WO2021051352A1 - 一种分离的抗原结合蛋白及其用途 - Google Patents

一种分离的抗原结合蛋白及其用途 Download PDF

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WO2021051352A1
WO2021051352A1 PCT/CN2019/106694 CN2019106694W WO2021051352A1 WO 2021051352 A1 WO2021051352 A1 WO 2021051352A1 CN 2019106694 W CN2019106694 W CN 2019106694W WO 2021051352 A1 WO2021051352 A1 WO 2021051352A1
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binding protein
antigen binding
amino acid
seq
acid sequence
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PCT/CN2019/106694
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English (en)
French (fr)
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张骅
华坚
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上药生物治疗（香港）有限公司
上海医药集团生物治疗技术有限公司
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Priority to CN201980082804.1A priority Critical patent/CN113164601B/zh
Priority to PCT/CN2019/106694 priority patent/WO2021051352A1/zh
Publication of WO2021051352A1 publication Critical patent/WO2021051352A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • This application relates to the field of biomedicine, in particular to an isolated antigen binding protein and its use.
  • Tumor is a disease that seriously threatens human health.
  • immunotherapy as a new therapy has shown great potential in tumor treatment.
  • T cell immunoglobulin and mucin molecule 3 are important immune checkpoint molecules discovered in recent years. They are different from other "immune checkpoint" molecules. They mainly induce sexually expressed on the surface of T cells in inflammation and tumor microenvironment. It is a type I membrane surface molecule, which is highly expressed in Th1 cells and generates inhibitory signals to cause the apoptosis of Th1 cells.
  • Gal-9 galectin 9
  • PtdSer phosphatidylserine
  • CEACAM1 carcino-embryonic antigen-related cellular adhesion molecule 1
  • selective activation of Tim-3 leading to immune escape is the main mechanism of drug resistance during PD-1/PD-L1 antibody immunotherapy.
  • the present application provides an isolated antigen binding protein, which includes at least one CDR in a heavy chain variable region VH and at least one CDR in a light chain variable region VL, wherein the VH comprises SEQ ID NO: 16 As shown in the amino acid sequence, the VL includes the amino acid sequence shown in SEQ ID NO: 15.
  • the isolated antigen binding protein described in this application has Tim-3 binding ability.
  • the isolated antigen binding protein described in this application has one or more of the following properties:
  • Tim-3 derived from human with a KD value of 10 nM or lower, wherein the KD value is measured by a surface plasmon resonance method
  • the isolated antigen-binding protein according to any one of claims 1-3, which comprises an antibody or an antigen-binding fragment thereof.
  • the antibodies include humanized and murine antibodies.
  • the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:4.
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:5.
  • the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:2.
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 3.
  • the isolated antigen-binding protein described in this application competes with a reference antibody for binding to the Tim-3 protein, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, so
  • the light chain variable region of the reference antibody includes LCDR1, LCDR2, and LCDR3, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 1;
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 2;
  • the LCDR3 It 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
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4;
  • the HCDR2 comprises SEQ The amino acid sequence shown in ID NO: 5;
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.
  • the VL includes the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO:7.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 8.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO:9.
  • the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 10.
  • the VL includes the amino acid sequence shown in SEQ ID NO: 15.
  • the isolated antigen binding protein described in this application includes an antibody light chain constant region, and the antibody light chain constant region includes a human Ig ⁇ constant region.
  • the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:17.
  • the isolated antigen binding protein described in this application comprises an antibody light chain LC, and the LC comprises the amino acid sequence shown in SEQ ID NO: 19.
  • the VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 11.
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 12.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 13.
  • the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 14.
  • the VH comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the isolated antigen binding protein described in this application includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region.
  • the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen binding protein described in this application comprises an antibody heavy chain HC
  • the HC comprises the amino acid sequence shown in SEQ ID NO:20.
  • this application also provides isolated one or more nucleic acid molecules, which encode the isolated antigen binding protein described in this application.
  • this application also provides a vector, which contains the nucleic acid molecule described in this application, or expresses the antigen binding protein described in this application.
  • this application also provides a cell, which comprises the nucleic acid molecule described in this application or the vector described in this application.
  • this application also provides a method for preparing the isolated antigen binding protein described in this application, and the method includes culturing the cells described in this application under conditions such that the isolated antigen binding protein described in this application is expressed. .
  • this application also provides a pharmaceutical composition, which comprises the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application and/or the cell described in this application, and Optionally a pharmaceutically acceptable carrier.
  • the application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of medicines, the Drugs are used to prevent, alleviate and/or treat tumors.
  • the tumor includes a solid tumor and/or hematological tumor.
  • the application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of a medicine. It is used to prevent, alleviate and/or treat Tim-3 related diseases.
  • this application also provides a method for inhibiting the binding of Tim-3 to PtdSer, the method comprising administering the isolated antigen binding protein described in this application.
  • this application also provides a method of preventing, alleviating and/or treating tumors, the method comprising administering the isolated antigen binding protein described in this application or the pharmaceutical composition described in this application to a subject in need .
  • the tumor includes a solid tumor and/or hematological tumor.
  • Figure 1 shows the binding of the isolated antigen binding protein Z2 described in this application to the cell line expressing human Tim-3;
  • Figure 2 shows the binding of the isolated antigen binding protein Z2 described in this application to the cell line expressing mouse Tim-3;
  • Figure 3 shows the binding of the comparative antibody to the cell line expressing mouse Tim-3
  • Figures 4A-4C show that the isolated antigen binding protein Z2 described in this application blocks the binding of human Tim-3 to the ligand PtdSer.
  • the present invention provides for the first time an isolated antigen binding protein comprising at least one CDR in the heavy chain variable region VH and at least one CDR in the light chain variable region VL, wherein the VH comprises SEQ ID NO: 16
  • the amino acid sequence shown in the VL includes the amino acid sequence shown in SEQ ID NO: 15.
  • the isolated antigen-binding protein described in this application can bind to human-derived Tim-3 with a KD value of 10 nM or lower. In addition, it can also specifically bind to human Tim-3 but not to mouse Tim-3. In addition, the isolated antigen binding protein described in this application can inhibit the binding of Tim-3 and PtdSer, and can also promote the secretion of IFN- ⁇ and/or TNF- ⁇ .
  • isolated generally refers to those obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • isolated antigen binding protein generally refers to a protein with antigen binding ability obtained from a natural state by artificial means.
  • the "isolated antigen binding protein” may comprise an antigen-binding portion and optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that facilitates the antigen-binding portion to bind to the 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, antibody-derived scaffolds containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic scaffolds containing, for example, biocompatible polymers.
  • peptide antibody mimics (“PAMs") and scaffolds based on antibody mimics using fibronectin components can be used as scaffolds.
  • KD usually refers to the "affinity constant” or "equilibrium dissociation constant", and refers to the titration measurement at equilibrium or through A value obtained by dividing the dissociation rate constant (K dissoc ) by the association rate constant (K assoc ).
  • the association rate constant (K assoc), dissociation rate constant (K dissoc) and the equilibrium dissociation constant (KD) represents binding proteins (e.g., according to the present application isolated antigen binding protein) of an antigen (e.g., Tim-3) of Binding affinity.
  • the K D value can be determined by surface plasmon resonance may be measured by the K D value is the Octet, may be used
  • Other experimental approaches and instruments such as a BIAcore (biomolecular interaction analysis) assay (e.g., from Instrument obtained by BIAcore International AB, aGE Healthcare company, Uppsala, Sweden).
  • KinExA KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho) can also be used to determine the K D value.
  • homology generally refers to the percentage of identical residues that are paired.
  • identity is calculated as follows: compare two optimally aligned sequences within a specific region; determine the number of positions where the same base or amino acid appears in the two sequences to obtain the matching position The number of locations; divide the number of such locations by the total number of locations in the segment being compared, and then multiply the quotient by 100.
  • the program used to determine homology compares the aligned sequences on an amino acid-to-amino acid basis, and the program can set different levels of stringency for the comparison (e.g., Same amino acids, conservative amino acid substitutions, etc.).
  • the two amino acids in question each belong to the same chemical category (i.e. acidic, non-polar/hydrophobic, uncharged polar and basic), they are considered to be mutually " Conservative substitution".
  • Conservative substitution if two amino acids in question each belong to the same chemical category (i.e. acidic, non-polar/hydrophobic, uncharged polar and basic), they are considered to be mutually " Conservative substitution".
  • a non-limiting example is that two different amino acids that are non-polar amino acids will be considered as "conservative substitutions" of each other, even if these two amino acids are not the same, and on the one hand are non-polar amino acids and on the other hand are bases.
  • conservative amino acid substitution also refers to the substitution of any amino acid for a given amino acid residue, where the substituted residue is so close to the given residue chemically that there is no result in terms of polypeptide function (for example, binding). Substantial reduction.
  • Tim-3 is the abbreviation of "T cell immunoglobulin and mucin-domain containing molecule 3", also known as TIM-3, HAVCR2, KIM- 3. TIMD3, and FLJ14428, which generally refer to a T helper cell type I specific cell surface protein that regulates macrophage activation and the severity of inflammatory conditions.
  • Tim-3 is also related to cancer, especially cancer stem cells. It is mainly inducibly expressed on the surface of T cells in inflammation and tumor microenvironment, and is highly expressed on Th1 cells, and generates inhibitory signals to cause Th1 cell apoptosis.
  • Gal-9 galectin 9
  • PtdSer phosphatidylserine
  • CEACAM1 carcino-embryonic antigen-related cellular adhesion molecule 1
  • Tim-3 may include human Tim-3 variants, isoforms, species homologs, and analogs that have at least one epitope in common with Tim-3.
  • the amino acid sequence of Tim-3 derived from human is shown in SEQ ID NO: 25
  • the amino acid sequence of Tim-3 derived from mouse is shown in SEQ ID NO: 26.
  • PtdSer generally refers to phosphatidylserine, which is a ligand of Tim-3.
  • the combination of Tim-3 and PtdSer can cause cell apoptosis or cause autoimmune diseases, allergic diseases and viral infection-related diseases.
  • the term "specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can be in a heterogeneous population of molecules (including biomolecules).
  • the existence of the target can determine the existence of the target.
  • an antibody that specifically binds a target (which may be an epitope) is an antibody that binds to the target with greater affinity, affinity, easier, and/or longer duration than it binds to other targets.
  • the extent to which the antibody binds 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).
  • the isolated antigen binding protein can bind to human-derived Tim-3 with a KD value of 10 nM or lower.
  • the isolated antigen binding protein can specifically bind to human Tim-3 but not to mouse Tim-3.
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include but does not require exclusive binding.
  • the term “inhibition” generally refers to reducing the growth rate of cells or the number of cells.
  • the isolated antigen binding protein described in this application can inhibit tumor growth and/or tumor cell proliferation.
  • the isolated antigen binding protein described in this application can inhibit the binding of Tim-3 and PtdSer.
  • IFN- ⁇ is a type of interferon (IFN), and IFN- ⁇ is a type II interferon and binds to a type II interferon antibody. IFN- ⁇ regulates a variety of biological functions, such as antiviral response, cell growth, immune response and tumor suppression.
  • TNF- ⁇ generally refers to tumor necrosis factor ⁇ (also known as cachexia), which is produced by many cell types (including monocytes and macrophages that respond to endotoxin or other stimuli) Of naturally occurring mammalian cytokines.
  • TNF- ⁇ is the main mediator of inflammatory, immunological and pathophysiological reactions (Grell, M. et al. (1995) Cell [Cell], 83:793-802).
  • TNF- ⁇ may include wild-type TNF- ⁇ , polymorphic variants of TNF- ⁇ , and functional equivalents of TNF- ⁇ from various species (for example, human, mouse, and monkey).
  • tumor generally refers to neoplasms or solid lesions formed by abnormal cell growth.
  • the tumor may be a solid tumor or a hematoma tumor.
  • variable domain generally refers to the amino terminal domain of an antibody heavy or light chain.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are usually the most varied parts of the antibody (relative to other antibodies of the same type) and contain antigen binding sites.
  • variable generally refers to the fact that certain segments of variable domains differ greatly in sequence between antibodies.
  • the V domain mediates antigen binding and determines the specificity of a specific antibody to its specific antigen.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved parts of the variable domains are called the framework regions (FR).
  • the variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a ⁇ -sheet configuration, connected by three CDRs, which form a circular connection, and in some cases form part of a ⁇ -sheet structure .
  • the CDRs in each chain are held in close proximity by the FR region, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fourth Edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, for example, antibodies are involved in antibody-dependent cytotoxicity.
  • antibody generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide that includes an antigen binding site, regardless of whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , Mutant and transplanted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain the antigen-binding function (e.g., specifically bind to Tim-3). Generally, 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 antibody is composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contains 10 antigen binding sites, while IgA antibody includes 2-5 that can be combined with J chain to form a multivalent The basic 4-chain unit of the combination.
  • the 4-chain unit is generally about 150,000 Daltons.
  • Each L chain is connected to the H chain by a covalent disulfide bond, and two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • Each H and L chain also has regularly spaced intra-chain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the ⁇ and ⁇ chains, and four CH domains for the ⁇ and ⁇ isotypes.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end.
  • VL corresponds to VH
  • CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • VH and VL pair together to form a single antigen binding site.
  • immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the gamma and alpha classes are further divided into subclasses.
  • humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1.
  • CDR generally refers to a region of an antibody variable domain, the sequence of which is highly variable and/or forms a structural definition loop.
  • an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3).
  • HCDR3 and LCDR3 show most of the diversity of the six CDRs, and in particular HCDR3 is considered to play a unique role in conferring fine specificity on antibodies.
  • variable domains of the natural heavy and light chains each contain four FR regions, namely, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).
  • VL of the isolated antigen binding protein described in this application may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the VH of the isolated antigen binding protein described in this application may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the term "antigen-binding fragment” generally refers to a fragment having antigen-binding activity.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the term "competitive binding” generally refers to that the antibody or fragment thereof interferes with the direct or indirect binding of the target/antigen (e.g., reference antibody) by another antibody (e.g., reference antibody). , Tim-3) ability.
  • the isolated antigen binding protein can compete with the reference antibody for binding to Tim-3.
  • the extent to which an antibody or fragment thereof can interfere with another antibody or fragment of the target, and therefore whether it can be considered as a block or competition according to the present invention can be determined using a competitive binding assay.
  • a particularly suitable quantitative competition assay uses FACS-based or AlphaScreen-based methods to measure the binding between a labeled (for example, His-labeled, biotinylated, or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof. Aspect of competition.
  • a labeled for example, His-labeled, biotinylated, or radiolabeled
  • a competing antibody or fragment thereof is, for example, one of the following: binds to a target in a competition test, so that during the test and in the presence of the second antibody or fragment thereof, the recorded value of the isolated antigen binding protein of the present invention Substitution reaches up to 100% of the maximum theoretical substitution (e.g., replacement by a cold (e.g., unlabeled) antibody or fragment thereof that needs to be blocked) obtained from the detected potential blocking antibody or fragment thereof in a given amount (For example, in FACS-based competition trials).
  • the competing antibody or fragment thereof has between 10% and 100%, such as between 50% and 100%, of the recorded substitution.
  • the term “directly connected” is opposite to the term “indirectly connected”, and the term “directly connected” generally refers to a direct connection.
  • the direct connection may be a case where the substances are directly connected without spacers.
  • the spacer may be a linker.
  • the linker may be a peptide linker.
  • the term “indirectly connected” generally refers to the situation where substances are not directly connected.
  • the indirect connection may be a case of connection through a spacer.
  • the C-terminus of L-FR1 and the N-terminus of LCDR1 may be directly or indirectly connected.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be transformed, transduced or transfected into the host cell, so that the genetic material elements it carries can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage And animal viruses.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papilloma viruses such as papilloma viruses
  • papillary polyoma vacuoles Virus such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the carrier may
  • the term "cell” generally refers to a single cell, cell line, or cell culture that can be or has been a recipient of a subject's plasmid or vector, which includes the nucleic acid molecule of the present invention or the nucleic acid molecule of the present invention. Carrier.
  • a cell can include the progeny of a single cell. Due to natural, accidental or deliberate mutations, the offspring may not necessarily be exactly the same as the original parent cell (in the form of the total DNA complement or in the genome).
  • the cells may include cells transfected in vitro with the vectors of the present invention.
  • the cells may be bacterial cells (e.g., E.
  • the cell is a mammalian cell.
  • the mammalian cell is a HEK293 cell.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, preferably a human patient.
  • the pharmaceutical composition described in this application may comprise the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application and/or the cell described in this application, and Optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also contain one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and/or preservatives. Preparations.
  • the acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • pharmaceutically acceptable carrier generally refers to any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration, and are generally safe and non-toxic. And it is neither biologically nor otherwise undesirable.
  • Tim-3 related diseases generally refers to diseases related to Tim-3 expressing cells. Such as cancer, autoimmune diseases and allergic diseases.
  • the Tim-3 related disease may be tumor and/or leukemia.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an isolated antigen binding protein, which includes at least one CDR in a heavy chain variable region VH and at least one CDR in a light chain variable region VL, wherein the VH comprises SEQ ID NO: 16 As shown in the amino acid sequence, the VL includes the amino acid sequence shown in SEQ ID NO: 15.
  • the isolated antigen binding protein may include HCDR1 in VH shown in SEQ ID NO:16.
  • the isolated antigen binding protein may include the HCDR2 in the VH whose amino acid sequence is as shown in SEQ ID NO: 16.
  • the isolated antigen binding protein may include HCDR3 in VH whose amino acid sequence is shown in SEQ ID NO: 16.
  • the isolated antigen binding protein may include LCDR1 in the VL shown in SEQ ID NO: 15 with an amino acid sequence.
  • the isolated antigen binding protein may comprise LCDR2 in VL shown in SEQ ID NO:15 with an amino acid sequence.
  • the isolated antigen binding protein may comprise LCDR3 in VL shown in SEQ ID NO:15 with an amino acid sequence.
  • the isolated antigen binding protein also includes the amino acid sequence shown in SEQ ID NO: 16 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% The identity of at least one CDR in the VH of the heavy chain variable region and the amino acid sequence shown in SEQ ID NO: 15 have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, At least one CDR in the light chain variable region VL with 99% identity.
  • the isolated antigen binding protein has Tim-3 binding ability.
  • the isolated antigen binding protein can bind to the amino acid sequence shown in SEQ ID NO: 25 with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% Epitopes within Tim-3 fragments of identity.
  • the isolated antigen binding protein may have one or more of the following properties:
  • Tim-3 derived from human with a KD value of 10 nM or lower, wherein the KD value is measured by a surface plasmon resonance method
  • the isolated antigen binding protein can bind to human-derived Tim-3 with a KD value of 10 nM or lower, wherein the KD value can be measured by a surface plasmon resonance method.
  • the KD value of the isolated antigen binding protein described in the present application in binding to Tim-3 derived from human may be ⁇ 10nM, ⁇ 9.5nM, ⁇ 9nM, ⁇ 8.9nM, ⁇ 8.8nM, ⁇ 8.7nM, ⁇ 8.6nM , ⁇ 8.5nM, ⁇ 8.4nM, ⁇ 8.3nM, ⁇ 8.2nM, ⁇ 8.1nM, ⁇ 8nM, ⁇ 7.9nM, ⁇ 7.8nM, ⁇ 7.7nM, ⁇ 7.6nM, ⁇ 7.5nM, ⁇ 7.4nM, ⁇ 7.3 nM, ⁇ 7.2nM, ⁇ 7.1nM, ⁇ 7nM, ⁇ 6.9nM,
  • the KD value can also be determined by FACS, ELISA, competitive ELISA or BIACORE or KINEXA.
  • the isolated antigen binding protein can specifically bind to human Tim-3 but not to mouse Tim-3, and the specific binding can be determined by FACS.
  • the half-maximum effect concentration (EC50) in the FACS measurement can be used to reflect the specific binding of the antigen-binding protein described in this application to human Tim-3.
  • the human Tim-3 may include the amino acid sequence shown in SEQ ID NO: 25
  • the mouse Tim-3 may include the amino acid sequence shown in SEQ ID NO: 26.
  • the isolated antigen binding protein can inhibit the binding of Tim-3 and PtdSer, and the inhibition can be measured by flow cytometry.
  • the isolated antigen binding protein can promote the secretion of IFN- ⁇ and/or TNF- ⁇ .
  • the isolated antigen binding protein described in this application increases the amount of IFN- ⁇ and/or TNF- ⁇ .
  • the type of the isolated antigen binding protein is the type of the isolated antigen binding protein
  • the isolated antigen binding protein may include an antibody or an antigen binding fragment thereof.
  • the isolated antigen binding protein described in this application may include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, and triabodies. , Tetrabodies, Fv fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab')2 fragments and camelized single domain antibodies.
  • the antibody may include a murine antibody.
  • Tim-3 when preparing the murine antibody, Tim-3 can be used to inject a test subject (such as a mouse), and then hybridomas expressing antibodies with desired sequences or functional properties can be isolated.
  • the murine antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chain or a variant thereof, or comprise murine IgGl, IgG2, IgG3 or IgG4 or its The heavy chain constant region of the variant.
  • the antibody may be a humanized antibody.
  • the isolated antigen-binding protein described in the present application may be immunospecifically binding to a related antigen (for example, human Tim-3) and comprise a framework (FR) region that basically has the amino acid sequence of a human antibody and a basic An antibody or a variant, derivative, analog or fragment thereof having the complementarity determining region (CDR) of the amino acid sequence of a non-human antibody.
  • CDR complementarity determining region
  • the humanized antibody may basically comprise all at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to non-human
  • the CDR regions of immunoglobulins (ie, antibodies) and all or substantially all of the framework regions are framework regions with consensus sequences of human immunoglobulins.
  • the humanized antibody also contains at least a portion of an immunoglobulin constant region (e.g., Fc), usually that of a human immunoglobulin.
  • a humanized antibody contains at least the variable domains of a light chain and a heavy chain.
  • the antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • humanized antibodies contain only humanized light chains. In some embodiments, a humanized antibody only contains a humanized heavy chain. In a specific embodiment, a humanized antibody only contains a humanized variable domain of a light chain and/or a humanized heavy chain.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the isolated antigen binding protein can compete with a reference antibody for binding to the Tim-3 protein, wherein the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the reference antibody
  • the light chain variable region of the antibody may include LCDR1, LCDR2, and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2; the LCDR3 It may include the amino acid sequence shown in SEQ ID NO: 3, the heavy chain variable region of the reference antibody may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 4; HCDR2 may include the amino acid sequence shown in SEQ ID NO: 5; the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 6.
  • the CDR region sequence in the VH and VL sequence can be determined according to the Kabat definition or Chothia definition (see, for example, Kabat , "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc . Natl. Acad. Sci. USA 86: 9268-9272 (1989)).
  • variable region sequence of the isolated antigen binding protein that is, the sequence of the VH or VL
  • the VH and VL sequences can also be determined according to the combined definition rule including the Kabat definition and Chothia definition CDR region sequence.
  • the VH may include HCDR1, HCDR2 and HCDR3, wherein the HCDR3 may include the amino acid sequence shown in SEQ ID NO:6.
  • the HCDR3 may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 6 sequence.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO:4.
  • the HCDR1 may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 4. sequence.
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO:5.
  • the HCDR2 may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 5. sequence.
  • the HCDR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 4
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 6. The amino acid sequence shown.
  • the VL may include LCDR1, LCDR2, and LCDR3, where the LCDR1 may include the amino acid sequence shown in SEQ ID NO:1.
  • the LCDR1 may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:1 sequence.
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2.
  • the LCDR2 may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 2. sequence.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • the LCDR3 may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 3. sequence.
  • LCDR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 1
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence shown.
  • HCDR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 4
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR3 may include SEQ ID NO: 6
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • the VL of the isolated antigen binding protein may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the L-FR1 may include the amino acid sequence shown in SEQ ID NO:7.
  • the L-FR1 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence.
  • the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO:7.
  • the L-FR2 may include the amino acid sequence shown in SEQ ID NO:8.
  • the L-FR2 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 8.
  • the L-FR3 may include the amino acid sequence shown in SEQ ID NO:9.
  • the L-FR3 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 9 The amino acid sequence.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may include the amino acid sequence shown in SEQ ID NO:9.
  • the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 10.
  • the L-FR4 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 10. The amino acid sequence.
  • the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 10.
  • L-FR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 7
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 8
  • L-FR3 may Containing the amino acid sequence shown in SEQ ID NO: 9
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 10.
  • the VH of the isolated antigen binding protein may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 11.
  • the H-FR1 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 11. The amino acid sequence.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 11.
  • the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 12.
  • the H-FR2 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 12. The amino acid sequence.
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 12.
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 13.
  • the H-FR3 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 13. The amino acid sequence.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 13.
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • the H-FR4 may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 14. The amino acid sequence.
  • the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • H-FR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 11
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 12
  • H-FR3 may include The amino acid sequence shown in SEQ ID NO: 13
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • L-FR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 7
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 8
  • L-FR3 may Including the amino acid sequence shown in SEQ ID NO: 9
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 10
  • H-FR1 may include the amino acid sequence shown in SEQ ID NO: 11
  • H-FR2 may include The amino acid sequence shown in SEQ ID NO: 12
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 13
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • the isolated antigen binding protein described in this application may comprise the variable region of the antibody light chain VL and the variable region of the antibody heavy chain VH.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 15
  • the VH may include the amino acid sequence shown in SEQ ID NO: 16.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 15
  • the VH may include the amino acid sequence shown in SEQ ID NO: 16.
  • the isolated antigen binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Ig ⁇ constant region.
  • the isolated antigen binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Ig ⁇ constant region.
  • the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO:17.
  • the antibody light chain constant region may include at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO: 17. Amino acid sequence of identity.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG2 heavy chain constant region.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG3 heavy chain constant region.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG4 heavy chain constant region.
  • the antibody heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 18.
  • the heavy chain constant region of the antibody may comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO: 18. Amino acid sequence of identity.
  • the isolated antigen binding protein may include an antibody light chain LC, and the LC may include the amino acid sequence shown in SEQ ID NO: 19.
  • the LC may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 19. sequence.
  • the isolated antigen binding protein may include an antibody heavy chain HC, and the HC may include the amino acid sequence shown in SEQ ID NO:20.
  • the HC may include amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO: 20 sequence.
  • the isolated antigen binding protein described in this application may comprise an antibody light chain and an antibody heavy chain.
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 19
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 20.
  • the light chain of the isolated antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 19, and the heavy chain may include the amino acid sequence shown in SEQ ID NO: 20.
  • HCDR1 of the isolated antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 4
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 6
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 3.
  • L-FR1 of the isolated antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 7
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 8
  • L-FR3 may include SEQ ID NO The amino acid sequence shown in :9
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 10
  • H-FR1 may include the amino acid sequence shown in SEQ ID NO: 11
  • H-FR2 may include SEQ ID NO:
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 13
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 16.
  • the isolated antigen binding protein may be Z2.
  • this application also provides isolated one or more nucleic acid molecules, which can encode the isolated antigen binding protein described in this application.
  • the isolated one or more nucleic acid molecules described in this application may be nucleotides, deoxyribonucleotides or ribonucleotides in isolated form of any length, or analogs isolated from their natural environment or artificially synthesized , But can encode the isolated antigen binding protein described in this application.
  • the nucleic acid molecules encoding the same isolated antigen binding protein described in this application are not unique.
  • this application also provides a vector, which may contain the nucleic acid molecule described in this application, or express the antigen binding protein described in this application.
  • the vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell.
  • the vector may include: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC) derived from P1; bacteriophage such as lambda phage or M13 phage Animal viruses and so on.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • the vector may contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain an origin of replication site.
  • the carrier may also include components that assist it to enter cells, such as viral particles, liposomes or protein coats, but not only these substances.
  • this application also provides a cell, which may contain the nucleic acid molecule described in this application or the vector described in this application.
  • the cell may include the progeny of a single cell. Due to natural, accidental or deliberate mutations, the offspring may not necessarily be exactly the same as the original parent cell (in the form of the total DNA complement or in the genome).
  • the cells may also include cells transfected in vitro with the vectors of the present invention.
  • the cell may be a bacterial cell (for example, Escherichia coli), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 Cell, NS0 cell or myeloma cell.
  • the cell may be a mammalian cell.
  • the mammalian cell may be a HEK293 cell.
  • the protein purification and separation method can be salting out, isoelectric point precipitation, low temperature organic solvent precipitation, dialysis and ultrafiltration, gel filtration, electrophoresis, ion exchange chromatography or affinity chromatography.
  • this application also provides a method for preparing the isolated antigen binding protein described in this application, and the method may include culturing the isolated antigen binding protein described in this application under conditions cells.
  • the isolated antigen binding protein described in this application can be expressed by the induction of exogenous genes (for example, IPTG induction).
  • this application also provides a pharmaceutical composition, which may comprise the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, and/or the cell described in this application. , And optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also include one or more (safe and effective amounts) pharmaceutically acceptable carriers, such as stabilizers, excipients, diluents, solubilizers, and surfactants. , Emulsifiers and/or preservatives.
  • pharmaceutically acceptable carriers such as stabilizers, excipients, diluents, solubilizers, and surfactants. , Emulsifiers and/or preservatives.
  • the acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with drug administration, and are generally safe and non-toxic. And it is neither biologically nor otherwise undesirable.
  • the pharmaceutical composition may comprise a route of parenteral administration and/or parenteral administration, such as subcutaneous, transdermal, intracavitary, intravenous, intraarterial, intrathecal, intratumor, intraperitoneal, And/or intranasal administration or direct injection into the tissue.
  • parenteral administration and/or parenteral administration such as subcutaneous, transdermal, intracavitary, intravenous, intraarterial, intrathecal, intratumor, intraperitoneal, And/or intranasal administration or direct injection into the tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition may be administered without interruption.
  • the uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015/
  • this application also provides the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and/or the drug described in this application.
  • the use of the composition in the preparation of a medicine which can be used to prevent, relieve and/or treat tumors.
  • this application also provides the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and/or the drug described in this application.
  • the use of the composition in the preparation of a medicament which can be used to prevent, alleviate and/or treat Tim-3 related diseases.
  • the present application also provides a method of preventing, alleviating and/or treating tumors, and the method may include administering the isolated antigen binding protein described in the present application to a subject in need.
  • the administration can be carried out in different ways, such as intravenous, intratumor, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application also provides methods for preventing, alleviating and/or treating Tim-3 related diseases, and the method may include administering the isolated antigen binding protein described in the present application to a subject in need.
  • the administration can be carried out in different ways, such as intravenous, intratumor, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the isolated antigen binding protein described in this application can be Used to prevent, alleviate or treat tumors.
  • the isolated antigen binding protein described in this application can be It is used to prevent, alleviate or treat Tim-3 related diseases.
  • the tumor may include solid tumors and/or hematological tumors.
  • the tumor may be leukemia.
  • the Tim-3 related diseases may include diseases related to cells expressing Tim-3. Such as cancer, autoimmune diseases and allergic diseases.
  • the Tim-3 related disease may be leukemia and/or tumor.
  • the subject may include humans and non-human animals.
  • the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • this application also provides a method for inhibiting the binding of Tim-3 to PtdSer, the method comprising administering the isolated antigen binding protein described in this application.
  • the administration can be carried out in different ways, such as intravenous, intratumor, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the isolated antigen binding protein described in the present application can also be administered to a subject in need with one or more effective amounts of therapeutic agents, and the one or more effective amounts of therapeutic agents can be These include chemotherapeutics, cytotoxic agents, immunosuppressants, steroids, antiemetics, cancer vaccines, analgesics, or another antibody.
  • therapeutic agents include chemotherapeutics, cytotoxic agents, immunosuppressants, steroids, antiemetics, cancer vaccines, analgesics, or another antibody.
  • the isolated antigen binding protein described in this application can also be fused with a toxin to produce an immunoconjugate, so that after it specifically binds to a cell expressing Tim-3 protein, it can function on the cell. Toxic activity, specifically kills cancer cells.
  • the isolated antigen binding protein described in this application can also be made into an oncolytic virus, which invades into tumor cells through cell surface molecules, and then uses specific receptors overexpressed in tumor cells as Targeting, invading the virus into tumor cells and performing subsequent functions.
  • the isolated antigen-binding protein described in this application can also be fused with an antibody capable of specifically binding to other antigens (that is, in addition to Tim-3) to generate bispecific antibodies, thereby simultaneously specific Combine two different antigens to achieve better tumor treatment effect.
  • the isolated antigen binding protein described in this application can bind to Tim-3 derived from human with a KD value of 10 nM or lower, wherein the KD value is determined by a surface plasmon resonance method;
  • the isolated antigen binding protein described in this application can specifically bind to human Tim-3 but not to mouse Tim-3, thereby having species specificity;
  • the isolated antigen binding protein described in this application can inhibit the binding of Tim-3 and PtdSer, block the Tim-3 signaling pathway, and thereby inhibit the growth of tumor cells;
  • the isolated antigen binding protein described in this application can promote the secretion of IFN- ⁇ and/or TNF- ⁇ , thereby generating immune stimulation, thereby inhibiting or eliminating tumors.
  • the following examples are only used to illustrate the protein molecules, preparation methods, and uses of the present application, and are not used to limit the scope of the present application.
  • the examples do not include detailed descriptions of traditional 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.
  • Example 1 The structure of the isolated antigen binding protein Z2 described in this application.
  • the light chain of the isolated antigen binding protein Z2 described in this application includes the amino acid sequence shown in SEQ ID NO: 19, and the heavy chain includes the amino acid sequence shown in SEQ ID NO: 20.
  • the HCDR1 of the isolated antigen binding protein Z2 includes the amino acid sequence shown in SEQ ID NO: 4
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO: 5
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO: 6
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 1
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 2
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 3.
  • L-FR1 of the isolated antigen binding protein Z2 includes the amino acid sequence shown in SEQ ID NO: 7
  • L-FR2 includes the amino acid sequence shown in SEQ ID NO: 8
  • L-FR3 includes SEQ ID NO: 9
  • L-FR4 includes the amino acid sequence shown in SEQ ID NO: 10
  • H-FR1 includes the amino acid sequence shown in SEQ ID NO: 11
  • H-FR2 includes the amino acid sequence shown in SEQ ID NO: 12
  • H-FR3 includes the amino acid sequence shown in SEQ ID NO: 13
  • H-FR4 includes the amino acid sequence shown in SEQ ID NO: 14.
  • the VL of the isolated antigen binding protein Z2 includes the amino acid sequence shown in SEQ ID NO: 15, and the VH includes the amino acid sequence shown in SEQ ID NO: 16.
  • the nucleotide sequence encoding the VL of the isolated antigen binding protein Z2 described in this application is shown in SEQ ID NO: 21, and the nucleotide sequence of the VH encoding the isolated antigen binding protein Z2 described in this application is shown in SEQ ID NO: 22, the nucleotide sequence encoding the light chain constant region of the isolated antigen binding protein Z2 described in this application is shown in SEQ ID NO: 23, which encodes the isolated antigen binding protein Z2 described in this application
  • the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO: 24.
  • each cycle includes antibody capture, analyte binding, and chip regeneration.
  • the antibodies were all diluted to 1 ⁇ g/ml, and injected into the 2, 3, and 4 channels at a flow rate of 10 ⁇ l/min for 40s, and each antibody was captured by the pre-coupled Protein A.
  • the capture amount was about 200RU.
  • the human Tim-3-hIg Fc fusion protein (that is, the human Tim-3 protein, American R&D system company, catalog number 2365-TM-050) was divided into 0nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM, 40nM The concentration gradient is injected into the four channels, the flow rate is 30ul/min, the injection time is 180s, and the dissociation time is 900s. Among them, the amino acid sequence of the human Tim-3 protein is shown in SEQ ID NO: 25. Finally, glycine (10mM, pH1.5) was injected at the same flow rate for 30s to regenerate the chip.
  • Biacore T200 analysis software 2.0 was used to analyze the experimental results, 1 channel was used as the reference channel for subtraction, and the analysis model was a 1:1 kinetic fitting model.
  • K assoc association rate constant
  • K dissoc the dissociation rate constant
  • KD affinity constant equal to K dissoc / K assoc.
  • FACS flow cytometry fluorescence sorting
  • iQue Screener flow cytometer purchased from IntelliCyt
  • PBS containing 0.1% BSA as a buffer for cell surface target antigens
  • antibody ie the isolated antigen binding protein Z2 or the comparative antibody described in this application binding affinity test
  • the amino acid sequence of human Tim-3 is as SEQ ID As shown in NO: 25
  • the amino acid sequence of mouse Tim-3 is as shown in SEQ ID NO: 26.
  • the comparative antibody is represented by Anti-mTim-3Ab, which is a commercial antibody (Rat IgG2a Clone#215008, purchased from R&D).
  • the specific detection process is as follows:
  • FIGs 1 to 3 The analysis results of the flow cytometry affinity binding experiment are shown in Figures 1 to 3, where Figure 1 shows the binding of the isolated antigen binding protein Z2 described in this application to the human Tim-3 cell line, and Figure 2 shows the application The combination of the isolated antigen binding protein Z2 and the expression mouse Tim-3 cell line.
  • Figure 3 shows the combination of the comparative antibody and the expression mouse Tim-3 cell line.
  • IgG in Figures 1 to 3 represents the isotype control antibody mouse IgG2b in the experiment, which is a commercial antibody (Mouse IgG2b Clone #133303, purchased from R&D).
  • the antigen binding protein Z2 has the biological activity of specifically binding to human Tim-3.
  • Example 4 The isolated antigen binding protein Z2 described in this application blocks the detection of the binding between human Tim-3 and PtdSer
  • Phosphatidylserine is another ligand of Tim-3, which is usually exposed on the cell membrane surface of apoptotic cells, binds to the Tim-3IgV domain, mediates the phagocytosis of apoptotic cells, promotes the clearance of apoptotic bodies and dendrites The cross-presentation of antigens from DCs.
  • Jurkat cells are diluted to 5 ⁇ 10 5 cells/ml and 5 ⁇ M Camptothecin is added, and the cells are co-cultured at 37°C for 5 hours to induce cell apoptosis and express phosphatidylserine. After that, it was washed twice with PBS plus 2% FSA.
  • apoptotic Jurkat cells per well were added to a 96-well plate.
  • the isolated antigen binding protein Z2 and isotype control antibody mouse IgG2b (Mouse IgG2b Clone#133303, purchased from R&D) described in this application were diluted with PBS to 20 ⁇ g/ml, or Tim-3-hIg Fc fusion protein ( That is, human Tim-3 protein, American R&D system company, article number 2365-TM-050) was diluted with PBS to 4 ⁇ g/ml, and Tim-3-hIg Fc fusion protein alone or Z2 and Tim-3-hIg Fc fusion The protein was co-mixed with the above-mentioned cells and incubated for 30 minutes at room temperature.

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Abstract

一种分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO：16所示的氨基酸序列，所述VL包含SEQ ID NO：15所示的氨基酸序列。还涉及编码所述分离的抗原结合蛋白的核酸分子、包含所述分离的抗原结合蛋白的载体、包含所述核酸分子或所述载体的细胞、制备所述分离的抗原结合蛋白的方法、药物组合物以及所述分离的抗原结合蛋白的用途。

Description

一种分离的抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种分离的抗原结合蛋白及其用途。
背景技术
肿瘤是一种严重威胁人类健康的疾病，近年来，免疫治疗作为一种新疗法，在肿瘤治疗中显示出了巨大的潜力。
T细胞免疫球蛋白及黏蛋白分子3(T cell immunoglobulin and mucin-domain containing molecule 3，Tim-3)是近年来发现的重要免疫检查点分子，不同于其他“免疫检查点”分子，其主要诱导性表达于炎症和肿瘤微环境中的T细胞表面。它是一种Ⅰ型膜表面分子，高表达于Th1细胞，并产生抑制信号从而导致Th1细胞的凋亡。在癌症与慢性病毒感染过程中，Tim-3与其配体半乳糖凝集素9(galectin 9，Gal-9)、磷脂酰丝氨酸(PtdSer)、高迁移率族蛋白1(high mobility group box 1 protein，HMGB1)、癌胚抗原相关细胞黏附分子1(carcino-embryonic antigen related cellular adhesion molecule 1，CEACAM1)相结合，继而引起T细胞的衰竭和凋亡，是导致肿瘤细胞发生免疫逃逸的重要原因之一。同时，选择性激活Tim-3从而导致免疫逃逸是PD-1/PD-L1抗体免疫治疗过程中发生耐药的主要机制。
发明内容
一方面，本申请提供一种分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO:16所示的氨基酸序列，所述VL包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，本申请所述的分离的抗原结合蛋白具有Tim-3结合能力。
在某些实施方式中，本申请所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
1)能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值通过表面等离子体共振法测定；
2)在FACS测定中，能够特异性结合人的Tim-3而不结合小鼠的Tim-3；
3)在ELISA测定中，能够抑制Tim-3与PtdSer的结合；和
4)促进IFN-γ和/或TNF-α的分泌。
根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
在某些实施方式中，所述抗体包括人源化和鼠源抗体。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述HCDR1包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述LCDR2包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述LCDR3包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，本申请所述的分离的抗原结合蛋白与参比抗体竞争结合所述Tim-3蛋白，其中所述参比抗体包含轻链可变区和重链可变区，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:1所示的氨基酸序列；所述LCDR2包含SEQ ID NO:2所示的氨基酸序列；所述LCDR3包含SEQ ID NO:3所示的氨基酸序列，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2包含SEQ ID NO:5所示的氨基酸序列；所述HCDR3包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
在某些实施方式中，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述VL包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，本申请所述的分离的抗原结合蛋白包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区。
在某些实施方式中，所述抗体轻链恒定区包含SEQ ID NO:17中所示的氨基酸序列。
在某些实施方式中，本申请所述的分离的抗原结合蛋白包含抗体轻链LC，且所述LC包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在某些实施方式中，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，本申请所述的分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
在某些实施方式中，所述抗体重链恒定区包含SEQ ID NO:18中所示的氨基酸序列。
在某些实施方式中，本申请所述的分离的抗原结合蛋白包含抗体重链HC，且所述HC包含SEQ ID NO:20所示的氨基酸序列。
另一方面，本申请还提供分离的一种或多种核酸分子，其编码本申请所述的分离的抗原结合蛋白。
另一方面，本申请还提供载体，其包含本申请所述的核酸分子，或，表达本申请所述的抗原结合蛋白。
另一方面，本申请还提供细胞，其包含本申请所述的核酸分子或本申请所述的载体。
另一方面，本申请还提供制备本申请所述的分离的抗原结合蛋白的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白表达的条件下，培养本申请所述的细胞。
另一方面，本申请还提供药物组合物，其包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载体。
另一方面，本申请还提供所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液肿瘤。
另一方面，本申请还提供所述分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗Tim-3相关的疾病。
另一方面，本申请还提供抑制Tim-3与PtdSer结合的方法，所述方法包括施用本申请所述的分离的抗原结合蛋白。
另一方面，本申请还提供预防、缓解和/或治疗肿瘤的方法，所述方法包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白或本申请所述的药物组合物。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液肿瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示本申请所述的分离的抗原结合蛋白Z2与表达人Tim-3细胞株的结合情况；
图2显示本申请所述的分离的抗原结合蛋白Z2与表达小鼠Tim-3细胞株的结合情况；
图3显示对比例抗体与表达小鼠Tim-3细胞株的结合情况；
图4A-4C显示本申请所述的分离的抗原结合蛋白Z2阻断人Tim-3与配体PtdSer的结合情况。
具体实施方式
本发明首次提供了一种分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO:16所示的氨基酸序列，所述VL包含SEQ ID NO:15所示的氨基酸序列。本申请所述分离的抗原结合蛋白能够以10nM或更低的KD值结合源自人的Tim-3，此外，其还能够特异性结合人的Tim-3而不结合小鼠的Tim-3。另外，本申请所述分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合，还可以促进IFN-γ和/或TNF-α的分泌。
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的抗原结合蛋白”通常指从天然状态下经人工手段获得的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的支架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白支架或具有移植的CDR或CDR衍生物的备选蛋白支架或人工支架。此类支架包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的支架以及包含例如生物相容性聚合物的完全合成的支架。参见例如Korndorfer等,2003,Proteins:Structure,Function,andBioinformatics,53(1):121-129(2003)；Roque等,Biotechnol.Prog.20:639-654(2004)。此外，肽抗体模拟物("PAMs")以及利用纤连蛋白组分基于抗体模拟物的支架可以用作支架。
在本申请中，术语“KD”(同样地，“K D”或“K D”)通常指“亲和常数”或“平衡解离常数”，并指在滴定测量中在平衡时、或者通过将解离速率常数(K dissoc)除以结合速率常数(K assoc)所获得的值。使用结合速率常数(K assoc)、解离速率常数(K dissoc)和平衡解离常数(KD)表示结合蛋白(例如本申请所述的分离的抗原结合蛋白)对抗原(例如Tim-3)的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。使用基于荧光的技术提供了高灵敏度以及在生理缓冲液中在平衡时检查样品的能力。例如，可以通过表面等离子体共振法测定所述K D值，也可以通过Octet测定所述K D值，也可以使用其他实验途径和仪器例如BIAcore(生物分子相互作用分析)测定(例如，可以从BIAcoreInternationalAB，aGEHealthcarecompany，Uppsala，瑞典获得的仪器)。另外，也可以使用可以从SapidyneInstruments(Boise，Idaho) 获得的KinExA(动态排阻测定(KineticExclusionAssay))测定所述K D值。
在本申请中，氨基酸或核苷酸序列的同源性可与术语“同一性”相互交换地使用。术语“同一性”通常是指配对的相同残基的百分比。“序列同一性百分数”是这样计算的：将两个进行最佳比对的序列在特定区域范围内进行比较；确定两个序列中出现相同碱基或氨基酸的位置的数量，以得到相匹配位置的数量；将这种位置的数量除以被比较区段中的位置总数，所得的商再乘以100。如上所述，用于确定同源性(或同一性)的程序在氨基酸-对-氨基酸的基础上比较被比对的序列，并且所述程序可对该比较设定严格性的不同水平(例如相同氨基酸、保守氨基酸置换等)。如本文中所使用的术语，如果所讨论的两个氨基酸各自属于相同的化学类别(即酸性、非极性/疏水性、不带电荷的极性和碱性)，则被认为是彼此的“保守置换”。非限制实例为，属于非极性氨基酸的两个不同的氨基酸将被认定是彼此的“保守置换”，即使这两个氨基酸不相同，而一方面为非极性氨基酸、与另一方面为碱性氨基酸时将不被认为是彼此的“保守置换”。Alberts、Johnson、Lewis、Raff、Roberts和Walter的《MolecularBi ology of the Cell》第4版(2002)第3.1栏，将氨基酸分成四个主要组别：酸性、非极性、不带电荷的极性和碱性。此分组在本发明的上下文中可用于确定特定氨基酸对所讨论的另一个氨基酸是否为保守取代的目的。上述主要组别可进一步再分类为例如小的非极性和大的非极性氨基酸、大的芳香族氨基酸等。术语“保守的氨基酸置换”还指对给定氨基酸残基的任一个氨基酸置换，其中该置换残基与给定的残基在化学上是如此的相近而在多肽功能(例如结合)结果方面无实质减少。
在本申请中，术语“Tim-3”是“T细胞免疫球蛋白及黏蛋白分子3(T cell immunoglobulin and mucin-domain containing molecule 3)”的缩写，也称作TIM-3，HAVCR2，KIM-3，TIMD3，和FLJ14428，其通常是指一种T辅助细胞Ⅰ型特异性细胞表面蛋白，其调节巨噬细胞活化和炎性状况的严重程度。Tim-3还与癌症，特别是癌干细胞有关，其主要诱导性表达于炎症和肿瘤微环境中的T细胞表面，高表达于Th1细胞，并产生抑制信号从而导致Th1细胞的凋亡。在癌症与慢性病毒感染过程中，Tim-3与其配体半乳糖凝集素9(galectin 9，Gal-9)、磷脂酰丝氨酸(PtdSer)、高迁移率族蛋白1(high mobility group box 1 protein，HMGB1)、癌胚抗原相关细胞黏附分子1(carcino-embryonic antigen related cellular adhesion molecule 1，CEACAM1)相结合，继而引起T细胞的衰竭和凋亡，是导致肿瘤细胞发生免疫逃逸的重要原因之一。同时，选择性激活Tim-3从而导致免疫逃逸是PD-1/PD-L1抗体免疫治疗过程中发生耐药的主要机制。Tim-3可以包括人Tim-3的变体，同等型，物种同系物，和与Tim-3具有至少一个共同表位的类似物。例如，源自人的Tim-3的氨基酸序列如SEQ ID NO:25所示，源自小鼠的Tim-3的氨基酸序列如SEQ ID NO:26所示。
在本申请中，术语“PtdSer”通常是指磷脂酰丝氨酸，为Tim-3的配体。Tim-3与PtdSer结合，会导致细胞凋亡或者引发自身免疫性疾病、过敏性疾病和病毒感染相关性疾病。
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，比如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在一个实施方案中，抗体结合无关靶标的程度小于抗体对靶标的结合的约10％，如例如通过放射免疫分析(RIA)测量的。例如，在本申请中，所述分离的抗原结合蛋白能够以10nM或更低的KD值与源自人的Tim-3相结合。又例如，所述分离的抗原结合蛋白能够特异性结合人的Tim-3而不结合小鼠的Tim-3。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在另一个实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“抑制”通常指降低细胞的生长速率或者细胞的数量。例如，本申请所述的分离的抗原结合蛋白，其能够抑制肿瘤生长和/或肿瘤细胞增殖。又例如，本申请所述的分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合。
在本申请中，术语“IFN-γ”是干扰素(IFN)的一种类型，IFN-γ是II型干扰素并且结合II型干扰素抗体。IFN-γ调控多种生物学功能，如抗病毒反应、细胞生长、免疫反应和肿瘤抑制。
在本申请中，术语“TNF-α”通常是指肿瘤坏死因子α(也称为恶病质素)，其是由许多细胞类型(包括应答内毒素或其他刺激的单核细胞和巨噬细胞)产生的天然存在的哺乳动物细胞因子。TNF-α是炎症性、免疫学和病理生理反应的主要介质(Grell,M.等人，(1995)Cell[细胞]，83:793-802)。在本申请中，TNF-α可以包括来自各种物种(例如人、小鼠和猴)的野生型TNF-α、TNF-α的多态性变体和TNF-α的功能等效物。
在本申请中，术语“肿瘤”通常指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或血液瘤肿瘤。
在本申请中，术语“可变结构域”通常指抗体重链或轻链的氨基末端结构域。重链和轻链的可变结构域可以分别称为“VH”和“VL”。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“可变”通常指在抗体之间可变结构域的某些区段在序列上存在很大差异的事实。V结构域介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性并非在整个可变结构域范围内均匀分布。相反，它集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分称为框架区(FR)。天然重链 和轻链的可变结构域各自包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991))。恒定结构域不直接参与抗体与抗原的结合，但显示出各种效应子功能，例如抗体参与抗体依赖性细胞毒性。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合Tim-3)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。取决于其重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。基于CH序列和功能方面的相对小的差异，将γ和α类进一步分成亚类，例如，人表达下述亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgK1。
在本申请中，术语“CDR”通常指抗体可变结构域的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和 在VL中三个(LCDR1、LCDR2、LCDR3)。在天然抗体中，HCDR3和LCDR3显示所述六个CDR的大多数多样性，并且特别地HCDR3被认为在赋予抗体的精细特异性方面起独特作用。参见，例如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)。实际上，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况功能正常且稳定。参见，例如,Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。
在本申请中，术语“FR”通常指抗体可变结构域的更高度保守的部分，其被称为框架区。通常，天然重链和轻链的可变结构域各自包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3，和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3，和L-FR4)。例如，本申请所述的分离的抗原结合蛋白的VL可以包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。本申请所述的分离的抗原结合蛋白的VH可以包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在本申请中，术语“抗原结合片段”通常指具有抗原结合活性的片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，术语“竞争结合”通常指抗体或其片段通过对另一种抗体(例如，参比抗体)进行变构调节，干扰另一种抗体直接地或间接地结合靶标/抗原(例如，Tim-3)的能力。例如，在本申请中，所述分离的抗原结合蛋白可以与参比抗体竞争结合Tim-3。此外，抗体或其片段能够干扰另一个抗体或其片段结合靶标的程度，且因此无论其是否可以被认为是根据本发明的阻断或竞争，都可以使用竞争性结合试验来确定。一种特别合适的定量竞争试验使用基于FACS或基于AlphaScreen方法测量标记的(例如，His标记的、生物素化的或放射性标记的)抗体或其片段和另一抗体或其片段之间在结合靶标方面的竞争。通常，竞争抗体或其片段为例如一种下述的：在竞争试验中结合靶标，使得在试验期间和在第二抗体或其片段的存在下，本发明的分离的抗原结合蛋白的所记录的取代达到由以给定量存在的所检测的潜在阻断抗体或其片段得到的最大理论取代(例如，由需要被阻断的冷(例如，未标记的)抗体或其片段取代)的至多100％(例如，在基于FACS的竞争试验中)。优选地，竞争抗体或其片段具有在10％至100％之间，比如在50％至100％之间的所记录的取代。
在本申请中，术语“直接相连”与术语“间接相连”相对，术语“直接相连”通常是指直接连接。例如，所述直接相连可以为物质间没有间隔子而直接相连的情况。所述间隔子可以是连接子。例如，所述连接子可以为肽连接子。术语“间接相连”通常是指物质间不直接相连的情况。例如，所述间接相连可以为通过间隔子而连接的情况。例如，在本申请所述的分离的抗原结合蛋白中，所述L-FR1的C末端与所述LCDR1的N末端可以直接或间接相连。
在本申请中，术语“分离的核酸分子”通常指任何长度的分离形式的核苷酸，脱氧核糖 核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本发明所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞或骨髓瘤细胞。在某些实施方案中，细胞为哺乳动物细胞。在某些实施方案中，哺乳动物细胞为HEK293细胞。
在本申请中，术语“药物组合物”通常指涉及适合施用于患者、优选人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载体。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载体”通常指与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂等，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在本申请中，术语“Tim-3相关的疾病”通常是指与表达Tim-3的细胞相关的疾病。例如癌症、自体免疫性疾病和过敏性疾病。在本申请中，Tim-3相关的疾病可以是肿瘤和/或白血病。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。
分离的抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO:16所示的氨基酸序列，所述VL包含SEQ ID NO:15所示的氨基酸序列。
例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO:16所示的VH中的HCDR1。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO:16所示的VH中的HCDR2。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO:16所示的VH中的HCDR3。又例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO:15所示的VL中的LCDR1。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO:15所示的VL中的LCDR2。例如，在本申请中，所述分离的抗原结合蛋白可以包含氨基酸序列如SEQ ID NO:15所示的VL中的LCDR3。
在本申请中，所述分离的抗原结合蛋白还包括与SEQ ID NO:16所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的重链可变区VH中的至少一个CDR，以及与SEQ ID NO:15所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的轻链可变区VL中的至少一个CDR。在本申请中，所述分离的抗原结合蛋白具有Tim-3结合能力。
在本申请中，所述分离的抗原结合蛋白可以结合与SEQ ID NO:25所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的Tim-3片段内的表位。
所述分离的抗原结合蛋白的性质
在本申请中，所述分离的抗原结合蛋白可以具有下述性质中的一种或多种：
1)能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值通过表面等离子体共振法测定；
2)在FACS测定中，能够特异性结合人的Tim-3而不结合小鼠的Tim-3；
3)在ELISA测定中，能够抑制Tim-3与PtdSer的结合；和
4)促进IFN-γ和/或TNF-α的分泌。
在本申请中，所述分离的抗原结合蛋白能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值可以通过表面等离子体共振法测定。例如，本申请所述的分离的抗原结合蛋白结合源自人的Tim-3的KD值可以为≤10nM、≤9.5nM、≤9nM、≤8.9nM、≤8.8nM、≤8.7nM、≤8.6nM、≤8.5nM、≤8.4nM、≤8.3nM、≤8.2nM、≤8.1nM、≤8nM、≤7.9nM、≤7.8nM、≤7.7nM、≤7.6nM、≤7.5nM、≤7.4nM、≤7.3nM、≤7.2nM、≤7.1nM、≤7nM、≤6.9nM、≤6.5nM、≤6nM、≤5.5nM、≤5nM、≤4.5nM、≤4nM、≤3.5nM、≤3nM、≤2.9nM、≤2.8nM、≤2.7nM、≤2.6nM、≤2.5nM、≤2.4nM、≤2.3nM、≤2.2nM、≤2.1nM、≤2Nm、≤1.5nM、≤1nM。
在本申请中，所述KD值还可以通过FACS、ELISA、竞争ELISA或BIACORE或KINEXA进行测定。
在本申请中，所述分离的抗原结合蛋白能够特异性结合人的Tim-3而不结合小鼠的Tim-3，所述特异性结合可以通过FACS测定。例如，可以通过FACS测定中的半最大效应浓度(EC50)来反映本申请所述分离的抗原结合蛋白特异性结合人的Tim-3的情况，例如，半最大效应浓度(EC50)越低说明特异性结合越好。
在本申请中，所述人的Tim-3可以包含如SEQ ID NO:25所示的氨基酸序列，所述小鼠的Tim-3可以包含如SEQ ID NO:26所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合，所述抑制情况可以通过流式细胞仪来测定。
在本申请中，所述分离的抗原结合蛋白能够促进IFN-γ和/或TNF-α的分泌。例如，本申请所述分离的抗原结合蛋白使IFN-γ和/或TNF-α的数量增加。
所述分离的抗原结合蛋白的种类
在本申请中，所述分离的抗原结合蛋白可以包括抗体或其抗原结合片段。例如，本申请所述的分离的抗原结合蛋白可以包括但不限于重组抗体、单克隆抗体、人抗体、人源化抗体、嵌合抗体、双特异性抗体、单链抗体、双抗体、三抗体、四抗体、Fv片段、scFv片段、Fab片段、Fab'片段、F(ab')2片段和骆驼化单结构域抗体。
在本申请中，所述抗体可以包括鼠源抗体。例如，制备所述鼠源抗体时可以用Tim-3注射试验对象(例如小鼠)，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在某些实施方案中，所述鼠源抗体或其抗原结合片段，还可以包含鼠源κ、λ链或其变体的轻链恒定区，或包含鼠源IgGl，IgG2，IgG3或IgG4或其变体的重链恒定区。
在本申请中，所述抗体可以为人源化抗体。换句话说，本申请所述的分离的抗原结合蛋 白，其可以为免疫特异性结合至相关抗原(例如人类Tim-3)且包含基本上具有人类抗体的氨基酸序列的框架(FR)区及基本上具有非人类抗体的氨基酸序列的互补决定区(CDR)的抗体或其变异体、衍生物、类似物或片段。此处的“基本上”在CDR的情况下是指CDR的氨基酸序列与非人类抗体CDR的氨基酸序列至少80％、至少85％、至少90％、至少95％、至少98％或至少99％同一。所述人源化抗体基本上可以包含所有至少一个且通常两个可变域(Fab、Fab′、F(ab′)2、FabC、Fv)，其中所有或基本上所有CDR区对应于非人类免疫球蛋白(即抗体)的CDR区且所有或基本上所有框架区为具有人类免疫球蛋白共有序列的框架区。优选地，人源化抗体还包含至少一部分免疫球蛋白恒定区(例如，Fc)，通常为人类免疫球蛋白的恒定区。在一些实施例中，人源化抗体含有轻链以及重链的至少可变域。抗体还可包括重链的CH1、铰链、CH2、CH3及CH4区。在一些实施例中，人源化抗体仅含人源化轻链。在一些实施例中，人源化抗体仅含人源化重链。在特定实施例中，人源化抗体仅含轻链和/或人源化重链的人源化可变域。
在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
参比抗体
在本申请中，所述分离的抗原结合蛋白可以与参比抗体竞争结合所述Tim-3蛋白，其中所述参比抗体可以包含轻链可变区和重链可变区，所述参比抗体的轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:1所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO:2所示的氨基酸序列；所述LCDR3可以包含SEQ ID NO:3所示的氨基酸序列，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:5所示的氨基酸序列；所述HCDR3可以包含SEQ ID NO:6所示的氨基酸序列。
CDR
在本申请中，对于所述分离的抗原结合蛋白的可变区序列(即所述VH或VL的序列)，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列(参阅例如Kabat,“Sequences of Proteins of ImmunologicalInterest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani etal.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。
在本申请中，对于所述分离的抗原结合蛋白的可变区序列(即所述VH或VL的序列)，还可以根据包含了Kabat定义和Chothia定义的Combined定义规则来确定VH和VL序列中CDR区序列。
在本申请中，所述VH可以包含HCDR1，HCDR2和HCDR3，其中所述HCDR3可以包含SEQ ID NO:6所示的氨基酸序列。
在本申请中，所述HCDR3可以包含与SEQ ID NO:6所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
在本申请中，所述HCDR1可以包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述HCDR1可以包含与SEQ ID NO:4所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
在本申请中，所述HCDR2可以包含SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述HCDR2可以包含与SEQ ID NO:5所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白的HCDR1可包含SEQ ID NO:4所示的氨基酸序列，HCDR2可包含SEQ ID NO:5所示的氨基酸序列，HCDR3可包含SEQ ID NO:6所示的氨基酸序列。
在本申请中，所述VL可以包含LCDR1，LCDR2和LCDR3，其中所述LCDR1可以包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述LCDR1可以包含与SEQ ID NO:1所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
在本申请中，所述LCDR2可以包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述LCDR2可以包含与SEQ ID NO:2所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
在本申请中，所述LCDR3可以包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述LCDR3可以包含与SEQ ID NO:3所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白的LCDR1可包含SEQ ID NO:1所示的氨基酸序列，LCDR2可包含SEQ ID NO:2所示的氨基酸序列，LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
又例如，本申请所述的分离的抗原结合蛋白的HCDR1可包含SEQ ID NO:4所示的氨基酸序列，HCDR2可包含SEQ ID NO:5所示的氨基酸序列，HCDR3可包含SEQ ID NO:6所示的氨基酸序列，且LCDR1可包含SEQ ID NO:1所示的氨基酸序列，LCDR2可包含SEQ ID NO:2所示的氨基酸序列，LCDR3可包含SEQ ID NO:3所示的氨基酸序列。
FR
在本申请中，所述分离的抗原结合蛋白的VL可以包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
在本申请中，所述L-FR1可以包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述L-FR1可以包含与SEQ ID NO:7所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述L-FR1的C末端可以与所述LCDR1的N末端直接或间接相连，且所述L-FR1可以包含SEQ ID NO:7所示的氨基酸序列。
在本申请中，所述L-FR2可以包含SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述L-FR2可以包含与SEQ ID NO:8所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2可以包含SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述L-FR3可以包含SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述L-FR3可以包含与SEQ ID NO:9所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3可以包含SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述L-FR4可以包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述L-FR4可以包含与SEQ ID NO:10所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4可以包含SEQ ID NO:10所示的氨基酸序列。
又例如，本申请所述的分离的抗原结合蛋白的L-FR1可包含SEQ ID NO:7所示的氨基酸序列，L-FR2可包含SEQ ID NO:8所示的氨基酸序列，L-FR3可包含SEQ ID NO:9所示的氨基酸序列，L-FR4可包含SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的所述VH可以包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在本申请中，所述H-FR1可以包含SEQ ID NO:11所示的氨基酸序列。
在本申请中，所述H-FR1可以包含与SEQ ID NO:11所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1 可以包含SEQ ID NO:11所示的氨基酸序列。
在本申请中，所述H-FR2可以包含SEQ ID NO:12所示的氨基酸序列。
在本申请中，所述H-FR2可以包含与SEQ ID NO:12所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:12所示的氨基酸序列。
在本申请中，所述H-FR3可以包含SEQ ID NO:13所示的氨基酸序列。
在本申请中，所述H-FR3可以包含与SEQ ID NO:13所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:13所示的氨基酸序列。
在本申请中，所述H-FR4可以包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述H-FR4可以包含与SEQ ID NO:14所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
例如，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4可以包含SEQ ID NO:14所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白的H-FR1可包含SEQ ID NO:11所示的氨基酸序列，H-FR2可包含SEQ ID NO:12所示的氨基酸序列，H-FR3可包含SEQ ID NO:13所示的氨基酸序列，H-FR4可包含SEQ ID NO:14所示的氨基酸序列。
又例如，本申请所述的分离的抗原结合蛋白的L-FR1可包含SEQ ID NO:7所示的氨基酸序列，L-FR2可包含SEQ ID NO:8所示的氨基酸序列，L-FR3可包含SEQ ID NO:9所示的氨基酸序列，L-FR4可包含SEQ ID NO:10所示的氨基酸序列，且H-FR1可包含SEQ ID NO:11所示的氨基酸序列，H-FR2可包含SEQ ID NO:12所示的氨基酸序列，H-FR3可包含SEQ ID NO:13所示的氨基酸序列，H-FR4可包含SEQ ID NO:14所示的氨基酸序列。
VL和VH
本申请所述的分离的抗原结合蛋白可包含抗体轻链可变区VL和抗体重链可变区VH。例如，所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述VH可包含SEQ ID NO:16所示的氨基酸序列。
例如，所述VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:16所示的氨基酸序列。
轻链和重链
在本申请中，所述分离的抗原结合蛋白可以包括抗体轻链恒定区，且所述抗体轻链恒定区可以包括人Igκ恒定区。
在本申请中，所述分离的抗原结合蛋白可以包括抗体轻链恒定区，且所述抗体轻链恒定区可以包括人Igλ恒定区。
在本申请中，所述抗体轻链恒定区可以包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述抗体轻链恒定区可以包含与SEQ ID NO:17所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG1重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG2重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG3重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG4重链恒定区。
在本申请中，所述抗体重链恒定区可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述抗体重链恒定区可以包含与SEQ ID NO:18所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体轻链LC，且所述LC可以包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述LC可以包含与SEQ ID NO:19所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体重链HC，且所述HC可以包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述HC可以包含与SEQ ID NO:20所示的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列。
本申请所述的分离的抗原结合蛋白可以包含抗体轻链和抗体重链。
例如，所述轻链可包含SEQ ID NO:19所示的氨基酸序列，且所述重链可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的轻链可包含SEQ ID NO:19所示的氨基酸序列， 且重链可包含SEQ ID NO:20所示的氨基酸序列。其中，所述分离的抗原结合蛋白的HCDR1可包含SEQ ID NO:4所示的氨基酸序列，HCDR2可包含SEQ ID NO:5所示的氨基酸序列，HCDR3可包含SEQ ID NO:6所示的氨基酸序列，且LCDR1可包含SEQ ID NO:1所示的氨基酸序列，LCDR2可包含SEQ ID NO:2所示的氨基酸序列，LCDR3可包含SEQ ID NO:3所示的氨基酸序列。其中，所述分离的抗原结合蛋白的L-FR1可包含SEQ ID NO:7所示的氨基酸序列，L-FR2可包含SEQ ID NO:8所示的氨基酸序列，L-FR3可包含SEQ ID NO:9所示的氨基酸序列，L-FR4可包含SEQ ID NO:10所示的氨基酸序列，且H-FR1可包含SEQ ID NO:11所示的氨基酸序列，H-FR2可包含SEQ ID NO:12所示的氨基酸序列，H-FR3可包含SEQ ID NO:13所示的氨基酸序列，H-FR4可包含SEQ ID NO:14所示的氨基酸序列。所述VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:16所示的氨基酸序列。例如，所述分离的抗原结合蛋白可以为Z2。
核酸分子、载体、细胞、制备方法和药物组合物
另一方面，本申请还提供了分离的一种或多种核酸分子，其可以编码本申请所述的分离的抗原结合蛋白。本申请所述的分离的一种或多种核酸分子可以为任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物，但可以编码本申请所述的分离的抗原结合蛋白。此外，考虑到同一种氨基酸具有两个或更多个密码子，即密码子的简并性，因此，编码同一种本申请所述的分离的抗原结合蛋白的核酸分子不是唯一的。
另一方面，本申请还提供了载体，其可以包含本申请所述的核酸分子，或，表达本申请所述的抗原结合蛋白。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
另一方面，本申请还提供了细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。在某些实施方式中，所 述细胞还可以包括用本发明所述的载体在体外转染的细胞。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞或骨髓瘤细胞。在某些实施方案中，所述细胞可以为哺乳动物细胞。在某些实施方案中，所述哺乳动物细胞可以为HEK293细胞。蛋白质的纯化分离方法可以为盐析法、等电点沉淀法、低温有机溶剂沉淀法、透析与超滤、凝胶过滤法、电泳法、离子交换层析法或亲和色谱法。
另一方面，本申请还提供了制备本申请所述的分离的抗原结合蛋白的方法，所述方法可以包括在使得本申请所述的分离的抗原结合蛋白表达的条件下，培养本申请所述的细胞。
在某些实施方式中，可以通过外源基因的诱导(例如IPTG诱导)，使本申请所述的分离的抗原结合蛋白进行表达。
另一方面，本申请还提供了药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载体。
在某些实施方案中，所述药物组合物还可以包含一种或多种(安全有效量的)药学上可接受的载体，例如稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方案中，所述药学上可接受的载体可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在某些实施方案中，所述药物组合物可以包含胃肠内施用和/或肠胃外施用途径，例如皮下、透皮、腔内、静脉内、动脉内、鞘内、瘤内、腹膜内、和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方案中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。
用途和应用
另一方面，本申请还提供了本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗肿瘤。
另一方面，本申请还提供了本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗Tim-3相关的疾病。
另一方面，本申请还提供了预防、缓解和/或治疗肿瘤的方法，所述方法可以包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
另一方面，本申请还提供了预防、缓解和/或治疗Tim-3相关的疾病的方法，所述方法可以包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
另一方面，本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物，其可以用于预防、缓解或治疗肿瘤。
另一方面，本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物，其可以用于预防、缓解或治疗Tim-3相关的疾病。
在本申请中，所述肿瘤可以包括实体瘤和/或血液肿瘤。例如，所述肿瘤可以为白血病。
在本申请中，所述Tim-3相关的疾病可以包括与表达Tim-3的细胞相关的疾病。例如癌症、自体免疫性疾病和过敏性疾病。在本申请中，Tim-3相关的疾病可以是白血病和/或肿瘤。
在本申请中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、牛、羊、兔、小鼠、大鼠或猴。
另一方面，本申请还提供了抑制Tim-3与PtdSer结合的方法，所述方法包括施用本申请所述的分离的抗原结合蛋白。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以与一种或多种有效量的治疗剂给与所需要的对象，所述一种或多种有效量的治疗剂可以包括化疗剂、细胞毒制剂、免疫抑制剂、类固醇、止吐药、癌症疫苗、镇痛药或另一种抗体。
在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以与毒素融合，以产生免疫缀合物，从而在特异性结合表达Tim-3蛋白的细胞后，在该细胞上发挥细胞毒性活性，特异性杀死癌细胞。
在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以制成溶瘤病毒，从而通过细胞表面分子入侵到肿瘤细胞中，然后以在肿瘤细胞中过度表达的特异性受体为靶向，将病毒入侵到肿瘤细胞中并行使后续的各项功能。
在某些实施方式中，本申请所述的分离的抗原结合蛋白还可以与能够特异性结合其他抗原(即除了Tim-3之外)的抗体融合，以产生双特异性抗体，从而同时特异性结合两种不同的抗原，以达到更好的肿瘤治疗效果。
本申请所述的分离的抗原结合蛋白至少具有一种下述的有益效果：
1)本申请所述的分离的抗原结合蛋白能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值通过表面等离子体共振法测定；
2)在FACS测定中，本申请所述的分离的抗原结合蛋白能够特异性结合人的Tim-3而不结合小鼠的Tim-3，从而具有种属特异性；
3)在ELISA测定中，本申请所述的分离的抗原结合蛋白能够抑制Tim-3与PtdSer的结合，阻断Tim-3信号通路，从而抑制肿瘤细胞生长；
4)本申请所述的分离的抗原结合蛋白能够促进IFN-γ和/或TNF-α的分泌，从而可以产生免疫刺激，进而抑制或消除肿瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的蛋白质分子、制备方法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。
实施例
实施例1.本申请所述的分离的抗原结合蛋白Z2的结构
本申请所述的分离的抗原结合蛋白Z2的轻链包含SEQ ID NO:19所示的氨基酸序列，且重链包含SEQ ID NO:20所示的氨基酸序列。其中，所述分离的抗原结合蛋白Z2的HCDR1包含SEQ ID NO:4所示的氨基酸序列，HCDR2包含SEQ ID NO:5所示的氨基酸序列，HCDR3包含SEQ ID NO:6所示的氨基酸序列，且LCDR1包含SEQ ID NO:1所示的氨基酸序列，LCDR2包含SEQ ID NO:2所示的氨基酸序列，LCDR3包含SEQ ID NO:3所示的氨基酸序列。此外，所述分离的抗原结合蛋白Z2的L-FR1包含SEQ ID NO:7所示的氨基酸序列，L-FR2包含SEQ ID NO:8所示的氨基酸序列，L-FR3包含SEQ ID NO:9所示的氨基酸序列，L-FR4包含SEQ ID NO:10所示的氨基酸序列，且H-FR1包含SEQ ID NO:11所示的氨基酸序列，H-FR2包含SEQ ID NO:12所示的氨基酸序列，H-FR3包含SEQ ID NO:13所示的氨基酸序列，H-FR4包含SEQ ID NO:14所示的氨基酸序列。此外，所述分离的抗原结合蛋白Z2的VL包含SEQ ID NO:15所示的氨基酸序列，且VH包含SEQ ID NO:16所示的氨基酸序列。
编码本申请所述的分离的抗原结合蛋白Z2的VL的核苷酸序列如SEQ ID NO:21所示，编码本申请所述的分离的抗原结合蛋白Z2的VH的核苷酸序列如SEQ ID NO:22所示，编码本申请所述的分离的抗原结合蛋白Z2的轻链恒定区的核苷酸序列如SEQ ID NO:23所示，编码本申请所述的分离的抗原结合蛋白Z2的重链恒定区的核苷酸序列如SEQ ID NO:24所示。
实施例2.本申请所述的分离的抗原结合蛋白Z2与人Tim-3的结合亲和力检测
2.1 Protein A的偶联
以1x HBS-EP+(GE Healthcare#BR-1006-69)为运行缓冲液，流速10μl/min,在CM5的4个通道上偶联Protein A(Thermo Fisher#21181)：1)设置注入时间800s，将50mM NHS和200mM EDC以1：1体积比混合后注入4个通道；2)用pH4.5的醋酸钠将Protein A稀释至20μg/ml，注入800s；3)注入1M乙醇胺800s，以封闭芯片表面剩余的活性羧基。封闭后继续用1x HBS-EP+缓冲液平衡仪器两小时，Protein A最终偶联量约为2000RU。
2.2动力学测试
设置多循环动力学模式，每个循环包括抗体的捕获、分析物的结合以及芯片的再生。将抗体均稀释至1μg/ml，以10μl/min的流速注入2、3、4通道40s，由预先偶联的Protein A捕获各抗体，捕获量约为200RU。分别将人Tim-3-hIg Fc融合蛋白(即人的Tim-3蛋白，美国R&D system公司,货号2365-TM-050)依次按照0nM、1.25nM、2.5nM、5nM、10nM、20nM、40nM的浓度梯度注入四个通道，流速为30ul/min，设置注入时间180s，解离时间900s。其中，人的Tim-3蛋白的氨基酸序列如SEQ ID NO:25所示。最后以同样流速注入甘氨酸(10mM，pH1.5)30s，以再生芯片。
2.3数据分析
用Biacore T200分析软件2.0对实验结果进行分析，1通道作为参比通道扣除，分析模型选用1：1动力学拟合模型。
结果如表1所示。实验结果表明，本申请所述的分离的抗原结合蛋白Z2与人Tim-3有较好的结合。
表1抗原结合蛋白Z2与人Tim-3结合的结合亲和力
抗原结合蛋白 K assoc(1/Ms) K dissoc(1/s) KD(nM) 同种型(Isotype)
Z2 3.1×10 4 8.7×10 -5 2.7 IgG2b,κ
表1中，K assoc：结合速率常数；K dissoc：解离速率常数；KD：亲和常数，等于K dissoc/K assoc。
实施例3.本申请所述的分离的抗原结合蛋白Z2与细胞表面Tim-3的结合检测
通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液进行细胞表面靶抗原(Tim-3，包括源自人的Tim-3和源自小鼠的Tim-3)与抗体(即本申请所述的分离的抗原结合蛋白Z2或对比例抗体)结合亲和力检测，其中，人的Tim-3的氨基酸序列如SEQ ID NO:25所示，小鼠的Tim-3的氨基酸序列如SEQ ID NO:26所示。对比例抗体用Anti-mTim-3Ab表示，其为商用抗体(Rat IgG2a Clone#215008，购自R&D)。具体检测过程如下：
1.使用缓冲液配置浓度为1*10 6cells/ml的靶细胞(即表达人Tim-3的转基因K562细胞，或表达小鼠Tim-3的转基因K562细胞)，加入96孔尖底板(corning 3894)，每孔30μl；
2.使用缓冲液配置检测抗体浓度为3μg/ml，并按3倍比稀释抗体，形成8个浓度梯度；
3.将配置好的不同浓度的抗体按30μl/孔加入已铺好的靶细胞中混匀；
4.4℃冰箱孵育1小时；
5.每孔加150μl缓冲液，300g离心5分钟，弃上清后将细胞震松散；
6.重复步骤5；
7.使用缓冲液按1:200配比配置荧光二抗(ab98593)，每孔30μl加入细胞中混匀，4°冰箱孵育30分钟；
8.每孔加150μl缓冲液，300g离心5分钟，弃上清后将细胞震松散；
9.重复步骤8；
10.每孔加35μl缓冲液混匀后用流式仪器检测；
11.用Graphpad软件对数据进行分析。
流式亲和力结合实验的分析结果如图1-图3所示，其中，图1显示本申请所述的分离的抗原结合蛋白Z2与表达人Tim-3细胞株的结合情况，图2显示本申请所述的分离的抗原结合蛋白Z2与表达小鼠Tim-3细胞株的结合情况，图3显示对比例抗体与表达小鼠Tim-3细胞株的结合情况。此外，图1-图3中的IgG表示实验中的同种型对照抗体小鼠IgG2b，其为商用抗体(Mouse IgG2b Clone#133303，购自R&D)。
从图1-图3可以看出，抗原结合蛋白Z2具有特异性结合人Tim-3的生物学活性。
实施例4.本申请所述的分离的抗原结合蛋白Z2阻断人Tim-3与PtdSer的结合检测
磷脂酰丝氨酸(PtdSer)是Tim-3的另一配体，通常暴露于凋亡细胞胞膜表面，与Tim-3IgV结构域结合，介导凋亡细胞吞噬，促进凋亡小体清除和树突状细胞(DCs)抗原交叉提呈。本申请将Jurkat细胞稀释成5×10 5细胞/ml并加入5μM Camptothecin,在37℃共培养5小 时以诱导细胞凋亡，表达磷脂酰丝氨酸。之后，用PBS加2％FSA洗涤2次。最后将凋亡的Jurkat细胞以每孔1×10 5个加入96孔板。将本申请所述的分离的抗原结合蛋白Z2和同种型对照抗体小鼠IgG2b(Mouse IgG2b Clone#133303，购自R&D)用PBS稀释成20μg/ml，或Tim-3-hIg Fc融合蛋白(即人的Tim-3蛋白，美国R&D system公司,货号2365-TM-050)用PBS稀释成4μg/ml，并分别让Tim-3-hIg Fc融合蛋白单独或Z2和Tim-3-hIg Fc融合蛋白共同混合与上述细胞在室温中共培养30分钟。培养结束后，离心，再用PBS加2％FSA洗涤2次。加入二抗APC-Conjugated anti-hIgG Fc Ab(R&D系统，#FAB110A)100μl，洗涤3次后进行流式细胞上机测试。检测结果如图4A-4C所示，从图4A-4C可以看出，只有在本申请所述的分离的抗原结合蛋白Z2存在的情况下，Tim-3-hIg Fc融合蛋白(即人的Tim-3蛋白)测不到凋亡细胞表达的相应受体磷脂酰丝氨酸(PtdSer)，而同种型对照小鼠抗体则无此效果。故本申请所述的分离的抗原结合蛋白Z2可以阻断人Tim-3与PtdSer的结合。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (42)

  1. 分离的抗原结合蛋白，其包括重链可变区VH中的至少一个CDR和轻链可变区VL中的至少一个CDR，其中所述VH包含SEQ ID NO:16所示的氨基酸序列，所述VL包含SEQ ID NO:15所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其具有Tim-3结合能力。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    1)能够以10nM或更低的KD值结合源自人的Tim-3，其中所述KD值通过表面等离子体共振法测定；
    2)在FACS测定中，能够特异性结合人的Tim-3而不结合小鼠的Tim-3；
    3)在ELISA测定中，能够抑制Tim-3与PtdSer的结合；和
    4)促进IFN-γ和/或TNF-α的分泌。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  5. 根据权利要求4所述的分离的抗原结合蛋白，其中所述抗体包括人源化和鼠源抗体。
  6. 根据权利要求4-5中任一项所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其中所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ ID NO:6所示的氨基酸序列。
  8. 根据权利要求7所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:4所示的氨基酸序列。
  9. 根据权利要求7-8中任一项所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:5所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其中所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:1所示的氨基酸序列。
  11. 根据权利要求10所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:2所示的氨基酸序列。
  12. 根据权利要求10-11中任一项所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:3所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的分离的抗原结合蛋白，其与参比抗体竞争结合所述Tim-3蛋白，其中所述参比抗体包含轻链可变区和重链可变区，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:1所示的氨基酸序列；所述LCDR2包含SEQ ID NO:2所示的氨基酸序列；所述LCDR3包含SEQ ID NO:3所示的氨基酸序列，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ  ID NO:4所示的氨基酸序列；所述HCDR2包含SEQ ID NO:5所示的氨基酸序列；所述HCDR3包含SEQ ID NO:6所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其中所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:7所示的氨基酸序列。
  16. 根据权利要求14-15中任一项所述的分离的抗原结合蛋白，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:8所示的氨基酸序列。
  17. 根据权利要求14-16中任一项所述的分离的抗原结合蛋白，其中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:9所示的氨基酸序列。
  18. 根据权利要求14-17中任一项所述的分离的抗原结合蛋白，其中所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4包含SEQ ID NO:10所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:15所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区。
  21. 根据权利要求20所述的分离的抗原结合蛋白，其中所述抗体轻链恒定区包含SEQ ID NO:17中所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其包含抗体轻链LC，且所述LC包含SEQ ID NO:19所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
  24. 根据权利要求23所述的分离的抗原结合蛋白，其中所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:11所示的氨基酸序列。
  25. 根据权利要求23-24中任一项所述的分离的抗原结合蛋白，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:12所示的氨基酸序列。
  26. 根据权利要求23-25中任一项所述的分离的抗原结合蛋白，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:13所示的氨基酸序列。
  27. 根据权利要求23-26中任一项所述的分离的抗原结合蛋白，其中所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:14所示的氨基酸序 列。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:16所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
  30. 根据权利要求29所述的分离的抗原结合蛋白，其中所述抗体重链恒定区包含SEQ ID NO:18中所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的分离的抗原结合蛋白，其包含抗体重链HC，且所述HC包含SEQ ID NO:20所示的氨基酸序列。
  32. 分离的一种或多种核酸分子，其编码权利要求1-31中任一项所述的分离的抗原结合蛋白。
  33. 载体，其包含根据权利要求32所述的核酸分子，或，表达权利要求1-31中任一项所述的抗原结合蛋白。
  34. 细胞，其包含根据权利要求32所述的核酸分子或根据权利要求33所述的载体。
  35. 制备权利要求1-31中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-31中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求34所述的细胞。
  36. 药物组合物，其包含权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32所述的核酸分子、权利要求33所述的载体和/或权利要求34所述的细胞，以及任选地药学上可接受的载体。
  37. 权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32所述的核酸分子、权利要求33所述的载体、权利要求34所述的细胞和/或权利要求36所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
  38. 根据权利要求37所述的用途，其中所述肿瘤包括实体瘤和/或血液肿瘤。
  39. 权利要求1-31中任一项所述的分离的抗原结合蛋白、权利要求32所述的核酸分子、权利要求33所述的载体、权利要求34所述的细胞和/或权利要求36所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗Tim-3相关的疾病。
  40. 抑制Tim-3与PtdSer结合的方法，所述方法包括施用权利要求1-31中任一项所述的分离的抗原结合蛋白。
  41. 预防、缓解和/或治疗肿瘤的方法，所述方法包括向有需要的受试者施用权利要求1-31中任一项所述的分离的抗原结合蛋白或权利要求36所述的药物组合物。
  42. 根据权利要求41所述的方法，其中所述肿瘤包括实体瘤和/或血液肿瘤。
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