CN113164601A - Isolated antigen binding protein and uses thereof - Google Patents

Abstract

An isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID No. 16 and at least one CDR in a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID No. 15. Also relates to nucleic acid molecules encoding the isolated antigen binding proteins, vectors comprising the isolated antigen binding proteins, cells comprising the nucleic acid molecules or the vectors, methods of making the isolated antigen binding proteins, pharmaceutical compositions, and uses of the isolated antigen binding proteins.

Description

Isolated antigen binding protein and uses thereof Technical Field

The application relates to the field of biomedicine, in particular to an isolated antigen binding protein and application thereof.

Background

Tumor is a disease seriously threatening human health, and in recent years, immunotherapy, as a new therapy, has shown great potential in tumor therapy.

T cell immunoglobulin and mucin molecule 3(T cell immunoglobulin and mucin-domain binding molecule 3, Tim-3) are important immune checkpoint molecules discovered in recent years, unlike other "immune checkpoint" molecules, which are predominantly inducibly expressed on the surface of T cells in the inflammatory and tumor microenvironment. It is a type I membrane surface molecule, is highly expressed in Th1 cells, and produces inhibitory signals to induce apoptosis of Th1 cells. In the process of cancer and chronic virus infection, Tim-3 is combined with a ligand galectin 9 (Gal-9), phosphatidylserine (PtdSer), high mobility group box 1 protein (HMGB 1) and carcino-embryonic antigen related cell adhesion molecule 1(carcino-embryonic antigen related cellular adhesion molecule 1, CEACAM1) to cause the failure and the escape of T cells, which is one of important reasons for generating immunity of tumor cells. Meanwhile, selective activation of Tim-3 to cause immune escape is the main mechanism for drug resistance in the course of immunotherapy with PD-1/PD-L1 antibody.

Disclosure of Invention

In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO. 16 and at least one CDR in a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 15.

In certain embodiments, an isolated antigen binding protein described herein has Tim-3 binding capacity.

In certain embodiments, an isolated antigen binding protein described herein has one or more of the following properties:

1) is capable of binding to human-derived Tim-3 with a KD value of 10nM or less, wherein said KD value is determined by surface plasmon resonance;

2) in a FACS assay, capable of specifically binding to human Tim-3 but not to mouse Tim-3;

3) in an ELISA assay, binding of Tim-3 to PtdSer can be inhibited; and

4) promote the secretion of IFN-gamma and/or TNF-alpha.

The isolated antigen binding protein of any one of claims 1-3, comprising an antibody or antigen binding fragment thereof.

In certain embodiments, the antibodies include humanized and murine antibodies.

In certain embodiments, the antigen-binding fragment comprises a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv and/or dAb.

In certain embodiments, the VH comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 6.

In certain embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 4.

In certain embodiments, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 5.

In certain embodiments, the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 1.

In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 2.

In certain embodiments, the LCDR3 comprises the amino acid sequence set forth in SEQ ID No. 3.

In certain embodiments, the isolated antigen binding protein described herein competes for binding to the Tim-3 protein with a reference antibody, wherein the reference antibody comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID No. 1; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 2; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 3, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO. 4; the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 5; the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 6.

In certain embodiments, the VL comprises the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

In certain embodiments, the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1 and the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 7.

In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2 and the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 8.

In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO 9.

In certain embodiments, the N-terminus of L-FR4 is linked directly or indirectly to the C-terminus of LCDR3 and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 10.

In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO. 15.

In certain embodiments, an isolated antigen binding protein described herein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Ig kappa constant region.

In certain embodiments, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 17.

In certain embodiments, an isolated antigen binding protein described herein comprises an antibody light chain LC, and the LC comprises the amino acid sequence set forth in SEQ ID NO. 19.

In certain embodiments, the VH comprises the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In certain embodiments, the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1 and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 11.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO 12.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO 13.

In certain embodiments, the N-terminus of H-FR4 is linked directly or indirectly to the C-terminus of HCDR3 and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 14.

In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO 16.

In certain embodiments, the isolated antigen binding protein described herein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.

In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 18.

In certain embodiments, an isolated antigen binding protein described herein comprises an antibody heavy chain HC, and the HC comprises the amino acid sequence set forth in SEQ ID No. 20.

In another aspect, the present application also provides an isolated one or more nucleic acid molecules encoding an isolated antigen binding protein described herein.

In another aspect, the present application also provides a vector comprising a nucleic acid molecule described herein, or expressing an antigen binding protein described herein.

In another aspect, the present application also provides a cell comprising a nucleic acid molecule described herein or a vector described herein.

In another aspect, the present application also provides a method of making an isolated antigen binding protein described herein, the method comprising culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In another aspect, the present application also provides a pharmaceutical composition comprising an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of a tumor.

In certain embodiments, the tumor comprises a solid tumor and/or a hematological tumor.

In another aspect, the present application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for preventing, alleviating and/or treating a Tim-3 associated disease.

In another aspect, the present application also provides a method of inhibiting the binding of Tim-3 to PtdSer, comprising administering an isolated antigen binding protein described herein.

In another aspect, the present application also provides a method of preventing, ameliorating and/or treating a tumor comprising administering to a subject in need thereof an isolated antigen binding protein described herein or a pharmaceutical composition described herein.

In certain embodiments, the tumor comprises a solid tumor and/or a hematological tumor.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows the binding of the isolated antigen binding protein Z2 described herein to a cell line expressing human Tim-3;

FIG. 2 shows the binding of the isolated antigen binding protein Z2 described herein to a cell line expressing mouse Tim-3;

FIG. 3 shows the binding of the comparative example antibody to a cell line expressing mouse Tim-3;

FIGS. 4A-4C show that the isolated antigen binding protein Z2 described herein blocks the binding of human Tim-3 to the ligand PtdSer.

Detailed Description

The present invention provides for the first time an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 16 and at least one CDR in a light chain variable region VL comprising the amino acid sequence shown in SEQ ID No. 15. The isolated antigen binding proteins described herein are capable of binding to human-derived Tim-3 with a KD value of 10nM or less, and in addition, are capable of specifically binding to human Tim-3 and not to mouse Tim-3. In addition, the isolated antigen binding proteins described herein can inhibit the binding of Tim-3 to PtdSer and can also promote the secretion of IFN- γ and/or TNF- α.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

The present application is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not affect the activity of the substance, mixed with artificial or synthetic substances.

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability obtained from a natural state by artificial means. The "isolated antigen binding protein" may comprise a portion that binds an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding portion to an antigen. The antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, scaffolds comprising antibody sources introduced, for example, with mutations to stabilize the three-dimensional structure of the antigen binding protein and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., Korndorfer et al, 2003, Proteins: Structure, Function, andBioinformatics,53(1): 121-; roque et al, Biotechnol.prog.20:639-654 (2004). In addition, peptide antibody mimetics ("PAMs") as well as scaffolds based on antibody mimetics utilizing fibronectin components can be used as scaffolds.

In the present application, the term "KD" (likewise, "K)_D"or" K_D") generally refers to an" affinity constant "or an" equilibrium dissociation constant, "and refers to a dissociation rate constant (K) at equilibrium, or by combining the dissociation rate constants, in a titration measurement_dissoc) Divided by the binding rate constant (K)_assoc) The obtained value. Using the binding Rate constant (K)_assoc) Dissociation rate constant (K)_dissoc) And the equilibrium dissociation constant (KD) represents the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) for an antigen (e.g., Tim-3). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine the sample at equilibrium in physiological buffer. For example, the K can be determined by surface plasmon resonance_DThe value of K can also be determined by Octet_DValues, other experimental pathways and instruments such as BIAcore (biomolecular interaction analysis) assays (e.g., instruments available from BIAcore international ab, algehalthica, Uppsala, sweden) may also be used. In addition, the K can also be measured using KinExA (dynamic exclusion assay) available from Sapidyne instruments (Boise, Idaho)_DThe value is obtained.

In the present application, homology of amino acid or nucleotide sequences is used interchangeably with the term "identity". The term "identity" generally refers to the percentage of identical residues that are paired. The "percent sequence identity" is calculated as: comparing the two optimally aligned sequences over a particular region; determining the number of positions in the two sequences at which the same base or amino acid occurs to obtain the number of matching positions; the number of such positions is divided by the total number of positions in the compared section and the resulting quotient is multiplied by 100. As described above, programs for determining homology (or identity) compare aligned sequences on an amino acid-to-amino acid basis, and the programs can set different levels of stringency for the comparison (e.g., identical amino acids, conservative amino acid substitutions, etc.). As the term is used herein, two amino acids in question are considered "conservative substitutions" for one another if they each belong to the same chemical class (i.e., acidic, non-polar/hydrophobic, uncharged polar and basic). As a non-limiting example, two different amino acids belonging to a non-polar amino acid will be considered "conservative substitutions" for each other, even if the two amino acids are not identical, but will not be considered "conservative substitutions" for each other when they are non-polar on the one hand, and basic on the other hand. Alberts, Johnson, Lewis, Raff, Roberts, and Walter, molecular Bi of the Cell, 4 th edition (2002), column 3.1, divide amino acids into four major groups: acidic, non-polar, uncharged polar and basic. This grouping can be used in the context of the present invention for the purpose of determining whether a particular amino acid is a conservative substitution for another amino acid in question. The above main groups can be further sub-classified into e.g. small non-polar and large non-polar amino acids, large aromatic amino acids, etc. The term "conservative amino acid substitution" also refers to any amino acid substitution for a given amino acid residue, wherein the substituted residue is so chemically similar to the given residue that there is no substantial decrease in the functional (e.g., binding) result of the polypeptide.

In the present application, the term "Tim-3" is an abbreviation for "T cell immunoglobulin and mucin molecule 3(T cell immunoglobulin and mucin-domain binding molecule 3)", also known as Tim-3, HAVCR2, KIM-3, TIMD3, and FLJ14428, which generally refers to a T helper type i specific cell surface protein that modulates macrophage activation and the severity of inflammatory conditions. Tim-3 is also associated with cancer, particularly cancer stem cells, which are predominantly inducibly expressed on the surface of T cells in the inflammatory and tumor microenvironment, highly expressed on Th1 cells, and produce inhibitory signals leading to apoptosis of Th1 cells. In the process of cancer and chronic virus infection, Tim-3 is combined with a ligand galectin 9 (Gal-9), phosphatidylserine (PtdSer), high mobility group box 1 protein (HMGB 1) and carcino-embryonic antigen related cell adhesion molecule 1(carcino-embryonic antigen related cellular adhesion molecule 1, CEACAM1) to cause the failure and the escape of T cells, which is one of important reasons for generating immunity of tumor cells. Meanwhile, selective activation of Tim-3 to cause immune escape is the main mechanism for drug resistance in the course of immunotherapy with PD-1/PD-L1 antibody. Tim-3 may include variants, isoforms, species homologs of human Tim-3, and analogs having at least one common epitope with Tim-3. For example, the amino acid sequence of human-derived Tim-3 is shown in SEQ ID NO. 25, and the amino acid sequence of mouse-derived Tim-3 is shown in SEQ ID NO. 26.

In this application, the term "PtdSer" generally refers to phosphatidylserine, which is a ligand for Tim-3. Tim-3 binds to PtdSer, leading to apoptosis or causing autoimmune diseases, allergic diseases and diseases associated with viral infections.

In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can be determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds a target (which may be an epitope) is an antibody that binds that target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). For example, in the present application, the isolated antigen binding protein is capable of binding to human-derived Tim-3 with a KD value of 10nM or less. As another example, the isolated antigen binding protein is capable of specifically binding to human Tim-3 but not to mouse Tim-3. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

In the present application, the term "inhibition" generally refers to a reduction in the growth rate of cells or the number of cells. For example, an isolated antigen binding protein described herein, which is capable of inhibiting tumor growth and/or tumor cell proliferation. For another example, an isolated antigen binding protein described herein can inhibit the binding of Tim-3 to PtdSer.

In the present application, the term "IFN- γ" is a type of Interferon (IFN), which is a type II interferon and binds to type II interferon antibodies. IFN- γ regulates a variety of biological functions such as antiviral response, cell growth, immune response, and tumor suppression.

In this application, the term "TNF- α" generally refers to tumor necrosis factor α (also known as cachectin), a naturally occurring mammalian cytokine produced by many cell types, including monocytes and macrophages in response to endotoxin or other stimuli. TNF- α is the major mediator of inflammatory, immunological and pathophysiological responses (Grell, M. et al, (1995) Cell [ Cell ], 83: 793-802). In the present application, TNF- α may include wild-type TNF- α from various species (e.g., human, mouse and monkey), polymorphic variants of TNF- α, and functional equivalents of TNF- α.

In the present application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor may be a solid tumor or a hematological tumor.

In the present application, the term "variable domain" generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain an antigen binding site.

In the present application, the term "variable" generally refers to the fact that certain segments of the variable domains differ greatly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection, and in some cases form part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F (ab')₂Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function (e.g., specifically bind to Tim-3). Typically, such fragments should include an antigen binding domain. The basic 4 chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 elementary heterotetramer units with another polypeptide called the J chain and contain 10 antigen binding sites, while IgA antibodies comprise 2-5 elementary 4 chain units that can aggregate in association with the J chain to form multivalent combinations. For IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for the alpha and gamma chains, respectively, and four CH domains for the mu and epsilon isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VHAnd CL corresponds to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The VH and VL pair together to form a single antigen-binding site. For the structure and properties of antibodies of different classes see, e.g., Basic and Clinical Immunology,8th Edition, Daniel P.Sties, Abba I.Terr and Tristram G.Parsolw (eds), Appleton&Lange, Norwalk, conn.,1994, page 71 and chapter 6. L chains from any vertebrate species can be classified into one of two distinctly different classes, termed κ and λ, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of its heavy Chain (CH) constant domain, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK 1.

In the present application, the term "CDR" generally refers to a region of an antibody variable domain whose sequence is highly variable and/or forms a structurally defined loop. Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR 3). In natural antibodies, HCDR3 and LCDR3 show the majority of diversity of the six CDRs, and in particular HCDR3 is thought to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al, Immunity 13:37-45 (2000); johnson and Wu, in Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camel antibodies consisting of only heavy chains function normally and are stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996).

In this application, the term "FR" generally refers to a more highly conserved portion of an antibody variable domain, which is referred to as the framework region. Typically, the variable domains of native heavy and light chains each comprise four FR regions, namely four in the VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in the VL (L-FR1, L-FR2, L-FR3, and L-FR 4). For example, the VL of an isolated antigen binding protein described herein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4. The VH of an isolated antigen binding protein described herein may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In the present application, the term "antigen-binding fragment" generally refers to a fragment having antigen-binding activity. In the present application, the antigen binding fragments may include Fab, Fab', F (ab)₂Fv fragment, F (ab')₂scFv, di-scFv and/or dAb.

In this application, the term "competitive binding" generally refers to the ability of an antibody or fragment thereof to interfere with the ability of another antibody to bind, directly or indirectly, a target/antigen (e.g., Tim-3) by allosteric modulation of the other antibody (e.g., a reference antibody). For example, in the present application, the isolated antigen binding protein can compete with a reference antibody for binding to Tim-3. Furthermore, the extent to which an antibody or fragment thereof is able to interfere with the binding of another antibody or fragment thereof to a target, and therefore whether it can be considered blocking or competing according to the present invention, can be determined using competitive binding assays. One particularly suitable quantitative competition assay measures competition between a labeled (e.g., His-tagged, biotinylated, or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof for binding to a target using FACS-based or AlphaScreen-based methods. Typically, the competing antibody or fragment thereof is, for example, one of the following: binding of the target in a competition assay such that the recorded substitutions of the isolated antigen binding protein of the invention reach up to 100% of the maximum theoretical substitution (e.g., substitution by cold (e.g., unlabeled) antibody or fragment thereof that needs to be blocked) by the detected potential blocking antibody or fragment thereof present in a given amount during the assay and in the presence of a second antibody or fragment thereof (e.g., in a FACS-based competition assay). Preferably, the competing antibody or fragment thereof has a recorded substitution of between 10% and 100%, such as between 50% and 100%.

In this application, the term "directly connected" is used in contrast to the term "indirectly connected," which generally refers to a direct connection. For example, the direct linkage may be a direct linkage without a spacer between the substances. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirectly linked" generally refers to a condition in which the substances are not directly linked to each other. For example, the indirect connection may be a connection via a spacer. For example, in the isolated antigen binding proteins described herein, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be linked directly or indirectly.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been the recipient of a subject plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may comprise cells transfected in vitro with a vector of the invention. The cell may be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell.

In the present application, the term "pharmaceutical composition" generally refers to a composition that is suitable for administration to a patient, preferably a human patient. For example, a pharmaceutical composition described herein, which can comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

In this application, the term "Tim-3 associated disease" generally refers to a disease associated with cells expressing Tim-3. Such as cancer, autoimmune diseases and allergic diseases. In the present application, the Tim-3 related disease may be a tumor and/or leukemia.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

Isolated antigen binding proteins

In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO. 16 and at least one CDR in a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 15.

For example, in the present application, the isolated antigen binding protein may comprise HCDR1 in the VH shown in SEQ ID NO: 16. For example, in the present application, the isolated antigen binding protein may comprise HCDR2 in the VH shown in SEQ ID NO: 16. For example, in the present application, the isolated antigen binding protein may comprise HCDR3 in the VH shown in SEQ ID NO: 16. As another example, in the present application, the isolated antigen binding protein may comprise LCDR1 in VL having the amino acid sequence set forth in SEQ ID NO. 15. For example, in the present application, the isolated antigen binding protein may comprise LCDR2 in VL having the amino acid sequence set forth in SEQ ID NO. 15. For example, in the present application, the isolated antigen binding protein may comprise LCDR3 in VL having the amino acid sequence set forth in SEQ ID NO. 15.

In the present application, the isolated antigen binding protein further comprises at least one CDR in the heavy chain variable region VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 16, and at least one CDR in the light chain variable region VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 15. In the present application, the isolated antigen binding protein has Tim-3 binding capacity.

In the present application, the isolated antigen binding protein may bind to an epitope within a fragment of Tim-3 that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID No. 25.

Properties of the isolated antigen binding protein

In the present application, the isolated antigen binding protein may have one or more of the following properties:

1) is capable of binding to human-derived Tim-3 with a KD value of 10nM or less, wherein said KD value is determined by surface plasmon resonance;

2) in a FACS assay, capable of specifically binding to human Tim-3 but not to mouse Tim-3;

3) in an ELISA assay, binding of Tim-3 to PtdSer can be inhibited; and

4) promote the secretion of IFN-gamma and/or TNF-alpha.

In the present application, the isolated antigen binding protein is capable of binding to human-derived Tim-3 with a KD value of 10nM or less, wherein the KD value can be determined by surface plasmon resonance. For example, an isolated antigen binding protein described herein binds to a value of.i.m-3 from a human being of less than or equal to 10nM, of less than or equal to 9.5nM, of less than or equal to 9nM, of less than or equal to 8.9nM, of less than or equal to 8.8nM, of less than or equal to 8.6nM, of less than or equal to 8.5nM, of less than or equal to 8.3nM, of less than or equal to 8.2nM, of less than or equal to 8.1nM, of less than or equal to 8nM, of less than or equal to 7.9nM, of less than or equal to 7.8nM, of less than or equal to 7.7nM, of less than or equal to 7.6nM, of less than or equal to 7.5nM, of less than or equal to 7.3nM, of less than or equal to 7.2, of less than or equal to 7.1nM, of less than or equal to 6.9nM, of less than or equal to 2nM, of less than or equal to 2.5nM, of less than or equal to 2nM, of less than or equal to 8.4.5 nM, of less than or equal to 2.3nM, of less than or equal to 2nM, of less than or equal to 8.7.7.7.7.7.7.7.7.7.7.7 nM, of less than or equal to 2nM, of less than or equal to 2.7.7.7.7.7.7.7.7.7.9 nM, of less than or equal to 2nM, e.5 nM, of less than or equal to 2nM, e, less than or equal to 2nM, e.7.5 nM, e, of less than or equal to 2nM, e, e.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7 nM, e, e.7.7.7.7.7.7.7.7.7.7.7 nM, e, less than or equal to 2nM, e, of less than or equal to 2nM, e, e.7.7.7.7.7.7.7.7.7.7.7.7.7.9, e, less than or equal to 2nM, e.

In the present application, the KD values can also be determined by FACS, ELISA, competition ELISA or BIACORE or KINEXA.

In the present application, the isolated antigen binding protein is capable of specifically binding to human Tim-3 and not to mouse Tim-3, as can be determined by FACS. For example, specific binding of an isolated antigen binding protein described herein to human Tim-3 can be reflected by a half maximal effect concentration in FACS assays (EC50), e.g., a lower half maximal effect concentration (EC50) indicates better specific binding.

In the present application, the human Tim-3 may comprise an amino acid sequence shown as SEQ ID NO. 25 and the mouse Tim-3 may comprise an amino acid sequence shown as SEQ ID NO. 26.

In the present application, the isolated antigen binding protein is capable of inhibiting the binding of Tim-3 to PtdSer, which inhibition can be measured by flow cytometry.

In the present application, the isolated antigen binding protein is capable of promoting secretion of IFN- γ and/or TNF- α. For example, the isolated antigen binding proteins described herein increase the amount of IFN- γ and/or TNF- α.

The class of isolated antigen binding proteins

In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof. For example, an isolated antigen binding protein described herein can include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, triabodies, tetrabodies, Fv fragments, scFv fragments, Fab 'fragments, F (ab')2 fragments, and camelized single domain antibodies.

In the present application, the antibody may comprise a murine antibody. For example, the murine antibody can be prepared by injecting a test subject (e.g., a mouse) with Tim-3 and then isolating a hybridoma that expresses the antibody with the desired sequence or functional property. In certain embodiments, the murine antibody, or antigen binding fragment thereof, may further comprise a light chain constant region of a murine kappa, lambda chain, or variant thereof, or a heavy chain constant region of a murine IgGl, IgG2, IgG3, or IgG4, or variant thereof.

In the present application, the antibody may be a humanized antibody. In other words, an isolated antigen binding protein described herein, which can be an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest (e.g., human Tim-3) and comprises Framework (FR) regions having substantially the amino acid sequence of a human antibody and Complementarity Determining Regions (CDRs) having substantially the amino acid sequence of a non-human antibody. By "substantially" in the context of a CDR is meant that the amino acid sequence of the CDR is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a CDR of a non-human antibody. The humanized antibody can comprise substantially all of at least one and typically two variable domains (Fab, Fab ', F (ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., antibody) and all or substantially all of the framework regions are framework regions having human immunoglobulin consensus sequences. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least a variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only humanized light chains. In some embodiments, the humanized antibody contains only humanized heavy chains. In particular embodiments, the humanized antibody contains only the humanized variable domains of the light chain and/or the humanized heavy chain.

In the present application, the antigen binding fragments may include Fab, Fab', F (ab)₂Fv fragment, F (ab')₂scFv, di-scFv and/or dAb.

Reference antibody

In the present application, the isolated antigen binding protein may compete with a reference antibody for binding to the Tim-3 protein, wherein the reference antibody may comprise a light chain variable region and a heavy chain variable region, the light chain variable region of the reference antibody may comprise LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 1; the LCDR2 can comprise an amino acid sequence shown as SEQ ID NO. 2; the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID No. 4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 5; the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 6.

CDR

In the present application, the CDR Sequences in the VH and VL Sequences can be determined according to the Kabat definition or Chothia definition for the variable region Sequences of the isolated antigen binding Proteins (see, e.g., Kabat, "Sequences of Proteins of immunological interest", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et., J.mol.biol.273: 927-.

In the present application, with respect to the variable region sequences of the isolated antigen binding proteins (i.e., the sequences of the VH or VL), the CDR region sequences in the VH and VL sequences can also be determined according to the combinatorial definition rules, which include the Kabat definition and the Chothia definition.

In the present application, the VH may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 6.

In the present application, the HCDR3 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 6.

In the present application, the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO. 4.

In the present application, the HCDR1 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 4.

In the present application, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 5.

In the present application, the HCDR2 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 5.

For example, the HCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 4, the HCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 5, and the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 6.

In the present application, the VL may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 1.

In the present application, the LCDR1 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 1.

In the present application, the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 2.

In the present application, the LCDR2 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 2.

In the present application, the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

In the present application, the LCDR3 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 3.

For example, the LCDR1 of the isolated antigen binding proteins described herein can comprise the amino acid sequence shown in SEQ ID NO. 1, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 2, and LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

As another example, the HCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 4, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 5, HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6, and LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 1, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 2, and LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3.

FR

In the present application, the VL of the isolated antigen binding protein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

In the present application, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 7.

In the present application, the L-FR1 may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO. 7.

For example, the C-terminus of the L-FR1 may be linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 7.

In the present application, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 8.

In the present application, the L-FR2 may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO. 8.

For example, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 8.

In the present application, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 9.

In the present application, the L-FR3 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO. 9.

For example, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 9.

In the present application, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 10.

In the present application, the L-FR4 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO. 10.

For example, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 10.

As another example, L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 7, L-FR2 can comprise the amino acid sequence shown as SEQ ID NO. 8, L-FR3 can comprise the amino acid sequence shown as SEQ ID NO. 9, and L-FR4 can comprise the amino acid sequence shown as SEQ ID NO. 10.

In the present application, the VH of the isolated antigen binding protein may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In the present application, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 11.

In the present application, the H-FR1 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO. 11.

For example, the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 11.

In the present application, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 12.

In the present application, the H-FR2 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO. 12.

For example, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 12.

In the present application, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 13.

In the present application, the H-FR3 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO. 13.

For example, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO 13.

In the present application, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 14.

In the present application, the H-FR4 may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO. 14.

For example, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 14.

For example, H-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 11, H-FR2 can comprise the amino acid sequence shown as SEQ ID NO. 12, H-FR3 can comprise the amino acid sequence shown as SEQ ID NO. 13, and H-FR4 can comprise the amino acid sequence shown as SEQ ID NO. 14.

As another example, L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 7, L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 8, L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 9, L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 10, and H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO. 11, H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 12, H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 13, and H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 14.

VL and VH

The isolated antigen binding proteins described herein can comprise an antibody light chain variable region VL and an antibody heavy chain variable region VH. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 15 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 16.

For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 15 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 16.

Light and heavy chains

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human Ig kappa constant region.

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human Ig λ constant region.

In the present application, the antibody light chain constant region may comprise the amino acid sequence shown in SEQ ID NO 17.

In the present application, the antibody light chain constant region may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO. 17.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG2 heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG3 heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG4 heavy chain constant region.

In the present application, the antibody heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO 18.

In the present application, the antibody heavy chain constant region may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO. 18.

In the present application, the isolated antigen binding protein may comprise an antibody light chain LC, and the LC may comprise the amino acid sequence shown in SEQ ID NO 19.

In the present application, the LC may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 19.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain HC, and the HC may comprise the amino acid sequence shown in SEQ ID No. 20.

In the present application, the HC may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 20.

An isolated antigen binding protein described herein can comprise an antibody light chain and an antibody heavy chain.

For example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 19 and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 20.

In the present application, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 19, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 20. Wherein the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 4, the HCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 5, the HCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 6, and the LCDR1 may comprise the amino acid sequence shown as SEQ ID NO. 1, the LCDR2 may comprise the amino acid sequence shown as SEQ ID NO. 2, and the LCDR3 may comprise the amino acid sequence shown as SEQ ID NO. 3. Wherein L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 7, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 8, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 9, L-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 10, and H-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 11, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 12, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 13, and H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 14. The VL may comprise the amino acid sequence shown in SEQ ID NO. 15 and the VH may comprise the amino acid sequence shown in SEQ ID NO. 16. For example, the isolated antigen binding protein may be Z2.

Nucleic acid molecules, vectors, cells, methods of preparation and pharmaceutical compositions

In another aspect, the present application also provides an isolated nucleic acid molecule or molecules that can encode an isolated antigen binding protein described herein. The isolated nucleic acid molecule or molecules described herein can be an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized, but can encode an isolated antigen binding protein described herein. Furthermore, nucleic acid molecules encoding the same isolated antigen binding protein described herein are not unique in view of the degeneracy of two or more codons, i.e., codons, for the same amino acid.

In another aspect, the present application also provides a vector, which may comprise a nucleic acid molecule described herein, or, express an antigen binding protein described herein. The vector may be used to express the genetic material element carried by the vector in a host cell by transformation, transduction or transfection of the host cell. For example, the carrier may include: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). As another example, the vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. In addition, the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In another aspect, the present application also provides a cell, which may comprise a nucleic acid molecule described herein or a vector described herein. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. In certain embodiments, the cell may further comprise a cell transfected in vitro with a vector of the invention. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell may be a mammalian cell. In certain embodiments, the mammalian cell can be a HEK293 cell. The method for purifying and separating the protein may be salting out method, isoelectric precipitation method, low temperature organic solvent precipitation method, dialysis and ultrafiltration, gel filtration method, electrophoresis method, ion exchange chromatography or affinity chromatography.

In another aspect, the present application also provides methods of making an isolated antigen binding protein described herein, which methods can include culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In certain embodiments, the isolated antigen binding proteins described herein can be expressed by induction of a foreign gene (e.g., IPTG induction).

In another aspect, the present application also provides a pharmaceutical composition that can comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may further comprise one or more (safe and effective amounts of) pharmaceutically acceptable carriers, such as suitable formulations of stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

In certain embodiments, the pharmaceutical composition may comprise a parenteral and/or parenteral route of administration, such as subcutaneous, transdermal, intracavity, intravenous, intraarterial, intrathecal, intratumoral, intraperitoneal, and/or intranasal administration or direct injection into a tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flow into the patient, as described in WO 2015/036583.

Use and application

In another aspect, the present application also provides the use of an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein, and/or a pharmaceutical composition as described herein, in the manufacture of a medicament for the prevention, amelioration and/or treatment of a tumor.

In another aspect, the present application also provides the use of an isolated antigen binding protein as described herein, a nucleic acid molecule as described herein, a vector as described herein, a cell as described herein, and/or a pharmaceutical composition as described herein, in the manufacture of a medicament for the prevention, amelioration, and/or treatment of a Tim-3 associated disease.

In another aspect, the present application also provides methods of preventing, ameliorating, and/or treating a tumor, which can comprise administering to a subject in need thereof an isolated antigen binding protein described herein. In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the present application also provides methods of preventing, ameliorating, and/or treating a Tim-3 associated disease, which can comprise administering to a subject in need thereof an isolated antigen binding protein described herein. In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and/or the pharmaceutical composition described herein can be used for preventing, ameliorating, or treating a tumor.

In another aspect, the isolated antigen binding proteins described herein, the nucleic acid molecules described herein, the vectors described herein, the cells described herein, and/or the pharmaceutical compositions described herein can be used to prevent, ameliorate, or treat a Tim-3 associated disease.

In the present application, the tumor may comprise a solid tumor and/or a hematological tumor. For example, the tumor may be a leukemia.

In the present application, the Tim-3 related disease may include a disease associated with a cell expressing Tim-3. Such as cancer, autoimmune diseases and allergic diseases. In the present application, the Tim-3 related disease may be leukemia and/or tumor.

In the present application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In another aspect, the present application also provides a method of inhibiting the binding of Tim-3 to PtdSer comprising administering an isolated antigen binding protein described herein. In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In certain embodiments, the isolated antigen binding proteins described herein can also be administered to a subject in need thereof with one or more effective amounts of a therapeutic agent, which can include a chemotherapeutic agent, a cytotoxic agent, an immunosuppressive agent, a steroid, an antiemetic, a cancer vaccine, an analgesic, or another antibody.

In certain embodiments, the isolated antigen binding proteins described herein can also be fused to a toxin to produce an immunoconjugate, thereby exerting cytotoxic activity on cells expressing Tim-3 protein upon specific binding to the cells, specifically killing cancer cells.

In certain embodiments, the isolated antigen binding proteins described herein can also be formulated as oncolytic viruses, such that invasion into tumor cells by cell surface molecules, and then targeting specific receptors that are overexpressed in tumor cells, allows viral invasion into tumor cells and subsequent functions.

In certain embodiments, the isolated antigen binding proteins described herein can also be fused to antibodies capable of specifically binding to other antigens (i.e., in addition to Tim-3) to create bispecific antibodies, thereby specifically binding to two different antigens simultaneously for better tumor therapy.

The isolated antigen binding proteins described herein have at least one of the following beneficial effects:

1) the isolated antigen binding proteins described herein are capable of binding to human-derived Tim-3 with a KD value of 10nM or less, wherein the KD value is determined by surface plasmon resonance;

2) in FACS assays, the isolated antigen binding proteins described herein are capable of specifically binding to human Tim-3 and not to mouse Tim-3, and thus are species-specific;

3) in an ELISA assay, the isolated antigen binding protein described herein can inhibit the binding of Tim-3 to PtdSer, blocking the Tim-3 signaling pathway, and thereby inhibiting tumor cell growth;

4) the isolated antigen binding proteins described herein can promote secretion of IFN- γ and/or TNF- α, thereby producing immune stimulation and inhibiting or eliminating tumors.

Without wishing to be bound by any theory, the following examples are only intended to illustrate the protein molecules, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells.

Examples

Example 1 Structure of the isolated antigen binding protein Z2 described herein

The light chain of the isolated antigen binding protein Z2 described herein comprises the amino acid sequence shown in SEQ ID NO 19 and the heavy chain comprises the amino acid sequence shown in SEQ ID NO 20. Wherein the HCDR1 of the isolated antigen binding protein Z2 comprises the amino acid sequence shown as SEQ ID NO. 4, the HCDR2 comprises the amino acid sequence shown as SEQ ID NO. 5, the HCDR3 comprises the amino acid sequence shown as SEQ ID NO. 6, and the LCDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, the LCDR2 comprises the amino acid sequence shown as SEQ ID NO. 2, and the LCDR3 comprises the amino acid sequence shown as SEQ ID NO. 3. Furthermore, L-FR1 of the isolated antigen binding protein Z2 comprises the amino acid sequence shown by SEQ ID NO. 7, L-FR2 comprises the amino acid sequence shown by SEQ ID NO. 8, L-FR3 comprises the amino acid sequence shown by SEQ ID NO. 9, L-FR4 comprises the amino acid sequence shown by SEQ ID NO. 10, and H-FR1 comprises the amino acid sequence shown by SEQ ID NO. 11, H-FR2 comprises the amino acid sequence shown by SEQ ID NO. 12, H-FR3 comprises the amino acid sequence shown by SEQ ID NO. 13, and H-FR4 comprises the amino acid sequence shown by SEQ ID NO. 14. Furthermore, the VL of the isolated antigen binding protein Z2 comprises the amino acid sequence shown in SEQ ID NO. 15 and the VH comprises the amino acid sequence shown in SEQ ID NO. 16.

The nucleotide sequence encoding the VL of the isolated antigen binding protein Z2 described herein is shown in SEQ ID NO 21, the nucleotide sequence encoding the VH of the isolated antigen binding protein Z2 described herein is shown in SEQ ID NO 22, the nucleotide sequence encoding the light chain constant region of the isolated antigen binding protein Z2 described herein is shown in SEQ ID NO 23, and the nucleotide sequence encoding the heavy chain constant region of the isolated antigen binding protein Z2 described herein is shown in SEQ ID NO 24.

Example 2. binding affinity assay of the isolated antigen binding protein Z2 described herein to human Tim-3

2.1 coupling of Protein A

Protein A (Thermo Fisher #21181) was coupled to 4 channels of CM5 at a flow rate of 10. mu.l/min using 1 XHBS-EP + (GE Healthcare # BR-1006-69) as running buffer: 1) set the injection time at 800s, 50mM NHS and 200mM EDC at 1: 1 volume ratio, and injecting the mixture into 4 channels; 2) diluting Protein A to 20 μ g/ml with sodium acetate of pH4.5, injecting for 800 s; 3) 1M ethanolamine 800s is injected to block the residual active carboxyl on the surface of the chip. After blocking, the instrument was equilibrated for two hours with 1 XHBS-EP + buffer and the final amount of Protein A coupled was about 2000 RU.

2.2 kinetic testing

A multi-cycle kinetic pattern is set up, each cycle comprising capture of the antibody, binding of the analyte and regeneration of the chip. Each antibody was diluted to 1. mu.g/ml, injected into 2, 3, 4 channels 40s at a flow rate of 10. mu.l/min, and captured by the pre-coupled Protein A in an amount of about 200 RU. Human Tim-3-hIg Fc fusion protein (i.e., human Tim-3 protein, R & D systems, USA, Cat. No. 2365-TM-050) was injected into four channels in the order of 0nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM, and 40nM concentration gradient with a flow rate of 30ul/min, an injection time of 180s, and a dissociation time of 900s, respectively. Wherein, the amino acid sequence of the human Tim-3 protein is shown as SEQ ID NO. 25. Finally, glycine (10mM, pH1.5) was injected at the same flow rate for 30s to regenerate the chip.

2.3 data analysis

The experimental results were analyzed with Biacore T200 analysis software 2.0, with channel 1 subtracted as the reference channel, and the analytical model selected from the group consisting of 1: 1 kinetic fitting model.

The results are shown in Table 1. Experimental results show that the isolated antigen binding protein Z2 has better binding with human Tim-3.

TABLE 1 binding affinities of antigen binding protein Z2 to human Tim-3

Antigen binding proteins	K assoc(1/Ms)	K dissoc(1/s)	KD(nM)	Isoform (Isotype)
Z2	3.1×10 4	8.7×10 -5	2.7	IgG2b,κ

In Table 1, K_assoc: an association rate constant; k_dissoc: an off rate constant; KD: affinity constant, equal to K_dissoc/K _assoc。

Example 3 binding assays of the isolated antigen binding protein Z2 described herein to cell surface Tim-3

The cell surface target antigen (Tim-3, including human-derived Tim-3 and mouse-derived Tim-3) was detected for binding affinity to an antibody (i.e., the isolated antigen-binding protein Z2 described herein or a comparative antibody) using a flow cytometric fluorescence sorting technique (FACS) using an iQue Screen flow instrument (available from IntelliCyt) using PBS containing 0.1% BSA as a buffer, wherein the amino acid sequence of human Tim-3 is shown in SEQ ID NO:25 and the amino acid sequence of mouse Tim-3 is shown in SEQ ID NO: 26. The control antibody was represented by Anti-mTim-3Ab, a commercial antibody (Rat IgG2a Clone #215008, from R & D). The specific detection process is as follows:

1. buffer was used to make up concentrations of 1x 10⁶cells/ml of target cells (i.e., transgenic K562 cells expressing human Tim-3, or transgenic K562 cells expressing mouse Tim-3) were added to a 96-well tip-and-bottom plate (corning 3894) at 30. mu.l per well;

2. using buffer solution to prepare detection antibody with the concentration of 3 mug/ml, and diluting the antibody according to 3 times of ratio to form 8 concentration gradients;

3. adding the prepared antibodies with different concentrations into the paved target cells according to 30 mul/hole and mixing evenly;

incubating for 1 hour at 4.4 ℃ in a refrigerator;

5. adding 150 mul of buffer solution into each hole, centrifuging for 5 minutes at 300g, removing supernatant, and shaking loose cells;

6. repeating the step 5;

7. preparing a fluorescent secondary antibody (ab98593) by using a buffer solution according to a ratio of 1:200, adding 30 mu l of the fluorescent secondary antibody into each hole, uniformly mixing, and incubating for 30 minutes in a 4-degree refrigerator;

8. adding 150 mul of buffer solution into each hole, centrifuging for 5 minutes at 300g, removing supernatant, and shaking loose cells;

9. repeating the step 8;

10. adding 35 mul of buffer solution into each hole, mixing uniformly, and detecting by using a flow type instrument;

11. data were analyzed using Graphpad software.

The results of the flow affinity binding assays are shown in FIGS. 1-3, where FIG. 1 shows the binding of the isolated antigen binding protein Z2 described herein to a cell line expressing human Tim-3, FIG. 2 shows the binding of the isolated antigen binding protein Z2 described herein to a cell line expressing mouse Tim-3, and FIG. 3 shows the binding of the comparative antibody to a cell line expressing mouse Tim-3. Furthermore, IgG in fig. 1-3 represents the experimental isotype control antibody Mouse IgG2b, which is a commercial antibody (Mouse IgG2b Clone #133303, available from R & D).

As can be seen from fig. 1-3, antigen binding protein Z2 has the biological activity of specifically binding to human Tim-3.

Example 4 the isolated antigen binding protein Z2 described herein blocks the detection of binding of human Tim-3 to PtdSer

Phosphatidylserine (PtdSer) is another ligand of Tim-3, usually exposed on the surface of apoptotic cell membranes, binds to the Tim-3IgV domain, mediates apoptotic cell phagocytosis, promotes apoptotic body clearance and Dendritic Cell (DCs) antigen cross-presentation. Jurkat cells were diluted to 5X 10 in this application⁵Cells/ml and 5. mu.M Camptothecin were added and co-cultured at 37 ℃ for 5 hours to induce apoptosis and express phosphatidylserine. Thereafter, wash 2 times with PBS plus 2% FSA. Finally, apoptotic Jurkat cells were plated at 1X 10 per well⁵One was added to a 96-well plate. The isolated antigen binding protein Z2 described herein and the isotype control antibody Mouse IgG2b (Mouse IgG2b Clone #133303, purchased from R)&D) Diluted to 20. mu.g/ml with PBS, or Tim-3-hIg Fc fusion protein (i.e., human Tim-3 protein, U.S. R)&D systems, cat # 2365-TM-050) was diluted to 4. mu.g/ml with PBS and the cells were co-cultured with Tim-3-hIg Fc fusion protein alone or Z2 and Tim-3-hIg Fc fusion protein, respectively, for 30 minutes at room temperature. After incubation, centrifugation was performed and washed 2 times with PBS plus 2% FSA. Adding a second antibody APC-Conjugated anti-hIgG Fc Ab (R)&System D, # FAB110A)100 μ l, washed 3 times and tested on flow cytometry. The results are shown in FIGS. 4A-4C, and it can be seen from FIGS. 4A-4C that only in the presence of the isolated antigen binding protein Z2 described hereinIn addition, the corresponding receptor phosphatidylserine (PtdSer) expressed by apoptotic cells was not detected by Tim-3-hIg Fc fusion protein (i.e., human Tim-3 protein), whereas the isotype control mouse antibody did not. Thus, the isolated antigen binding protein Z2 described herein can block the binding of human Tim-3 to PtdSer.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims (42)

1. An isolated antigen binding protein comprising at least one CDR from a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO. 16 and at least one CDR from a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 15.
2. The isolated antigen binding protein of claim 1, having Tim-3 binding capability.
3. The isolated antigen binding protein of any one of claims 1-2, having one or more of the following properties:

   1) is capable of binding to human-derived Tim-3 with a KD value of 10nM or less, wherein said KD value is determined by surface plasmon resonance;

   2) in a FACS assay, capable of specifically binding to human Tim-3 but not to mouse Tim-3;

   3) in an ELISA assay, binding of Tim-3 to PtdSer can be inhibited; and

   4) promote the secretion of IFN-gamma and/or TNF-alpha.
4. The isolated antigen binding protein of any one of claims 1-3, comprising an antibody or antigen binding fragment thereof.
5. The isolated antigen binding protein of claim 4, wherein the antibody comprises humanized and murine antibodies.
6. The isolated antigen binding protein of any of claims 4-5, wherein the antigen binding fragment comprises a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv, and/or dAb.
7. The isolated antigen binding protein of any of claims 1-6, wherein the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO 6.
8. The isolated antigen binding protein of claim 7, wherein said HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 4.
9. The isolated antigen binding protein of any of claims 7-8, wherein the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO 5.
10. The isolated antigen binding protein of any of claims 1-9, wherein the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 1.
11. The isolated antigen binding protein of claim 10, wherein the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO 2.
12. The isolated antigen binding protein of any of claims 10-11, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID No. 3.
13. The isolated antigen binding protein of any one of claims 1-12, which competes for binding to the Tim-3 protein with a reference antibody, wherein the reference antibody comprises a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID No. 1; the LCDR2 comprises an amino acid sequence shown in SEQ ID NO. 2; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 3, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO. 4; the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 5; the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 6.
14. The isolated antigen binding protein of any of claims 1-13, wherein the VL comprises the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.
15. The isolated antigen binding protein of claim 14, wherein the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1 and L-FR1 comprises the amino acid sequence set forth in SEQ ID No. 7.
16. The isolated antigen binding protein of any of claims 14-15, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence set forth in SEQ ID No. 8.
17. The isolated antigen binding protein of any of claims 14-16, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence set forth in SEQ ID No. 9.
18. The isolated antigen binding protein of any of claims 14-17, wherein the N-terminus of L-FR4 is linked directly or indirectly to the C-terminus of the LCDR3 and the L-FR4 comprises the amino acid sequence set forth in SEQ ID No. 10.
19. The isolated antigen binding protein of any of claims 1-18, wherein said VL comprises the amino acid sequence set forth in SEQ ID No. 15.
20. The isolated antigen binding protein of any one of claims 1-19, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human Ig kappa constant region.
21. The isolated antigen binding protein of claim 20, wherein the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 17.
22. The isolated antigen binding protein of any of claims 1-21, comprising an antibody light chain LC, and the LC comprises the amino acid sequence set forth in SEQ ID No. 19.
23. The isolated antigen binding protein of any of claims 1-22, wherein said VH comprises the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.
24. The isolated antigen binding protein of claim 23, wherein the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1 and the H-FR1 comprises the amino acid sequence set forth in SEQ ID No. 11.
25. The isolated antigen binding protein of any of claims 23-24, wherein said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence set forth in SEQ ID No. 12.
26. The isolated antigen binding protein of any of claims 23-25, wherein said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence set forth in SEQ ID No. 13.
27. The isolated antigen binding protein of any of claims 23-26, wherein the N-terminus of H-FR4 is linked directly or indirectly to the C-terminus of HCDR3 and the H-FR4 comprises the amino acid sequence set forth in SEQ ID No. 14.
28. The isolated antigen binding protein of any of claims 1-27, wherein said VH comprises the amino acid sequence set forth in SEQ ID No. 16.
29. The isolated antigen binding protein of any one of claims 1-28, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.
30. The isolated antigen binding protein of claim 29, wherein the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 18.
31. The isolated antigen binding protein of any of claims 1-30, comprising an antibody heavy chain HC, and the HC comprises the amino acid sequence set forth in SEQ ID No. 20.
32. An isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-31.
33. A vector comprising the nucleic acid molecule of claim 32, or expressing the antigen binding protein of any one of claims 1-31.
34. A cell comprising the nucleic acid molecule of claim 32 or the vector of claim 33.
35. A method of making the isolated antigen binding protein of any one of claims 1-31, the method comprising culturing the cell of claim 34 under conditions such that the isolated antigen binding protein of any one of claims 1-31 is expressed.
36. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-31, the nucleic acid molecule of claim 32, the vector of claim 33, and/or the cell of claim 34, and optionally a pharmaceutically acceptable carrier.
37. Use of the isolated antigen binding protein of any one of claims 1-31, the nucleic acid molecule of claim 32, the vector of claim 33, the cell of claim 34, and/or the pharmaceutical composition of claim 36 in the manufacture of a medicament for the prevention, amelioration, and/or treatment of a tumor.
38. The use of claim 37, wherein the tumor comprises a solid tumor and/or a hematological tumor.
39. Use of the isolated antigen binding protein of any one of claims 1-31, the nucleic acid molecule of claim 32, the vector of claim 33, the cell of claim 34, and/or the pharmaceutical composition of claim 36 in the manufacture of a medicament for preventing, ameliorating, and/or treating a Tim-3 associated disease.
40. A method of inhibiting Tim-3 binding to PtdSer comprising administering the isolated antigen binding protein of any one of claims 1-31.
41. A method of preventing, ameliorating and/or treating a tumor comprising administering to a subject in need thereof the isolated antigen binding protein of any one of claims 1-31 or the pharmaceutical composition of claim 36.
42. The method of claim 41, wherein the tumor comprises a solid tumor and/or a hematological tumor.

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