of the condition, and cure of the condition. In the case of cancer, “treating” may mean weakening or slowing the growth, proliferation, or metastasis of a tumor or malignant cells, or any combination thereof.

[0063] As used herein, the term “effective dose” refers to the amount of an active compound or material, composition, or dosage form containing an active compound that is effective in producing some desired therapeutic effect commensurate with a reasonable benefit-to-risk ratio when administered according to a desired therapeutic regimen. For example, when used in connection with the treatment of a disease or condition, “effective dose” refers to an amount or concentration of antibody or its antigen-binding moiety that is effective in treating the disease or condition.

[0064] As used herein, the term “pharmaceutically acceptable” means that its vehicle, diluent, excipient, and / or salt are chemically and / or physically compatible with the other components in the formulation and physiologically compatible with the recipient.

[0065] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to 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.

[0066] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that can enhance the immune response to an antigen or change the type of immune response in an organism when delivered to the organism together with or pre-delivered to the organism with an antigen. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0067] Anti-TIGIT antibody In some embodiments, the disclosure provides an antibody or an antigen-binding portion or variant thereof that binds to TIGIT (such as human, mouse, or cynomolgus monkey TIGIT) with sufficient affinity to substantially or completely inhibit the biological activity of TIGIT.

[0068] In some embodiments, the anti-TIGIT antibodies disclosed herein are antibodies produced in rats immunized with the TIGIT antigen protein. In some embodiments, the anti-TIGIT antibodies disclosed herein are chimeric antibodies. In some embodiments, the anti-TIGIT antibodies disclosed herein are humanized antibodies. The antigen-binding moiety of the antibody may be Fab, Fab', F(ab')2, a single-chain variable fragment (scFv), or a diabody.

[0069] In some embodiments, humanized anti-TIGIT antibodies are provided herein in which the HCDR and LCDR sequences of human immunoglobulin are replaced with HCDR and LCDR sequences obtained from a non-human species (such as rat) having desired specificity, affinity, and / or capabilities. In some further embodiments, specific framework ("FR") residues of human immunoglobulin are reverse-mutated with corresponding non-human residues. In some other embodiments, the humanized antibody may contain residues not found in either the parental rat antibody or the human immunoglobulin. Further modifications to the CDR and framework residues can remove potential post-translational modifications and improve antibody performance, such as binding affinity.

[0070] Various methods for humanizing non-human antibodies are known in the art. For example, humanized antibodies may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called "import" residues and are typically obtained from "import" variable domains. Humanized antibodies that bind to TIGIT can be prepared 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. Patents 6,180,370, 6,054,927, 5,869,619, 5,861,155, 5,712,120, and 4,816,567).

[0071] The antibodies disclosed herein can bind with high affinity to at least one of human, mouse, and cynomolgus monkey TIGIT. The binding of the antibodies disclosed to TIGIT can be evaluated using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies disclosed can also be determined by monitoring the binding of the antibodies to cells expressing the TIGIT protein, such as by flow cytometry. In some embodiments, the antibodies are tested by a flow cytometry assay in which the antibodies are reacted with human TIGIT-expressing cell lines, such as HEK293 cells transfected to express TIGIT on the cell surface. Additionally or alternatively, the binding of the antibodies, such as the binding reaction rate (e.g., K), can be evaluated. D The value can be tested using a BIAcore binding assay.

[0072] In some embodiments, the antibody or its antigen-binding moiety, when measured by SPR, is 1 × 10⁻⁶ -9 M or less, 5×10 -10 M or less, 1×10 -10 M or less, 5×10-11 Less than M, 4×10 -11 Less than M, 3×10 -11 Less than M, 2.5×10 -11 Less than M, or 2×10 -11 K less than M D binds to human TIGIT. In some embodiments, the antibody or antigen-binding portion thereof can specifically bind to cynomolgus TIGIT and mouse TIGIT in addition to human TIGIT. For example, the antibody or antigen-binding portion thereof binds to human TIGIT-expressing cells with an EC50 of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, for example 0.23 nM or less as measured by FACS, and binds to cynomolgus TIGIT-expressing cells with an EC50 of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, for example 0.18 nM or less, and binds to mouse TIGIT-expressing cells with an EC50 of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, for example 0.33 nM or less.

[0073] 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, the anti-TIGIT antibody can block signal transduction via PVR, PVRL2, and / or PVRL3 to restore the functional responses (e.g., proliferation, cytokine production, killing of target cells) of T cells to antigen stimulation from a dysfunctional state.

[0074] In some embodiments, the anti-TIGIT antibodies provided herein inhibit the interaction between TIGIT and ligand CD155. In some embodiments, the anti-TIGIT antibodies provided herein inhibit the interaction between TIGIT and ligand CD112. In some embodiments, the anti-TIGIT antibodies provided herein inhibit the interaction between TIGIT and ligand CD113. In some embodiments, the anti-TIGIT antibodies provided herein inhibit the interaction between TIGIT and one or more of the ligands CD155, CD112, and CD113.

[0075] In some embodiments, the ability of an anti-TIGIT antibody to inhibit the interaction between TIGIT and CD155, CD112, or CD113 is evaluated 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. The assay may be carried out in various formats, including, but is not limited to, ELISA assays, flow cytometry, surface plasmon resonance (SPR) assays (e.g., Biacore®), or biolayer interferometry (e.g., ForteBio Octet®).

[0076] Anti-TIGIT antibodies containing CDR In some embodiments, the present disclosure provides an isolated antibody or its antigen-binding moiety, the antibody or its antigen-binding moiety is A) One or more heavy chain CDRs (HCDRs) selected from the group consisting of: A) HCDR1 containing an amino acid sequence different from SEQ ID NO: 1, or a sequence with two or fewer amino acids added, deleted, or substituted from SEQ ID NO: 1; HCDR2 containing an amino acid sequence different from SEQ ID NO: 2, or a sequence with two or fewer amino acids added, deleted, or substituted from SEQ ID NO: 2; and HCDR3 containing an amino acid sequence different from SEQ ID NO: 3, or a sequence with two or fewer amino acids added, deleted, or substituted from SEQ ID NO: 3; B) One or more light chain CDRs (LCDRs) selected from the group consisting of: LCDR1 containing an amino acid sequence different from SEQ ID NO: 4, or SEQ ID NO: 4 due to the addition, deletion, or substitution of two or fewer amino acids; LCDR2 containing an amino acid sequence different from any of SEQ ID NOs: 5, 7, 8, and 9, or any of SEQ ID NOs: 5, 7, 8, and 9 due to the addition, deletion, or substitution of two or fewer amino acids; and LCDR3 containing an amino acid sequence different from SEQ ID NO: 6, or SEQ ID NO: 6 due to the addition, deletion, or substitution of two or fewer amino acids; or C) One or more HCDRs from A) and one or more LCDRs from B) Includes.

[0077] In some embodiments, CDR identification follows the Contact definition introduced by the group of Dr. Andrew CRMartin (http: / / www.bioinf.org.uk / abs / ).

[0078] In some embodiments, the present disclosure provides an isolated antibody or its antigen-binding moiety comprising HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in any of SEQ ID NOs. 5, 7, 8, and 9, and LCDR3 shown in SEQ ID NO: 6.

[0079] The framework area and the scope of the CDR can be precisely identified using methods known in the art, for example, by definitions of Kabat, Chothia, AbM, Contact, IMGT (all well known in the art), and any combination thereof. For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877, Chothia, C. et al. al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Antibodies”, chapter 5, 2007. Furthermore, see hgmp.mrc.ac.uk and bioinf.org.uk / abs. Correspondence or alignment between numberings based on 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 Comp Immunol. (2003) 27:55-77).

[0080] As those skilled in the art will understand, the exact numbering and arrangement of CDRs may differ depending on the numbering system. However, regardless of the numbering method employed, it should be understood that the disclosure of a variable heavy chain sequence and / or variable light chain sequence includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each variable region is a disclosure of the CDRs (e.g., HCDR1, HCDR2, and HCDR3). The fact that two antibodies have the same VH and VL means that their CDRs are identical when determined by the same method (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering methods known in the art). Even the same antibody disclosed herein may have different CDR sets when determined by different numbering methods.

[0081] Variable regions and CDRs in antibody sequences can be identified according to common rules developed in the art (e.g., the numbering systems of Kabat, AbM, Chothia, Contact, and IMGT) or by aligning the sequence against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are available on the "Abysis" website (www.bioinf.org.uk / abs, maintained by AC Martin of the Department of Biochemistry and Molecular Biology, University College London, UK) and the VBASE2 website (www.vbase2.org, described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005)), and can be accessed through these sites. Sequences may also be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB. See the chapter "Protein Sequence and Structure Analysis of Antibody Variable Domains" in the Antibody Engineering Lab Manual by Dr. Andrew CRMartin (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs). The Abysis database website further includes general rules developed for identifying CDRs, which can be used in accordance with the teachings herein.

[0082] In some embodiments, the anti-TIGIT antibody disclosed herein comprises a VH region and a VL region, where the VH region comprises FRW1-HCDR1-FRW2-HCDR2-FRW3-HCDR3-FRW4 (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 the VL region comprises FRW1-LCDR1-FRW2-LCDR2-FRW3-LCDR3-FRW4 (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).

[0083] In some embodiments, the framework region is derived from human germline, such as human immunoglobulin (i.e., is human germline). In some embodiments, specific residues within the human germline framework region are reverse-mutated with corresponding residues in the parental non-human antibody. In some embodiments, the FR region may include substitutions of one or more individual FR residues that improve antibody performance, such as binding affinity, isomerization, and immunogenicity. In some specific embodiments, FRW3 of the human germline VH region contains Val and Trp at positions 78 and 94 (according to Kabat numbering). In some specific embodiments, FRW1 of the human germline VL region contains one or more of Gln at position 1, Ala at position 2, and Val at position 3 (according to Kabat numbering). The Kabat numbering system is typically used when referring to residues within the variable region (generally light chain residues 1-107 and heavy chain residues 1-113) (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-terminus and C-terminus of the VH and / or VL regions may be cleaved to contain only partial FRW1 and / or partial FRW4. In some embodiments, the CDR and FR regions are PTM removal optimized.

[0084] In some embodiments, anti-TIGIT antibodies 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 are provided herein. In some embodiments, anti-TIGIT antibodies 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 are provided herein. In some embodiments, anti-TIGIT antibodies 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 are provided herein. In some embodiments, anti-TIGIT antibodies 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 are provided herein.

[0085] In some embodiments, anti-TIGIT antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 10 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 12. In some embodiments, anti-TIGIT antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 11 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 13. In some embodiments, anti-TIGIT antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 10 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 14. In some embodiments, anti-TIGIT antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 11 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 15. In some embodiments, anti-TIGIT antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 11 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 16. In some embodiments, anti-TIGIT antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 10 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 17. In some embodiments, anti-TIGIT antibodies are provided herein that include at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 10 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 18.

[0086] In some specific embodiments, the VH region contains the amino acid Val(V) at position 78 and Trp(W) at position 94. Additionally or alternatively, the VL region contains the amino acid "QAV" (Gln-Ala-Val) at positions 1-3.

[0087] Anti-TIGIT antibody containing heavy chain variable region and light chain variable region In some embodiments, the isolated antibody or its antigen-binding moiety includes a heavy chain variable region (VH) and a light chain variable region (VL). VH is: (i) One amino acid sequence from sequence numbers 10-11; (ii) an amino acid sequence having the same CDR set as one of sequence numbers 10-11 and having at least 85%, 90%, or 95% identity in the framework region; or (iii) Amino acid sequences having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acid additions, deletions, and / or substitutions in the framework region compared to one of the amino acid sequences of sequence numbers 10-11. including and / or, VL is: (i) One amino acid sequence from sequence numbers 12-18; (ii) an amino acid sequence having the same CDR set as one of sequence numbers 12-18 and having at least 85%, 90%, or 95% identity in the framework region; or (iii) Amino acid sequences having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acid additions, deletions, and / or substitutions in the framework region compared to one of the amino acid sequences of sequence numbers 12-18. Includes.

[0088] The percentage of identity between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput.Appl.Biosci.,4:11-17(1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J.Mol.Biol.48:444-453(1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either the Blossum62 matrix or the PAM250 matrix, and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6.

[0089] Additionally or alternatively, the protein sequences of this disclosure can be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) described in Altschul, et al. (1990) J.MoI. Biol. 215:403-10. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. To obtain gapped alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST program and the Gapped BLAST program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0090] In some further embodiments, the isolated antibody or its antigen-binding moiety may include conservative amino acid substitutions or modifications in the variable regions of the heavy and / or light chains. In the art, it is understood that certain conservative sequence modifications can be made that do not remove antigen binding. For example, see Brummell et al. (1993) Biochem 32:1180-8, de Wildt et al. (1997) Prot.Eng.10:835-41, Komissarov et al. (1997) J.Biol.Chem.272:26864-26870, Hall et al. (1992) J.Immunol.149:1605-12, Kelley and O' Connell (1993) Biochem.32:6862-35, Adib-Conquy et al. (1998) Int.Immunol.10:341-6, and Beers et al. (2000) Clin.Can.Res.6:2835-43.

[0091] As used herein, the term “conservative substitution” refers to an amino acid substitution that will not adversely affect or alter the essential properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which one 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)). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with alkaline side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charged 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 with 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) (these are incorporated herein by reference)).

[0092] In certain embodiments, the isolated antibody or its antigen-binding moiety includes a heavy chain variable region containing or comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region containing or comprising the amino acid sequence of SEQ ID NO: 15.

[0093] In other embodiments, the amino acid sequences of the heavy chain variable region and / or 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 respective sequences shown above.

[0094] Anti-TIGIT antibodies with specific properties The antibodies of this disclosure are characterized by their specific functional features or properties. Based on the mechanism of action against the target, the in vitro functional properties and pharmacological activity of the antibodies were thoroughly evaluated at both the molecular and cellular levels. In some embodiments, the isolated antibody or its antigen-binding moiety has one or more of the following properties: (a) Specifically binds to at least one of the following: human TIGIT protein, cynomolgus monkey TIGIT protein, and mouse TIGIT protein, and has a K content of less than 1 nM as determined by, for example, SPR. D It can bind to human TIGIT; (b) Does not cross-bind with TIGIT homologous proteins; (c) Block the binding of TIGIT to its ligands CD155, CD112, and CD113; (d) Activate immune cells such as T cells and NK cells; (e) Induce the ADCC effect on TIGIT-expressing CHOK1 cells; and (f) When confirmed in an in vivo mouse model, when combined with an anti-PD-1 agent, it shows significantly superior efficacy in cancer treatment compared to the benchmark antibody.

[0095] The dual blocking of TIGIT and PD-1 can reverse immunosuppression. As shown herein, the anti-TIGIT antibodies of this disclosure exhibit synergistic effects with anti-PD-1 agents (e.g., anti-PD-1 antibodies) or anti-PD-L1 agents (e.g., anti-PD-L1 antibodies).

[0096] In some preferred embodiments, the antibodies of this disclosure may be used in combination with additional therapeutic agents, such as anticancer agents, including anticancer antibodies and chemotherapeutic agents. The additional therapeutic agents may be antagonists or inhibitors of T cell co-suppressive molecules, agonists of T cell co-activating molecules, or immunostimulatory cytokines.

[0097] In some embodiments, the additional therapeutic agent is an antibody that binds to a protein selected from CD25, PD-1, PD-L1, Tim3, Lag3, CTLA4, 41BB, 0X40, CD3, CD40, CD47M, GM-CSF, CSF1R, TLR, STING, RIGI, TAM receptor kinase, NKG2A, NKG2D, GD2, TIGIT, EGFR, PDGFRa, SLAMF7, VEGF, CTLA-4, CD20, cCLB8, KIR, and CD52. In some embodiments, additional therapeutic agents are selected from anti-CD25 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-Tim3 antibody, anti-Lag3 antibody, anti-CTLA4 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-CD3 antibody, anti-CD40 antibody, anti-CD47M antibody, anti-CSF1R antibody, anti-TLR antibody, anti-STING antibody, anti-RIGI antibody, anti-TAM receptor kinase antibody, anti-NKG2A antibody, anti-NKG2D antibody, anti-GD2 antibody, anti-EGFR antibody, anti-PDGFR-a antibody, anti-SLAMF7 antibody, anti-VEGF antibody, anti-CTLA-4 antibody, anti-CD20 antibody, anti-cCLB8 antibody, anti-KIR antibody, and anti-CD52 antibody. In some embodiments, additional therapeutic agents are selected from SEA-CD40, avelumab, durvalumab, nivolumab, pembrolizumab, pizilizumab, atezolizumab, Hul4.18K322A, Hu3F8, dinituximab, trastuzumab, cetuximab, olaratumab, necitumumab, elotuzumab, ramucirumab, pertuzumab, ipilimumab, bevacizumab, rituximab, obinutuzumab, siltuximab, ofatumumab, lirirumab, and alemtuzumab.

[0098] Fc area The anti-TIGIT antibodies and antigen-binding moieties provided herein further comprise an immunoglobulin constant region including an Fc region such as a human IgG1, IgG2, IgG3, or IgG4 Fc region (natural type or 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, or at least about 90% homology, e.g., at least about 95% homology, with the natural sequence Fc region. 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 certain embodiments, the anti-TIGIT antibodies disclosed herein comprise a wild-type human IgG1 Fc region. The variant Fc region may comprise one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA) that alter antibody-dependent cell-mediated cytotoxicity (ADCC) or other effector function. In some embodiments, the Fc region may include changes in one or more amino acids (e.g., insertions, deletions, or substitutions) that result in a modified Fc region in which the binding interaction between Fc and FcRn or FcγR is altered.

[0099] In certain embodiments, the Fc region is an IgG4 Fc region containing the S228P mutation (according to EU numbering, as in Kabat et al.) which prevents Fab arm exchange and stabilizes the IgG4 molecule. In certain embodiments, the Fc region is an IgG1 Fc region containing the LALA mutation, i.e., the L234A and L235A mutations. The LALA mutation is perhaps the most commonly used mutation to interfere with antibody effector function, for example, by eliminating Fc binding to specific FcγR and reducing PBMC and monocyte-mediated ADCC activity. The “EU numbering system” or “EU index” is generally used to refer to residues within the constant domain of the immunoglobulin heavy chain (e.g., the EU index reported by Kabat et al. above). “Kabat-like EU numbering” or “Kabat-like EU index” refers to the residue numbering of a human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0100] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals (e.g., XenoMouse®), or any combination thereof. For example, monoclonal antibodies can be produced using hybridomas and biochemical and genetic engineering techniques recognized in the art, such as 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 stThe details are described in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988, and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, 563-681 (Elsevier, NY, 1981), and each of these references is incorporated herein by reference in its entirety. The selected binding sequences can be further modified, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, or to produce multispecific antibodies, and it should be understood that 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. The preparation of hybridomas is well known in the art. For example, see Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0101] Nucleic acid molecules encoding antibodies in this disclosure In some embodiments, 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 disclosed herein.

[0102] The nucleic acids of this disclosure can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice possessing human immunoglobulin genes), the cDNA encoding the light and heavy chains of the antibodies produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0103] Isolated nucleic acids encoding the VH region can be converted into full-length heavy chain genes by operably ligating 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) cited above), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant regions may be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but more preferably IgG1 or IgG4 constant regions.

[0104] Isolated nucleic acids encoding the VL region can be converted into full-length light chain genes (and Fab light chain genes) by operably ligating 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. cited above), and DNA fragments containing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa constant region or a lambda constant region.

[0105] Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques to convert, for example, a variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operably ligated to another DNA fragment encoding another protein, such as an antibody constant region or a mobile linker. The term "operably ligated" as used in this context is intended to mean that the two DNA fragments are ligated in such a way that the amino acid sequences encoded by these two DNA fragments remain in frame.

[0106] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding the heavy chain variable region of an isolated antibody disclosed herein.

[0107] In some specific embodiments, the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated antibody and includes a nucleic acid sequence selected from the group consisting of: (A) A nucleic acid sequence encoding the heavy chain variable region shown in any one of sequence numbers 10-11; (B) Nucleic acid sequence shown in Sequence ID No. 21; or (C) A nucleic acid sequence hybridized to the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions.

[0108] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding the light chain variable region of an isolated antibody disclosed herein.

[0109] In some specific embodiments, the isolated nucleic acid molecule encodes the light chain variable region of the isolated antibody and includes a nucleic acid sequence selected from the group consisting of: (A) A nucleic acid sequence encoding the light chain variable region shown in any one of sequence numbers 12-18; (B) Nucleic acid sequence shown in Sequence ID No. 22; or (C) A nucleic acid sequence hybridized to the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions.

[0110] For example, the nucleic acid molecule includes SEQ ID NOs: 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 NOs: 21 or 22. In some specific embodiments, the identity percentage is based on the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0111] Exemplary high-stringency conditions include hybridization at 45°C in 5×SSPE and 45% formamide, and final washing at 65°C in 0.1×SSC. In the art, it is understood that equivalent stringency conditions can be achieved by varying the temperature and buffer or salt concentration, as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3~6.4.10. Modification of hybridization conditions can be determined empirically or precisely calculated based on probe length and guanosine / cytosine (GC) base pairing ratio. Hybridization conditions can be calculated as described in Sambrook, et al. (Eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47~9.51.

[0112] host cell The host cells disclosed herein may be any cells suitable for expressing the antibodies disclosed herein, such as yeast, bacteria, fungi, plants, and animal cells, and preferably mammalian cells. Examples of mammalian host cells for expressing the antibodies disclosed herein include Chinese hamster ovary (CHO cells) (e.g., dhfr CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with a DHFR selection marker as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621, etc.), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. In particular, when used in conjunction with NSO myeloma cells, other expression systems include the GS gene expression system disclosed in International Publication No. 87 / 04462, International Publication No. 89 / 01036, and European Patent No. 338,841.Additionally, the following cells were used: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL1651), human fetal kidney cell line (293 or 293 cells subcloned for proliferation in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), and human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB8065), mouse mammary tumor cells (MMT060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68), MRC5 cells, FS4 cells, mouse myeloma cells, e.g., NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC Examples include CRL1581), rat myeloma cells, e.g., YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL1662), PER.C6 cells, and human hepatocellular carcinoma cell lines (Hep G2). CHO cells are one of the cell lines that can be used herein, with CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555(1986)), and Lec13 being exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44, or CHO-DP12 host cells, the ability to fucosylate expressed proteins may be modified to be deficient.In some embodiments, the host cells herein are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, NSO cells, and lymphocytes.

[0113] Suitable prokaryotes for this purpose include bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as the genera Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as Bacillus subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0114] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains, such as Schizosaccharomyces pombe, Clibellomyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotlerans, and K. marxianus; Yarrowia (European Patent No. 402,226); Pichia pastris (European Patent No. 183,070); Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa; Schwanniomyces, for example, Schwanniomyces Hosts of *Occidentalis* and filamentous fungi, such as *Nepeta crassa*, *Penicolus*, *Tripocladium*, and *Aspergillus*, such as *A. nidulans* and *A. niger*, are commonly available and useful herein.

[0115] 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 sufficient to be expressed in the host cells or secreted into the culture medium in which the host cells are cultured. The antibody can be recovered from the culture medium using standard protein purification methods.

[0116] Pharmaceutical composition In some embodiments, this disclosure relates to a pharmaceutical composition comprising at least one antibody or antigen-binding moiety disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a pharmaceutical composition comprising a nucleic acid encoding an antibody or antigen-binding moiety disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a pharmaceutical composition comprising cells expressing an antibody or antigen-binding moiety disclosed herein and a pharmaceutically acceptable carrier.

[0117] Composition components The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of this disclosure may also be administered in combination therapy with another immunostimulant, anticancer agent, antiviral agent, or vaccine. Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspension / dispersant agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, and various combinations thereof.

[0118] Suitable components include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl gallate. For example, a composition comprising an antibody or antigen-binding fragment of the present disclosure may contain one or more antioxidants (such as methionine) to suppress the oxidation of the antibody or its antigen-binding fragment. Suppression of oxidation can prevent or reduce a decrease in binding affinity, thereby increasing antibody stability and extending shelf life. Accordingly, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants (such as methionine). The disclosure further provides various methods by which an antibody or its antigen-binding fragment is mixed with one or more antioxidants (such as methionine) to prevent oxidation of the antibody or its antigen-binding fragment, thereby extending their shelf life and / or increasing their activity.

[0119] Further examples of pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as plant-derived fixative oils such as cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate buffer or citrate buffer; antioxidants such as sodium bisulfate; and local anesthetics. Examples of pharmaceuticals include procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN®-80), metal ion sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers, including phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride, may be added to the pharmaceutical composition in a multi-dose container. Suitable excipients include, for example, water, physiological saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or pharmaceuticals such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0120] Administration, formulation, and dosage The pharmaceutical compositions of this disclosure may be administered in vivo to the target subject as needed by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The compositions may be formulated in the form of solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols, but not limited to these. The appropriate formulation and route of administration may be selected according to the intended use and treatment regimen.

[0121] Formulations suitable for intravenous administration include hard or soft gelatin capsules, pills, coated tablets, elixirs, suspensions, syrups, or inhalants, as well as controlled-release forms thereof.

[0122] 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 provided (e.g., in liposomes or other microparticles). Such liquids may further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the recipient's blood (or other relevant body fluids). Examples of excipients include, for example, water, alcohol, polyols, glycerol, and vegetable oils. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosing regimens, including dosage, timing, and repetition, will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0123] The frequency of administration may be determined and adjusted throughout the course of treatment, based on reducing the number of proliferative or tumorigenic cells, maintaining a reduction in such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dose administered may be adjusted or attenuated to manage potential side effects and / or toxicity. Alternatively, a sustained-release formulation of the therapeutic composition in question may be appropriate.

[0124] Those skilled in the art will understand that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit with any risks or adverse side effects. The dosage level to be selected will depend on various factors, including, but are not limited to, the activity of the specific compound, the route of administration, the timing of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the patient's species, sex, age, weight, condition, overall health, and medical history. The dosage is generally selected to achieve a local concentration at the site of action that produces the desired effect without causing significant adverse side effects, but the amount of compound and the route of administration are ultimately at the discretion of the physician, veterinarian, or clinician.

[0125] In general, the antibody or its antigen-binding moiety of this disclosure can be administered in a variety of doses. These include approximately 5 μg / kg body weight to approximately 40 mg / kg body weight per dose, approximately 50 μg / kg body weight to approximately 5 mg / kg body weight per dose, and approximately 100 μg / kg body weight to approximately 10 mg / kg body weight per dose. Other ranges include approximately 100 μg / kg body weight to approximately 20 mg / kg body weight per dose and approximately 0.5 mg / kg body weight to approximately 20 mg / kg body weight per dose. In certain embodiments, the dose is at least approximately 100 μg / kg body weight, at least approximately 250 μg / kg body weight, at least approximately 750 μg / kg body weight, at least approximately 3 mg / kg body weight, at least approximately 5 mg / kg body weight, and at least approximately 10 mg / kg body weight.

[0126] In any case, the antibody or its antigen-binding portion of this disclosure is preferably administered as needed to the target. The frequency of administration can be determined by a person skilled in the art, such as the attending physician, taking into consideration the condition being treated, the age of the target, the severity of the condition, and the general health status of the target.

[0127] In certain preferred embodiments, a therapeutic course comprising the antibody or its antigen-binding moiety of the Disclosure would involve multiple administrations of a selected formulation over a period of several weeks or months. More specifically, the antibody or its antigen-binding moiety of the Disclosure may be administered once daily, every two days, every four days, once a week, every ten days, every two weeks, every three weeks, once a month, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be understood that the dosage may be changed or the intervals adjusted based on the patient's response and clinical practice.

[0128] Dosages and regimens may also be empirically determined for the therapeutic compositions of the Disclosure in individuals receiving one or more doses. For example, an individual may be given an escalating dose of the therapeutic composition prepared as described herein. In selected embodiments, doses may be gradually increased, decreased, or attenuated based on empirically determined or observed side effects or toxicity. To evaluate the efficacy of the selected composition, markers of specific diseases, disorders, or conditions may be tracked as described above. In the case of cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques, improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples, measurement of indirect tumor markers (e.g., PSA in prostate cancer) or oncoplastic antigens identified according to the methods described herein, reduction of pain or paralysis, improvement of speech, vision, respiration, or other impairments related to the tumor, increased appetite, or improvement in quality of life as measured by approved tests, or extension of survival.

[0129] Suitable formulations for parenteral administration (e.g., intravenous injection) may contain the antibody or its antigen-binding moiety disclosed herein at concentrations ranging from approximately 10 μg / ml to approximately 100 mg / ml. It will be apparent to those skilled in the art that the dosage of the antibody or its antigen-binding moiety disclosed herein will vary depending on the individual, the type of neoplasm, the stage of the neoplasm, whether the neoplasm has begun to spread to other parts of the individual, past and concomitant treatments, and the dosage of any therapeutic agents used in combination with the antibody disclosed herein.

[0130] Uses of this disclosure The antibodies, antibody compositions, and methods of this disclosure have numerous in vitro and in vivo uses, including detection of TIGIT or enhancement of the immune response. For example, these molecules can be administered in vitro or ex vivo to cells in culture, or to human subjects, for example, in vivo, to enhance immunity in various situations. The immune response can be modulated, for example, enhanced, stimulated, or upregulated.

[0131] For example, the target population includes human patients who require enhancement of the immune response. This method is particularly suitable for treating human patients with disorders that can be treated by enhancing the immune response (e.g., T cell-mediated immune response). In certain embodiments, this method is particularly suitable for in vivo cancer treatment. When the anti-TIGIT antibody is administered together with another agent, such as an anti-PD-1 agent, the two can be administered in any order or simultaneously.

[0132] 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 that enable the antibody or portion to form a complex with TIGIT. The formation of the complex is then detected, and if there is a difference in complex formation between the sample and the control sample, it can be determined that TIGIT antigen is present in the sample. Furthermore, TIGIT can be purified by immunoaffinity purification using the anti-TIGIT antibody of this disclosure.

[0133] Treatment of diseases such as cancer In some embodiments, the Disclosure provides a method for treating a disorder or disease in a mammal, comprising administering a therapeutically effective amount of an anti-TIGIT antibody or its antigen-binding moiety disclosed herein, preferably together with a PD-1 / PD-L1 antagonist, to a subject in need of treatment (e.g., a human). The disorder or disease includes, but is not limited to, proliferative disorders (such as cancer), immunodeficiencies, inflammatory diseases, or infections. For example, the disorder may be cancer.

[0134] In some embodiments, the cancer is characterized by high expression of CD112, CD113, or CD155. In some embodiments, the cancer is characterized by an abundance of TIGIT-expressing T cells or natural killer (NK) cells.

[0135] Cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, lung cancer, such as non-small cell lung cancer (NSCLC) (NSCLC includes squamous cell NSCLC or non-squamous cell NSCLC, which include locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC) or recurrent or metastatic NSCLC (e.g., stage IV) This includes NSCLC), lung adenocarcinoma or squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-resistant non-muscle-invasive bladder cancer (NMIBC)); urinary tract cancer; hepatocellular carcinoma; breast cancer (e.g., TIGIT+ breast cancer and triple-negative breast cancer (TNBC) (estrogen receptor-negative (ER-), progesterone receptor-negative (PR-), and TIGIT-negative (TIGIT-)); colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer or renal cancer (e.g., renal cell carcinoma (RCC)); prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; superficial spreading melanoma , melanoma including lentigo malignant melanoma, acral lentiginous melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (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-incisional nuclear NHL; giant tumor NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Walde This includes Ström's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorders (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal angiogenesis associated with nevus syndromes, edema (such as that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and associated metastases.

[0136] As a co-inhibitory receptor on various immune cells, TIGIT is associated with a variety of cancers, whether malignant or benign, primary or secondary, which can be treated or prevented in the manner provided herein. The anti-TIGIT antibodies disclosed herein are preferably administered in combination with another anticancer agent, preferably a PD-1 / PD-L1 antagonist, such as an anti-PD-1 antibody. The cancer may be a solid tumor or a hematological malignancy. Examples of such cancers include lung cancers such as bronchogenic cancers (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondropathic hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyoma; osteochondroma, chondroma, chondroblastoma, chondromyxofibroma, osteoid, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, and Ewing's tumor. Bone cancers such as Ewing's sarcoma and reticular cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), undifferentiated astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, Schwannoma, ependymoma, meningioma, pituitary adenoma, pineal glandoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermoid cystoma, and hemangioma; colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colon polyps, and Cancers of the digestive system, such as colorectal cancer; liver cancers, such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolaminar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers, such as renal adenocarcinoma, renal cell carcinoma, hyperephroma, 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; benign prostate cancer. Cancers of the male reproductive system, such as hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonic 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 (papillary carcinoma, follicular carcinoma, undifferentiated carcinoma, or medullary carcinoma, etc.); pheochromocytoma (adrenal gland); non-cancerous proliferation of the parathyroid gland; and pancreatic cancer.In certain embodiments, the cancer is colon cancer.

[0137] In some other embodiments, the disorder or disease to be treated or prevented is an immune-related disorder. The immune-related disorder may be associated with T-cell dysfunction. In some embodiments, T-cell dysfunction is characterized by reduced responsiveness to antigen stimulation. In some embodiments, T-cell dysfunction is characterized by T-cell anergy, or by reduced cytokine secretion, reduced proliferation, or reduced cytolytic activity. In some embodiments, T-cell dysfunction is characterized by T-cell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the immune-related disorder is selected from the group consisting of unresolved acute infections, chronic infections, and reduced tumor immunity.

[0138] Stimulation of the immune response In some embodiments, the Disclosure further provides a method for enhancing (e.g., stimulating) an immune response in a subject, comprising administering an antibody of the Disclosure or an antigen-binding moiety thereof to the subject so as to enhance the immune response in the subject. For example, the subject is a mammal. In certain embodiments, the subject is a human.

[0139] The term "enhance the immune response," or its grammatical variations, means stimulating, inducing, increasing, improving, or enhancing any response of the mammalian immune system. The immune response can be a cellular response (i.e., cell-mediated, e.g., cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated), and can be a primary or secondary immune response. An example of enhancing the immune response is CD4 +Enhancement of the immune response can be evaluated 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. Typically, the methods of the disclosure enhance the immune response in mammals compared to the immune response in untreated mammals or mammals not treated with the methods disclosed herein. In one embodiment, an antibody or its antigen-binding moiety is used to enhance the human immune response to a microbial pathogen (such as a virus). In another embodiment, an antibody or its antigen-binding moiety is used to enhance the human immune response to a vaccine. In one embodiment, the method enhances the cellular immune response, particularly the cytotoxic T cell response. In another embodiment, the cellular immune response is a T helper cell response. In yet another embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-2 production. Antibodies or their antigen-binding portions may be used to enhance the human immune response to microbial pathogens (such as viruses) or vaccines.

[0140] Antibodies or their antigen-binding portions may be used alone as monotherapy, or in combination with chemotherapy, radiotherapy, targeted therapy, or cellular immunotherapy.

[0141] Combination with chemotherapy Antibodies or their antigen-binding portions may be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0142] The terms “anticancer agent” or “antiproliferative agent” mean any agent that can be used to treat cell proliferation disorders such as cancer, and include, but are not limited to, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, weight-reducing agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapy agents. It will be understood that such anticancer agents may include conjugates and may be conjugated with the antibodies of this disclosure before administration. More specifically, in certain embodiments, an engineered conjugate is provided by linking a selected anticancer agent to an unpaired cysteine ​​of an engineered antibody. Thus, such an engineered conjugate is explicitly intended to be within the scope of this disclosure. In some other embodiments, the anticancer agent is given in combination with an antibody-drug conjugate containing a different therapeutic agent.

[0143] As used herein, the term “cytotoxic agent” means a substance that is toxic to cells, reduces or inhibits cellular function, and / or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-sarcin, restrictosin), plants (e.g., abrin, lysine, modesin, biscumin, pokeweed antiviral protein, saporin, geronin, momorizin, tricosanthin, barley toxin, Chinese tallow tree protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitors, curcin, crotin, soapwort inhibitors, geronin, mitejerin, restrictosin, phenomycin, neomycin, and trichothecene), or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase, DNase I (including its fragments and / or variants)).

[0144] For the purposes of this disclosure, “chemotherapeutic agents” include chemical compounds (e.g., cytotoxic agents or cell proliferation inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents often target intracellular processes necessary for cell growth or division and are therefore generally particularly effective against cancer cells that grow and divide rapidly. For example, vincristine depolymerizes microtubules and, consequently, inhibits cells from entering mitosis. Generally, chemotherapeutic agents may include any chemical agents that inhibit or are designed to inhibit cancer cells or cells that are likely to become cancerous or produce tumorigenic offspring (e.g., TICs). Such agents are often administered and are most effective in combination with regimens such as CHOP or FOLFIRI.

[0145] Examples of anticancer agents that may be used in combination with the antibodies of this disclosure (either as components of site-directed complexes or in an unbound state) include alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylamelamines, acetogenins, camptothecin, bryostatin, calistatin, CC-1065, cryptophycin, dorastatin, duocalmycin, eleuterobin, pancratistatin, sarcodicin, spongistin, nitrogen mustard, antibiotics, enediyne antibiotics, dinemycin, bisphosphonates , Esperamycin, pigment protein enediin antibiotic chromophore, Acrasinomycin, Actinomycin, Ausramycin, Azaserin, Bleomycin, Cactinomycin, Carabicin, Carminomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5-Oxo-L-Norleucine, ADRIAMYCIN®, Doxorubicin, Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin, Mycophenolate Nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin, antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folate analogs, purine analogs, androgens, anti-adrenal agents, folate supplements such as folic acid, acegraton, aldofosphamide glycoside, aminolevulinic acid, enyl Uracil, Amsacrin, Bestrabusil, Bisanthren, Edatraxate, Dehofamine, Demecolsin, Diadicone, Elhornitine, Erliptinium Acetate, Epothiron, Etoglucid, Gallium Nitrate, Hydroxyurea, Lentinan, Ronidinin, Mytansinoid, Mitoguazone, Mitoxanthrone, Mopidammol, Nitraerine, Pentostatin, Fenamet, Pirarubicin, Rosoxanthrone, Podophyllic Acid, 2-Ethylhydrazide, Procarbazine, PSK (Registered Trademark) Polysaccharide Complex (JHS Natural Products, Eugene, Oregon), Lazoxane, Rhizoxin, Schizophyllan, Spirogermanium, Tenuazonic Acid, Triadicone, 2,2',2"-Trichlorotriethylamine, Trichothecenes (especially T-2 toxin, Beraclin A, Loridine A, and Angidin), Urethane, Vindesine, Dacarbazine, Mannomustine, Mitobronitol, Mitractol, Pipobroman, Gacitosine, Arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxoid, Chlorambucil, GEMZAR® Gemcitabine, 6-Thiogunine, Mercaptopurine, Methotrexate, Platinum Analogue, Vinblastine, Platinum, Etoposide (VP-16), Ifosfamide, Mitoxantrone, Vincristine, NAVE Examples of pharmaceutically acceptable substances include, but are not limited to, LBINE® vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeroda, ibandronate, irinotecan (Camptosar, CPT-11), the topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoids, capecitabine, combretastatin, leucovorin, oxaliplatin, inhibitors of PKC-alpha, Raf, H-Ras, EGFR, and VEGF-A that reduce cell proliferation, and any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition also includes anti-hormone agents that act to control or inhibit hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, an enzyme that controls estrogen production in the adrenal gland, and anti-androgens, as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog), antisense oligonucleotides, ribozymes, such as VEGF expression inhibitors and TIGIT expression inhibitors, vaccines, PROLEUKIN, (R) rIL-2, LURTOTECAN (R) Topoisomerase 1 inhibitor, ABARELIX (R) This also includes rmRH, vinorelbine, and esperamycin, as well as any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0146] Combined use with radiation therapy This disclosure also provides combinations of antibodies or their antigen-binding moieties with radiotherapy (i.e., any mechanism for inducing localized DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combination therapies using directed delivery of radioisotopes to tumor cells are also envisioned, and the antibodies of this disclosure may be used in conjunction with targeted anticancer agents or other targeting means. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy may be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy may be administered as a single dose or as a series of doses.

[0147] Pharmaceutical packs and kits Pharmaceutical packs and kits are also provided, comprising one or more containers containing one or more doses of antibody or its antigen-binding moiety. In certain embodiments, a unit dosage form is provided, comprising a predetermined amount of a composition containing, for example, an antibody or its antigen-binding moiety, together with or without one or more additional agents. In other embodiments, such a unit dosage form is supplied as a single-use pre-filled syringe for injection. In yet another embodiment, the composition contained in the unit dosage form may include, and / or be formulated within a stable and effective pH range, including, physiological saline, sucrose, and buffering agents such as phosphates. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder that is reconstituted upon addition of a suitable liquid, such as sterile water or physiological saline. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, for example, sucrose and arginine, but not limited to the following. Any label on or accompanying the container indicates that the encapsulated antibody is used to treat a selected neoplastic disease condition.

[0148] This disclosure also provides kits comprising single or multi-dose units of an antibody and, optionally, one or more anticancer agents. The kits comprise a container and labels or accompanying documents on or associated with the container. Suitable containers include, for example, bottles, vials, and syringes. Containers may be formed from a variety of materials, such as glass or plastic, and may contain a pharmaceutically effective amount of the antibody of this disclosure. In some embodiments, the container includes a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper puncturable by a subcutaneous needle). Such kits generally comprise a pharmaceutically acceptable formulation of the antibody in a suitable container and, optionally, one or more anticancer agents in the same or different containers. The kits may also comprise other pharmaceutically acceptable formulations for either diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding moiety of the present disclosure, such kits may include one or more of various anticancer agents, such as chemotherapeutic agents or radiotherapeutic agents, anti-angiogenic agents, anti-metastatic agents, targeted anticancer agents, cytotoxic agents, and / or other anticancer agents. In some embodiments, the kit may include an anti-PD-1 antibody.

[0149] More specifically, the kit may have a single container containing the antibody or its antigen-binding moiety with or without additional components, or it may have separate containers for each desired drug. If concomitant therapeutic agents are provided for binding, a single solution may be pre-mixed in molar equivalent combinations or such that one component outweighs the other. Alternatively, the antibody and optional anticancer agents of the kit may be maintained separately in separate containers before administration to the patient. The kit may also include second / third container means for sterile, pharmaceutically acceptable buffers or other diluents, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution.

[0150] If the components of this kit are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, and sterile aqueous solutions or physiological saline are particularly preferred. However, the components of this kit may also be provided as dry powders. If the reagents or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent.

[0151] As briefly stated above, the kit may also include means for administering the antibody or its antigen-binding portion and optional components to a patient, for example, one or more needles, IV bags or syringes, or eyedroppers, pipettes or other such instruments that can inject or introduce the formulation into an animal or apply it to a diseased area of ​​the body. The kit of the disclosure will also typically include means for tightly sealing and housing vials and other components for commercial sale, for example, injection-molded or blow-molded plastic containers that hold the desired vials or other instruments.

[0152] Sequence List Overview A sequence listing containing several nucleic acid and amino acid sequences is attached to this application. Tables A, B, and C below provide an overview of the included sequences.

[0153] W3642-1.433.11 is a parent hybridoma clone, and humanization of it yields the W3642-1.433.11-z11 clone, while PTM removal yields 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 set; W3642-1.433.11-z11-p1-IgG4.SP and W3642-1.433.11-z11-p1-uIgG1L differ only in their constant region; W3642-1.433.11-p1-xIgG4.SP, W3642-1.433.11-p2-xIgG4.SP, and W3642-1.433.11-p3-xIgG4.SP are antibodies containing different substitutions in LCDR2 for PTM removal.

[0154] [Table 1]

[0155] [Table 2-1] [Table 2-2]

[0156] [Table 3] [Examples]

[0157] The disclosure, broadly described above, will be more readily understood by referring to the following examples, which are provided as illustrations but are not intended to limit the disclosure. The examples are not intended to represent all or only experiments that have been conducted.

[0158] Example 1 Preparation of antigens, benchmark antibodies, and cell lines 1.1 Antigen Preparation W364-hPro1.ECD.His is the extracellular domain of human TIGIT (NP_776160.2) with a polyhistidine tag at the C-terminus; W364-hPro1.ECD.hFc is the extracellular domain of human TIGIT (NP_776160.2) with the Fc region of human IgG1 at the C-terminus; W364-mPro1.ECD.His is the mouse TIGIT (NP_776160.2) with a polyhistidine tag at the C-terminus. W364-mPro1.ECD.hFc is the extracellular domain of mouse TIGIT (NP_001139797.1) with a human IgG1 Fc region at its C-terminus; W364-hPro1L1.ECD.hFc is the extracellular domain of human CD155 (NP_006496.3) with a human IgG1 Fc region at its C-terminus. W364-hPro1L1.ECD.mFc is the extracellular domain of human CD155 (NP_006496.3) with a mouse IgG1 Fc region at its C-terminus. These antigens were purchased from vendors or prepared in-house.

[0159] 1.2 Preparation of Benchmark Antibody (BMK) The amino acid sequences encoding the variable domains of the anti-TIGIT reference antibodies WBP364-BMK1, WBP364-BMK4, and WBP364-BMK6 were synthesized according to the sequences disclosed in their respective patents. This information is summarized in Table 1.

[0160] [Table 4]

[0161] 1.3 Preparation of cell pools / cell lines The human TIGIT-expressing cell pool W364-293F.hPro1.pool was created using 293F cells transfected with full-length human TIGIT (NP_776160.2). The human TIGIT-expressing cell line W364-CHOK1.hPro1.2A11 was created using CHOK1 cells transfected with full-length human TIGIT (NP_776160.2). The cynomolgus monkey TIGIT-expressing cell pool W364-FlpinCHO.cynoPro1.pool was created using FlpinCHO cells transfected with full-length cynomolgus monkey TIGIT (XP_015300911.1). The mouse TIGIT-expressing cell pool W364-FlpinCHO.mPro1.pool was created using FlpinCHO cells transfected with full-length mouse TIGIT (NP_001139797.1).

[0162] Example 2 Production of chimeric antibodies and humanized antibodies 2.1 Immunization Two female SD rats, 6-8 weeks old, were purchased from Shanghai SLAC Experiment Animal Co., Ltd. and raised in an IACUC-certified animal facility. These two animals were alternately immunized with W364-hPro1.ECD.His and W364-mPro1.ECD.His.

[0163] 2.2 Detection of serum titer Anti-human / mouse TIGIT antibody titers in serum samples were determined by ELISA. Microplates were coated with 1 μg / mL of W364-hPro1.ECD.hFc or W364-mPro1.ECD.hFc in 100 μL of coating buffer (Na2CO3 / NaHCO3, pH 9.2) per well and incubated overnight at 4°C. On the day of the assay, diluted rat serum samples (first 1:100 dilution using 1×PBS / 2%BSA, then 3-fold dilution) and negative controls were added to plates blocked with 1×PBS / 2%BSA for 1 hour, and then incubated at room temperature for 1 hour. After washing three times with 1×PBST (PBS containing 0.05% Tween®-20), HRP-labeled goat anti-rat IgG Fc (Bethyl, cat#A110-236P) was added and incubated at room temperature for 1 hour. After removing unbound substances, the TMB (3,3',5,5'-tetramethylbenzidine) substrate was added, and the reaction was stopped with 2M HCl. Absorbance at 450 nm was detected using a microplate spectrophotometer.

[0164] Table 2 shows the serum titers of immunized SD rats. After the final boost with W364-hPro1.ECD.His and W364-mPro1.ECD.His, the two animals were euthanized, and their lymph nodes were harvested and fused.

[0165] [Table 5]

[0166] 2.3 Creation of Hybridomas Lymph nodes were collected from immunized rats under sterile conditions, dissociated, and prepared as single-cell suspensions. B cells were isolated from the lymph nodes and mixed with myeloma cells SP2 / 0 in a 1:1.2 ratio. Electrofusion was performed using a BTX 2001 Electro cell manipulator according to an optimized electrofusion procedure. After fusion, the cells were transferred to 96-well plates containing DMEM medium supplemented with 20% FBS and 1% HAT selective reagent (1.2 × 10⁶). 4Cells were cultured in a 96-well assay plate at 37°C and 5% CO2, and monitored regularly. When the clones reached approximately 80% confluence in the wells, 100 μL of the supernatant was transferred from the tissue culture plate to a 96-well assay plate for antibody screening.

[0167] 2.4 Antibody screening and subcloning As a high-throughput screening process, we performed 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.

[0168] Positive cell lines in the logarithmic growth phase were diluted to 200-300 cells per 1.5 mL of semi-solid HAT medium. The cell suspension was gently mixed for 5-10 seconds using a vortex shaker, and then seeded into 6-well plates. The plates were kept at 37°C and 5% CO2 for 7-8 days. Once cell clusters had grown, visible single colonies were harvested and seeded into 96-well plates containing DMEM medium supplemented with 10% fetal bovine serum. After 2-3 days, the supernatant of each clone was collected and screened again to obtain positive hybridoma single clones.

[0169] 2.5 Sequencing of Hybridomas RNA was isolated from monoclonal hybridoma cells using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa Bio Inc.) according to the manufacturer's instructions. cDNA was amplified using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Inc.) according to the manufacturer's instructions. Subsequently, PCR amplification was performed using the obtained cDNA as a template with primers specific to the target gene. The PCR product was inserted into a pMD18-T vector, and the ligation product was sent to GENEWIZ for sequencing.

[0170] Fifty positive cell lines were selected and subcloned using primary and secondary binding screening, as well as TIGIT / PVR blocking. After confirming monoclonal antibody status, 39 positive hits were selected and sequenced, and three of these were converted to human IgG.

[0171] 2.6 Array Optimization 2.6.1 Production of Chimeric Antibodies The amino acid sequences of the VH and VL domains were codon-optimized for expression in mammals. After synthesizing the codon-optimized DNA sequences using GENEWIZ, they were subcloned into pcDNA expression vectors containing the constant region 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 before the supernatant was collected. Antibodies were purified from the supernatant using a Protein A column.

[0172] The W3642-1.433.11 clone was identified. Its variable domain sequence is shown in Table B. A chimeric antibody of W3642-1.433.11 containing a heavy chain in which rat VH is fused to the human IgG4 constant region and a light chain in which rat VL is fused to the human Ig lambda was named W3642-1.433.11-xIgG4.SP.

[0173] 2.6.2 PTM removal In the parent antibody W3642-1.433.11, the amino acid residue "DG" at the VLCDR2 boundary was identified as a potentially unstable residue with a risk of isomerization. After eliminating the risk of post-translational modification (PTM) by introducing mutations, the binding reaction rate of the antibody to human TIGIT was measured using SPR analysis. Mutation of residue "DG" to EG, QG, or SG did not significantly alter the binding affinity. The results are shown in Table 3.

[0174] [Table 6]

[0175] 2.6.3 Humanization of rat antibodies Humanization of W3642-1.433.11 was primarily performed using CDR grafts. CDRs were identified according to the Contact definition (http: / / www.bioinf.org.uk / abs / ) introduced by the group of Dr. Andrew CRMartin. The VH and VL sequences of W3642-1.433.11 were BLAST matched against the human germline V gene database, and the human IGVH and IGVL with the highest homology to W3642-1.433.11 were selected as humanization templates. The CDRs of the VH and VL domains of W3642-1.433.11 were grafted onto the humanization template framework to construct the germline VH and VL domain sequences.

[0176] We performed several "reverse mutations" within the framework to convert amino acids in the germline sequence to the corresponding amino acids in the original rat sequence. We also created a hybrid chimeric antibody (W3642-1.433.11-hyAbL) consisting of rat VH fused to the human IgG4 constant region and germline VL fused to human Ig lambda.

[0177] To confirm whether the humanized mutant retained antigen-binding activity, surface plasmon resonance (SPR) was performed using a Biacore 8K instrument (Cytiva). In short, a goat anti-human IgG Fc antibody (JacksonImmunoResearch, cat#109-005-098) was immobilized on a CM5 biosensor chip (GE, cat#29-1496-03). Antibodies in the supernatant of pilot-scale transfections were captured with the goat anti-human IgG Fc antibody. The antigen W364-hPro1.ECD.His was injected into a running buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at a flow rate of 100 μL / min for a 120-second binding phase, followed by a 900-second dissociation phase. The dissociation rate (koff) was calculated using a simple one-to-one Langmuir binding model. The results are shown in Table 4.

[0178] [Table 7]

[0179] Two mutant strains, W3642-1.433.11-z10-p1-IgG4.SP and W3642-1.433.11-z11-p1-IgG4.SP, which were created by combining humanization and PTM removal, showed high affinity binding to human TIGIT when measured by Biacore. The affinity of W3642-1.433.11-z11-p1-IgG4.SP was approximately twice that of W3642-1.433.11-z10-p1-IgG4.SP. The results are shown in Table 5.

[0180] [Table 8]

[0181] An antibody in the corresponding IgG1 format for W3642-1.433.11-z11-p1-IgG4.SP was prepared and named W3642-1.433.11-z11-p1-uIgG1L, or simply W3642.

[0182] Example 3 In vitro characterization 3.1 Human TIGIT Binding Assay W364-293F.hPro1.pool(1×10 5 Cells were incubated with various concentrations of anti-TIGIT antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, AlexaFluor647-labeled goat anti-human IgG (JacksonImmunoResearch cat#109-605-098), was added, and the cells were incubated with the antibody at 4°C in the dark 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. The cells were then washed and resuspended in 1×PBS / 1%BSA. Cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0183] Figure 1 shows the binding results of W3642-1.433.11-z11-p1-uIgG1L to W364-293F.hPro1.pool cells. These results indicate that W3642-1.433.11-z11-p1-uIgG1L can strongly bind to human TIGIT-expressing cells, and its binding affinity is equivalent to that of the reference antibody. Table 7 summarizes the antibody binding process.

[0184] 3.2 TIGIT-binding assay in cynomolgus monkeys W364-FlpinCHO.cynoPro1.pool(1×10 5 Cells were incubated with various concentrations of anti-TIGIT antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, AlexaFluor647-labeled goat anti-human IgG (JacksonImmunoResearch cat#109-605-098), was added, and the cells were incubated with the antibody at 4°C in the dark 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. The cells were then washed and resuspended in 1×PBS / 1%BSA. Cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0185] Figure 2 shows the binding results of W3642-1.433.11-z11-p1-uIgG1L to W364-FlpinCHO.cynoPro1.pool cells. These results indicate that W3642-1.433.11-z11-p1-uIgG1L can strongly bind to cynomolgus monkey TIGIT-expressing cells, and its binding affinity is equivalent to that of the reference antibody. Table 7 summarizes the antibody binding process.

[0186] 3.3 Mouse TIGIT binding assay W364-FlpinCHO.mPro1.pool(1×10 5Cells were incubated with various concentrations of anti-TIGIT antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, AlexaFluor647-labeled goat anti-human IgG (JacksonImmunoResearch cat#109-605-098), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. The anti-human TIGIT antibody WBP364-BMK6, known to cross-react with mouse TIGIT, was used as a positive control. Human IgG1 isotype antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS / 1%BSA. Cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0187] Figure 3 shows the binding results of W3642-1.433.11-z11-p1-uIgG1L to the extracellular domain of mouse TIGIT. These results indicate that W3642-1.433.11-z11-p1-uIgG1L can strongly bind to mouse TIGIT-expressing cells, and its binding affinity is higher than that of the reference antibody WBP364-BMK6. WBP364-BMK1 and WBP364-BMK4 did not show binding to mouse TIGIT-expressing cells. Table 7 summarizes the antibody binding.

[0188] 3.4 Human TIGIT affinity assay The affinity of anti-TIGIT antibodies to recombinant human TIGIT was determined by surface plasmon resonance (SPR) using a Biacore 8K instrument (Cytiva). Goat anti-human IgG Fc antibody (JacksonImmunoResearch cat#109-005-098) was immobilized on a CM5 biosensor chip (GE cat#29-1496-03), and the anti-TIGIT antibody was captured with the goat anti-human IgG Fc antibody. For reaction rate measurement, a binding phase was performed by injecting a series of concentrations of W364-hPro1.ECD.His into a running buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at a flow rate of 30 μL / min at 25°C, followed by a dissociation phase for a certain period. Binding rates (kon) and dissociation rates (koff) were calculated using a simple 1:1 Languir binding model. The equilibrium dissociation constant (kD) was calculated as the koff / kon ratio.

[0189] Table 6 shows the binding affinity of W3642-1.433.11-z11-p1-uIgG1L to the extracellular domain of human TIGIT. W3642-1.433.11-z11-p1-uIgG1L binds to human TIGIT with an affinity of 2.39E-11M KD.

[0190] [Table 9]

[0191] 3.5 TIGIT Paralogous Protein Binding Assay Plates were pre-coated overnight at 4°C with 1 μg / mL of W364-hPro1.ECD.His, recombinant human CD28, CTLA-4, PD-1, ICOS, or PVRIG extracellular domain in 50 μL of coating buffer per well. After blocking with 200 μL of 1×PBS / 2%BSA, 50 μL of test antibody was added to the plate at concentrations of 1 nM, 0.1 nM, or 0.01 nM, and the plate was incubated at room temperature for 1 hour. After incubation, the plate was washed three times with 1×PBST. HRP-labeled goat anti-human IgG antibody (Bethyl cat#A80-304P) diluted with 1×PBS / 2%BSA was added, and the plate was incubated at room 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 antibodies were used as isotype controls. After washing six times with 1×PBST, 100 μL of TMB substrate was dispensed and allowed to develop color, then 100 μL of 2M HCl was added to stop the reaction. Absorbance was read at 450 nm using an M5e microplate reader (Molecule Devices).

[0192] Figure 4 shows the binding results of W3642-1.433.11-z11-p1-uIgG1L to the TIGIT paralogous protein. From these results, it can be seen that W3642-1.433.11-z11-p1-uIgG1L specifically binds to TIGIT without cross-reactivity with human CD28, ICOS, PVRIG, PD-1, or CTLA-4.

[0193] 3.6 Human TIGIT / Ligand Blocking Assay The ability of anti-TIGIT antibodies to block human TIGIT / PVR interactions was tested by FACS. W364-CHOK1.hPro1.2A11 cells were washed with 1×PBS / 1%BSA and loaded into 96-well round-bottom plates at a rate of 1×10⁶ cells per well. 5 Cells were seeded. Excess buffer in the wells was removed by centrifugation. Serially diluted anti-TIGIT antibody (2× concentration) and 4 μg / mL (2× concentration) W364-hPro1L1.ECD.mFc were pre-mixed in a 1:1 volume ratio, and 100 μL of the antibody / ligand mixture was added to each well. The plates were incubated at 4°C for 1 hour. After washing with 1× PBS / 1% BSA, the secondary antibody, PE-labeled goat anti-mouse IgG (Bethyl cat#A90-239PE), was added, and the cells were incubated with the antibody at 4°C in the dark 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. The cells were then washed and resuspended in 1× PBS / 1% BSA. Cellular cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0194] Furthermore, the ability of anti-TIGIT antibodies to block human TIGIT / CD112 and TIGIT / CD113 interactions was tested by FACS.

[0195] Figures 5-7 show the binding blocking results for human TIGIT and its ligands (CD155, CD112, and CD113). These results demonstrate that W3642-1.433.11-z11-p1-uIgG1L effectively blocks the binding of human CD155, CD112, and CD113 to TIGIT. Table 7 summarizes the antibody blocking activity. Ligand binding rates are given as binding % = MFI. 試料 / MFI max The calculation was performed using a multiplier of 100%. However, MFI (Multi-Factor Indication) max This was defined as MFI in the absence of antibodies.

[0196] 3.7 Jurkat TIGIT / NFAT-luciferase reporter gene assay (RGA) Jurkat cells co-expressing human TIGIT and NFAT-luciferase reporters were stimulated by co-culturing them with CHOK1 cells co-expressing human PVR and TCR activator, thereby binding them to T cell receptors. CHOK1 / PVR / TCR activator cells were placed in 96-well plates in a 2x10⁶ format. 4 Cells were seeded at a cell / well density and cultured overnight at 37°C in 5% CO2. On day 2, after removing the supernatant and non-adherent cells, serially diluted anti-TIGIT antibody and Jurkat / TIGIT / NFAT-luciferase cells (2 × 10⁶ cells) were incubated. 4 Cells were added to the plate (per well) and co-cultured at 37°C and 5% CO2 for 5-6 hours. After incubation, the reconstituted luciferase substrate (Promega cat#E6130) was added to each well and mixed thoroughly. Luciferase levels were read using an EnVision microplate reader (PerkinElmer).

[0197] Figure 8 shows the results of W3642-1.433.11-z11-p1-uIgG1L reversing the suppression of NFAT signaling induced by TIGIT / PVR interaction in RGA. This result indicates that W3642-1.433.11-z11-p1-uIgG1L can enhance TCR / NFAT activation by suppressing the TIGIT / PVR pathway. Table 7 shows an overview of the antibody RGA activity.

[0198] 3.8 Jurkat Functional Assay HT1080 is a human fibrosarcoma cell line that expresses human PVR, PVRL2, and PVRL3. Jurkat cells overexpressing human TIGIT were stimulated by co-culturing them with HT1080 cells overexpressing human TCR activator, thereby binding them to T cell receptors. 1 × 10 4 Jurkat / TIGIT cells were divided into 5 × 10¹⁶ cells in the presence of serially diluted anti-TIGIT antibodies. 3The cells were co-cultured with HT1080 / TCR activator cells at 37°C and 5% CO2 for 2 days. After incubation, the supernatant was collected and IL-2 levels were measured by ELISA (capture antibody R&D cat#MAB602, detection antibody R&D cat#BAF202). Absorbance was detected using an M5e microplate reader (Molecule Devices).

[0199] TIGIT-expressing Jurkat cells were co-cultured with HT1080 / TCR activator cells in the presence of the antibody, and IL-2 in the supernatant was quantified by ELISA. The results show that W3642-1.433.11-z11-p1-uIgG1L can dose-dependently enhance Jurkat cell activation. The data are shown in Figure 9. Table 7 summarizes the antibody-induced promotion of IL-2 secretion.

[0200] [Table 10]

[0201] 3.9 Human Primary NK Cell Activation Assay HT1080 cells expressing human PVR, PVRL2, and PVRL3 were used as target cells, and human primary NK cells isolated from human PBMCs were used as effector cells. 1 × 10⁶ cells preloaded with EuTDA (PerkinElmer cat#AD0116) were used. 4 1 × 10¹ HT1080 cells were subjected to serial dilution in the presence of anti-TIGIT antibody. 4 Human primary NK cells were co-cultured with target cells at 37°C and 5% CO2 for 2 hours. Subsequently, the culture supernatant was tested for target cell lysis using time-resolved fluorescence spectroscopy according to the manufacturer's instructions (PerkinElmer cat#AD0116). Fluorescence was detected using an EnVision microplate reader (PerkinElmer).

[0202] Human NK cells were co-cultured with HT1080 cells in the presence of the antibody. The results showed that W3642-1.433.11-z11-p1-uIgG1L could dose-dependently enhance NK cell-killing activity. The data are shown in Figure 10.

[0203] 3.10 Antibody-dependent cell-mediated cytotoxicity assay To evaluate the ADCC effect of anti-TIGIT antibodies against TIGIT-expressing cells, human TIGIT-engineered W364-CHOK1.hPro1.2A11 cells were used as target cells, and human primary NK cells isolated from human PBMCs were used as effector cells. 1 × 10 4 Individual target cells and various concentrations of anti-TIGIT antibody were pre-incubated in a 96-well plate at 37°C and 5% CO2 for 30 minutes, and then 5 × 10⁶ cells were incubated. 4 One newly isolated human NK cell was added to each well. The plate was incubated at 37°C and 5% CO2 for 6 hours. Target cell lysis was measured using an LDH-based cytotoxicity detection kit (Roche cat#04744926001) according to the manufacturer's instructions. Absorbance was detected using an M5e microplate reader (Molecule Devices).

[0204] The results of the ADCC assay are shown in Figure 11. The results show that W3642-1.433.11-z11-p1-uIgG1L can induce the ADCC effect in a dose-dependent manner in TIGIT-expressing CHOK1 cells, and its potency is similar to that of the reference antibody.

[0205] 3.11 Antibody serum stability assay Fresh human serum was isolated from a healthy donor. Anti-TIGIT antibody was diluted in the serum. The sample was dispensed into five tubes and incubated at 37°C. Subsequently, samples were collected on days 0, 1, 4, 7, and 14, rapidly frozen, and stored in a freezer set to -70°C until analysis. The binding activity of the samples was evaluated by FACS according to the method described in Section 3.1.

[0206] Figure 12 shows the binding of W3642-1.433.11-z11-p1-uIgG1L incubated in serum to W364-293F.hPro1.pool cells. Antibodies incubated with serum for up to two weeks maintained binding activity and very similar EC50 levels to fresh antibodies. The results indicate that W3642-1.433.11-z11-p1-uIgG1L is stable in human serum at 37°C for at least two weeks.

[0207] 3.12 Antibody Thermal Stability Assay Conformational stability is a crucial property for promising antibodies. Conformational stability can be evaluated by measuring thermal stability using differential scanning fluorescence (DSF), a method that can detect changes in protein folding. DSF measures the unfolding transition temperature (Tm) of a protein based on changes in the fluorescence intensity of the environmentally sensitive dye SYPRO Orange.

[0208] DSF was performed using a Quant Studio 7 Flex real-time PCR instrument (Applied Biosystems) in each formulation buffer. SYPRO Orange dye (Invitrogen cat#S6651) was added to the antibody, and the mixture was transferred to a 96-well plate. The plate was placed in a Quant Studio® 7 Flex real-time PCR instrument, with the temperature range set to 26°C to 95°C and the heating rate set to 0.9°C / min. The first two temperatures during protein unfolding were recorded as Tm1 and Tm2. These two values ​​were calculated according to the melting curve using Quant Studio® real-time PCR software (v1.3).

[0209] The DSF thermogram of the W3642-1.433.11-z11-p1-uIgG1L antibody shows two transitions. The first has a low melting point (Tm1), and the second has a high melting point (Tm2), at 69.0°C and 79.0°C, respectively. The results are shown in Table 8.

[0210] [Table 11]

[0211] Example 4 In vivo characteristic evaluation 4.1 In vivo efficacy of combination therapy with anti-TIGIT antibodies and anti-PD-1 antibodies To investigate the antitumor activity of anti-TIGIT antibodies in an MC38 syngeneic mouse model, tumor cells were inoculated into human TIGIT transgenic h-TIGIT C57BL / 6 mice (Jiangsu GemPharmatech Co., Ltd.). Wild-type MC38 tumor cells (1 × 10⁶) suspended in 0.1 mL DPBS were used. 6 Tumors were induced by subcutaneous injection of ) into the right forearm axilla of h-TIGIT C57BL / 6 mice. The average tumor size was approximately 73 mm. 3 Upon reaching this point, tumor-bearing animals were randomly divided into six study groups. Each group consisted of eight mice. The study design is shown in Table 9. In some groups, a monoclonal anti-PD-1 antibody ("anti-mPD-1" disclosed in WO2018053709 with clone ID 2E5) was used in combination with an anti-TIGIT antibody.

[0212] All antibodies were administered intraperitoneally to tumor-bearing mice twice a week. Body weight and tumor volume were measured twice a week. All animal handling, care, and treatment procedures in the study followed guidelines approved by the Shanghai Bio-model's Institutional Animal Care and Use Committee (IACUC), in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice were euthanized according to predetermined health criteria, and the study concluded 24 days after the first dose.

[0213] [Table 12]

[0214] This study was to determine the antitumor activity of W3642-1.433.11-z11-p1-uIgG1L in h-TIGIT mice inoculated with MC38 tumor cells. During the entire experiment, the tumor growth and body weight of all mice were carefully monitored, and the tumor size was measured and recorded twice a week. The tumor growth inhibition rate (TGI) was calculated and analyzed at the optimal treatment time point (24 days after grouping, and the first dose was administered on the same day as grouping). The results of tumor volume are shown in Figure 13 and summarized in Tables 10 and 11. The results of body weight changes are shown in Figure 14, and it can be seen that there were no abnormal body weight changes in each group.

[0215] According to the results, although W3642-1.433.11-z11-p1-uIgG1L alone had a weak tumor inhibitory effect, it enhanced the effectiveness of the combined anti-mPD-1 antibody. Compared with the combination of WBP364-BMK1 and anti-mPD-1 antibody, the combination of W3642 and anti-mPD-1 antibody had significantly better tumor inhibition results from the start to the end of treatment.

[0216]

Table 13

[0217]

Table 14

[0218] Those skilled in the art will further understand that the present disclosure can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the present disclosure only discloses its exemplary embodiments, it should be understood that other variations are also within the scope of the present disclosure. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims to indicate the scope and content of the present invention.

Claims

1. Heavy chain CDR (HCDR) 1 containing the amino acid sequence of SEQ ID NO: 1; HCDR2 containing the amino acid sequence of SEQ ID NO: 2; HCDR3 containing the amino acid sequence of SEQ ID NO: 3; Light chain CDR (LCDR) 1 containing the amino acid sequence of SEQ ID NO: 4; LCDR2 containing any of the amino acid sequences of SEQ ID NOs: 7, 5, 8, and 9; LCDR3 containing the amino acid sequence of SEQ ID NO: 6 An isolated antibody containing or its antigen-binding moiety.

2. It includes a heavy chain variable region (VH) and a light chain variable region (VL), The above VH is: (i) The amino acid sequence shown in any of SEQ ID NOs: 10 to 11; or (ii) An amino acid sequence that is at least 85%, 90%, or 95% identical to any of sequence numbers 10-11 Includes, The above VL is: (i) The amino acid sequence shown in any of SEQ ID NOs: 12-18; or (ii) An amino acid sequence that is at least 85%, 90%, or 95% identical to any of sequence numbers 12-18 The isolated antibody or antigen-binding moiety according to claim 1, comprising:

3. An isolated antibody or antigen-binding moiety according to claim 1 or 2, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

15.

4. The isolated antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the isolated antibody further comprises a human IgG constant region.

5. The isolated antibody or antigen-binding moiety according to claim 4, wherein the above-mentioned human IgG constant region is the human IgG1, IgG4, IgG2, or IgG3 constant region or a variant thereof.

6. The isolated antibody or antigen-binding moiety according to any one of claims 1 to 5, wherein the antibody comprises a human IgG4 Fc region or a human IgG1 Fc region having an S228P substitution.

7. The isolated antibody or antigen-binding moiety thereof according to any one of claims 1 to 6, wherein the antibody is a chimeric antibody or a humanized antibody.

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

9. A vector comprising an isolated nucleic acid molecule as described in claim 8.

10. A host cell comprising the vector according to claim 9.

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

12. A method for producing an antibody or its antigen-binding portion as defined in any one of claims 1 to 7, A step of culturing host cells containing the above-mentioned antibody or an expression vector encoding its antigen-binding portion under appropriate conditions; A step of collecting the above antibody or its antigen-binding portion from a cell culture. A method that includes this.

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

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

15. The method according to 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, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach 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 tumors, mesothelioma, myeloma, and sarcoma.

16. The method according to claim 14, wherein the above-mentioned immune-related disorder is T-cell dysfunction or infection.

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

18. A combination of an isolated antibody or its antigen-binding moiety according to any one of claims 1 to 7 and an anti-PD-1 antibody.

19. Use of an antibody or its antigen-binding moiety, either alone or in combination with an anti-PD-1 antibody, as defined in any one of claims 1 to 7, in the manufacture of a pharmaceutical product for treating or preventing cancer or an immune-related disorder.

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

21. An antibody or antigen-binding moiety as defined in any one of claims 1 to 7, for use in the treatment or prevention of cancer or immune-related disorders.

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

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