WO2021175191A1 - Anticorps anti-tim-3 et son utilisation - Google Patents

Anticorps anti-tim-3 et son utilisation Download PDF

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WO2021175191A1
WO2021175191A1 PCT/CN2021/078473 CN2021078473W WO2021175191A1 WO 2021175191 A1 WO2021175191 A1 WO 2021175191A1 CN 2021078473 W CN2021078473 W CN 2021078473W WO 2021175191 A1 WO2021175191 A1 WO 2021175191A1
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amino acid
acid sequence
antibody
seq
tim
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Chinese (zh)
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匡智慧
荆华
陈炳良
刘军建
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Innovent Biologics Suzhou Co Ltd
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Innovent Biologics Suzhou Co Ltd
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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
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies

Definitions

  • the present invention relates to novel antibodies and antibody fragments that specifically bind to TIM-3, and compositions containing the antibodies or antibody fragments.
  • the present invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, host cells containing them, and related uses.
  • the present invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments (especially in combination with PD-1 pathway antibodies).
  • T cell immunoglobulin and mucin domain-3 (TIM3), also known as hepatitis A virus cell receptor 2 (HAVCR2), is a type I transmembrane protein that serves as a target for second-generation immune checkpoint drugs One is expressed on the surface of a variety of immune cells, such as T cells, NK cells, and macrophages. Like many inhibitory receptors (eg, PD-1 and CTLA-4), TIM3 expression is associated with many types of chronic diseases, including cancer.
  • HAVCR2 hepatitis A virus cell receptor 2
  • PtdSer Phosphatidylserine
  • TIM3 Galectin-9, HMGB1, Semaphorin-4A, CEACAM-1, ILT-4 and Phosphatidylserine (PtdSer or PS).
  • PtdSer is an important cell membrane component and is usually located in the inner lobules of the cell membrane. But when the cell undergoes apoptosis, PtdSer is redistributed and exposed to the outer membrane. This redistribution is also observed in many tumor cell lines. The binding of TIM3 to PtdSer may be essential for phagocytosis and cross-presentation.
  • TIM3 has a close relationship with the inhibitory receptor PD-1.
  • many tumor-specific T cells express PD-1 and TIM3, and these T cells have been shown to be more dysfunctional compared to T cells expressing only PD-1 or TIM3. See Fourcade J et al., J Exp Med. 207: 2175-2186 (2010).
  • Tim-3 signaling pathway plays a negative regulatory role in immune regulation and inhibits the anti-tumor T cell response. Therefore, blocking the Tim-3 signaling pathway can significantly improve the anti-tumor effect of T cells.
  • T cells expressing Tim-3 may exhibit a depletion phenotype characterized by impaired cytotoxic function, effector cytokine production, and proliferation.
  • anti-Tim-3 antibodies can restore anti-tumor immunity in some murine cancer models.
  • Antibodies against human Tim-3 are known.
  • WO2016161270 describes a humanized antibody against human Tim-3.
  • a number of anti-tumor experiments against human Tim-3 antibodies have been carried out clinically.
  • no antibodies targeting Tim-3 have been approved for therapeutic use in humans.
  • the present invention provides a novel Tim-3 antibody that binds Tim-3 with higher affinity, especially human and cynomolgus Tim-3, which can enhance the ability of PD-1 pathway antibodies to activate T cells and enhance PD-1 Anti-tumor activity of pathway antibodies.
  • anti-Tim-3 antibody combined with anti-PD-1 antibody can achieve better anti-tumor effects than PD-1 antibody alone. Therefore, the combination therapy of anti-Tim-3 antibody and anti-PD-1 antibody may not only improve the curative effect of anti-PD-1/anti-PD-L1 antibody, but also hope to overcome the resistance caused by anti-PD-1/anti-PD-L1 antibody treatment. It has been proved that the anti-Tim-3 monoclonal antibody combined with the anti-PD-1 monoclonal antibody of the present invention has better efficacy than the PD-1 single drug in vivo and in vitro.
  • the present invention relates to the following embodiments:
  • An antibody or antigen-binding fragment thereof that binds to TIM-3 which comprises
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO:1;
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 2 or 12;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 5 or 14;
  • LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 6;
  • LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 7;
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 19;
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 20;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 23;
  • LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 24;
  • LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25;
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO:1;
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 4 or 13;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 5 or 14;
  • LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 6;
  • LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 7;
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 19;
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 22;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 24;
  • LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25.
  • amino acid sequence comprising SEQ ID NO: 8, 15 or 26 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
  • amino acid sequence or consists of said amino acid sequence
  • amino acid sequence comprising SEQ ID NO: 9, 16 or 27 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
  • amino acid sequence or consists of said amino acid sequence
  • (iii) Contains amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 26 Sequence or heavy chain variable region consisting of the amino acid sequence, and/or comprising the amino acid sequence shown in SEQ ID NO: 27 with at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence with 96%, 97%, 98% or 99% identity or a light chain variable region composed of said amino acid sequence.
  • amino acid sequence comprising SEQ ID NO: 10, 17 or 28 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity or consisting of said amino acid sequence; or
  • amino acid sequence comprising SEQ ID NO: 11, 18 or 29 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity or consisting of said amino acid sequence; or
  • amino acid sequence having 1-20 amino acid changes compared with the amino acid sequence shown in SEQ ID NO: 11, 18, or 29, or consisting of the amino acid sequence.
  • TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, which comprises a heavy chain and/or a light chain, wherein
  • the heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 10 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 11 , 95%, 96%, 97%, 98%, or 99% identical amino acid sequence or consist of said amino acid sequence;
  • the heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 17 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 18 , 95%, 96%, 97%, 98%, or 99% identical amino acid sequence or consist of said amino acid sequence;
  • the heavy chain contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 28 % Identity of the amino acid sequence or consists of the amino acid sequence, and/or the light chain contains at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 29 , 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consist of said amino acid sequence.
  • TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, which comprises a heavy chain and/or a light chain, wherein
  • the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 11;
  • the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 18;
  • the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and/or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29.
  • TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1 or IgG2 or IgG3 or IgG4.
  • TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 10, wherein the antibody is a monoclonal antibody.
  • TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody.
  • the antibody is a bispecific or multispecific antibody molecule, preferably, the bispecific antibody molecule is also compatible with PD-1, PD- L1 or PD-L2 binding.
  • a method for preparing an antibody or antigen-binding fragment thereof that binds to TIM-3 comprising: The host cell of embodiment 17 is cultured under conditions, and the antibody or antigen-binding fragment thereof is optionally isolated. Optionally, the method further comprises recovering the antibody or antigen-binding fragment thereof that binds to TIM-3 from the host cell .
  • An immunoconjugate comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 and other substances, such as a cytotoxic agent.
  • a pharmaceutical composition comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or the immunoconjugate of embodiment 19, and optionally one or more other treatments Agents, such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.
  • Agents such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.
  • a pharmaceutical combination comprising the TIM-3 binding antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or the immunoconjugate of embodiment 19, and a PD-1 pathway antibody such as an anti-PD-1 antibody Or anti-PD-L1 antibody or anti-PD-L2 antibody, and optionally one or more other therapeutic agents, such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
  • a PD-1 pathway antibody such as an anti-PD-1 antibody Or anti-PD-L1 antibody or anti-PD-L2 antibody
  • other therapeutic agents such as chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
  • a method for preventing or treating a tumor in a subject comprising administering to the subject an effective amount of the TIM-3 binding antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14, or The immunoconjugate of embodiment 19, or the pharmaceutical composition of embodiment 20.
  • a PD-1 pathway antibody such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, to the subject.
  • a method of preventing or treating a tumor in a subject comprising administering to the subject an effective amount of the pharmaceutical combination of embodiment 21.
  • the cancer has elevated levels (e.g., nucleic acid or protein levels) of TIM-3 and/or elevated levels (E.g. at the nucleic acid or protein level) PD-L1 or PD-1 or PD-L2.
  • elevated levels e.g., nucleic acid or protein levels
  • elevated levels E.g. at the nucleic acid or protein level
  • the tumor is a tumor that is resistant to treatment with PD-1 pathway antibodies, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies .
  • PD-1 pathway antibodies such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies .
  • the method further comprises administering to the patient one or more therapies, such as treatment modalities and/or other therapeutic agents, preferably, the treatment modalities include surgical treatment and/ Or radiotherapy, other therapeutic agents are selected from chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
  • therapies such as treatment modalities and/or other therapeutic agents
  • the treatment modalities include surgical treatment and/ Or radiotherapy
  • other therapeutic agents are selected from chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
  • a method for detecting TIM-3 in a sample comprising
  • Figure 1 shows the determination of the binding ability of the chimeric antibody of the present invention to the human Tim-3CHO-S stably transfected cell line by flow cytometry.
  • Figure 2 shows the determination of the binding ability of the humanized antibody of the present invention to the human Tim-3CHO-S stably transfected cell line by flow cytometry.
  • Figure 3 shows the determination of the binding ability of the humanized antibody of the present invention to CD4+T by flow cytometry.
  • Figure 4 shows the determination of the ability of the humanized antibody of the present invention to block Tim-3 from binding to PtdSer by the MOA method.
  • Figure 5 shows the ability of the humanized antibody of the present invention to activate CD4+ T cells to release IL-2.
  • Figure 6 shows the results of the NK cell activation experiment, where A shows the percentage of cells expressing NKG2D on the cell surface, and B shows the percentage of CD107a expressing on the cell surface
  • Figure 7 shows the results of the in vivo drug efficacy test of the humanized antibody molecule of the present invention.
  • Figure 8 shows the effect of the antibody of the present invention on the body weight of mice.
  • Figure 9 shows the detection of the Tm of the antibody Hz4-3.6 of the present invention by the DSF method.
  • Figure 10 shows the accelerated stability of the antibody Hz4-3.6 of the present invention.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” when used, unless otherwise specified, it also covers the combination of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence it is also intended to encompass the antibody variable region composed of the specific sequence.
  • anti-TIM-3 antibody refers to antibodies that can bind with sufficient affinity ( Human or cynomolgus) TIM-3 or variants and isotypes thereof so that the antibody can be used as a diagnostic and/or therapeutic agent that targets (human or cynomolgus) TIM-3.
  • the degree of binding of the anti-TIM-3 antibody to non-(human or cynomolgus) TIM-3 protein is less than about 10% of the binding of the antibody to (human or cynomolgus) TIM-3.
  • RIA radioimmunoassay
  • MSD biofilm thin-layer interferometry
  • SPR surface plasmon resonance
  • Antibody fragment refers to a molecule that is different from an intact antibody, which contains a part of an intact antibody and binds to the antigen to which the intact antibody binds.
  • Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibodies (such as scFv); single-domain antibodies; Specific antibodies or fragments thereof; camelid antibodies; and bispecific antibodies or multispecific antibodies formed from antibody fragments.
  • epitope refers to a portion of an antigen (e.g., TIM-3) that specifically interacts with an antibody molecule.
  • an "antibody that binds to the same or overlapping epitope” as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody in a competition assay. The binding of the antigen, on the contrary, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen in the competition assay.
  • An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the antibody to its antigen in a competition assay.
  • Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Determination (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253).
  • An antibody that shows the same or similar binding affinity and/or specificity as the reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody Affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ( "Antigen contact point”) area.
  • CDR is mainly responsible for binding to antigen epitopes.
  • the CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • the residues of each CDR are as follows.
  • the CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).
  • the CDR of the heavy chain variable region of the antibody of the present invention is determined according to the following rules
  • VH CDR1 is determined in accordance with Abm rules, and VHCDR2 and VHCDR3 are provided in accordance with Kabat rules; or
  • VH CDR1, 2, and 3 are determined in accordance with Abm's rules.
  • the CDR of the light chain variable region of the antibody of the invention is determined according to the Kabat rule.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Antibodies with different specificities have different CDRs (under the same assignment system).
  • CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding.
  • the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of the CDR.
  • the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined by Kabat or Chothia can be replaced by conserved amino acid residues.
  • Antibody in the form of IgG refers to the form of IgG to which the constant region of the heavy chain of the antibody belongs.
  • the heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different.
  • an antibody in the form of IgG4 means that its heavy chain constant region is derived from IgG4.
  • a “humanized” antibody refers to an antibody comprising amino acid residues derived from non-human CDR and amino acid residues derived from human FR.
  • a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of the non-human antibody, and all Or substantially all FRs correspond to those of human antibodies.
  • the humanized antibody optionally may comprise at least a portion of the constant region of an antibody derived from a human antibody.
  • a "humanized form" of an antibody refers to an antibody that has been humanized.
  • Human antibody refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source, using a human antibody library or other human Antibody coding sequence. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antigen binding site to bind to a specific antigen can be by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as, for example, by radioimmunoassay (RIA) or biofilm thin-layer interferometry or MSD. Measurement method or surface plasmon resonance (SPR) measurement.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD biofilm thin-layer interferometry
  • SPR surface plasmon resonance
  • an “immunoconjugate” is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • PD-1 pathway antibody includes antibodies that specifically bind to PD-1 or its binding partner, such as PD-L1 or PD-L2.
  • the PD-1 pathway antibody reduces, blocks, inhibits, eliminates, or interferes with signal transduction derived from the interaction of PD-1 with one or more of its ligands (such as PD-L1, PD-L2) .
  • the PD-1 pathway antibody is a PD-1 antibody, a PD-L1 antibody, or a PD-L2 antibody.
  • anti-PD-1/PD-L1/PD-L2 antibody As used herein, the terms “anti-PD-1/PD-L1/PD-L2 antibody”, “anti-PD-1/PD-L1/PD-L2”, “PD-1/PD-L1/PD-L2 antibody” or “An antibody that binds to PD-1/PD-L1/PD-L2” refers to an antibody that can bind to the PD-1/PD-L1/PD-L2 protein or a fragment thereof with sufficient affinity.
  • the anti-PD-1/PD-L1/PD-L2 antibody binds to a non-PD-1/PD-L1/PD-L2 protein to a lesser degree than the antibody binds to PD-1/PD-L1/ About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of PD-L2 binding, as, for example, by radioimmunoassay (RIA) or biofilm thin-layer interferometry or MSD measurement or surface plasmon resonance (SPR) measurement.
  • RIA radioimmunoassay
  • MSD biofilm thin-layer interferometry
  • SPR surface plasmon resonance
  • therapeutic agent encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressive agents). ).
  • cytotoxic agent used in the present invention refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical compounds useful in the treatment of cancer or diseases of the immune system.
  • small molecule drugs refers to low molecular weight organic compounds capable of regulating biological processes.
  • Small molecules are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As a therapeutic agent, small molecules can penetrate cells better than large molecules, are less susceptible to degradation, and are less likely to trigger an immune response.
  • immunomodulator refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immunosuppressive agents.
  • immunosuppressive agents are therapeutic agents used to suppress or prevent the activity of the immune system in immunosuppressive therapy.
  • the “functional Fc region” possesses the "effector function” of the native sequence Fc region.
  • effector functions include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor; BCR), and the like.
  • Such effector functions generally require that the Fc region be combined with a binding domain (e.g., antibody variable domain), and can be assessed using a variety of assays, such as those disclosed herein.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • an effective amount refers to the amount or dose of the antibody or fragment or conjugate or composition or combination of the present invention, which, after being administered to a patient in single or multiple doses, produces the desired effect in patients in need of treatment or prevention .
  • “Therapeutically effective amount” refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effect of the antibody or antibody fragment or its conjugate or composition or combination is less than the therapeutically beneficial effect.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg tumor volume) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% And still more preferably at least about 80% or 90%.
  • prophylactically effective amount refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • host cell refers to cells into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations. Included herein is the mutant progeny with the same function or biological activity that is screened or selected in the initially transformed cell.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused.
  • the label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of enzymatic labeling, can catalyze a chemical change of a detectable substrate compound or composition.
  • the term is intended to cover the direct labeling of the probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody and the indirect labeling of the probe or antibody by reaction with another reagent that is directly labelled.
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., , Mice and rats). In some embodiments, the individual or subject is a human.
  • an “isolated” antibody is an antibody that has been separated from a component of its natural environment.
  • the antibody is purified to more than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) confirmed.
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC
  • isolated nucleic acid encoding anti-TIM-3 antibody or fragments thereof refers to one or more nucleic acid molecules that encode antibody heavy or light chains (or fragments thereof), including such in a single vector or separate vectors Nucleic acid molecules, and such nucleic acid molecules that are present at one or more locations in the host cell.
  • the sequences are aligned for optimal comparison purposes (for example, the first and second amino acid sequences or nucleic acid sequences may be used for optimal alignment. Gaps can be introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • Mathematical algorithms can be used to achieve sequence comparison between two sequences and calculation of percent identity.
  • the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used. Available), use Blossum 62 matrix or PAM250 matrix and gap weight 16, 14, 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6, to determine the difference between two amino acid sequences Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 are used. Length weights 1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossom 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • hybridizes under stringent conditions e.g., under low stringency, medium stringency, high stringency, or very high stringency conditions
  • stringent conditions e.g., under low stringency, medium stringency, high stringency, or very high stringency conditions
  • instructions for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in the references and either method can be used.
  • the specific hybridization conditions mentioned in this article are as follows: 1) Low stringency hybridization conditions are in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by at least 50°C (for low stringency conditions, you can Increase the washing temperature to 55°C) Wash twice in 0.2X SSC, 0.1% SDS; 2) Medium stringency hybridization conditions are about 45°C in 6X SSC, and then at 60°C in 0.2X SSC, 0.1% Wash one or more times in SDS; 3) High-stringency hybridization conditions are in 6X SSC at about 45°C, and then wash one or more times in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) Very high stringency hybridization conditions are washing one or more times in 0.5M sodium phosphate, 7% SDS at 65°C, and then in 0.2X SSC, 0.1% SDS at 65°C.
  • the very high stringency condition (4) is the preferred condition and one that should be used unless otherwise specified.
  • anti-tumor effect refers to a biological effect that can be exhibited by various means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass solid tumors and liquid tumors.
  • cancer and “cancerous” refer to or describe a physiological condition in mammals that is usually characterized by unregulated cell growth.
  • cancers suitable for treatment by the antibodies of the invention include colon cancer, rectal cancer, colorectal cancer, including metastatic forms of those cancers.
  • tumor refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancerous and cancerous cells and tissues.
  • pharmaceutical adjuvant refers to diluents, adjuvants (for example Freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. administered together with the active substance.
  • composition refers to a composition that is present in a form that allows the biological activity of the active ingredients contained therein to be effective, and does not contain other substances that have unacceptable toxicity to the subject to which the composition is administered. Ingredients.
  • non-fixed combination means that the active ingredients (for example, (i) anti-TIM-3 antibody or antigen-binding fragment thereof, and (ii) PD-1 pathway antibody or antigen-binding fragment thereof) are simultaneously, without Specific time limits or sequential administration to the patient at the same or different time intervals, wherein such administration provides a preventive or therapeutically effective level of two or more active agents in the patient.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof and the PD-1 pathway antibody or antigen-binding fragment thereof used in the pharmaceutical combination are administered at a level not exceeding the level when they are used alone.
  • fixed combination means that two or more active agents are simultaneously administered to a patient in the form of a single entity.
  • the dosage and/or time interval of two or more active agents are selected so that the combined use of each part can produce an effect greater than that achieved by using any one component alone in the treatment of diseases or conditions.
  • Each component may be in the form of a separate preparation, and the preparation form may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (e.g. radiation therapy or surgery) to treat the diseases described herein.
  • administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of active ingredients.
  • administration includes co-administration of the respective active ingredients in multiple or separate containers (e.g., tablets, capsules, powders and liquids). The powder and/or liquid can be reconstituted or diluted to the desired dose before administration.
  • such administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in the treatment of the conditions or conditions described herein.
  • treatment refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of drugs before the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.
  • tissue/patient/individual sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from the subject's pregnancy or development at any time.
  • Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and so on.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention binds to TIM-3 (e.g., human TIM-3 or cynomolgus TIM-3) with high affinity.
  • TIM-3 It is human TIM-3.
  • the antibody or antigen-binding fragment thereof of the present invention has a higher binding affinity to human or cyno TIM-3 than known TIM-3 antibodies, such as the TIM-3 antibody of WO2016161270, for example, referred to herein as It is an anti-TIM-3 antibody of TSR-022.
  • the affinity of antibodies is determined by biofilm thin-layer interferometry or surface plasmon resonance methods, such as Biacore.
  • the anti-TIM-3 antibody of the present invention in the following equilibrium dissociation constant (K D) human TIM-3 binding, the K D of less than about 10 nM, preferably less than or equal to about 6nM, 5.5 nM, 5nM, 4.5nM, 4nM, 3.5nM, 3nM, 2.5nM, and in some embodiments, the K D is between the aforementioned values (inclusive).
  • K D equilibrium dissociation constant
  • the anti-TIM-3 antibody of the present invention in the following equilibrium dissociation constant (K D) in combination with cynomolgus TIM-3, the K D of less than about 11 nM, preferably less than or equal to about 5nM , 4.5nM, 4nM, 3.5nM, 3nM, 2.5nM, 2nM, 1.5nM, in some embodiments, the K D is between the above-mentioned values (inclusive).
  • the antibody or antigen-binding fragment thereof of the present invention binds to Tim-3 on the cell surface.
  • Tim-3 is overexpressed on the cell surface.
  • the cell is a CHO cell, such as CHO-S.
  • the cells are NK cells.
  • the binding is detected using flow cytometry.
  • the antibody of the present invention binds to TIM overexpressed on CHO cells with an EC50 less than or equal to about 1.5nM, 1.4nM, 1.3nM, 1.2nM, 1.1nM, 1nM, 0.9nM, 0.8nM, 0.7nM -3.
  • the antibodies of the invention or antigen-binding fragments thereof bind to CD4+ T cells.
  • the antibody or antigen-binding fragment thereof of the invention blocks the binding of TIM-3 to its ligand.
  • the ligand is PtdSer, such as PtdSer on the surface of apoptotic cells.
  • the cells are L363 cells.
  • the combination of the antibody or its antigen-binding fragment of the present invention and the PD-1 pathway antibody or its antigen-binding fragment can effectively activate CD4+ T cells, preferably better than the PD-1 pathway antibody alone, more preferably It is better than PD-1 pathway antibody combined with other anti-TIM-3 antibodies (such as TSR-022) to activate.
  • the PD-1 pathway antibody is an anti-PD-1 antibody, such as IBI308.
  • the antibody or antigen-binding fragment thereof of the present invention can effectively activate NK cells. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention can enhance the expression of NKG2D and/or CD107a on the surface of NK cells.
  • the antibody or antigen-binding fragment thereof of the present invention can be used in the treatment of cancer in combination with the PD-1 pathway antibody or antigen-binding fragment thereof.
  • its efficacy is better than that of the PD-1 pathway antibody alone.
  • the antibodies or antigen-binding fragments of the present invention and the PD-1 pathway antibody or antigen-binding fragments can effectively inhibit tumor growth, and the tumor inhibition rate is greater than or equal to about 60%, 65%, or 70% .
  • the antibody or antigen-binding fragment thereof of the present invention has good thermal stability.
  • the thermal stability of the antibody or antigen-binding fragment thereof of the present invention is detected by differential scanning fluorescence method.
  • the T m is greater than or equal to 65°C, 66°C, 67°C, 68°C, or 69°C.
  • the antibody or antigen-binding fragment thereof of the present invention has good long-term thermal stability and accelerated stability. In one embodiment, the antibody or fragment thereof of the present invention maintains a purity of at least 95%, 96%, 97%, 97.5% for at least 7, 8, 9, 10, 11, 12, 13, 14 days, and its binding There was no significant change in the ability of cells expressing human Tim-3.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the VH comprises
  • the three CDR regions contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequences in the three CDR regions.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the VL comprises:
  • the three CDR regions contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequences in the three CDR regions.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein
  • the VH includes
  • the three CDR regions contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequences in the three CDR regions.
  • VL comprises an amino acid sequence selected from SEQ ID NO: 9, 16 or 27, or consists of said amino acid sequence.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises the three complementarity determining regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 8, 15 or 26, and the HCDR as shown in SEQ ID NO: 8, 15 or 26.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein
  • the VH includes complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence of SEQ ID NO:1 or 19, or consists of the amino acid sequence, or HCDR1 includes the amino acid sequence of SEQ ID NO:1
  • the amino acid sequence of or 19 has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequence
  • HCDR2 contains an amino acid selected from SEQ ID NO: 2 or 4 or 12 or 13 or 20 or 22
  • the sequence, or consists of the amino acid sequence, or HCDR2 contains one, two or three changes (preferably amino acid substitutions, Preferably conservative substitution) amino acid sequence
  • HCDR3 includes the amino acid sequence of SEQ ID NO: 3 or 21 or consists of the amino acid sequence, or HCDR3 includes the amino acid sequence of SEQ ID NO: 3 or 21 that has one, two, or The amino acid sequence of three changes (preferably amino acid substitutions, preferably conservative substitutions);
  • said VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, where LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or 14 or 23, or LCDR1 comprises the same sequence as SEQ ID NO: : Compared with the amino acid sequence of 5 or 14 or 23, the amino acid sequence has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions); LCDR2 includes the amino acid sequence of SEQ ID NO: 6 or 24 or is composed of the amino acid sequence Sequence composition, or LCDR2 includes an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 6 or 24; LCDR3 includes an amino acid sequence selected from SEQ ID NO: 7 Or the amino acid sequence of 25 or consists of the amino acid sequence, or LCDR3 contains an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO:
  • the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 2 or 12;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 5 or 14;
  • LCDR3 includes the amino acid sequence of SEQ ID NO: 7 or consists of the amino acid sequence.
  • the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 20;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 24;
  • LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25.
  • the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO:1;
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 4 or 13;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 3;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 5 or 14;
  • LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 6;
  • the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 19;
  • HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 22;
  • HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 21;
  • the VL includes LCDR1, LCDR2 and LCDR3, wherein
  • LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 23;
  • LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 24;
  • LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 25.
  • the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH includes
  • the VL includes
  • the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a complementarity determining region ( CDR) HCDR1, HCDR2 and HCDR3 and said VL comprises (CDR) LCDR1, LCDR2 and LCDR3, wherein the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in said antibody or antigen-binding fragment thereof is as follows (table A) shows:
  • Table A Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in the antibody of the present invention or its antigen-binding fragment
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,
  • amino acid sequence comprising SEQ ID NO: 8, 15 or 26 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
  • amino acid sequence or consists of said amino acid sequence
  • amino acid sequence of an amino acid change (preferably an amino acid substitution, more preferably an amino acid conservative substitution) is composed of the amino acid sequence, and preferably, the amino acid change does not occur in the CDR region;
  • amino acid sequence comprising SEQ ID NO: 9, 16 or 27 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% An identical amino acid sequence or consist of said amino acid sequence;
  • amino acid sequence of an amino acid change (preferably an amino acid substitution, more preferably an amino acid conservative substitution) is composed of the amino acid sequence, and preferably, the amino acid change does not occur in the CDR region.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,
  • amino acid sequence (iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO: 8 or 15
  • the amino acid sequence (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region;
  • amino acid sequence (iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO: 9 or 16
  • the amino acid sequence (preferably amino acid substitution, more preferably amino acid conservative substitution) is composed of the amino acid sequence, and preferably, the amino acid change does not occur in the CDR region.
  • the present invention provides an anti-TIM-3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof
  • VH heavy chain variable region
  • VL light chain variable region
  • Table B Exemplary combinations of heavy chain variable region VH and light chain variable region VL in the antibody or antigen-binding fragment of the present invention
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and/or light chain, wherein
  • amino acid sequence comprising SEQ ID NO: 10, 17 or 28 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or a 99% identical amino acid sequence or consist of said amino acid sequence;
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the Amino acid changes do not occur in the variable region of the heavy chain;
  • amino acid sequence comprising SEQ ID NO: 11, 18 or 29 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or a 99% identical amino acid sequence or consist of said amino acid sequence;
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the Amino acid changes do not occur in the variable region of the light chain.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and/or light chain, wherein
  • amino acid sequence comprising SEQ ID NO: 10 or 17 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % Identity amino acid sequence or consists of said amino acid sequence;
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or composed of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the amino acid change Does not occur in the variable region of the heavy chain;
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change Does not occur in the variable region of the light chain.
  • Table C Exemplary combinations of heavy and light chains in the antibodies of the present invention or antigen-binding fragments thereof
  • the heavy chain and/or light chain of the anti-TIM-3 antibody or fragment thereof of the present invention further comprises a signal peptide sequence, for example, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 35).
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described in the present invention occur in regions outside the CDR (for example, in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain.
  • substitutions are conservative substitutions.
  • Conservative substitution refers to the replacement of an amino acid by another amino acid in the same category, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid. Replacement. Exemplary permutations are shown in Table D below:
  • the substitution occurs in the CDR region of the antibody.
  • the obtained variant has a modification (e.g., improvement) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties that are substantially retained of the parent antibody.
  • An exemplary substitution variant is an affinity matured antibody.
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants to change one or more functional properties of the antibody, such as serum half-life, Complement binding, Fc receptor binding, and/or antigen-dependent cytotoxicity.
  • An Fc region variant may include a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.
  • the antibody described herein introduces substitution mutations in the Fc region to improve the binding ability to human FcRn, in order to extend its half-life in vivo.
  • cysteine-engineered antibodies such as "thioMAbs", in which one or more residues of the antibody are replaced with cysteine residues.
  • Cysteine engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.
  • the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art.
  • the part suitable for antibody derivatization includes, but is not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerin), polyvinyl alcohol, and mixtures thereof.
  • PEG poly
  • the anti-TIM-3 antibody or antigen-binding fragment thereof of the present invention has one or more of the following characteristics:
  • Inhibit e.g., competitively inhibit the binding of the antibody (e.g. CH4-3, Hz4-3.6 or CH5-17) of the present invention to TIM-3;
  • the anti-TIM-3 antibody of the present invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4.
  • the anti-TIM-3 antibody is a monoclonal antibody.
  • the anti-TIM-3 antibody is a human antibody.
  • the anti-TIM-3 antibody is a chimeric antibody.
  • At least part of the framework sequence of the anti-TIM-3 antibody is a human consensus framework sequence.
  • the anti-TIM-3 antibody of the present invention also encompasses its antibody fragments, preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibodies (such as scFv) or ( Fab') 2 , single domain antibody, double antibody (dAb) or linear antibody.
  • antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibodies (such as scFv) or ( Fab') 2 , single domain antibody, double antibody (dAb) or linear antibody.
  • nucleic acid of the present invention and the host cell containing it
  • the invention provides a nucleic acid encoding any of the above anti-TIM-3 antibodies or fragments thereof.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector, such as pcDNA3.1.
  • a host cell comprising the nucleic acid or the vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (such as CHO cells (such as CHO-S or ExpiCHO) or 293 cells (such as 293F)) or other cells suitable for preparing antibodies or antigen-binding fragments thereof .
  • the host cell is prokaryotic.
  • the invention provides a nucleic acid encoding any anti-TIM-3 antibody or fragment thereof described herein.
  • the nucleic acid may include a nucleic acid encoding the amino acid sequence of the light chain variable region and/or the heavy chain variable region of an antibody, or a nucleic acid encoding the amino acid sequence of the light chain and/or heavy chain of the antibody.
  • the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 8-11, 15-18, 26-29, or a nucleic acid that encodes an amino acid sequence selected from SEQ ID NO: 8-11, 15.
  • the amino acid sequence shown in any one of -18, 26-29 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% The identity of the amino acid sequence of the nucleic acid.
  • the amino acid sequence of the nucleic acid has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence of the nucleic acid sequence.
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include but are not limited to viruses, plasmids, cosmids, lambda phage or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • the vector is pTT5 vector or pcDNA3.1.
  • a host cell comprising the vector.
  • Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • For the expression of antibody fragments and polypeptides in bacteria see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, and see also Charlton, Methods in Molecular Biology, Volume 248 (BKCLo, editor, Humana Press, Totowa, NJ, 2003) , Pages 245-254, which describe the expression of antibody fragments in E. coli).
  • the antibody can be separated from the bacterial cell paste in the soluble fraction and can be further purified.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains where the glycosylation pathway has been "humanized” result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • a mammalian cell line modified to be suitable for growth in suspension can be used.
  • useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293HEK or 293F or 293 cells, such as, for example, Graham et al., J. Gen Virol. 36:59 (1977)) and so on.
  • Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl.
  • the present invention provides a method for preparing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment), wherein the method includes a method suitable for expressing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment).
  • the host cell is cultured under the condition of nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated.
  • the method further comprises recovering the antibody molecule of the invention or a fragment thereof (preferably an antigen-binding fragment) from the host cell.
  • a method for preparing an antibody molecule of the present invention comprises, under conditions suitable for expression of the antibody, culturing the antibody (for example, any one polypeptide chain and/or multiple polypeptide chains)
  • the nucleic acid or the host cell containing the expression vector of the nucleic acid, as provided above, and the antibody is optionally recovered from the host cell (or host cell culture medium).
  • the nucleic acid encoding the antibody (such as the antibody described above, such as any one polypeptide chain and/or multiple polypeptide chains) is isolated and inserted into one or more vectors for use in the host Further cloning and/or expression in the cell.
  • Such nucleic acids are easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains).
  • the antibody molecules prepared as described herein can be purified by known existing techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be obvious to those skilled in the art.
  • the purity of the antibody molecule of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, and the like.
  • the anti-TIM-3 antibodies provided herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities by a variety of assays known in the art.
  • the antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot, and the like.
  • the binding to TIM-3 can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, it is measured using radioimmunoassay (RIA) or biofilm thin-layer interferometry or MSD assay or surface plasmon resonance (SPR).
  • a competition assay can be used to identify antibodies that compete with any of the anti-TIM-3 antibodies disclosed herein for binding to TIM-3.
  • such competitive antibodies bind to the same or overlapping epitopes (e.g., linear or conformational epitopes) as bound by any of the anti-TIM-3 antibodies disclosed herein.
  • epitopes bound by antibodies are known, for example, see Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) for an exemplary method.
  • the present invention also provides an assay method for identifying anti-TIM-3 antibodies with biological activity.
  • Biological activities can include, for example, binding to TIM-3 (for example, binding to human and/or cynomolgus TIM-3), binding to cell surface TIM-3, binding to T cells, and blocking TIM-3 and its ligands Function, activation of T cells and activation of NK cells.
  • An antibody having such biological activity in vivo and/or in vitro is also provided.
  • the antibodies of the invention are tested for such biological activity.
  • the present invention also provides methods for identifying properties of antibodies, such as properties related to druggability.
  • properties related to druggability include, for example, thermal stability, such as long-term thermal stability and accelerated stability.
  • the immunoconjugate of the present invention can be used to replace or supplement the anti-TIM-3 antibody to perform any of the aforementioned assays.
  • the present invention provides immunoconjugates comprising any of the anti-TIM-3 antibodies provided herein and other substances, such as therapeutic agents, including chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small Molecular drugs or immunomodulators (e.g. anti-inflammatory or immunosuppressive agents).
  • the other substance is, for example, a cytotoxic agent, which includes any agent that is harmful to cells.
  • the present invention provides a composition comprising any anti-TIM-3 antibody or fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof described herein, preferably the composition is a pharmaceutical composition.
  • the composition further comprises pharmaceutical excipients.
  • the composition for example, a pharmaceutical composition, comprises the anti-TIM-3 antibody of the present invention or a fragment or immunoconjugate thereof, and a combination of one or more other therapeutic agents.
  • the pharmaceutical preparation of the antibody is preferably in the form of a lyophilized preparation or an aqueous solution.
  • the pharmaceutical composition or preparation of the present invention may also contain more than one active ingredient that is required for the specific indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other.
  • active ingredients such as chemotherapeutics, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulators.
  • the active ingredients are suitably present in combination in an amount effective for the intended use.
  • the other antibody is a PD-1 pathway antibody.
  • kit of the present invention contains in the same package:
  • the PD-1 pathway antibodies include anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies.
  • the tumor (e.g., cancer) patient has (e.g., elevated levels, e.g., nucleic acid or protein levels) TIM-3.
  • the tumor (e.g., cancer) patient has (e.g., elevated levels, e.g., nucleic acid or protein levels) PD-L1 or PD-1 or PD-L2.
  • the tumor (e.g. cancer) patient has both (e.g. elevated levels, e.g. nucleic acid or protein levels) TIM-3 and (e.g. elevated levels, e.g. nucleic acid or protein levels) PD -L1 or PD-1 or PD-L2.
  • the tumor treatment will benefit from TIM-3 that inhibits nucleic acid or protein levels. In some embodiments, the tumor treatment benefits from blocking the binding of TIM-3 to its ligand, such as phosphatidylserine.
  • the tumor treatment will benefit from
  • the anti-TIM-3 antibody of the present invention enhances CD4+ T cell function, for example, by increasing CD4+ T cell proliferation and/or increasing CD4+ T cell cytokine production.
  • the cytokine is an interleukin, such as IL-2.
  • the tumor is tumor immune escape. In some embodiments, the tumor is cancer.
  • the present invention provides the use of antibody molecules or fragments or immunoconjugates or pharmaceutical compositions or pharmaceutical combinations or kits in the production or preparation of drugs for the purposes described herein, for example, For the prevention or treatment of related diseases or disorders mentioned herein.
  • the antibody molecules of the present invention or fragments or immunoconjugates or pharmaceutical compositions thereof can also be combined with PD-1 pathway antibodies, and optionally one or more other therapies such as treatment modalities and/or Other therapeutic agents are administered in combination for the purposes described herein, for example for the prevention and/or treatment of related diseases or disorders mentioned herein.
  • the treatment modality includes surgery; radiation therapy, localized irradiation or focused irradiation, and the like.
  • immunomodulators include immunosuppressive agents or anti-inflammatory agents.
  • the antibody combinations described herein may be administered separately, for example, as separate antibodies, or when linked (for example, as a bispecific or trispecific antibody molecule).
  • Such combination therapies encompass combined administration (for example, two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case, additional therapeutic agents and/or agents may be administered.
  • the administration of the antibody of the invention occurs before, at the same time, and/or afterwards.
  • the route of administration of the pharmaceutical composition is according to known methods, for example, oral, intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional Approach; through a sustained release system or through an implanted device.
  • the composition may be administered by bolus injection or by continuous infusion or by implantation device.
  • composition may also be applied topically via an implanted membrane, sponge, or another suitable material on which the desired molecule is absorbed or encapsulated.
  • an implanted device when used, the device can be implanted into any suitable tissue or organ, and the desired molecule can be delivered via diffusion, timed release bolus, or continuous administration.
  • any anti-TIM-3 antibody or antigen-binding fragment thereof provided herein can be used to detect the presence of TIM-3 in a biological sample.
  • detection includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule complexed magnetic beads, ELISA assays Method, PCR-technology (for example, RT-PCR).
  • the biological sample is blood, serum, or other liquid samples of biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is from a lesion associated with a hyperproliferative or cancerous lesion.
  • an anti-TIM-3 antibody or antigen-binding fragment thereof for use in a diagnosis or detection method is provided.
  • a method of detecting the presence of TIM-3 in a biological sample comprises detecting the presence of TIM-3 protein in a biological sample.
  • TIM-3 is human TIM-3 or cynomolgus TIM-3.
  • the method includes subjecting a biological sample to an anti-TIM-3 antibody or antigen-binding fragment thereof as described herein under conditions that allow the anti-TIM-3 antibody or antigen-binding fragment thereof to bind to TIM-3 Contact and detect whether a complex is formed between the anti-TIM-3 antibody or its antigen-binding fragment and TIM-3.
  • the formation of the complex indicates the presence of TIM-3.
  • the method can be an in vitro or in vivo method.
  • the anti-TIM-3 antibody or antigen-binding fragment thereof is used to select subjects suitable for treatment with the anti-TIM-3 antibody or antigen-binding fragment thereof, for example, where TIM-3 is used to select the The subject's biomarker.
  • the antibodies of the invention can be used to diagnose tumors, such as cancer, for example to evaluate (eg, monitor) the treatment or progression, diagnosis, and/or staging of the diseases described herein in an individual.
  • a labeled anti-TIM-3 antibody or antigen-binding fragment thereof is provided.
  • Labels include, but are not limited to, labels or parts that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and parts that are indirectly detected, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.
  • the sample is obtained prior to treatment with the anti-TIM-3 antibody or antigen-binding fragment thereof. In some embodiments, the sample is obtained before other therapies are used. In some embodiments, the sample is obtained during or after treatment with other therapies. In some embodiments, the sample is obtained before, during, or after treatment with the PD-1 pathway antibody or antigen-binding fragment thereof.
  • the sample is formalin fixed, paraffin coated (FFPE).
  • FFPE formalin fixed, paraffin coated
  • the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.
  • TIM-3 is detected before treatment, for example, before initiating treatment or before some treatment after the treatment interval.
  • a method of treating the disease of the present invention comprising: testing a subject (e.g., sample) (e.g., subject sample) for the presence of TIM-3, thereby determining TIM- 3 value, compare the TIM-3 value with the control value, and if the TIM-3 value is greater than the control value, then administer a therapeutically effective amount of an anti-TIM-3 antibody, optionally in combination with one or more other therapies, to the subject Or an antigen-binding fragment thereof (for example, an anti-TIM-3 antibody or antigen-binding fragment thereof as described herein), thereby treating the disease.
  • a subject e.g., sample
  • an antigen-binding fragment thereof for example, an anti-TIM-3 antibody or antigen-binding fragment thereof as described herein
  • Hybridoma technology is to fuse two kinds of cells while maintaining the main characteristics of both. These two kinds of cells are mouse spleen cells immunized with antigen and mouse myeloma cells.
  • the main feature of mouse spleen cells (B lymphocytes) immunized with specific antigens is its antibody secretion function, but they cannot be continuously cultured in vitro.
  • Mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, they have The so-called immortality. Under the action of the selective medium, only hybrid cells fused with B cells and myeloma cells can have the ability to continue culture, forming cell clones that have both the function of antibody secretion and the maintenance of cell immortality.
  • mice were immunized with human Tim-3 protein, and then spleen cells of the mice were fused with myeloma cells to obtain hybridoma cells capable of expressing positive antibodies.
  • Preparation of the electrofusion dish Thoroughly soak the electrofusion dish with 70% ethanol, and dry it in an ultra-clean table for later use.
  • mice were sacrificed by cervical dislocation, the body surface was disinfected with 75% alcohol for 5 minutes, and then placed on the mouse dissecting board in the ultra-clean bench, lying on the left side, and fixing the limbs with a 7-gauge needle. Open the abdominal cavity aseptically and take out the spleen, wash it with basal medium (the configuration method is as follows), and carefully remove the surrounding connective tissue. The spleen was then transferred to another petri dish containing basal medium. Press the spleen with an elbow needle, insert a hole on the spleen with a small needle, and squeeze with tweezers to fully release the spleen cells to make a spleen cell suspension. The cell suspension was filtered through a 70 ⁇ M cell sieve and washed with 30ml basal medium, and centrifuged at 1200rpm for 6min.
  • Lysis of red blood cells Remove the supernatant, and resuspend the cells with 10ml RBC Lysis Buffer (GIBCO, A10492-01). Then add 20ml RBC Lysis Buffer. The suspension was allowed to stand for 5 minutes and then centrifuged at 1100 rpm for 6 minutes. After removing the supernatant, resuspend the cells in 10ml basal medium, then add 30ml basal medium and centrifuge at 1100rpm for 6min. After removing the supernatant, the cells were resuspended in 20 ml of basal medium and counted.
  • Electrofusion Resuspend mouse myeloma SP2/0 cells (ATCC, CRL-1581) in 20ml basal medium and count them.
  • the SP2/0 and spleen cells were mixed at a ratio of 1:2 to 1:1, and centrifuged at 1000 rpm for 6 min. After removing the supernatant, the mixed cells were resuspended in 10 ml fusion buffer (BTXpress, 47-0001). Then add 15 ml of fusion buffer, centrifuge at 1000 rpm for 5 min, and remove the supernatant. After repeating the above steps, reselect the cells with an appropriate amount of fusion buffer, and adjust the mixed cell density to 1 ⁇ 10 7 cells/ml.
  • the parameters of the electrofusion instrument are set as follows. Add 2ml of cell suspension to each electrofusion dish for electrofusion.
  • Plating after electrofusion cells are allowed to stand for 5 min at room temperature in an electrofusion dish. Transfer the cells to a centrifuge tube, and dilute the cells to 1 to 2 ⁇ 10 4 cells/ml with the selection medium (the configuration method is as follows). Add 100 ⁇ l of cell suspension to each well of a 96-well plate. The selection medium was replaced on the 7th day after fusion. Screening is performed after the 10th day of culture (or longer, depending on the cell growth status). FACS (C6 (BD Biosciences)) was used to screen out hybridoma cells expressing specific anti-Tim-3 antibodies.
  • Cryopreservation of cells Observe the cell status, and when the cells grow well and the viability is >90%, centrifuge at 1000 rpm for 5 minutes to remove the supernatant. Resuspend the cells with cryopreservation solution (45.5% FBS (Hyclone), 44.5% RPMI-1640 (Hyclone), 10% DMSO (SIGMA)) to 1 ⁇ 10 7 cells/ml, aliquot into cryopreservation tubes, and put Store in a program cooling box at -80°C.
  • cryopreservation solution 45.5% FBS (Hyclone), 44.5% RPMI-1640 (Hyclone), 10% DMSO (SIGMA)
  • the present invention uses molecular biology technology to obtain the antibody sequence of the anti-Tim-3 antibody produced by the hybridoma cell in Example 1, and uses it to construct a human-mouse chimeric antibody.
  • RNA extraction Centrifuge the fresh cells obtained in Example 1 at 300g for 5min, remove the supernatant, add 500 ⁇ l LY buffer (Biomiga, R6311-02) to the pellet (add 20 ⁇ l ⁇ -mercaptoethanol per 1ml before use), and mix well To clarify. Add to the DNA removal tube, centrifuge at 13000 rpm for 2 min, and collect the flow-through. Add 100% ethanol to the flow-through liquid at a ratio of 1/2, and mix 5 times until it is clear.
  • LY buffer Biomiga, R6311-02
  • reaction system I After incubating at 65°C for 5 min, quickly place on ice to cool. Add the following reverse transcription system to reaction system I, the total amount is 20 ⁇ l:
  • PCR amplifies the variable regions of the heavy chain and light chain respectively, and the PCR reaction system is as follows:
  • VH primers of the mouse anti-Tim-3 antibody (Primer Mix 1, mixed in the following proportions to obtain Primer Mix 1 for subsequent VH PCR amplification):
  • VL primers of the mouse anti-Tim-3 antibody (Primer Mix 2, after mixing in the following proportions, obtain Primer Mix 2 for subsequent PCR amplification of VL.):
  • the PCR reaction conditions are as follows:
  • VH and VL regions of the murine anti-Tim-3 antibody produced by the hybridoma in Example 1 that have been sequenced were amplified by PCR.
  • the PCR system is as follows:
  • Primer Mix 1 is used; for VL chain amplification, Primer Mix 2 is used.
  • the gel is cut to recover the PCR amplified product.
  • the homologous recombination system is as follows:
  • the recombinant product was transformed into TOP10 (Tiangen, CB104-02) competent, and a single clone was selected for sequencing, the clone containing the plasmid with the correct insertion direction was selected as the positive clone, and the positive clone was saved.
  • a plasmid containing anti-Tim-3 antibody was extracted from the positive clone obtained above.
  • the chromatography column used for purification was treated with 0.1M NaOH for 2 hours, and the glass bottles were washed with distilled water and then dried at 180°C for 4 hours to obtain a purification column. Before purification, centrifuge the collected cell material at 4500 rpm for 30 min, and discard the cells. The supernatant was filtered with a 0.22 ⁇ m filter. Use 10 column volumes of binding buffer (sodium phosphate 20mM.NaCl 150mM, pH7.0) to equilibrate the Protein A column (Hitrap Mabselect Sure 5*5ml, GE, 11-0034-95). Add the filtered supernatant to the purification column and equilibrate with 10 column volumes of binding buffer.
  • binding buffer sodium phosphate 20mM.NaCl 150mM, pH7.0
  • the two chimeric antibodies (CH4-3 and CH5-17) obtained in the present invention please refer to the CDR sequence, the variable region of the light chain and the variable region of the heavy chain, the amino acid sequence of the light chain and the heavy chain, and the sequence numbers. Attached sequence list.
  • control antibody used in the present invention comes from the TIM-3 antibody of the patent WO2016161270 of Tesaro, Inc., hereinafter referred to as TSR-022, the negative control application antibody IgG1, the sequence of which is shown in the attached sequence table.
  • TSR-022 the negative control application antibody IgG1
  • the expression and purification method of the control is the same as the antibody of the present invention.
  • Example 3 Determination of the binding kinetics between the chimeric antibody of the present invention and the antigen by the biofilm thin-layer interference technique
  • the equilibrium dissociation constant (K D ) of the antibody of the present invention bound to human Tim-3 is determined by the biofilm thin-layer interferometry (BLI) technique.
  • BLI biofilm thin-layer interferometry
  • the K D values of the chimeric antibodies CH4-3, CH5-17 and human Tim-3 were 2.02E-09M and 5.36E-09M, respectively.
  • the antibody in this study Have similar or better K D values.
  • the cDNA encoding human Tim-3 (SEQ ID NO: 34) was cloned into the pCHO1.0 vector (Invitrogen) and transfected into CHO-S cells (Invitrogen) to produce CHO-S cells overexpressing human Tim-3 ( CHOS-hTim-3).
  • the CHOS-hTim-3 cells were counted and diluted to 2 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to the U-shaped bottom 96-well plate. Centrifuge at 300g for 5 minutes to remove the cell culture medium.
  • the samples (respectively chimeric antibodies CH4-3, CH5-17, and positive control antibody TSR-022) (antibody dilution method: the highest antibody concentration is 400nM, three-fold dilution in PBS, a total of 12 concentrations were tested ) Add the U-shaped plate and resuspend the cells, 100 ⁇ l/well, and let stand on ice for 30 min. Centrifuge at 400g for 5 min to remove the supernatant, and wash the cells twice with PBS.
  • the antibody CH4-3 chimeric antibody obtained in Example 2 was humanized. And go through the following steps for humanization:
  • the humanized antibody Hz4-3.6 sequence obtained above was cloned into pcDNA3.1 (Invitrogen) to obtain plasmid DNA.
  • the ExpiCHO TM Expression system (Gibco, A29133) was used to produce protein. Specifically: ExpiCHO cells (Gibco) were passaged according to the required transfection volume, and the cell density was adjusted to 3.5 ⁇ 10 6 cells/ml the day before transfection. . On the day of transfection, the cell density was adjusted to 6 ⁇ 10 6 cells/ml.
  • the transfection buffer reagent OptiPRO SFM (Gibco, 12309019) at 8% of the transfected cell volume, and calculate the total amount of plasmid needed for the transfected cells at 0.8 ⁇ g/ml (the ratio of light to heavy chain is 1: 1)
  • Use a 0.22 ⁇ m filter membrane to filter the transfection buffer containing the obtained plasmid DNA into another new 50ml centrifuge tube, add ExpiFectamineTMCHO reagent (Gibco ,100033022), mix thoroughly, mix the transfection reagent with the plasmid DNA and slowly add it to the cells immediately, gently shake the flask while adding, and control the incubation time of the transfection reagent with the plasmid not to exceed 5min; 36.5°C, 8% CO 2 , shaker culture.
  • Enhancer (Gibco, 100033019) was added at a cell volume of 6 ⁇ l/ml, and Feed (Gibco, A29101-01) was added to a cell volume of 300 ⁇ l/ml.
  • the cells were cultured at 36.5° C., 120 rpm, and 8% CO 2 . When the culture is continued to the 6th day or when the cell viability is ⁇ 60%, the cell supernatant is collected for purification.
  • the chromatography column used for purification was treated with 0.1M NaOH for 2 hours, and the glass bottles were washed with distilled water and then dried at 180°C for 4 hours to obtain a purification column. Before purification, centrifuge the collected cell material at 4500 rpm for 30 min, and discard the cells. Filter the supernatant with a 0.22 ⁇ l filter. Use 10 column volumes of binding buffer (sodium phosphate 20mM.NaCl 150mM, pH7.0) to equilibrate the pre-packed Protein A column (Hitrap Mabselect Sure 5*5ml, GE, 11-0034-95). Add the filtered supernatant to the purification column and equilibrate with 10 column volumes of binding buffer.
  • binding buffer sodium phosphate 20mM.NaCl 150mM, pH7.0
  • Example 7 Determination of the binding kinetics of the humanized antibody of the present invention with the cynomolgus monkey antigen by the biofilm thin-layer interference technique
  • the equilibrium dissociation constant (K D ) of the antibody of the present invention bound to the cynomolgus monkey Tim-3 was determined by the biofilm thin-layer interferometry technique (BLI).
  • BLI method affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p.270-8).
  • the K D value of the humanized antibody Hz4-3.6 is 1.04E-08M.
  • the antibody in this study has a similar K D value.
  • the surface plasmon resonance method was used to determine the equilibrium dissociation constant (K D ) of the antibody of the present invention binding to human Tim-3. Based on the SPR principle, when a beam of polarized light is incident on the end face of the prism at a certain angle, a surface plasmon wave will be generated at the interface between the prism and the gold film, causing free electrons in the metal film to resonate, that is, surface plasmon resonance.
  • SPR surface plasmon resonance method
  • Biacore (GE Healthcare, T200) was used to determine the K D of a humanized antibody.
  • the specific method is as follows: After the antibody (Hz4-3.6 and TSR-022 at the same concentration) is captured on the chip by the anti-human Fc antibody, the antigen is detected The binding and dissociation with the captured antibody obtains affinity and kinetic constants.
  • the method includes chip preparation and affinity detection. In the measurement process, 10 ⁇ HBS-EP+(BR-1006-69, GE Healthcare) diluted by 10 times was used as the experimental buffer.
  • the chip preparation process uses an amino coupling kit (BR-1006-33, GE Healthcare), and the anti-human Fc antibody is coupled to the surface of the CM5 chip (29-1496-03, GE Healthcare).
  • the anti-human Fc antibody was diluted in 10mM Acetate (pH 5.0) and injected into the dual channel of the CM5 chip to make the protein covalently coupled to the surface of the chip channel with a coupling height of about 6000RU. Finally, 1M ethanolamine was injected to block the remaining activated sites.
  • Each cycle of affinity detection includes capturing antibody, binding a concentration of antigen and chip regeneration. First, the diluted antibody was captured on the fourth channel of the CM5 chip at a flow rate of 10 ⁇ l/min, and the capture time was 60s. Channel 3 was the blank control channel.
  • the antigen human Tim-3 (SINO, 10390-H08H-50)
  • SINO human Tim-3
  • 10mM Glycine pH 1.5 (BR-1003-54, GE Healthcare) was used to regenerate the chip.
  • the data results use Biacore T200 analysis software, and the analysis model used is a 1:1 combination model for kinetic analysis.
  • Detection steps 400g, 5min, centrifugation, remove the cell culture medium, resuspend CHOS-hTim-3 cells in PBS, after counting, adjust the cell density to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/ml to the U-shaped bottom 96-well plate hole. Add the antibody to be tested, three-fold gradient dilution, and let stand on ice for 30 min. 300g, 5min remove the supernatant, wash the cells with PBS once. 300g, 5min remove PBS, add 100 ⁇ l 1:200 dilution of PE-anti-human Fc antibody (SOUTHERN BIOTECH, 2040-09) to each well. Incubate on ice for 30 min in the dark. 400g, 5min, remove the supernatant, and wash the cells twice with PBS. Resuspend the cells with 100 ⁇ l PBS and detect by flow cytometry (BD, FACSCELESTA).
  • Resuscitate human PBMC cells (ALLCELLS, PB005F), stand for 3 hours to adhere to the wall and become monocytes, add 10ml AIM Medium CTS (GIBCO, A3021002) medium, add IL4 (20ng/ml) (R&D, 204-IL), GM-CSF (10ng/ml) (R&D, 215-GM) to induce monocytes to differentiate into DC cells, culture By day 5, add TNF ⁇ (1000U/ml, 10ng/ml) (R&D, 210-TA), RhIL-1 ⁇ (5ng/ml) (R&D, 201-LB), RhIL-6 ( 10ng/ml) (R&D, 206-IL), 1 ⁇ M PGE (Tocris, 2296), in a carbon dioxide incubator at 37°C and 5% CO 2 for 2 days, as a mature DC for mixed lymphocyte reaction (MLR) cell;
  • MLR mixed lymphocyte reaction
  • Anti-Tim-3 antibodies can bind to Tim-3 molecules expressed on the surface of NK cells, and then mediate the activation of NK cells.
  • the activation of NK cells was reflected by detecting the activation of NKG2D and CD107a on the surface of NK cells to detect the activation activity of antibodies on NK cells.
  • the antibody Hz4-3.6 can effectively enhance the expression of NKG2D and CD107a on the cell surface characterized by NK activation, and the enhancement effect is similar to the control antibody TSR-022.
  • Mouse MC38 cells were purchased from Shanghai Heyuan Biotech (CAT#:HYC3401), and routinely subcultured in strict accordance with the instructions for subsequent in vivo experiments. Collect the cells by centrifugation, resuspend the cells in sterile PBS and adjust the cell density to 5 ⁇ 10 6 cells/ml. On day 0, 0.2ml of the cell suspension was subcutaneously inoculated into the right abdominal region of human Tim-3 knock-in mice to establish the MC38-hTim-3 tumor-bearing mouse model.
  • mice with tumor volume ranging from 25.23mm 3 to 108.66mm 3 were selected and divided into groups according to tumor volume (7 mice per group), dosage and method of administration As shown in Table 4, h-IgG (purchased from EQUITECH-BIO) was used as a negative control and was administered on the 7, 10, 14, 17, and 21 days after vaccination, and the tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration, and the relative tumor inhibition rate (TGI%) was calculated on the 25th day after inoculation. The calculation formula is as follows:
  • TGI% 100%*(control group tumor volume—treatment group tumor volume)/(control group tumor volume—control group tumor volume before administration).
  • An electronic balance is used to determine body weight.
  • Differential scanning fluorimetry can provide information about the stability of the protein structure according to the fluorescence change process in the protein map, detect the configuration change of the protein, and obtain the melting temperature (Tm) of the protein.
  • Tm melting temperature
  • the DSF method was used to determine the T m value of the antibody of the present invention.
  • the antibody Hz4-3.6 of the present invention was diluted to 1 mg/ml with PBS solution.
  • the experimental results are shown in Table 6 and Figure 9 below.
  • the T m value of the antibody of the present invention is >65°C, therefore, it has better thermal stability.
  • the humanized antibody Hz4-3.6 described herein has a T m greater than 65°C, and has good thermal stability.
  • the humanized antibody Hz4-3.6 described herein was placed at 40°C for 14 days, and its monomer main peak ratio decreased by only 0.65%, and the ability to bind to cells expressing human Tim-3 did not change significantly.
  • the results show that the humanized antibody Hz4-3.6 described herein has better accelerated stability.

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Abstract

L'invention concerne un anticorps ou un fragment de liaison à l'antigène de celui-ci qui se lie spécifiquement à TIM-3, et une composition contenant l'anticorps ou un fragment de liaison à l'antigène de celui-ci. L'invention concerne également un acide nucléique de codage correspondant, une cellule hôte, et l'utilisation de l'anticorps ou de son fragment de liaison à l'antigène dans la thérapie et le diagnostic.
PCT/CN2021/078473 2020-03-02 2021-03-01 Anticorps anti-tim-3 et son utilisation Ceased WO2021175191A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118165111A (zh) * 2024-05-15 2024-06-11 中国人民解放军军事科学院军事医学研究院 针对Tim-3的抗体

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103079644A (zh) * 2010-06-11 2013-05-01 协和发酵麒麟株式会社 抗tim-3抗体
CN107001475A (zh) * 2014-11-06 2017-08-01 豪夫迈·罗氏有限公司 抗tim3抗体及使用方法
CN108794630A (zh) * 2017-12-18 2018-11-13 镇江爱必梦生物科技有限公司 鼠抗人tim3蛋白单克隆抗体制备及其免疫组化用途
CN110267988A (zh) * 2016-12-08 2019-09-20 伊莱利利公司 用于与抗pd-1抗体组合的抗tim-3抗体
WO2020041520A1 (fr) * 2018-08-21 2020-02-27 Albert Einstein College Of Medicine Anticorps monoclonaux contre tim-3 humain
TW202009241A (zh) * 2018-08-28 2020-03-01 大陸商江蘇恆瑞醫藥股份有限公司 一種ｔｉｍ３抗體醫藥組成物及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103079644A (zh) * 2010-06-11 2013-05-01 协和发酵麒麟株式会社 抗tim-3抗体
CN107001475A (zh) * 2014-11-06 2017-08-01 豪夫迈·罗氏有限公司 抗tim3抗体及使用方法
CN110267988A (zh) * 2016-12-08 2019-09-20 伊莱利利公司 用于与抗pd-1抗体组合的抗tim-3抗体
CN108794630A (zh) * 2017-12-18 2018-11-13 镇江爱必梦生物科技有限公司 鼠抗人tim3蛋白单克隆抗体制备及其免疫组化用途
WO2020041520A1 (fr) * 2018-08-21 2020-02-27 Albert Einstein College Of Medicine Anticorps monoclonaux contre tim-3 humain
TW202009241A (zh) * 2018-08-28 2020-03-01 大陸商江蘇恆瑞醫藥股份有限公司 一種ｔｉｍ３抗體醫藥組成物及其用途

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118165111A (zh) * 2024-05-15 2024-06-11 中国人民解放军军事科学院军事医学研究院 针对Tim-3的抗体

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