WO2021169948A1 - Tim3结合分子及其应用 - Google Patents

Tim3结合分子及其应用 Download PDF

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WO2021169948A1
WO2021169948A1 PCT/CN2021/077454 CN2021077454W WO2021169948A1 WO 2021169948 A1 WO2021169948 A1 WO 2021169948A1 CN 2021077454 W CN2021077454 W CN 2021077454W WO 2021169948 A1 WO2021169948 A1 WO 2021169948A1
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antibody
seq
amino acid
tim3
acid sequence
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French (fr)
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吴炯
杨斌
潘燕峰
夏玉龙
唐英杰
金昕
孙馨
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苏州恒康生命科学有限公司
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a molecule that binds to TIM3.
  • T cell exhaustion is mediated by several immune checkpoint inhibitory receptors (such as PD1, TIM3, CTLA-1 and LAG3, etc.).
  • immune checkpoint inhibitory receptors such as PD1, TIM3, CTLA-1 and LAG3, etc.
  • the interaction of TIM3 and PD1 signaling pathways plays a role in T cell exhaustion.
  • Immune checkpoints play an important role in maintaining autoimmune tolerance and preventing the immune system from attacking their own organs. Many cancers achieve immune evasion through the imbalance of immune checkpoint protein expression. By blocking immune checkpoints, restoring the body's own anti-tumor immune response, and using the body's immune function to eliminate cancer cells in the body has always been one of the research directions of oncologists.
  • Inhibitory monoclonal antibodies targeting CTLA-1 and PD-1 at immune checkpoints have achieved objective effects in the clinical treatment of various tumors such as melanoma, kidney cancer, and lung cancer.
  • tumors such as melanoma, kidney cancer, and lung cancer.
  • immune checkpoints such as Tim-3
  • the present invention provides a TIM3 binding molecule comprising one or more amino acid sequences selected from: the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 7, The amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence shown in SEQ ID NO: 14, the amino acid sequence shown in SEQ ID NO: 15 and the amino acid sequence shown in SEQ ID NO: 16.
  • the TIM3 binding molecule of the present invention comprises the amino acid sequence shown in SEQ ID NO: 5 and/or the amino acid sequence shown in SEQ ID NO: 13.
  • the present invention relates to an antibody that binds to TIM3, comprising: light chain CDR1 (LCDR1), comprising the amino acid sequence shown in SEQ ID NO: 6 or at least 90%, 91%, 92%, 93% of the sequence , 94%, 95%, 96%, 97%, 98%, 99% homology of amino acid sequence, light chain CDR2 (LCDR2), including the amino acid sequence shown in SEQ ID NO: 7 or at least 90% of the sequence %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology of amino acid sequence, light chain CDR3 (LCDR3), including SEQ ID NO: 8
  • the amino acid sequence or an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the sequence, the heavy chain CDR1 (HCDR1 ) contains the amino acid sequence shown in SEQ ID NO: 14 or is at least 90%, 91%, 92%, 9
  • the present invention provides an antibody that binds to TIM3, comprising the amino acid sequence of the variable region of the light chain as shown in SEQ ID NO: 5 or at least 90%, 91%, 92%, 93% of the sequence. %, 94%, 95%, 96%, 97%, 98%, 99% homology of the amino acid sequence, and/or the heavy chain variable region amino acid sequence shown in SEQ ID NO: 13 or has A heavy chain variable region amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
  • the TIM3 binding molecule is a TIM3 antagonist protein. In one embodiment, the TIM3 binding molecule is a TIM3 antagonist antibody or antibody fragment. In one embodiment, the TIM3 binding molecule is a fusion protein that blocks the TIM3 signaling pathway.
  • the TIM3 binding molecule, antibody or antigen-binding fragment thereof has the activity of promoting immune response and activating immune cells, such as activating T cells.
  • the above-mentioned molecule or antibody that binds to TIM3 is a monospecific molecule or antibody or antibody fragment that binds to TIM3.
  • the above-mentioned TIM3 binding molecule or antibody is a multispecific antibody or antibody fragment.
  • the multispecific antibody is a bispecific antibody.
  • the bispecific antibody comprises a second binding domain that binds to a second biomolecule, wherein the second biomolecule is a cell surface antigen, such as a tumor antigen, for example selected from: CD3, CD20, FcRH5, Tumor antigens for HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1 and TenB2.
  • a tumor antigen for example selected from: CD3, CD20, FcRH5, Tumor antigens for HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET, Steap1 and TenB2.
  • the present invention relates to an immunoconjugate comprising a therapeutic agent linked to the above-mentioned TIM3-binding molecule or antibody, the therapeutic agent being, for example, a cytotoxic agent.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned TIM3 binding molecule, antibody, immunoconjugate, and a pharmaceutically acceptable carrier.
  • the present invention relates to a product comprising a container containing the above-mentioned pharmaceutical composition and a package insert, wherein the package insert describes the usage of the pharmaceutical composition.
  • the above-mentioned article further comprises one or more containers containing one or more other drugs.
  • the other drug is an immunostimulatory antibody or a chemotherapeutic agent.
  • the present invention relates to an isolated nucleic acid molecule, comprising a nucleotide sequence encoding one or more amino acid sequences selected from: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and/or SEQ ID NO: 13.
  • the present invention also relates to the use of these isolated nucleic acid molecules in the preparation of immunotherapy-related drugs or cells (for example, CAR-T cells, TCR-T cells).
  • immunotherapy-related drugs or cells for example, CAR-T cells, TCR-T cells.
  • the present invention relates to vectors and host cells containing the aforementioned isolated nucleic acid molecules, and their use in preparing molecules and antibodies that bind to TIM3.
  • the present invention relates to a method for promoting an immune response in a subject, the method comprising contacting the immune cells of the subject with the above-mentioned TIM3-binding molecule or antibody, thereby promoting the immune response of the subject.
  • the subject is a tumor-carrying subject or a virus-carrying subject.
  • the present invention relates to a method for inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject the above-mentioned molecule or antibody that binds to TIM3.
  • the present invention relates to a method of treating a viral infection in a subject, the method comprising administering to the subject the above-mentioned molecule or antibody that binds to TIM3.
  • the above-mentioned molecules or antibodies that bind to TIM3 are used in combination with one or more other drugs, for example, with other antibodies (including anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA -4 antibody and other TIM3 antibody), anticancer drugs or antiviral drugs used in combination.
  • the present invention relates to the use of the above-mentioned TIM3-binding molecule or antibody for the preparation of medicines.
  • the present invention relates to:
  • the TIM3 binding molecule of claim 1 or 2 which is a TIM3 antagonist antibody or antibody fragment.
  • the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence that has at least 90% homology with the sequence,
  • the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence that has at least 90% homology with the sequence, and/or
  • the light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence that has at least 90% homology with the sequence;
  • variable region of the heavy chain contains:
  • the heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that has at least 90% homology with the sequence,
  • the heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that has at least 90% homology with the sequence, and/or
  • the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence that has at least 90% homology with the sequence.
  • the antibody of claim 5 comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5 or comprises the amino acid sequence shown in SEQ ID NO: 5
  • the amino acid sequence shown is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % Homology to the amino acid sequence.
  • the antibody of claim 6, comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or comprises the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence shown is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % Homology to the amino acid sequence.
  • the antibody of claim 9 which is Fab, Fab'-SH, Fv, scFv, or (Fab') 2 .
  • the antibody of claim 14, wherein the multispecific antibody is a bispecific antibody.
  • the bispecific antibody comprises a second binding domain that binds a second biomolecule, wherein the second biomolecule is a cell surface antigen.
  • An immunoconjugate comprising a therapeutic agent linked to the antibody of any one of claims 5-18.
  • An immunologically active polypeptide comprising the light chain variable region and/or heavy chain variable region of the antibody of any one of claims 5-12.
  • An article of manufacture comprising a container containing the pharmaceutical composition of claim 23 and a package insert, wherein the package insert describes the use of the pharmaceutical composition.
  • An isolated nucleic acid comprising a nucleotide sequence encoding any amino acid sequence selected from: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 38.
  • An isolated nucleic acid comprising a nucleotide sequence encoding the light chain variable region and/or heavy chain variable region of the antibody of any one of claims 5-12.
  • a vector comprising the isolated nucleic acid of claim 27 or 28.
  • a host cell comprising the vector of claim 30.
  • a method for preparing the antibody of any one of claims 5-12, comprising culturing the host cell of claim 30 and recovering the antibody that binds to TIM3.
  • a method for promoting an immune response in a subject comprising administering to the subject a therapeutically effective amount of the TIM3-binding molecule of any one of claims 1 to 4, and any one of claims 5-18.
  • a method for inhibiting the growth of tumor cells in a subject comprising administering to the subject a therapeutically effective amount of the TIM3-binding molecule of any one of claims 1 to 4, any one of claims 5-18
  • the antibody is selected from one or more of the following: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, and other TIM3 antibodies.
  • Figures 1A-1B show the Western blot analysis results of anti-human TIM3 monoclonal antibody (clone 3G11).
  • the left picture (A) is the incubation with TIM3 monoclonal antibody (1:2000)
  • the right picture (B) is the incubation with anti-human Fc monoclonal antibody, while using anti-human GAPDH as a spot control.
  • A is 293-6E cells transfected with cell lysate encoding TIM3(22-202aa)-Fc;
  • B is 293-6E cells transfected with cell lysate encoding TIM1(21-290aa)-Fc;
  • C is 293 -6E cells were transfected with cell lysate encoding TIM4(25-314aa)-Fc;
  • D is 293-6E cells transfected with cell lysate encoding an empty vector plasmid (control).
  • Fig. 3 shows the result of immunostaining and detection of IF (immunofluorescence) used to identify the TIM3 monoclonal antibody that specifically recognizes human foreign-transfected TIM3.
  • IF immunofluorescence
  • Figure 4 shows the result of using human peripheral blood to do flow cytometric detection of TIM3 expression to determine whether TIM3 monoclonal antibody recognizes endogenously expressed TIM3.
  • the whole blood of the sample used in the flow cytometry results is from the donor number D2015, D2015-Isotype is the negative control of the same subtype antibody; D2015-5B4, -6C7, -3G11A (3G11) are TIM3 monoclonal antibodies 5B4, 6C7, 3G11 staining ; D2015-BD is the TIM3 monoclonal antibody positive control (BD product).
  • Figures 5A-5B show the results of using ELISA to determine the affinity of TIM3 monoclonal antibody.
  • the upper figure (A) is the result of TIM3 monoclonal antibody 3G11
  • the lower figure (B) is the result of TIM3 monoclonal antibody 5B4.
  • Figure 6 shows the results of using real-time fluorescent quantitative nucleic acid amplification detection system (QPCR) to detect the real-time expression of IL-2 and IFNgamma to determine the activation of T cell activity by TIM3 monoclonal antibodies 3G11 and 5B4.
  • QPCR quantitative nucleic acid amplification detection system
  • Ctrl mIgG (10ug/ml); TIM3 mAb clones, each 10ug/ml; Anti-Tim3, positive control (Biolegend); Gal-9: galectin-9, 1ug/ml;
  • Ctrl-Gal no Galectin- Control IgG isotype of 9.
  • Figure 9 is the result of using TIM3 monoclonal antibodies 3G11 and 5B4 to enhance the cytotoxicity of CIK (cytokine induced killer cells) on leukemia cells.
  • E:T refers to the ratio of effector cells to target cells.
  • Figure 10 shows the results of the inhibitory effect of TIM3 monoclonal antibody alone or in combination with PD1 monoclonal antibody on the growth of CT26 colon cancer cells.
  • antibody herein is used in a broad sense to encompass various antibody structural molecules that bind to TIM3 and include one or more of the CDR domains disclosed herein, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific Antibodies (such as bispecific antibodies) and antibody fragments (such as Fv, Fab, Fab', Fab'-SH, F(ab') 2 ), linear antibodies and single-chain antibody molecules (such as scFv), etc., as long as they exhibit all The required binding activity with TIM3 is sufficient.
  • Those skilled in the art can fuse one or more CDR domains disclosed in the present invention with one or more other polypeptide sequences to prepare functional fusion proteins or polypeptide molecules that bind to TIM3 molecules, such as vaccines, cell membrane receptor antagonists , Signal pathway regulators and chimeric antigen receptor molecules, etc.
  • one or more CDR domains disclosed in the present invention can be used to prepare a TIM3 CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) molecule.
  • TIM3 CAR-T Chimeric Antigen Receptor T-Cell Immunotherapy
  • the modifier "monoclonal” in the term “monoclonal antibody” means that the antibody is obtained from a substantially homogeneous antibody population and contains only traces of naturally occurring mutations or occurs during the preparation of monoclonal antibodies. Mutation. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes, each monoclonal antibody in the monoclonal antibody preparation is directed against a single epitope on the antigen.
  • the monoclonal antibodies of the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci.
  • Antibody fragment refers to a portion of a complete antibody that contains the antigen binding or variable regions of the complete antibody. Antibody fragments such as Fab, Fab', F(ab') and Fv fragments; diabodies; single-chain antibody molecules, such as single-chain Fv (scFv) molecules. Papain digestion of the antibody produces two identical antigen-binding fragments, called “Fab” fragments, each with an antigen-binding site and a residual "Fc” fragment. Pepsin treatment produces F(ab') 2 fragments that have two antigen binding sites and can still cross-link antigens.
  • Single-chain Fv or “scFv” antibody fragments are antibody fragments in the form of a single polypeptide chain, including the VH and VL regions of the antibody. Generally, the scFv polypeptide also contains a polypeptide linker between the VH and VL regions.
  • (d) A combination of (a), (b) and/or (c), including HVR (CDR region) amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49 -56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).
  • HVR CDR region amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49 -56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).
  • a “chimeric antibody” is an antibody having at least a portion of the heavy chain variable region and at least a portion of the light chain variable region derived from one species and at least a portion of the constant region derived from another species.
  • the chimeric antibody may comprise murine variable regions and human constant regions.
  • human common framework is a framework representing the amino acid residues most commonly present in the selection of human immunoglobulin VL or VH framework sequences.
  • human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences.
  • the sequence subgroup is as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), Volume 1-3.
  • the subgroup is subgroup ⁇ I as described by Kabat et al. (supra).
  • the subgroup is subgroup III as described by Kabat et al. (supra).
  • sequence identity refers to the percentage of identical amino acid residues or nucleotide residues between the two sequences. If the two sequences to be compared are different in length, the sequence “homology” or “identity” preferably refers to the nucleotide residues in the shorter sequence that are identical to the amino acid residues or nucleotide residues in the longer sequence. Percentage of the base. Sequence identity can be routinely determined by using sequence analysis software commonly used in the art, such as the Wisconsin sequence analysis package.
  • Bind refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise indicated, as used herein, "binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between binding partner members (eg, antibody and antigen). The affinity of a molecule X to its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • Tumor antigen refers to new antigens and overexpressed antigens in the process of cell canceration.
  • tumor antigens can be newly produced proteins in the process of carcinogenesis, special degradation products of proteins, proteins with structural changes, proteins with hidden epitopes exposed, abnormal aggregation of various membrane protein molecules or abnormally high expression of embryonic antigens Or differentiation antigen.
  • TAA tumor specific antigen
  • TAA tumor-associated antigen
  • a molecule or antibody that binds to TIM3 binds to cells (such as tumor cells) expressing TIM3 to inhibit or restrict the cells, such as inhibiting the growth of tumor cells.
  • Fusion protein refers to a protein molecule formed by linking different proteins or polypeptides. Different proteins or polypeptides can be linked by chemical methods, or can be achieved by recombinant expression of DNA sequences from different sources by gene recombination technology.
  • the "fusion protein” prepared by recombinant expression of DNA sequences from different sources through gene recombination technology is sometimes also called "chimeric protein”.
  • Immunoactive peptide refers to a polypeptide or peptide that has the activity of stimulating the body's immune response, such as a polypeptide or peptide that has the ability to stimulate the proliferation of lymphocytes, secretion of cytokines, or enhance the killing or phagocytosis of antigens.
  • Immuno response refers to the process by which immune cells recognize antigens, activate proliferation, differentiate, and form effector cells or effector molecules to respond to antigens or eliminate antigens after contacting antigens (such as self-antigens, antigenic foreign bodies, mutant cells or tumor cells) .
  • antigens such as self-antigens, antigenic foreign bodies, mutant cells or tumor cells
  • cytotoxic agent refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes; chemotherapeutic agents or drugs (e.g.
  • methotrexate adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), adriamycin, Fallen, mitomycin C, chlorambucil, daunorubicin or other inserts); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as bacteria, fungi, plants Or animal-derived small molecule toxins or enzymatically active toxins, including fragments and/or variants thereof; various anti-tumor drugs or anti-cancer agents known in the art.
  • an “immunoconjugate” is a conjugate of an antibody and one or more heterologous molecules (including but not limited to cytotoxic agents).
  • terapéuticaally effective amount refers to the amount of the drug (for example, the TIM 3 binding molecule or antibody of the present invention) in an amount sufficient to produce a therapeutic effect in the subject when administered to the subject.
  • the administration of the TIM 3 binding molecule or antibody of the present invention to the subject to treat tumors (cancer) as an example, giving the subject a "therapeutically effective amount" of the TIM 3 binding molecule or antibody can reduce tumor cells (cancer cells); shrink tumors Cells (cancer cells); and/or inhibit tumor growth to a certain extent.
  • tumor (cancer) treatment the effect can be determined by measuring the size of the tumor. Those skilled in the art can easily determine the therapeutically effective amount according to conventional methods.
  • package insert is used to refer to the instructions usually included in the commercial packaging of therapeutic products, which contain information about indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings about the use of such therapeutic products information.
  • the present invention relates to anti-TIM3 antibodies.
  • the present invention provides an anti-TIM3 antibody comprising at least 1, 2, 3, 4, 5 or 6 hypervariable regions (HVR) or complementarity determining regions (CDR ) Binding domain: (a) HVR-L1 (also called light chain CDR1), which contains the amino acid sequence shown in SASSSVSSSHLY (SEQ ID NO: 6) or is at least 90%, 95%, 96%, Amino acid sequence with 97%, 98%, 99% homology; (b) HVR-L2 (also known as light chain CDR2), which contains the amino acid sequence shown in GTSNLAS (SEQ ID NO: 7) or is similar to this sequence Amino acid sequences with at least 90%, 95%, 96%, 97%, 98%, 99% homology; (c) HVR-L3 (also called light chain CDR3), which includes HQWSSFPLT (SEQ ID NO: 8) ) Or an amino acid sequence with at least 90%, 95%, 96%, 97%
  • the light chain variable (VL) domain (region) possessed by the anti-TIM3 antibody may include an amino acid sequence having at least 80% homology (e.g., at least 81%) to the sequence shown in VDIVLTQTPAIMSASPGEKVTLTCSASSSVSSSHLYWYQQKPGSSPKLWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSFPLTFGAGPSHL (SEQ ID NO: 5) %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology), and/or its variable heavy (VH) domain (region) contains at least the amino acid sequence shown in LIGAC-AWGFSETLLCTSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTKYSASVKGRFTISRDYSQSILYLQMNTLTAEDSATYFCARD
  • the anti-TIM3 antibody comprises the amino acid sequences of the light chain variable region and the heavy chain variable region shown in SEQ ID NO: 5 and SEQ ID NO: 13, respectively.
  • the antibodies provided herein are antibody fragments.
  • Antibody fragments include but are not limited to Fab, Fab', Fab'-SH, (Fab') 2 , Fv and scFv fragments and other fragments described below.
  • Fab fragment antigen binding protein
  • Fab' fragment antigen binding protein
  • Fv fragment antigen binding protein
  • scFv fragments see, for example, WO 93/16185.
  • Bifunctional antibodies are antibody fragments with two antigen binding sites, which can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161. For trifunctional antibodies and tetrafunctional antibodies, see, for example, Hudson et al., Nat. Med. 9:129-134 (2003).
  • Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody.
  • the single domain antibody is a human single domain antibody (see, for example, US Patent 6,248,516 B1).
  • Antibody fragments can be produced by various techniques, including but not limited to proteolytic digestion of intact antibodies and recombinant host cell (such as E. coli or phage) production.
  • the antibodies provided herein are human antibodies.
  • Human antibodies can be produced using various techniques known in the art.
  • Human antibodies can be prepared by administering immunogens to modified transgenic animals and then attacking with antigens to prepare complete human antibodies or complete antibodies with human variable regions.
  • Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus or exists outside the chromosomes or is randomly integrated into the animal chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated.
  • U.S. Patent Nos. 6,075,181 and 6,150,584 description of XENOMOUSETM technology
  • U.S. Patent No. 5,770,429 U.S. Patent No. 7,041,870
  • U.S. Application Publication No. US No. 2007/0061900 The human variable regions derived from intact antibodies produced by such animals can be further modified, for example by combining them with different human constant regions.
  • Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described, see, for example, Boerner et al., J. Immunol., 147:86 (1991). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Other methods include, for example, U.S. Patent No.
  • Human antibodies can also be prepared by isolating Fv clone variable domain sequences selected from a phage display library of human origin. Such variable domain sequences can then be combined with the desired human constant domains.
  • the antibodies of the present invention with high affinity can be isolated by screening combinatorial libraries for antibodies with TIM3 binding activity.
  • various methods for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. Such methods can be found in, for example, Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).
  • the anti-TIM3 antibodies provided herein are multispecific antibodies, such as bispecific antibodies.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.
  • one binding specificity is for TIM3 and the other binding specificity is for any other antigen (e.g., a second biomolecule, such as a cell surface antigen, such as a tumor antigen).
  • the bispecific anti-TIM3 antibody can target TIM3 and tumor antigens, such as CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79B, EDAR, GFRA1, MRP4, RET , Steap1 or TenB2 has binding specificity.
  • Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.
  • the antibody of the present invention encompasses amino acid sequence variants of the anti-TIM3 antibody of the present invention.
  • antibody variants prepared to further improve the binding affinity and/or other biological properties of the antibody may be required.
  • Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the required characteristics, such as binding properties to the TIM3 antigen.
  • Amino acids can be grouped according to the properties of the common side chain:
  • the present invention covers antibody variants containing non-conservative mutations and/or conservative mutations obtained according to the antibodies disclosed in the present invention, as long as the variants still have the required TIM3 binding activity.
  • substitutional variant involves an antibody variant that replaces one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody).
  • a parent antibody e.g., a humanized antibody or a human antibody
  • the resulting variants selected for further research will be modified (e.g. improved) relative to the parent antibody in terms of certain biological properties (e.g. increased affinity) and/or will substantially retain certain biological properties of the parent antibody. nature.
  • Exemplary substitution variants are affinity matured antibodies, which can be conveniently produced using, for example, phage display-based affinity maturation techniques such as those described herein.
  • one or more HVR (CDR) residues are mutated and the mutated antibody is displayed on the phage, and the mutated antibody is screened for a specific biological activity (such as binding affinity).
  • substitutions, insertions, or deletions may occur in one or more HVRs (CDRs), as long as such changes do not substantially impair the ability of the antibody to bind to TIM3.
  • CDRs HVRs
  • conservative changes that do not substantially reduce binding affinity can be made in HVR (CDR).
  • such changes can be outside the antigen contact residues in the HVR, for example, conservative or non-conservative amino acid substitutions can occur at 1, 2, 3, 4, and 5 amino acid residues in the FR region.
  • the host cell comprises (e.g., transformed to have): (1) a vector comprising a nucleic acid that encodes the amino acid sequence of the VL of the antibody and the amino acid sequence of the VH of the antibody; or (2 ) A first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding the amino acid sequence of the VH of the antibody.
  • the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • the nucleic acid encoding the antibody is isolated (e.g., as described above) and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic cells or eukaryotic cells described herein.
  • prokaryotic cells or eukaryotic cells described herein antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • antibody fragments and polypeptides in bacteria see, for example, U.S. Patent No. 5,648,237. After expression, the antibody in the soluble fraction can be isolated from the bacterial cytoplasm and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors, including glycosylation pathways that have been "humanized” to produce partially or fully human glycosylation patterns.
  • Antibodies to fungal and yeast strains See Li et al., Nat. Biotech. 24:210-215 (2006).
  • Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used to bind insect cells, especially for transfecting Spodoptera frugiperda cells.
  • Plant cell cultures can also be used as hosts.
  • US Patent No. 6,417,429 describes PLANTIBODIESTM technology for producing antibodies in transgenic plants.
  • Vertebrate cells can also be used as hosts.
  • mammalian cell lines suitable for growth in suspension may be suitable.
  • suitable mammalian host cell lines are monkey kidney CV1 cell lines (COS-7) transformed with SV40; human embryonic kidney cell lines (such as 293 cells); baby hamster kidney cells (BHK); mouse Serto Li cells (such as TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells; MRC 5 cells; Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and Myeloma cell lines, such as Y0, NS0, and Sp2/0.
  • COS-7 monkey kidney CV1 cell lines
  • human embryonic kidney cell lines such as 293 cells
  • BHK baby hamster kidney cells
  • the present invention also provides immunoconjugates comprising the anti-TIM3 antibody herein combined with one or more cytotoxic agents, such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein Toxins, bacterial, fungal, enzymatically active toxins of plant or animal origin or fragments thereof) or radioisotopes.
  • cytotoxic agents such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein Toxins, bacterial, fungal, enzymatically active toxins of plant or animal origin or fragments thereof) or radioisotopes.
  • the immunoconjugate is an antibody-drug conjugate (ADC), in which the antibody binds to one or more drugs, including but not limited to maytansine, orlistatin, dolastatin, Methotrexate, vindesine, taxane, trichothecene and CC1065.
  • ADC antibody-drug conjugate
  • Conjugates of antibodies and cytotoxic agents can be produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinyl Imino-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imide ester Bifunctional derivatives (such as dimethyl adipate hydrochloride), active esters (such as disuccinimide suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis( (P-azidobenzoyl) hexamethylene diamine), dual nitrogen derivatives (such as bis(p-diazobenzoyl) ethylene diamine), diisocyanates (such as toluene 2,6-diisocyanate), and double-reactive fluorine compounds (Such as 1,5-difluoro-2,4
  • Exemplary freeze-dried antibody formulations are described in U.S. Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006/044908.
  • an article containing the antibody or pharmaceutical composition of the present invention includes a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Such containers can be formed of various materials, such as glass or plastic.
  • the container contains the composition of the present invention itself or a combination of the composition and another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a bottle with a stopper pierceable by a hypodermic injection needle). Vial).
  • At least one active agent in the composition is an antibody of the invention.
  • the label or package insert indicates that the composition is used to treat the selected tumor.
  • mice 3 Balb/c mice in each group, weighing 20 grams/mouse. A total of 2 groups of mice were immunized.
  • Immunogen The nucleotide sequence encoding the extracellular region of TIM3 (amino acids 1-201 or amino acids 31-180) was digested by PCR, and inserted into the vectors pcDNA3.1 and PET-32a (Biovector, catalog number). :3683689), the plasmid pcDNA3.1-TIM3 (1-201aa) and the recombinant protein of extracellular region of TIM3 (amino acids 31-180) were prepared.
  • SuperImmune TM Use plasmid pcDNA3.1-TIM3 (1-201aa) to prepare 0.6 ⁇ g/ ⁇ l PBS solution, according to the literature (Immune adherence and the processing of soluble complement-fixing antibody/DNA immune complexes in mice The super immune technique (SupperImmuneTM ) described in JC Edberg, L Tosic, RP Taylor-Clinical immunology and immunopathology, 1989-Elsevier) immunized mice. The immunization is carried out every two weeks. After 3 immunizations, 20-30 ⁇ l of blood is taken from the tail of the mouse, and the serum is separated by centrifugation, and the antibody titer after immunization is detected by ELISA.
  • the first group was immunized with pcDNA3.1-TIM3 (1-201aa) for 5 times, and the second group was immunized with pcDNA3.1-TIM3 (1-201aa) for 3 times, and then boosted with the above-mentioned TIM3 extracellular region recombinant protein 1-2 times.
  • mice The spleens of the immunized mice were taken and prepared into single suspension cells for fusion with SP2/0 cells, and then cloned and screened.
  • step (4) Add 1ml PEG4000 to the mixed cells in step "3", first slowly add dropwise to the centrifuge tube and rotate the centrifuge tube slowly, and then slightly increase the dropping rate (the PEG4000 must be added within 1 minute). Let it stand for 1.5 minutes, and finally add D-Hank’s solution to 45ml immediately. The operation of this step (4) is completed within 3 minutes.
  • each fusion of 8 96-well plates, each group of immunized mice performed a total of 5 fusions. After each fusion, the positive clones were subcloned three times in succession.
  • Screening Use CHO cells stably transfected to express TIM3 protein to screen clones, and use CHO cells as a negative control.
  • the clones screened in this way ensure the recognition of eukaryotic expressed TIM3.
  • a total of 47 clones were obtained during the entire screening. Among the 47 clones, the 10 with the highest titer were selected for further biochemical and immunological identification.
  • Protein denaturation Put the lysed cells in a dry heat device at 100°C and heat for 5-10 minutes.
  • Collect the lysate Centrifuge the cooked protein at 4°C and 12000 rpm for 10 min. Pipette the supernatant into small EP tubes, 60 ⁇ L per tube, store at -20°C to avoid repeated freezing and thawing.
  • Sample loading Thaw the processed sample, centrifuge for 2 minutes and mix well, take the upper layer and load the sample at night.
  • Electrophoresis The voltage is 200V, and the electrophoresis is about 50 minutes.
  • TIM3 antibody to be tested on a shaker, slowly shake at 70 rpm, room temperature for 1-2 h or 4° C. overnight; rinse with PBST 3 times.
  • Goat anti-mouse secondary antibody shake slowly on a shaker at room temperature for 1 hour; rinse with PBST 3 times.
  • A is 293-6E cells transfected with cell lysate encoding TIM3(22-202aa)-Fc;
  • B is 293-6E cells transfected with cell lysate encoding TIM1(21-290aa)-Fc;
  • C is 293 -6E cells were transfected with cell lysate encoding TIM4(25-314aa)-Fc;
  • D is 293-6E cells transfected with cell lysate encoding an empty vector plasmid (control). It can be seen from the Western blot analysis results of Figure 1 that the anti-human TIM3 monoclonal antibody (clone 3G11) only specifically recognizes human TIM3, but not its family members human TIM1 and human TIM4.
  • TIM3 monoclonal antibody recognizes human endogenous and exogenous TIM3
  • TIM3 1-205aa-GFP
  • B Jurkat cells
  • C 293A cells
  • D TIM3 (1-205aa)-GFP
  • the upper part of the figure was incubated with TIM3 monoclonal antibody (1:2000), the middle part of the figure was incubated with GFP antibody, and the lower part was incubated with GAPDH antibody as a spotting control.
  • the results showed that TIM3 monoclonal antibody can recognize humans.
  • the internal source can also recognize the external source TIM3.
  • TIM3 monoclonal antibody specifically recognizes human transfected TIM3
  • Immunostaining detection steps A. Fixation: Wash the cell slides gently with PBS, fix with 4% paraformaldehyde at room temperature for 15-20 minutes, and wash twice with PBS. B. Blocking: 400 ⁇ l blocking buffer/well, blocking for 45min at room temperature. C. TIM3 monoclonal antibody: stand at room temperature for 1 hour or overnight in a refrigerator at 4°C. D. Goat anti-mouse secondary antibody: incubate for 1h in the dark. E.
  • 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole, DAPI) staining: discard the secondary antibody, wash with washing buffer and aspirate the remaining liquid, add DAPI to work Solution, keep it at room temperature and avoid light for 2-5min.
  • TIM3 monoclonal antibody (3G11) diluted 1:1000, incubated for 1 hour at room temperature, incubated with secondary antibody labeled with Alexa 568 for 1 hour, observed and photographed under the Confocol microscope.
  • the staining of the upper row of cells shows that HEK293 cells are only transfected with the vector skeleton (empty vector); the staining of the next row of cells shows that HEK293 cells are transfected with a eukaryotic expression vector encoding TIM3.
  • TIM3 monoclonal antibody (3G11) diluted 1:1000, incubated for 1 hour at room temperature, incubated with secondary antibody labeled with Alexa 568 for 1 hour, observed and photographed under the Confocol microscope.
  • DAPI staining stains the nucleus. The results show that TIM3 monoclonal antibody specifically recognizes human exogenously transfected TIM3.
  • Example 3 Monoclonal antibody recognizes endogenous TIM3 detection
  • the coating protein is the TIM3 extracellular region recombinant protein 1.0ug/ml prepared above.
  • the TIM3 monoclonal antibody was used for ELISA assay with gradient dilution. Calculate the EC50 value.
  • TIM3 mAbs 3G11 and 5B4 have good affinity, and the measured EC50 values are 0.733nM and 0.82nM, respectively. The result is shown in Figure 5.
  • Blocking TIM3 monoclonal antibody activates T cell activity
  • TIM3 monoclonal antibody clones 3G11 and 5B4 have the function of blocking the binding of TIM3 receptor and Galectin 9 and activating T cells. The result is shown in Figure 6.
  • Blocking TIM3 monoclonal antibody enhances the cytotoxic effect of CIK on leukemia cells
  • CIK cytokine induced killer cells
  • the activated CIK cells were used as effector cells (Effector) and leukemia cells U937 (Target) were mixed and cultured in vitro.
  • TIM3 the killing effect of effector cells on target cells is detected.
  • lactate dehydrogenase LDH By detecting the release of lactate dehydrogenase LDH to determine the percentage of specific lysed target cells.
  • E:T refers to the ratio of effector cells to target cells.
  • the experimental results show that TIM3 monoclonal antibodies 3G11 and 5B4 can enhance the killing effect of CIK on tumor cells.
  • Example 6 Humanized expression and detection of TIM3 monoclonal antibody
  • Sequence description serial number Encoding 3G11 monoclonal antibody light chain variable region nucleotide sequence SEQ ID NO: 1 Encoding 3G11 monoclonal antibody light chain CDR1 nucleotide sequence SEQ ID NO: 2 Encoding 3G11 monoclonal antibody light chain CDR2 nucleotide sequence SEQ ID NO: 3 Encoding 3G11 monoclonal antibody light chain CDR3 nucleotide sequence SEQ ID NO: 4 3G11 monoclonal antibody light chain variable region amino acid sequence SEQ ID NO: 5 3G11 monoclonal antibody light chain CDR1 amino acid sequence SEQ ID NO: 6 3G11 monoclonal antibody light chain CDR2 amino acid sequence SEQ ID NO: 7 3G11 monoclonal antibody light chain CDR3 amino acid sequence SEQ ID NO: 8 Encoding 3G11 monoclonal antibody heavy chain variable region nucleotide sequence SEQ ID NO: 9 Encoding 3G11 monoclonal antibody heavy chain CDR1 nucleotide sequence SEQ ID NO: 10 Encoding
  • the six CDR regions of the heavy and light chains of the 3G11 monoclonal antibody were embedded into the constant framework of human IgG1 and LC kappa to prepare the humanized TIM3 monoclonal antibody 3G11.
  • the humanized chimeric antibody VL light chain plasmid pTT-LC (EcoR1-Leader-Sal1-VL-BsiW1-LC kappa) and the VH heavy chain plasmid pTT-HC (EcoR1-Leader-Sal1-VH-Nhe1- CH1-CH2-CH3)
  • the plasmid was transfected in HEK293-6E according to the proportion, and 100 ⁇ l of supernatant was taken for testing after culture.
  • the ELISA results obtained are as follows:
  • TIM3 monoclonal antibody (3G11) expressed after humanization (chimerization or semi-humanization) can specifically recognize the TIM3 recombinant protein as before humanization.
  • Example 7 In vivo experiment on the inhibition of tumor growth by TIM3 monoclonal antibody

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Abstract

提供一种结合TIM3的分子，包含重链可变区和轻链可变区，能够特异性识别内源和外源TIM3，可用于治疗包括血液肿瘤，例如白血病和淋巴瘤在内的不同癌症。

Description

TIM3结合分子及其应用 技术领域
本发明属于生物医药领域，具体涉及结合TIM3的分子。
背景技术
T细胞免疫球蛋白及粘蛋白家族-3(T-cell immunoglobulin and mucin-domain containing-3，Tim3)蛋白，是Tim家族的成员，又名HAVCR2(Hepatitis A virus cellular receptor-2)蛋白，是由HAVCR2基因编码的细胞膜受体。研究发现，Tim-3表达于活化的CD4 +Th1、Th17、CD8 +Tc1细胞上,并通过负性调控上述细胞的功能维持免疫耐受。
本领域已知，T细胞耗竭是由几种免疫检查点抑制性受体(如PD1、TIM3、CTLA-1和LAG3等)所介导，TIM3和PD1信号通路相互作用在T细胞耗竭中起到重要作用。免疫检查点对于维持自身免疫耐受、避免免疫系统在对自身器官的攻击起到重要作用，而多种癌症正是通过免疫检查点蛋白表达失调来实现免疫逃避的。通过阻断免疫检查点，恢复机体自身抗肿瘤免疫应答，借助机体免疫功能来清除体内癌细胞一直是肿瘤学家研究的方向之一。
针对于免疫检查点CTLA-1和PD-1的抑制性单抗药物已经在多种肿瘤如黑色素瘤、肾癌、肺癌等临床治疗中取得了客观疗效。然而，还是有许多病人不能从这两个靶标治疗中受益，因此，人们期望寻找新的针对免疫检查点，例如Tim-3的药物，从而通过抑制免疫检查点实现对包括肿瘤在内的疾病的治疗。
发明内容
本发明涉及与TIM3结合的分子，例如抗体或其抗原结合片段，其在表达TIM3的细胞，例如肿瘤细胞上快且强内在化，在治疗不同的疾病，例如肿瘤，尤其是血液肿瘤，像白血病和淋巴瘤方面有显著的治疗效果。而且，它们在混合淋巴细胞反应(MLR)中显示强免疫刺激性细胞因子释放， 因此作为免疫刺激性药物有明显的应用价值。
一方面，本发明提供了一种TIM3结合分子，包含选自如下的一种或多种氨基酸序列：如SEQ ID NO：6所示的氨基酸序列，如SEQ ID NO：7所示的氨基酸序列，如SEQ ID NO：8所示的氨基酸序列，如SEQ ID NO：14所示的氨基酸序列，如SEQ ID NO：15所示的氨基酸序列和如SEQ ID NO：16所示的氨基酸序列。在一个实施方案中，本发明的TIM3结合分子包含如SEQ ID NO：5所示的氨基酸序列和/或如SEQ ID NO：13所示的氨基酸序列。
一方面，本发明涉及一种结合TIM3的抗体，包含：轻链CDR1(LCDR1)，包含SEQ ID NO：6所示的氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的氨基酸序列，轻链CDR2(LCDR2)，包含SEQ ID NO：7所示的氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的氨基酸序列，轻链CDR3(LCDR3)，包含SEQ ID NO：8所示的氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的氨基酸序列，重链CDR1(HCDR1)，包含SEQ ID NO：14所示的氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的氨基酸序列，重链CDR2(HCDR2)，包含SEQ ID NO：15所示的氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的氨基酸序列，和重链CDR3(HCDR3)，包含SEQ ID NO：16所示的氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的氨基酸序列。在一个实施方案中，本发明提供了一种结合TIM3的抗体，包含如SEQ ID NO：5所示的轻链可变区氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的氨基酸序列，和/或如SEQ ID NO：13所示的重链可变区氨基酸序列或与该序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的重链可变区氨基酸序列。
在一个实施方案中，所述结合TIM3的分子为TIM3拮抗蛋白。在一个实施方案中，所述结合TIM3的分子为TIM3拮抗性抗体或抗体片段。在一个实施方案中，所述结合TIM3的分子为阻断TIM3信号通路的融合 蛋白。
在一个实施方案中，上述结合TIM3的分子为结合TIM3的抗体或其抗原结合片段，例如鼠抗体、嵌合抗体、人抗体或人源化抗体或其抗原结合片段。在一非限制性实施方式中，所述的结合TIM3的抗体或其抗原结合片段为单克隆抗体、scFv、Fab片段、Fab’片段、F(ab)’片段、双特异性抗体、免疫缀合物或其组合。在一非限制性实施方式中，所述的结合TIM3的分离的抗体或其片段特异性识别内源和外源TIM3。
在一非限制性实施方式中，所述的结合TIM3的分子、抗体或其抗原结合片段具有促进免疫应答、激活免疫细胞，例如激活T细胞的活性。
在一个实施方案中，上述结合TIM3的分子或抗体为结合TIM3的单特异性分子或抗体或抗体片段。在一个实施方案中，上述结合TIM3的分子或抗体为多特异性抗体或抗体片段。在一个实施方案中，所述多特异性抗体为双特异性抗体。在一个实施方案中，所述双特异性抗体包含结合第二生物分子的第二结合域，其中所述第二生物分子为细胞表面抗原，例如肿瘤抗原，例如选自：CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1和TenB2的肿瘤抗原。
一方面，本发明涉及一种免疫缀合物，包括与上述与TIM3结合的分子或抗体连接的治疗剂，所述治疗剂例如为细胞毒性剂。
一方面，本发明涉及一种药物组合物，包含上述结合TIM3的分子、抗体、免疫缀合物，以及药学上可接受的载体。
一方面，本发明涉及一种制品，包含内装有上述药物组合物的容器和包装插页，其中所述包装插页说明该药物组合物的用法。
在一个实施方案中，上述制品还包含内装有一种或多种其它药物的一个或多个容器。在一个实施方案中，所述其它药物为免疫刺激性抗体或化疗剂。
一方面，本发明涉及一种分离的核酸分子，包含编码选自如下的一个或多个氨基酸序列的核苷酸序列：SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16。
一方面，本发明涉及一种分离的核酸分子，包含编码选自如下的氨基酸序列的核苷酸序列：SEQ ID NO：5和/或SEQ ID NO：13。
一方面，本发明还涉及这些分离的核酸分子在制备免疫治疗相关药物或细胞(例如CAR-T细胞、TCR-T细胞)中的用途。
本发明涉及包含上述分离的核酸分子的载体、宿主细胞，以及它们在制备与TIM3结合的分子和抗体方面的用途。
一方面，本发明涉及一种促进受试者免疫应答的方法，该方法包括使所述受试者免疫细胞与上述结合TIM3的分子或抗体接触，从而促进受试者免疫应答。在一个实施方案中，所述受试者为携带肿瘤的受试者或为携带病毒之受试者。
一方面，本发明涉及一种抑制受试者中肿瘤细胞生长的方法，该方法包括给予该受试者上述结合TIM3的分子或抗体。
一方面，本发明涉及一种治疗受试者中病毒感染的方法，该方法包括给予该受试者上述结合TIM3的分子或抗体。在一些实施方案中，上述结合TIM3的分子或抗体与一种或多种其它药物联合使用，例如与其它抗体(包括抗PD-1抗体、抗PD-L1抗体、抗PD-L2抗体、抗CTLA-4抗体和其它TIM3抗体)、抗癌药物或抗病毒药物联合使用。
一方面，本发明涉及上述TIM3结合的分子或抗体用于制备药物的用途。
具体地，本发明涉及：
1.一种结合TIM3的分子，包含如下所示的一种或多种氨基酸序列：
如SEQ ID NO：6所示的氨基酸序列，
如SEQ ID NO：7所示的氨基酸序列，
如SEQ ID NO：8所示的氨基酸序列，
如SEQ ID NO：14所示的氨基酸序列，
如SEQ ID NO：15所示的氨基酸序列，
如SEQ ID NO：16所示的氨基酸序列。
2.权利要求1的结合TIM3的分子，其为TIM3拮抗蛋白。
3.权利要求1或2的结合TIM3的分子，其为TIM3拮抗性抗体或抗体片段。
4.权利要求1-3的结合TIM3的分子，其为阻断TIM3信号通路的融合蛋白。
5.一种结合TIM3的抗体，包含轻链可变区和重链可变区，其中所述 轻链可变区包含：
轻链CDR1(LCDR1)，包含SEQ ID NO：6所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，
轻链CDR2(LCDR2)，包含SEQ ID NO：7所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，和/或
轻链CDR3(LCDR3)，包含SEQ ID NO：8所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列；
重链可变区包含：
重链CDR1(HCDR1)，包含SEQ ID NO：14所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，
重链CDR2(HCDR2)，包含SEQ ID NO：15所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，和/或
重链CDR3(HCDR3)，包含SEQ ID NO：16所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列。
6.权利要求5所述的抗体，包含轻链可变区和重链可变区，其中所述轻链可变区包含SEQ ID NO：5所示的氨基酸序列或包含与SEQ ID NO：5所示的氨基酸序列有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性的氨基酸序列。
7.权利要求6所述的抗体，包含轻链可变区和重链可变区，其中所述重链可变区包含SEQ ID NO：13所示的氨基酸序列或包含与SEQ ID NO：13所示的氨基酸序列有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性的氨基酸序列。
8.权利要求5-7任一项所述的抗体，其为嵌合抗体、人源化抗体或人类抗体。
9.权利要求5-7任一项所述的抗体，其为结合TIM3的抗体片段。
10.权利要求9的抗体，其为Fab、Fab'-SH、Fv、scFv或(Fab') 2。
11.权利要求5-7任一项所述的抗体，其为全长抗体。
12.权利要求11所述的抗体，其为IgG抗体。
13.权利要求5-12任一项所述的抗体，其为结合TIM3的单特异性抗 体或抗体片段。
14.权利要求5-12任一项所述的抗体，其为多特异性抗体或抗体片段。
15.权利要求14所述的抗体，其中所述多特异性抗体为双特异性抗体。
16.权利要求15的抗体，其中所述双特异性抗体包含结合第二生物分子的第二结合域，其中所述第二生物分子为细胞表面抗原。
17.权利要求16所述的抗体，其中所述细胞表面抗原为肿瘤抗原。
18.权利要求17所述的抗体，其中所述肿瘤抗原选自：CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1和TenB2。
19.一种免疫缀合物，包括与权利要求5-18任一项的抗体连接的治疗剂。
20.权利要求19的免疫缀合物，其中所述治疗剂为化疗药物。
21.权利要求20的免疫缀合物，其中所述治疗剂为细胞毒性剂。
22.一种免疫活性多肽，包含权利要求权5-12任一项所述的抗体的轻链可变区和/或重链可变区。
23.一种药物组合物，包含权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽，以及药学上可接受的载体。
24.一种制品，包含内装有权利要求23的药物组合物的容器和包装插页，其中所述包装插页说明该药物组合物的用法。
25.权利要求24的制品，还包含内装有一种或多种其它药物的一个或多个容器。
26.权利要求25的制品，其中所述其它药物为抗体、激素类药物或化疗剂。
27.一种分离的核酸，包含编码选自如下的任一氨基酸序列的核苷酸序列：SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO： 46、SEQ ID NO：47和SEQ ID NO：48。
28.一种分离的核酸，包含编码权利要求权5-12任一项所述的抗体的轻链可变区和/或重链可变区的核苷酸序列。
29.一种载体，包含权利要求27或28的分离的核酸。
30.一种宿主细胞，包含权利要求30所述的载体。
31.权利要求30所述的宿主细胞，其中所述宿主细胞为哺乳动物细胞。
32.一种制备权利要求5-12任一项所述的抗体的方法，包括培养权利要求30所述的宿主细胞并回收所述结合TIM3的抗体。
33.一种促进受试者免疫应答的方法，包括给药所述受试者治疗有效量的权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽。
34.权利要求33的方法，其中所述受试者为携带肿瘤的受试者。
35.权利要求33的方法，其中该受试者为携带病毒之受试者。
36.一种抑制受试者中肿瘤细胞生长的方法，包括给药所述受试者治疗有效量的权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽。
37.一种治疗受试者中病毒感染的方法，包括给药所述受试者治疗有效量的权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽。
38.权利要求33-37任一项的方法，其中所述权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽与一种或多种其它药物联合使用。
39.权利要求38的方法，其中所述其它药物选自如下的一种或多种：抗体、抗癌药物或抗病毒药物。
40.权利要求39的方法，其中所述抗体选自如下的一种或多种：抗PD-1抗体、抗PD-L1抗体、抗PD-L2抗体、抗CTLA-4抗体和其它TIM3抗 体。
附图说明
图1A-1B为抗人TIM3单抗(克隆3G11)的蛋白质印迹分析结果。其中，左图(A)为与TIM3单抗(1：2000)的孵育，右图(B)为与抗人Fc单抗孵育，同时用抗人GAPDH作为点样对照。图中，A为293-6E细胞转染编码TIM3(22-202aa)-Fc的细胞裂解物；B为293-6E细胞转染编码TIM1(21-290aa)-Fc的细胞裂解物；C为293-6E细胞转染编码TIM4(25-314aa)-Fc的细胞裂解物；D为293-6E细胞转染编码空载体质粒的细胞裂解物(对照)。
图2为用蛋白免疫印迹检查抗人TIM3单抗(3G11)识别以下裂解物中的TIM3表达的结果。其中，A为激活的人T细胞(用CD3单抗激活后的T细胞)；B为Jurkat细胞；C为293A细胞；D为用编码TIM3(1-205aa)-GFP的质粒转染的293A细胞，上图部分用TIM3单抗孵育(1：2000)，图中间部分用GFP抗体孵育，下图部分用GAPDH抗体孵育作为点样对照。
图3为用于鉴定TIM3单抗特异性识别人外源转染的TIM3的免疫染色检测IF(免疫荧光)的结果。其中，上一排细胞染色为HEK293细胞只转染了载体骨架(空载体)；下一排细胞染色为HEK293细胞转染了编码TIM3的真核表达载体。
图4为用人外周血做TIM3表达的流式检测来判断TIM3单抗是否识别内源表达的TIM3的结果。其中，流式结果所用的样品的全血来自献血者号码D2015，D2015–Isotype为同亚型抗体阴性对照；D2015-5B4、-6C7、-3G11A(3G11)为TIM3单抗5B4、6C7、3G11染色；D2015-BD为TIM3单抗阳性对照(BD产品)。
图5A-5B为利用ELISA测定TIM3单抗的亲和力的结果。上图(A)为TIM3单抗3G11的结果，下图(B)为TIM3单抗5B4的结果。
图6为利用实时荧光定量核酸扩增检测系统(QPCR)检测IL-2和IFNgamma实时表达情况来判断TIM3单抗3G11和5B4激活T细胞活性的结果。其中，Ctrl:mIgG(10ug/ml)；TIM3 mAb克隆,每个10ug/ml；Anti-Tim3,阳性对照(Biolegend)；Gal-9:galectin-9,1ug/ml；Ctrl-Gal:没有Galectin-9的对照IgG同种型。
图7为利用QPCR检测IL-2和IFNgamma实时表达情况来判断TIM3 单抗3G11呈剂量依赖性激活T细胞的结果。其中，Ctrl-Gal:没有Galectin-9的对照IgG同种型。
图8为利用QPCR检测IL-2和IFNgamma实时表达情况来判断TIM3单抗5B4呈剂量依赖性激活T细胞的结果。其中，Ctrl-Gal:没有Galectin-9的对照IgG同种型。
图9为利用TIM3单抗3G11和5B4增强CIK(cytokine induced killer cells，细胞因子诱导的杀伤细胞)对白血病细胞的细胞毒性作用的结果。其中，E:T是指效应细胞与靶细胞的比率。
图10为TIM3单克隆抗体单独或与PD1单克隆抗体合用对CT26结肠癌细胞生长的抑制作用的结果。
具体实施方式
1.定义
本文中的术语“抗体”以广义使用，涵盖结合TIM3的、包含本文所公开的一个或多个CDR结构域的各种抗体结构分子，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)以及抗体片段(例如Fv、Fab、Fab'、Fab'-SH、F(ab') 2)、线性抗体和单链抗体分子(例如scFv)等，只要其展现所需要的与TIM3的结合活性即可。
本领域技术人员可以将本发明公开的一个或多个CDR结构域与一种或多种其它多肽序列融合，制备成结合TIM3分子的功能性融合蛋白或多肽分子，例如疫苗、细胞膜受体拮抗剂、信号通路调节剂和嵌合抗原受体分子等等。例如，可以利用本发明公开的一个或多个CDR结构域制备成TIM3 CAR-T(Chimeric Antigen Receptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法)分子。这些基于本发明公开的序列内容衍生、制备的融合蛋白分子也涵盖在本发明的保护范围内。
本文中所使用的术语“单克隆抗体”中的修饰语“单克隆”是指该抗体获自实质上均质的抗体群体，仅含有微量的天然存在的突变或在单克隆抗体制备过程中出现的突变。与典型地包括针对不同表位的不同抗体的多克隆抗体制剂相比，单克隆抗体制剂中的各单克隆抗体针对抗原上的单一表位。本发明的单克隆抗体可通过多种技术制造，包括但不限于杂交瘤方 法、重组DNA方法、噬菌体展示方法及利用含有所有或部分人类免疫球蛋白基因座的转基因动物的方法制造。
术语“全长抗体”、“完整抗体”指具有基本上与天然抗体结构相似的结构的抗体，该术语在本文中可互换使用。
抗体的“类”指其重链拥有的恒定域或恒定区的类型。抗体有5大类：IgA、IgD、IgE、IgG、和IgM，并且这些中的几种可以进一步分成亚类(同种型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1、和IgA2。与不同类免疫球蛋白对应的重链恒定域分别称作α、δ、ε、γ、和μ。
“抗体片段”指包含完整抗体的抗原结合区或可变区的完整抗体的一部分。抗体片段例如Fab，Fab’，F(ab’)和Fv片段；双抗体(diabodies)；单链抗体分子，例如单链Fv(scFv)分子。木瓜蛋白酶消化抗体产生两个相同的抗原结合片段，称为“Fab”片段，各具有一个抗原结合位点和一残余的“Fc”片段。胃蛋白酶处理产生F(ab’) 2片段，其具有两个抗原结合位点且仍能交联抗原。
“Fab”片段含有轻链的恒定区和重链的第一恒定区(CH1)。Fab‘片段不同于Fab段之处在于其在重链CH1结构域羧基未端添加了几个残基，包括抗体铰链区的一个或多个半胱氨酸。Fab’-SH指恒定区的半胱氨酸具有自由巯基的Fab’。F(ab’) 2抗体片段最初产生为成对的Fab’片段，此成对Fab’段之间有铰链半胱氨酸。
“单链Fv”或“scFv”抗体片段以单一多肽链形式存在的抗体片段，包含抗体的VH和VL区。通常，scFv多肽还包含VH和VL区之间的多肽接头。
“Fv”是含有完整抗原识别和结合位点的最小抗体片段。在双链Fv中，该区域由非共价键紧密联接的轻链和重链可变区的二聚体组成。在单链Fv中，重链和轻链可变区可通过一柔性肽接头共价连接，因而轻链和重链能以类似于双链Fv中的“二聚体”结构相连，其中每个可变区的3个CDR互相作用形成VH-VL二聚体表面的抗原结合位点。6个CDR共同赋予了抗体的抗原结合特异性。
术语“可变区”或“可变域”是指与抗体与抗原结合有关的抗体重链或轻链结构域。天然抗体的重链及轻链(分别为VH及VL)的可变域一般具有类似结构，其中各域包含4个保守框架区(FR)及3个互补决定区(CDR 区)。(参见例如Kindt等人，Kuby Immunology，第6版。单一VH或VL域即可足以赋予抗原结合特异性。
如本文中所使用的术语“高变区”或“HVR”是指抗体可变域中具有序列高变区(也称为“互补决定区”或”CDR”)和/或形成结构确定的环(“高变环”)和/或含有抗原接触残基(“抗原触点”)的各区。一般而言，抗体包含6个HVR(CDR区)：3个处于VH中(H1、H2、H3)且3个处于VL中(L1、L2、L3)。本文中的例示性HVR(CDR区)包括：
(a)在氨基酸残基26-32(L1)、50-52(L2)、91-96(L3)、26-32(H1)、53-55(H2)及96-101(H3)处出现的高变环(Chothia及Lesk,J.Mol.Biol.196:901-917(1987))；
(b)在氨基酸残基24-34(L1)、50-56(L2)、89-97(L3)、31-35b(H1)、50-65(H2)及95-102(H3)处出现的HVR(CDR区)(Kabat等人,Sequences of Proteins of Immunological Interest,第5版，Public Health Service,National Institutes of Health,Bethesda,MD(1991))；
(c)在氨基酸残基27c-36(L1)、46-55(L2)、89-96(L3)、30-35b(H1)、47-58(H2)及93-101(H3)处出现的抗原触点(MacCallum等人,J.Mol.Biol.262:732-745(1996))；及
(d)(a)、(b)和/或(c)的组合，包括HVR(CDR区)氨基酸残基46-56(L2)、47-56(L2)、48-56(L2)、49-56(L2)、26-35(H1)、26-35b(H1)、49-65(H2)、93-102(H3)及94-102(H3)。
除非另外指出，否则HVR(CDR区)残基及可变域中的其他残基(例如FR残基)在本文中根据Kabat等人(同上)进行编号。
“嵌合抗体”是具有来源于一种物种的至少一部分重链可变区和至少一部分轻链可变区以及来源于另一物种的至少一部分恒定区的抗体。例如，在一个实施方案中，嵌合抗体可包含鼠可变区和人恒定区。
“人源化”抗体指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施方案中，人源化抗体会包含至少一个，通常两个基本上整个可变域，其中所有或基本上所有HVR(例如CDR)对应于非人抗体的那些，且所有或基本上所有FR对应于人抗体的那些。任选地，人源化抗体可以至少包含自人抗体衍生的抗体恒定区的一部分。抗体(例如非人抗体)的“人源化形式”指已经经历人源化的抗体。
“人类共同框架”为代表在选择人类免疫球蛋白VL或VH框架序列中最常存在的氨基酸残基的框架。一般而言，人类免疫球蛋白VL或VH序列选自于可变域序列的亚组。一般而言，序列亚组为如Kabat等人，Sequences of Proteins of Immunological Interest，第5版，NIH Publication 91-3242，Bethesda MD(1991)，第1-3卷中的亚组。在一个实施方案中，对于VL而言，所述亚组为如Kabat等人(同上)描述的亚组κI。在一个实施方案中，对于VH而言，所述亚组为如Kabat等人(同上)描述的亚组III。
“人类抗体”也可称为“人抗体”、“全人源抗体”或“全人抗体”，为其氨基酸序列对应于由人类产生的或人类细胞产生的氨基酸序列的抗体。此人类抗体定义特定地排除了包含非人类抗原结合残基的人源化抗体。人类抗体可使用本领域中已知的各种技术制备，包括噬菌体展示库技术等。
“双特异性抗体”或称“双功能抗体”是具有两个不同的重/轻链对和两个不同的结合位点的人工杂合抗体。可通过多种方法，包括杂交瘤融合或Fab’片段的连接来制备双特异抗体。
两氨基酸序列或核苷酸序列间的“同源性”或“同一性”指两序列间氨基酸残基或核苷酸残基完全相同的百分数。如果待相互比较的两序列长度有别，则序列“同源性”或“同一性”优选是指较短序列中与较长序列氨基酸残基或核苷酸残基完全相同的核苷酸残基的百分数。序列同一性可以通过使用本领域常用的序列分析软件，诸如Wisconsin序列分析包等常规确定。
“亲和力”是指分子(例如抗体)的单一结合位点与其结合伴侣(例如抗原)之间的非共价相互作用总和的强度。除非另有指示，否则如本文中所使用，“结合亲和力”是指固有结合亲和力，其反映出结合伴侣成员(例如抗体与抗原)之间的1:1相互作用。分子X对其伴侣Y的亲和力一般可由解离常数(Kd)表示。亲和力可通过本领域中已知的常用方法加以测量，包括本文中所描述的那些方法。用于测量结合亲和力的特定说明性及例示性实施方案描述于下文中。
“肿瘤抗原”指细胞癌变过程中新出现的抗原以及过度表达的抗原物质。例如，肿瘤抗原可以是癌变过程中新产生的蛋白、蛋白的特殊降解产 物、结构发生变化的蛋白、隐蔽的抗原表位暴露的蛋白，多种膜蛋白分子的异常聚集或异常高表达的胚胎抗原或分化抗原。其中肿瘤细胞所特有的抗原称为“肿瘤特异性抗原(TSA)”；而并非某一种肿瘤所特有，在其他肿瘤细胞或正常细胞上也存在的抗原分子通常被称为“肿瘤相关抗原(TAA)”，例如胚胎性蛋白、糖蛋白抗原、鳞状细胞抗原。
“拮抗”是指一种物质被另一种物质所阻抑、制约的现象。例如，一种与TIM3结合的分子或抗体与表达TIM3的细胞(例如肿瘤细胞)结合后对该细胞产生的阻抑或制约，例如抑制肿瘤细胞的生长的现象。
“融合蛋白”指不同的蛋白质或多肽连成的一个蛋白分子。不同的蛋白质或多肽可以通过化学方法连接，也可通过基因重组技术将来源不同的DNA序列重组表达来实现。将通过基因重组技术将来源不同的DNA序列重组表达制备的“融合蛋白”有时也称为“嵌合蛋白”。
“免疫活性肽(多肽)”指具有刺激机体免疫应答活性的多肽或肽，例如具有刺激机体淋巴细胞增殖、细胞因子分泌或增强对抗原的杀伤或吞噬作用的多肽或肽。
“免疫应答”指免疫细胞在接触抗原(例如自身抗原、抗原性异物、突变细胞或肿瘤细胞)后，识别抗原、自身活化增殖、分化、形成效应细胞或效应分子以应对抗原或清除抗原的过程。
如本文中所使用的术语“细胞毒性剂”是指抑制或预防细胞功能和/或引起细胞死亡或破坏的物质。细胞毒性剂包括但不限于放射性同位素；化学治疗剂或药物(例如氨甲蝶呤、亚德里米星、长春花生物碱(长春新碱、长春花碱、依托泊苷)、阿霉素、美法仑、丝裂霉素C、苯丁酸氮芥、道诺红菌素或其他插入剂)；生长抑制剂；酶及其片段，诸如核分解酶；抗生素；毒素，诸如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体；本领域已知的各种抗肿瘤药物或抗癌剂。
“免疫缀合物”为抗体与一个或多个异源分子(包括但不限于细胞毒性剂)的缀合物。
“肿瘤”指各种恶性或良性肿瘤的统称。“恶性肿瘤”和“癌症”在本文可互换使用。
“受试者”或“个体”为哺乳动物。哺乳动物包括但不限于驯化动物、灵长类动物及啮齿动物(例如小鼠及大鼠)。在某些实施方案中，所述受试者或个体为人类。
短语“治疗有效量”指药物的量(例如本发明TIM 3结合分子或抗体)的量在向受试者给药时足以在所述受试者体内产生治疗效果的量。以给药受试者本发明的TIM 3结合分子或抗体治疗肿瘤(癌症)为例，给受试者“治疗有效量”的TIM 3结合分子或抗体可减少肿瘤细胞(癌细胞)；缩小肿瘤细胞(癌细胞)；和/或一定程度地抑制肿瘤生长。就肿瘤(癌症)治疗而言，可以通过测定肿瘤的大小来确定效果。本领域技术人员按照常规方法能很容易确定治疗有效量。
术语“包装插页”用于指通常包括在治疗产品的商业包装中的说明书，其含有关于适应症、用法、剂量、给药、组合疗法、禁忌症和/或关于使用此类治疗产品的警告的信息。
2.抗体、制备方法、组合物及制品
1)抗体
本发明涉及抗TIM3抗体。在某些实施方案中，本发明提供一种抗TIM3抗体，其包含含有至少1、2、3、4、5或6个选自以下的高变区(HVR)或称为互补决定区(CDR)的结合域：(a)HVR-L1(也可称为轻链CDR1)，其包含SASSSVSSSHLY(SEQ ID NO：6)所示的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(b)HVR-L2(也可称为轻链CDR2)，其包含GTSNLAS(SEQ ID NO：7)所示的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(c)HVR-L3(也可称为轻链CDR3)，其包含HQWSSFPLT(SEQ ID NO：8)所示的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(d)HVR-H1(也可称为重链CDR1)，其包含GFTFTDY(SEQ ID NO：14)所示的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(e)HVR-H2(也可称为重链CDR2)，其包含RNKANGYT(SEQ ID NO：15)所示的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列；及(f)HVR-H3(也可称为重链CDR3)，其包含DLDY(SEQ ID NO： 16)所示的氨基酸序列或与该序列至少90％、95％、96％、97％、98％、99％同源性的氨基酸序列。在一些情况下，抗TIM3抗体所具有的轻链可变(VL)域(区)可包括与VDIVLTQTPAIMSASPGEKVTLTCSASSSVSSSHLYWYQQKPGSSPKLWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSFPLTFGAGPSHL(SEQ ID NO：5)所示的氨基酸序列具有至少80％序列同源性(例如至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性)的氨基酸序列，和/或其重链可变(VH)域(区)包含与LIGAC-AWGFSETLLCTSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTKYSASVKGRFTISRDYSQSILYLQMNTLTAEDSATYFCARDLDYWGQGTFLTVSSKK(SEQ ID NO：13)所示的氨基酸序列具有至少80％序列同源性(例如至少81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同源性)的氨基酸序列。
在一些实施方案中，抗TIM3抗体包含分别如SEQ ID NO：5和SEQ ID NO：13所示的轻链可变区和重链可变区氨基酸序列。
2)抗体片段
在某些实施方案中，本文中所提供的抗体为抗体片段。抗体片段包括但不限于Fab、Fab’、Fab’-SH、(Fab’) 2、Fv及scFv片段及下文所描述的其他片段。关于某些抗体片段的综述，参见Hudson等人，Nat.Med.9:129-134(2003)。关于scFv片段参见例如WO 93/16185。
双功能抗体为具有两个抗原结合位点的抗体片段，其可为二价或双特异性的。参见例如EP 404,097；WO 1993/01161。三功能抗体及四功能抗体可参见例如Hudson等人，Nat.Med.9:129-134(2003)中。
单域抗体为包含抗体的所有或部分重链可变域或所有或部分轻链可变域的抗体片段。在某些实施方案中，单域抗体为人类单域抗体(参见例如美国专利6,248,516 B1)。
抗体片段可通过各种技术产生，包括但不限于完整抗体的蛋白水解消化以及重组宿主细胞(例如大肠杆菌或噬菌体)产生。
3)嵌合抗体及人源化抗体
在某些实施方案中，本文中所提供的抗体为嵌合抗体。嵌合抗体的制备可参见例如美国专利4,816,567。
在某些实施方案中，嵌合抗体为人源化抗体。典型地，非人类抗体经人源化以降低对人类的免疫原性，同时保留亲本非人类抗体的特异性及亲和力。一般而言，人源化抗体包含一个或多个可变域，其中全部HVR(CDR)区，或其部分源自于非人类抗体，且FR(或其部分)源自于人类抗体序列。人源化抗体任选地包含人类恒定区的至少一部分。在一些实施方案中，可用来自于非人类抗体的相应残基取代人源化抗体中的一些FR残基，以修复或改善抗体的亲和力。
人源化抗体及其制造方法可参照美国专利第5,821,337号、第7,527,791号、第6,982,321号及第7,087,409号。
4)人类抗体
在某些实施方案中，本文中所提供的抗体为人类抗体。人类抗体可使用本领域中已知的各种技术产生。
人类抗体可通过将免疫原施用给经修饰的转基因动物，然后用抗原攻击制备完整人类抗体或具有人类可变区的完整抗体。此类动物典型地含有所有或部分人类免疫球蛋白基因座，其置换内源免疫球蛋白基因座或存在于染色体外或随机整合于动物染色体中。在此类转基因小鼠中，内源免疫球蛋白基因座一般已灭活。关于由转基因动物获得人类抗体的方法，参见例如美国专利第6,075,181号及第6,150,584号(描述XENOMOUSETM技术)；美国专利第5,770,429号；美国专利第7,041,870号(描述K-M技术)；及美国申请公布第US 2007/0061900号。得自于由此类动物产生的完整抗体的人类可变区可经进一步修饰，例如将其与不同的人类恒定区进行组合。
人类抗体也可通过基于杂交瘤的方法制造。已描述用于产生人类单克隆抗体的人类骨髓瘤及小鼠-人类杂交骨髓瘤细胞株，参见例如Boerner等人，J.Immunol.,147:86(1991)。经由人类B细胞杂交瘤技术产生的人类抗体也描述于Li等人，Proc.Natl.Acad.Sci.USA,103:3557-3562(2006)中。其他方法包括例如美国专利第7,189,826号(描述由杂交瘤细胞株产生单克隆人类IgM抗体)及Ni，Xiandai Mianyixue,26(4):265-268(2006)(描述人类-人类杂交瘤)中所描述的方法。
人类抗体也可通过分离选自人类来源噬菌体展示库的Fv克隆可变域序列来制备。接着可将此类可变域序列与所要的人类恒定域组合。
具体地，可通过针对具有结合TIM3活性的抗体筛选组合库来分离具有高亲和力的本发明抗体。例如，本领域中已知用于产生噬菌体展示库及针对具有所要结合特征的抗体筛选此类库的多种方法。此类方法可查阅例如Lee等人，J.Immunol.Methods 284(1-2):119-132(2004)。
在某些噬菌体展示方法中，通过聚合酶链反应(PCR)单独克隆VH及VL基因谱系，且随机重组于噬菌体库中，接着针对抗原-结合噬菌体进行筛选。噬菌体典型地将抗体片段呈现为单链Fv(scFv)片段或Fab片段。描述人类抗体噬菌体库的专利包括例如：美国专利第5,750,373号及美国专利第2005/0079574号。
自人类抗体库分离的抗体或抗体片段在本文中被视为人类抗体或人类抗体片段。
5)多特异性抗体
在任何上述方面中，本文中所提供的抗TIM3抗体为多特异性抗体，例如双特异性抗体。多特异性抗体为对至少两个不同的位点具有结合特异性的单克隆抗体。在某些实施方案中，一个结合特异性针对TIM3，而另一结合特异性针对任何其他抗原(例如第二生物分子，例如细胞表面抗原，例如肿瘤抗原)。相应地，双特异性抗TIM3抗体可对TIM3及肿瘤抗原，例如CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1或TenB2有结合特异性。双特异性抗体可制备为全长抗体或抗体片段。
用于制造多特异性抗体的技术包括但不限于具有不同特异性的两个免疫球蛋白重链-轻链配对的重组共表达，参见WO 93/08829、WO2009/08025及WO 2009/089004A1等。
6)抗体变体
本发明的抗体涵盖本发明抗TIM3抗体的氨基酸序列变体。例如，可能需要进一步改善抗体的结合亲和力和/或其他生物学性质而制备的抗体变体。抗体的氨基酸序列变体可通过向编码该抗体的核苷酸序列中引入适当修饰来制备。此类修饰包括例如抗体氨基酸序列内的残基缺失和/或插入 和/或取代。可对缺失、插入及取代进行任何组合以获得最终的构建体，条件是最终的构建体具有所需要的特征，例如与TIM3抗原的结合特性。
在某些实施方案中，提供具有一个或多个氨基酸取代的抗体变体。可以在HVR(CDR)区和/或FR区的一个或多个位点进行取代获得取代型突变体(包括保守取代突变体或非保守取代突变体)。
可根据共同侧链性质对氨基酸进行分组：
(1)疏水性：正亮氨酸、Met、Ala、Val、Leu、Ile；
(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；
(3)酸性：Asp、Glu；
(4)碱性：His、Lys、Arg；
(5)影响链取向的残基：Gly、Pro；
(6)芳族：Trp、Tyr、Phe。
保守取代定义为同一组氨基酸之间的取代，非保守取代定义为不同类别之一的氨基酸被另一类别的氨基酸的取代。可将氨基酸取代引入本发明的抗体中且针对所希望获得的活性(例如保留/改善抗原结合或改善ADCC或CDC)来筛选产物，获得本发明的抗体变体。
本发明涵盖根据本发明公开的抗体获得的含有非保守突变和/或保守突变的抗体变体，只要该变体仍然具有所需要的TIM3结合活性。
一种类型的取代型变体涉及取代亲本抗体(例如人源化抗体或人类抗体)的一个或多个高变区残基的抗体变体。一般而言，选择用于进一步研究的所得变体将在某些生物学性质(例如增加亲和力)方面相对于亲本抗体得以修饰(例如改善)和/或将实质上保留亲本抗体的某些生物学性质。例示性取代型变体为亲和力成熟抗体，其可使用例如基于噬菌体展示的亲和力成熟技术(诸如本文中所描述的那些)便利地产生。简言之，使一个或多个HVR(CDR)残基突变且将该突变的抗体展现于噬菌体上，且针对特定生物活性(例如结合亲和力)对突变的抗体进行筛选。
在某些实施方案中，一个或多个HVR(CDR)内可发生取代、插入或缺失，只要此类变化实质上不削弱抗体结合TIM3的能力即可。例如，可在HVR(CDR)中进行实质上不降低结合亲和力的保守变化。例如，此类变化可在HVR中的抗原接触残基以外，例如可在FR区的1个1、2个、3个、4个、5个氨基酸残基处发生保守或非保守氨基酸取代。
7)重组方法
本发明的抗TIM3抗体可使用重组方法来制备，例如，如美国专利第4,816,567号中所描述。在一个实施方案中，提供编码本文中所描述的抗TIM3抗体的经分离核酸。此类核酸可编码抗体的VL氨基酸序列和/或VH的氨基酸序列。在另一实施方案中，提供包含此类核酸的一种或多种载体(例如表达载体)。在另一实施方案种，提供包含此类核酸的宿主细胞。在一个此类实施方案中，宿主细胞包含(例如，经转化而具有)：(1)包含编码包含该抗体的VL的氨基酸序列及包含该抗体的VH的氨基酸序列的核酸的载体；或(2)包含编码包含该抗体的VL的氨基酸序列的核酸的第一载体及包含编码包含该抗体的VH的氨基酸序列的核酸的第二载体。在一个实施方案中，该宿主细胞为真核细胞，例如中国仓鼠卵巢(CHO)细胞或淋巴样细胞(例如Y0、NS0、Sp20细胞)。在一个实施方案中，提供制造抗TIM3抗体的方法，其中该方法包括在适合表达该抗体的条件下培养如上文所提供的包含编码该抗体的核酸的宿主细胞，及任选地自该宿主细胞(或宿主细胞培养基)回收该抗体。
为了重组产生抗TIM3抗体，分离编码抗体的核酸(例如，如上所描述)且插入一个或多个载体中以便在宿主细胞中进行进一步克隆和/或表达。此类核酸可使用常规程序(例如，通过使用能够特异性结合编码该抗体的重链及轻链的基因的寡核苷酸探针)容易地分离并测序。
适用于克隆或表达抗体编码载体的宿主细胞包括本文中所描述的原核细胞或真核细胞。例如，可在细菌中产生抗体，尤其在不需要糖基化及Fc效应功能时。对于在细菌中表达抗体片段及多肽，参见例如美国专利第5,648,237号所述。在表达之后，可自细菌细胞浆中分离处于可溶性部分中的抗体且可进一步纯化。
除原核生物以外，诸如丝状真菌或酵母的真核微生物也为抗体编码载体的合适克隆或表达宿主，包括糖基化途径已“人源化”从而产生具有部分或完全人类糖基化模式的抗体的真菌及酵母菌株。参见Li等人,Nat.Biotech.24:210-215(2006)。
适用于表达糖基化抗体的宿主细胞也可来源于多细胞生物体(无脊椎动物及脊椎动物)。无脊椎动物细胞的实例包括植物及昆虫细胞。已鉴别 许多可用于结合昆虫细胞，尤其用于转染草地贪夜蛾(Spodoptera frugiperda)细胞的杆状病毒菌株。
植物细胞培养物也可用作宿主。例如美国专利第6,417,429号描述了用于在转基因植物中产生抗体的PLANTIBODIESTM技术。
脊椎动物细胞也可用作宿主。例如，适于在悬浮液中生长的哺乳动物细胞株可能适用。适用哺乳动物宿主细胞株的其他实例为经SV40转化的猴类肾CV1细胞株(COS-7)；人类胚肾细胞株(例如293细胞)；幼仓鼠肾细胞(BHK)；小鼠塞尔托利细胞(例如TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人类子宫颈癌细胞(HELA)；犬类肾细胞(MDCK)；布法罗大鼠肝细胞(BRL3A)；人类肺细胞(W138)；人类肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞；MRC 5细胞；中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞；及骨髓瘤细胞株，诸如Y0、NS0及Sp2/0。
8)免疫缀合物
本发明也提供包含本文中的抗TIM3抗体与一种或多种细胞毒性剂结合的免疫缀合物，此类细胞毒性剂为诸如化学治疗剂或化学治疗药物、生长抑制剂、毒素(例如蛋白毒素、细菌、真菌、植物或动物来源的酶活性毒素或其片段)或放射性同位素。
在一个实施方案中，免疫缀合物为抗体-药物缀合物(ADC)，其中抗体与一种或多种药物结合，包括但不限于美登素、奥利司他汀、多拉司他汀、氨甲蝶呤、长春地辛、紫杉烷、单端孢霉毒素(trichothecene)及CC1065。
在另一实施方案中，免疫缀合物包含如本文中所描述的抗TIM3抗体与酶活性毒素或其片段的缀合物，该酶活性毒素包括但不限于白喉A链、白喉毒素的非结合活性片段、外毒素A链及单端孢霉毒素等。
在另一实施方案中，免疫缀合物包含如本文中所描述的抗TIM3抗体与放射性原子结合形成的放射性缀合物。多种放射性同位素可用于产生放射性缀合物。实例包括At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32、Pb 212及Lu的放射性同位素。
抗体与细胞毒性剂的缀合物可使用多种双官能蛋白偶合剂制造，诸如N-丁二酰亚胺基-3-(2-吡啶基二硫)丙酸酯(SPDP)、丁二酰亚胺基-4-(N-顺丁烯二酰亚胺基甲基)环己烷-1-甲酸酯(SMCC)、亚胺基硫杂环戊烷(IT)、酰亚胺酯的双官能衍生物(诸如己二酸二甲酯盐酸盐)、活性酯(诸如辛二酸 二丁二酰亚胺酯)、醛(诸如戊二醛)、双叠氮基化合物(诸如双(对叠氮基苯甲酰)己二胺)、双重氮衍生物(诸如双(对重氮苯甲酰)乙二胺)、二异氰酸酯(诸如甲苯2,6-二异氰酸酯)及双活性氟化合物(诸如1,5-二氟-2,4-二硝基苯)。
9)药物制剂
本发明的抗TIM3抗体的药物制剂通过将具有所要纯度的抗体与一种或多种任选的药学上可接受的载体混合呈冻干制剂或水溶液形式来制备。药学上可接受的载体在所采用的剂量及浓度下一般对受体无毒，且包括但不限于：缓冲剂，诸如磷酸盐、柠檬酸盐及其他有机酸；抗氧化剂，包括抗坏血酸及甲硫氨酸；防腐剂(诸如十八烷基二甲基苯甲基氯化铵；氯化六烃季铵；苯扎氯铵；苄索氯铵；苯酚、丁醇或苯甲醇；对羟基苯甲酸烷基酯，诸如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；及间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，诸如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯啶酮；氨基酸，诸如甘氨酸、谷酰氨酸、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，诸如EDTA；糖，诸如蔗糖、甘露醇、岩藻糖或山梨糖醇；成盐相对离子，诸如钠；金属配合物(例如锌-蛋白配合物)；和/或非离子表面活性剂，诸如聚乙二醇(PEG)。
例示性冻干抗体制剂描述于美国专利第6,267,958号中。水性抗体制剂包括美国专利第6,171,586号及WO2006/044908中所描述的那些。
本文中的制剂也可含有超过一种对于所治疗的特定适应症而言必需存在的活性成分，优选具有不会对彼此造成不利影响的互补活性的活性成分。例如，可能需要进一步提供额外治疗剂(例如化疗剂、细胞毒性剂、生长抑制剂和/或抗激素剂)。此类活性成分适合以组合形式以对预定目的有效的量存在。
10)制品
在本发明的另一方面中，提供含有本发明抗体或药物组合物的制品。该制品包含容器及处于该容器上或与该容器相关联的标签或包装插页。合适的容器包括例如瓶、小瓶、注射器、IV溶液袋等。此类容器可由各种材料形成，诸如玻璃或塑料。该容器容纳本发明组合物自身或该组合物与另 一组合物的组合，且可具有无菌进入口(例如，该容器可为静脉内溶液袋或具有可由皮下注射针刺穿的瓶塞的小瓶)。该组合物中的至少一种活性剂为本发明的抗体。该标签或包装插页指示该组合物用于治疗选定肿瘤。此外，该制品可包含(a)其中含有组合物的第一容器，其中该组合物包含本发明的抗体；及(b)其中含有组合物的第二容器，其中该组合物包含另一肿瘤治疗药物或另一抗体。本发明的此实施方案中的制品可进一步包含指示此类组合物可用于治疗肿瘤的包装插页。可选地，或另外，该制品可进一步包括第二(或第三)容器，该容器包含药学上可接受的缓冲剂，诸如抑菌注射用水(BWFI)、磷酸盐缓冲盐水、林格氏溶液及右旋糖溶液。其可进一步包括自商业及使用者的立场来看合乎需要的其他材料，包括其他缓冲剂、稀释剂、过滤器、针及注射器。
以下将对本发明做以详细说明。
以下是本发明的方法和组合物的实施例。应理解鉴于上文提供的一般性描述，可以实践各种其它实施方案。
实施例1：抗TIM3单克隆抗体的制备
(一)小鼠的免疫接种
1、动物：Balb/c小鼠每组3只，体重20克/只。总共免疫2组小鼠。
2、免疫原：编码TIM3胞外区(第1-201位氨基酸或第31-180位氨基酸)的核苷酸序列通过PCR，酶切，分别插入载体pcDNA3.1和PET-32a(Biovector，货号：3683689)中，制备出质粒pcDNA3.1-TIM3(1-201aa)和TIM3胞外区重组蛋白(第31-180位氨基酸)。
3、超级免疫(SuperImmune TM)：用质粒pcDNA3.1-TIM3(1-201aa)制备成0.6μg/μl PBS溶液，根据文献(Immune adherence and the processing of soluble complement-fixing antibody/DNA immune complexes in mice JC Edberg,L Tosic,RP Taylor-Clinical immunology and immunopathology,1989-Elsevier)中记载的超级免疫技术(SupperImmune TM)对小鼠进行免疫。每隔两周免疫一次，免疫3次后从小鼠尾部取血20-30μl，离心分离血清，用以ELISA检测免疫后抗体的滴度。如果抗体滴度高于1:10000，即加强免疫一次后进行融合。如果没有达到此滴度，需要继续免疫直到滴度高于1:10000。免疫过程：第一组用pcDNA3.1-TIM3(1-201aa)免疫5 次，第二组用pcDNA3.1-TIM3(1-201aa)免疫3次后，用上述TIM3胞外区重组蛋白加强免疫1-2次。
(二)融合筛选
取上述经过免疫的小鼠的脾脏，制备成单个悬浮细胞，用于与SP2/0细胞融合，然后进行克隆筛选。
脾与骨髓瘤细胞融合：
(1)处于对数生长期的SP2/0细胞弃培养上清，加D-Hank’s液收集细胞，1000rpm离心5min，弃上清，加45ml D-Hank’s溶液，制备成细胞悬液。
(2)将上述经过免疫的小鼠的脾脏，制备成单个悬浮细胞，将该脾细胞加至上述SP2/0悬液中(SP2/0：脾细胞＝1：5-10)充分混匀，1000rpm离心5min。
(3)弃尽上清，用手弹离心管底部，使细胞分散。
(4)加1ml PEG4000至步骤“3”混合细胞中，先缓慢逐滴加至离心管并不断慢慢转动离心管，再稍微加快滴速(必须在1分钟内将PEG4000加完)。静置1.5min，最后立即加D-Hank’s液至45ml。该步骤(4)的操作在3分钟内完成。
(5)1000rpm离心5min，迅速颠倒离心管弃上清，用吸管将细胞团吹散，再加D-Hank’s液至45ml，颠倒混匀，1000rpm离心5min。
(6)弃上清，用吸管将细胞吹散，再加含10％小牛血清的HAT1640培养基，以100μl/孔，分散至预先制备的96孔板中。
(7)细胞融合后7-8天开始更换HT1640培养基。
每次融合8块96孔板，每组免疫的小鼠总共进行了5次融合。每次融合后，再对阳性克隆进行3次连续亚克隆。
筛选：利用稳定转染表达TIM3蛋白的CHO细胞筛选克隆，用CHO细胞作为阴性对照。这样筛选的克隆确保识别真核表达的TIM3。
整个筛选得到了总共47株克隆，在47株克隆中选择滴度最高的10株做进一步的生物化学和免疫学鉴定。
实施例2：抗TIM3单克隆抗体的鉴定
1、选择滴度最高的10株克隆，做一系列生物化学和免疫学鉴定，结 果见下表1(+表示阳性；-表示阴性)。
表1
Figure PCTCN2021077454-appb-000001
在上述10个筛选的克隆中，选择3G11和5B4克隆进行如下进一步的研究。
2、对人源TIM3识别的特异性
蛋白质免疫印迹检测流程：
A.上样裂解物准备
(1)收集生长至80％左右状况良好的细胞，弃尽培养基，加样品贮存液，充分混匀后收集细胞；
(2)超声破碎细胞8分钟至样品无粘稠状；
(3)将样品EP管100℃干热10min，冷却后12000rpm离心10min，吸取上清60μL分装保存至-20℃，避免反复冻融。
(4)蛋白变性：将裂解好的细胞放入干热器100℃加热保温5-10min。
(5)收集裂解物：将煮好的蛋白于4℃，12000rpm离心10min。吸取上清分装到小EP管中，每管60μL，－20℃保存，避免反复冻融。
B.上样：取已处理好的样品解冻，离心2min混匀，取上层清夜上样。
C.电泳：电压200V，电泳50分钟左右。
D.转膜：电压60V，1-2h。
E.封闭：在摇床上，以70rpm室温缓慢摇动1h。
F.待检测的TIM3抗体：在摇床上，以70rpm缓慢摇动，室温1-2h或4℃过夜；PBST漂洗3次。
G.羊抗小鼠二抗：室温摇床缓慢摇动1h；PBST漂洗3次。
H.显影。
图1为抗人TIM3单抗(克隆3G11)的蛋白质印迹分析结果。其中，左图(A)为与TIM3单抗(1：2000)的孵育，右图(B)为与抗人Fc单抗孵育，同时用抗人GAPDH作为点样对照。图中，A为293-6E细胞转染编码TIM3(22-202aa)-Fc的细胞裂解物；B为293-6E细胞转染编码TIM1(21-290aa)-Fc的细胞裂解物；C为293-6E细胞转染编码TIM4(25-314aa)-Fc的细胞裂解物；D为293-6E细胞转染编码空载体质粒的细胞裂解物(对照)。从图1的蛋白质印迹分析结果可以看出，抗人TIM3单抗(克隆3G11)只特异性识别人TIM3，而不识别其同家族成员人TIM1和人TIM4。
3、TIM3单抗识别人内源和外源TIM3
通过与上述相同流程的蛋白免疫印迹检测用抗TIM3单抗(3G11)检测TIM3在A：激活的T细胞；B：Jurkat细胞；C：293A细胞；D：用编码TIM3(1-205aa)-GFP的质粒转染的293A细胞的细胞裂解液中的表达。如图2所示，上图部分用TIM3单抗孵育(1：2000)，图中间部分用GFP抗体孵育，下图部分用GAPDH抗体孵育作为点样对照，其结果表明TIM3单抗既能够识别人内源也能识别外源TIM3。
4、TIM3单抗特异性识别人外源转染的TIM3
免疫染色检测步骤：A.固定：PBS轻洗细胞爬片，4％多聚甲醛室温固定15～20min，PBS洗两次。B.封闭：400μl封闭缓冲液/孔，室温封闭45min。C.TIM3单抗：室温静置1h或4℃冰箱内过夜。D.羊抗小鼠二抗：避光孵育1h。E.4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)染色：弃二抗，冲洗缓冲液洗涤并吸尽残液，加DAPI工作液，室温避光静置2-5min。F.封片观察：弃DAPI，用PBS、双蒸水各洗5min后吸干残液，将爬片反扣于滴有抗淬灭封片剂 的载玻片上，封片，观察。
其结果如图3所示。TIM3单抗(3G11)用1：1000稀释，室温孵育1个小时，用Alexa 568标记的二抗孵育1个小时，Confocol显微镜下观察拍照。图3中，上一排细胞染色为HEK293细胞只转染了载体骨架(空载体)；下一排细胞染色为HEK293细胞转染了编码TIM3的真核表达载体。TIM3单抗(3G11)用1：1000稀释，室温孵育1个小时，用Alexa 568标记的二抗孵育1个小时，Confocol显微镜下观察拍照。DAPI染色是染细胞核。结果表明TIM3单抗特异性识别人外源转染的TIM3。
实施例3：单抗识别内源性TIM3检测
用人外周血做TIM3表达的流式检测来判断TIM3单抗是否识别内源表达的TIM3。实验步骤如下：1)从5-10mL全血细胞中制备PBMC。2)如有必要，可通过加入红细胞裂解缓冲液裂解红细胞。3)将PBMC细胞缓慢转移至15ml无菌管(标记)。4)缓慢加入5毫升洗涤缓冲液(室温)。5)500g离心5分钟。6)弃上清液。7)重新悬浮细胞，然后加入6-10毫升洗涤缓冲液。8)温和混合细胞，然后将2mL分吸到另外3-5个15ml试管(每个试管至少包含1x10 6个细胞)中，然后标记，在室温下以500g离心5分钟。9)使用移液管滗析上清液以除去管开口端上的剩余上清液(管中剩余100μl)，并且在每个管中加入11μl 1:10稀释的小鼠抗人TIM3 mAb，或11μl 1:10稀释的小鼠抗GFP mAb作为对照。在室温下孵育60分钟。10)将2-3mL洗涤缓冲液加入每个管中，并在室温下以500g离心5分钟。11)使用移液管滗析上清液以除去管开口端的剩余上清液(管中剩余100μl)，并在每个管中加入11μl 1:40稀释的山羊抗小鼠IgG(H+L)二抗Alexa Fluor 568(Life Technology批号：1793903)在室温下在黑暗中孵育30分钟。12)将2mL洗涤缓冲液加入每个管中，并在室温下以500g离心5分钟。13)倾倒上清液，加入200μL1％多聚甲醛的PBS溶液。涡旋以重悬沉淀，并且在流式细胞术分析之前在4℃下在黑暗中储存。在24小时内分析。
流式结果表明：多种TIM3单抗克隆(3G11、5B4和6C7)识别人单核细胞内源表达的TIM3，结果如图4。
实施例4：TIM3单抗的亲和力测定
包被蛋白为上述制备的TIM3胞外区重组蛋白1.0ug/ml。用呈梯度稀释的TIM3单抗做ELISA测定。计算EC50值。TIM3单抗3G11和5B4具有良好的亲和力，所测得的EC50值分别为0.733nM和0.82nM。结果如图5所示。
实施例5：TIM3单抗的功能测定
1.阻断性TIM3单抗激活T细胞活性
将正常人PBMC在补充有10％胎牛血清，重组IL2(Cat#68-8779-82,ThermoFisher,USA)50U/ml和抗CD3抗体(Cat#317301,OKT,BioLegend,San Diego,USA)的RPMI中培养3天以上调Tim3的表达。将抗-CD3刺激的PBMC与10μg/ml抗-Tim3单克隆抗体3G11和5B4或IgG同种型对照或10μg/ml抗-Tim3阳性对照(Cat#345001(F38-2E2),BioLegend,San Diego,USA)在37度温育过夜以阻断Tim3与Gal-9的相互作用。第二天加入Galectin-9(Gal-9，为Tim3配体)(Cat#754802,BioLegend,San Diego,USA)(1ug/ml)孵育2小时，通过QPCR测定IL2和IFNgamma的mRNA，来检测IL-2和IFNgamma实时表达情况。Ctrl为对照，没有加入Galectin 9；Ctrl+Gal为对照加入了Galectin 9。
本实验采取检测IL-2和IFNg的实时表达(QPCR，Real-time Quantitative PCR Detecting System，即实时荧光定量核酸扩增检测系统)来判断TIM3单抗的生物学效应。实验结果表明，TIM3单抗克隆3G11和5B4具有阻断TIM3受体与Galectin 9的结合，激活T细胞的功能。结果如图6所示。
2.TIM3单抗呈现剂量依赖性激活T细胞的效应
实验采取了三个不同剂量的TIM3单抗(2、10、50ug/ml)，实验流程同上。实验结果表明，TIM3单抗3G11和5B4呈剂量依赖性地阻断TIM3与配体Galectin 9的结合，激活T细胞功能。结果分别如图7和图8所示。
3.阻断性TIM3单抗增强CIK对白血病细胞的细胞毒性作用
体外培养人外周血T细胞获得CIK(cytokine induced killer cells，细胞因子诱导的杀伤细胞),利用这个培养激活的CIK细胞作为效应细胞(Effector)与白血病细胞U937(Target)在体外混合培养，在加入TIM3的情况下检测效应细胞对靶细胞的杀伤作用。通过检测乳酸脱氢酶LDH的 释放来判断特异性裂解靶细胞的百分率。结果如图9所示。E:T是指效应细胞与靶细胞的比率。实验结果表明，TIM3单抗3G11和5B4具有增强CIK对肿瘤细胞的杀伤作用。
实施例6：TIM3单抗的人源化表达和检测
利用Sanger双脱氧终止测序法对TIM3单抗3G11克隆进行测序，测序结果参见下表。
序列说明 序列号
编码3G11单克隆抗体轻链可变区核苷酸序列 SEQ ID NO：1
编码3G11单克隆抗体轻链CDR1核苷酸序列 SEQ ID NO：2
编码3G11单克隆抗体轻链CDR2核苷酸序列 SEQ ID NO：3
编码3G11单克隆抗体轻链CDR3核苷酸序列 SEQ ID NO：4
3G11单克隆抗体轻链可变区氨基酸序列 SEQ ID NO：5
3G11单克隆抗体轻链CDR1氨基酸序列 SEQ ID NO：6
3G11单克隆抗体轻链CDR2氨基酸序列 SEQ ID NO：7
3G11单克隆抗体轻链CDR3氨基酸序列 SEQ ID NO：8
编码3G11单克隆抗体重链可变区核苷酸序列 SEQ ID NO：9
编码3G11单克隆抗体重链CDR1核苷酸序列 SEQ ID NO：10
编码3G11单克隆抗体重链CDR2核苷酸序列 SEQ ID NO：11
编码3G11单克隆抗体重链CDR3核苷酸序列 SEQ ID NO：12
3G11单克隆抗体重链可变区氨基酸序列 SEQ ID NO：13
3G11单克隆抗体重链CDR1氨基酸序列 SEQ ID NO：14
3G11单克隆抗体重链CDR2氨基酸序列 SEQ ID NO：15
3G11单克隆抗体重链CDR3氨基酸序列 SEQ ID NO：16
将3G11单克隆抗体重链和轻链的六个CDR区嵌入人IgG1和LC kappa恒定骨架中制备人源化TIM3单抗3G11。具体地，构建人源化嵌合抗体VL轻链质粒pTT-LC(EcoR1-Leader-Sal1-VL-BsiW1-LC kappa)和VH重链质粒pTT-HC(EcoR1-Leader-Sal1-VH-Nhe1-CH1-CH2-CH3)，将质粒按照比例在HEK293-6E中进行转染，培养后取100μl上清进行测试，所得ELISA结 果如下：
Figure PCTCN2021077454-appb-000002
以上结果显示，人源化(嵌合体，或者半人源化)后表达的TIM3单抗(3G11)与人源化前一样可以特异识别TIM3重组蛋白。
实施例7：TIM3单抗对肿瘤生长的抑制的体内实验
为了测试TIM3(3G11)单克隆抗体在体内抑制肿瘤生长的能力，使用人CT26结肠癌细胞在NSG小鼠上接种的肿瘤移植模型。将50μL RPMI1640培养基(含2×10 6个人CT26结肠癌细胞)与50μL基底膜基质胶混合后，第0天各自对NSG小鼠皮下注射。在第6天、第9天、第12天、第15天，分别对小鼠腹腔注射100μg不同的抗体，分别是TIM3单克隆抗体、PD1单克隆抗体(nivolumab(Opdivo,Bristol-Myers Squibb GmbH&Co)、TIM3单克隆抗体+PD1单克隆抗体(TIM3单克隆抗体和PD1单克隆抗体联合施用)、对照抗体(小鼠IgG，Sigma，#I5381)。实验过程中，测定小鼠肿瘤的尺寸，其中肿瘤的体积采用以下公式计算：肿瘤体积＝1/2×肿瘤长度×肿瘤宽度 2。
不同抗体对小鼠肿瘤生长的抑制结果如图10所示，由图可以看出，与对照组(小鼠IgG)相比，TIM3抗体单独使用非常显著地抑制了CT26结肠癌生长。值得强调的是TIM3抗体比PD1抗体单独使用抗癌效果更加的明显。与TIM3或PD1抗体单独使用相比，TIM3联合PD1抗体治疗起到协同抗癌作用，进一步显著抑制肿瘤生长并持续性的缩小肿瘤负荷。
尽管以上结合附图对本发明的实施方案进行了描述，但本发明并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性 和指导性的实例，不构成对本申请权利要求范围的限制。本领域的普通技术人员在本说明书的启示下和在不脱离本发明权利要求所保护的范围的情况下，还可以做出很多种取代和变化，这些均属于本发明要求保护的范围。

Claims (40)

  1. 一种结合TIM3的分子，包含如下所示的一种或多种氨基酸序列：
    如SEQ ID NO：6所示的氨基酸序列，
    如SEQ ID NO：7所示的氨基酸序列，
    如SEQ ID NO：8所示的氨基酸序列，
    如SEQ ID NO：14所示的氨基酸序列，
    如SEQ ID NO：15所示的氨基酸序列，
    如SEQ ID NO：16所示的氨基酸序列。
  2. 权利要求1的结合TIM3的分子，其为TIM3拮抗蛋白。
  3. 权利要求1或2的结合TIM3的分子，其为TIM3拮抗性抗体或抗体片段。
  4. 权利要求1-3的结合TIM3的分子，其为阻断TIM3信号通路的融合蛋白。
  5. 一种结合TIM3的抗体，包含轻链可变区和重链可变区，其中所述轻链可变区包含：
    轻链CDR1(LCDR1)，包含SEQ ID NO：6所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，
    轻链CDR2(LCDR2)，包含SEQ ID NO：7所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，和/或
    轻链CDR3(LCDR3)，包含SEQ ID NO：8所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列；
    重链可变区包含：
    重链CDR1(HCDR1)，包含SEQ ID NO：14所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，
    重链CDR2(HCDR2)，包含SEQ ID NO：15所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列，和/或
    重链CDR3(HCDR3)，包含SEQ ID NO：16所示的氨基酸序列或与该序列有至少90％同源性的氨基酸序列。
  6. 权利要求5所述的抗体，包含轻链可变区和重链可变区，其中所述轻链可变区包含SEQ ID NO：5所示的氨基酸序列或包含与SEQ ID  NO：5所示的氨基酸序列有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性的氨基酸序列。
  7. 权利要求6所述的抗体，包含轻链可变区和重链可变区，其中所述重链可变区包含SEQ ID NO：13所示的氨基酸序列或包含与SEQ ID NO：13所示的氨基酸序列有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性的氨基酸序列。
  8. 权利要求5-7任一项所述的抗体，其为嵌合抗体、人源化抗体或人类抗体。
  9. 权利要求5-7任一项所述的抗体，其为结合TIM3的抗体片段。
  10. 权利要求9的抗体，其为Fab、Fab'-SH、Fv、scFv或(Fab') 2。
  11. 权利要求5-7任一项所述的抗体，其为全长抗体。
  12. 权利要求11所述的抗体，其为IgG抗体。
  13. 权利要求5-12任一项所述的抗体，其为结合TIM3的单特异性抗体或抗体片段。
  14. 权利要求5-12任一项所述的抗体，其为多特异性抗体或抗体片段。
  15. 权利要求14所述的抗体，其中所述多特异性抗体为双特异性抗体。
  16. 权利要求15的抗体，其中所述双特异性抗体包含结合第二生物分子的第二结合域，其中所述第二生物分子为细胞表面抗原。
  17. 权利要求16所述的抗体，其中所述细胞表面抗原为肿瘤抗原。
  18. 权利要求17所述的抗体，其中所述肿瘤抗原选自：CD3、CD20、FcRH5、HER2、LYPD1、LY6G6D、PMEL17、LY6E、CD19、CD33、CD22、CD79A、CD79B、EDAR、GFRA1、MRP4、RET、Steap1和TenB2。
  19. 一种免疫缀合物，包括与权利要求5-18任一项的抗体连接的治疗剂。
  20. 权利要求19的免疫缀合物，其中所述治疗剂为化疗药物。
  21. 权利要求20的免疫缀合物，其中所述治疗剂为细胞毒性剂。
  22. 一种免疫活性多肽，包含权利要求权5-12任一项所述的抗体的轻 链可变区和/或重链可变区。
  23. 一种药物组合物，包含权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽，以及药学上可接受的载体。
  24. 一种制品，包含内装有权利要求23的药物组合物的容器和包装插页，其中所述包装插页说明该药物组合物的用法。
  25. 权利要求24的制品，还包含内装有一种或多种其它药物的一个或多个容器。
  26. 权利要求25的制品，其中所述其它药物为抗体、激素类药物或化疗剂。
  27. 一种分离的核酸，包含编码选自如下的任一氨基酸序列的核苷酸序列：SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：46、SEQ ID NO：47和SEQ ID NO：48。
  28. 一种分离的核酸，包含编码权利要求权5-12任一项所述的抗体的轻链可变区和/或重链可变区的核苷酸序列。
  29. 一种载体，包含权利要求27或28的分离的核酸。
  30. 一种宿主细胞，包含权利要求30所述的载体。
  31. 权利要求30所述的宿主细胞，其中所述宿主细胞为哺乳动物细胞。
  32. 一种制备权利要求5-12任一项所述的抗体的方法，包括培养权利要求30所述的宿主细胞并回收所述结合TIM3的抗体。
  33. 一种促进受试者免疫应答的方法，包括给药所述受试者治疗有效量的权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽。
  34. 权利要求33的方法，其中所述受试者为携带肿瘤的受试者。
  35. 权利要求33的方法，其中该受试者为携带病毒之受试者。
  36. 一种抑制受试者中肿瘤细胞生长的方法，包括给药所述受试者治疗有效量的权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽。
  37. 一种治疗受试者中病毒感染的方法，包括给药所述受试者治疗有效量的权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽。
  38. 权利要求33-37任一项的方法，其中所述权利要求1-4任一项所述的结合TIM3的分子、权利要求5-18任一项所述的抗体或权利要求19-21任一项所述的免疫缀合物、或权利要求22的免疫活性多肽与一种或多种其它药物联合使用。
  39. 权利要求38的方法，其中所述其它药物选自如下的一种或多种：抗体、抗癌药物或抗病毒药物。
  40. 权利要求39的方法，其中所述抗体选自如下的一种或多种：抗PD-1抗体、抗PD-L1抗体、抗PD-L2抗体、抗CTLA-4抗体和其它TIM3抗体。
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