WO2019129137A1 - 抗lag-3抗体及其用途 - Google Patents

抗lag-3抗体及其用途 Download PDF

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Publication number
WO2019129137A1
WO2019129137A1 PCT/CN2018/124315 CN2018124315W WO2019129137A1 WO 2019129137 A1 WO2019129137 A1 WO 2019129137A1 CN 2018124315 W CN2018124315 W CN 2018124315W WO 2019129137 A1 WO2019129137 A1 WO 2019129137A1
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Prior art keywords
antibody
lag
amino acid
seq
binding
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PCT/CN2018/124315
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English (en)
French (fr)
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缪小牛
胡化静
曾竣玮
刘军建
刘晓林
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信达生物制药(苏州)有限公司
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Priority claimed from CN201811561512.XA external-priority patent/CN109970856B/zh
Application filed by 信达生物制药(苏州)有限公司 filed Critical 信达生物制药(苏州)有限公司
Priority to KR1020207017696A priority Critical patent/KR20200104299A/ko
Priority to EP18893532.4A priority patent/EP3733702A4/en
Priority to US16/759,722 priority patent/US11732044B2/en
Priority to CA3080830A priority patent/CA3080830A1/en
Priority to JP2020535990A priority patent/JP2021508467A/ja
Priority to AU2018396877A priority patent/AU2018396877B2/en
Publication of WO2019129137A1 publication Critical patent/WO2019129137A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to novel antibodies and antibody fragments that specifically bind to LAG-3 and compositions containing the antibodies or antibody fragments. Furthermore, the invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, and host cells comprising the same, and related uses. Furthermore, the invention relates to the therapeutic and diagnostic use of these antibodies and antibody fragments. In particular, the invention relates to the combination of these antibodies and antibody fragments with other therapies, such as therapeutic modalities or therapeutic agents, such as anti-PD-1 or anti-PD-L1 antibodies.
  • Lymphocyte activating gene 3 (LAG-3), also known as CD223, is a type I transmembrane protein encoded by the LAG3 gene in humans.
  • LAG-3 is a CD4-like protein expressed on the surface of T cells (especially activated T cells), natural killer cells, B cells, and plasmacytoid dendritic cells.
  • LAG-3 has been shown to be a negative costimulatory receptor, an inhibitory receptor.
  • LAG-3 binds to MHC class II molecules (Baixeras et al (1992) J. Exp. Med. 176: 327-337; Huard et al (1996) Eur. J. Immunol, 26: 1180-1186), in antigen presenting cells ( APC) constitutively expresses a high level of molecular family at the surface of dendritic cells, macrophages, and B cells.
  • APC antigen presenting cells
  • LAG-3 function is dependent on binding to class II MHC and signaling through its cytoplasmic domain. Direct binding of LAG-3 to MHC class II molecules has been suggested to play a role in down-regulating antigen-dependent stimulation of CD4+ T lymphocytes (Huard et al. (1994) Eur. J. Immunol.
  • CD8+ T cells depleted after chronic viral infection express multiple inhibitory receptors (eg, PD-1, CD160 and 2B4).
  • LAG-3 is expressed at high levels after LCMV infection and shows that blocking the PD-1/PD-L1 pathway and blocking LAG-3 significantly reduce viral load in chronically infected mice (Blackburn et al., Nat Immunol (2009) ) 10:29-37). It has also been shown that the combined inhibition of the PD-1/PD-L1 pathway and the LAG-3 blocker provides anti-tumor efficacy (Jing et al, Journal for Immuno Therapy of Cancer (2015) 3:2).
  • anti-LAG-3 antibodies particularly humanized or human antibodies, that modulate their activity. Such antibodies are better used to treat tumors as well as other diseases such as infections. It would also be desirable to have new ones that can be used in combination with other therapies (eg, therapeutic agents, such as anti-PD-1 or anti-PD-L1 antibodies) for the treatment of tumors, particularly metastatic or refractory tumors, or for the treatment of infections, such as chronic infections.
  • therapies eg, therapeutic agents, such as anti-PD-1 or anti-PD-L1 antibodies
  • Anti-LAG-3 antibody e.g, anti-mouse LAG-3 antibodies that can be used in mouse models to facilitate the study of the in vivo biological activity of antibodies.
  • antibody molecules that bind to LAG-3, such as fully human antibody molecules or humanized antibody molecules. Also provided are nucleic acids encoding the antibodies or antibody fragments thereof, expression vectors, host cells and methods for producing antibody molecules. Immunoconjugates, multispecific or bispecific antibody molecules comprising an anti-LAG-3 antibody molecule, and pharmaceutical compositions are also provided.
  • the anti-LAG-3 antibody molecules disclosed herein can be used to treat, prevent, and/or diagnose neoplastic diseases and infections, alone or in combination with other therapies, such as therapeutic agents (eg, anti-PD-1 antibodies or anti-PD-L1 antibodies) or treatments. Sexual disease.
  • compositions and methods for detecting LAG-3 and methods of treating various diseases, including tumors and/or infectious diseases, using anti-LAG-3 antibody molecules are also provided.
  • an antibody of the invention or a fragment thereof (specific) binds to LAG-3.
  • an antibody or fragment thereof (specifically) of the invention binds to human LAG-3 or mouse LAG-3.
  • an anti-LAG-3 antibody or fragment thereof of the invention binds LAG-3 (eg, human LAG-3) with high affinity, eg, binds to LAG-3 with the following equilibrium dissociation constant (K D ), the K D of less than about 10OnM, preferably less than or equal to about 50 nM, more preferably less than or equal to about 20 nM, more preferably less than or equal to about 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM or 2nM Most preferably, the K D is less than or equal to about 1 nM, 0.9 nM, 0.8 nM or 0.7 nM.
  • K D equilibrium dissociation constant
  • the anti-LAG-3 antibodies of the invention are in the range of 0.1-20 nM, preferably 0.5-20 nM, more preferably 0.5-10 nM, 0.5-8 nM, 0.5-5 nM, most preferably 0.5-1 nM, 0.5-0.8. nM, 0.5-0.7nM, 0.6-0.7nM K D binding of LAG-3.
  • LAG-3 is human LAG-3.
  • the LAG-3 is mouse LAG-3.
  • antibody binding affinity is determined using a bio-optical interference assay (eg, Fortebio affinity measurement) assay.
  • an antibody or fragment thereof of the invention binds to a cell expressing human LAG-3, eg, at less than or equal to about 3.3 nM, 3 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM , EC50 of 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM or 0.5 nM.
  • the binding is determined by flow cytometry (eg, FACS).
  • the human LAG-3 expressing cell is a human LAG-3 expressing 293 cell (eg, HEK293 cell).
  • an antibody or fragment thereof of the invention binds to a cell expressing mouse LAG-3, eg, with an EC50 of less than or equal to about 15000 nM, 14000 nM, or 13000 nM. In some embodiments, an antibody or fragment thereof of the invention binds to a cell expressing mouse LAG-3, eg, with an EC50 of less than or equal to about 50 nM, such as an EC50 of about 40-50 nM, an EC50 of about 40-45 nM, or An EC50 of approximately 42 nM. In some embodiments, the binding is determined by flow cytometry (eg, FACS). In some embodiments, the cell expressing mouse LAG-3 is a Chinese hamster ovary (CHO) cell expressing mouse LAG-3.
  • CHO Chinese hamster ovary
  • the antibody or fragment thereof of the present invention inhibit the activity of LAG-3-related, for example, less than or equal to about 20nM, 10nM, 9nM, 8nM, 7nM, 6nM 5nM or the IC 50, preferably about 1-6nM , 1-5nM, 4nM, 4.5nM, 5nM , 5.1nM, 5.2nM, 5.3nM, 5.4nM, 5.5nM, 5.6nM, 5.7nM, 5.8nM, 5.9nM or 6nM the IC 50.
  • the related activity of LAG-3 is the binding of an MHC class II molecule to LAG-3.
  • the antibody or fragment of the invention is less than or equal to about 20nM, 10nM, 9nM, 8nM, 7nM, 6nM 5nM or the IC 50, preferably about 1-6nM, 1-5nM, 4nM, 4.5nM , 5nM, 5.1nM, 5.2nM, 5.3nM , 5.4nM, 5.5nM, 5.6nM, 5.7nM, 5.8nM, 5.9nM or 6nM IC 50 inhibition of LAG-3 and MHC II expression on a cell MHC class II molecules
  • the MHC class II molecule is HLA-DR.
  • the cell is a CHO cell.
  • inhibition of the associated activity of LAG-3 by the antibody or fragment thereof of the invention is measured by flow cytometry (e.g., FACS).
  • an antibody or fragment thereof of the invention binds to endogenous LAG-3 on the surface of activated CD4+ and/or CD8+ T cells, eg, at less than or equal to about 35 pM, 30 pMnM, 25 pM, 20 pM, 15 pM, EC 14pM 13pM, or 50, preferably about 1-20pM, 5-20pM, 5-15pM, 10-15pM, 11-13pM, 10pM, 11pM, 12pM 13pM, or the EC 50.
  • the activated CD4+ T cells are activated human CD4+ T cells.
  • the above binding is determined by flow cytometry (e.g., FACS). In some embodiments, flow cytometry is performed in an Accuri C6 system.
  • an antibody or fragment thereof of the invention inhibits one or more activities of LAG-3, for example, resulting in one or more of the following: increased antigen-dependent stimulation of CD4+ T lymphocytes; T cell proliferation Increased; increased expression of activated antigen (eg, CD25); increased expression of cytokines (eg, increased interferon-interferon; cytokine, interleukin-2 (IL-2) or interleukin-4 (IL-4)); Increased expression of chemokines (eg, CCL3, CCL4, or CCL5); decreased repression activity of Treg cells; increased T cell homeostasis; increased tumor infiltrating lymphocytes; or reduced immune evasion of cancer cells.
  • activated antigen eg, CD25
  • cytokines eg, increased interferon-interferon; cytokine, interleukin-2 (IL-2) or interleukin-4 (IL-4)
  • chemokines eg, CCL3, CCL4, or CCL5
  • an anti-LAG-3 antibody or fragment thereof of the invention is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • an anti-LAG-3 antibody of the invention is effective to treat a tumor (eg, cancer) or an infectious disease (eg, a chronic infection).
  • a tumor eg, cancer
  • an infectious disease eg, a chronic infection
  • the anti-LAG-3 antibodies of the invention are capable of treating tumors, particularly metastatic or refractory tumors, in combination with anti-PD-1 or anti-PD-L1 antibodies.
  • the tumor is a cancer.
  • the tumor is a gastrointestinal tumor.
  • the cancer is colon cancer.
  • the heavy and/or light chain of an anti-LAG-3 antibody or fragment thereof of the invention further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 48).
  • the antibodies of the invention also encompass variants of the amino acid sequence of an anti-LAG-3 antibody, as well as antibodies that bind to the same epitope as any of the anti-LAG-3 antibodies or fragments thereof described above.
  • an antibody or antibody fragment (preferably an antigen-binding fragment) that binds to LAG-3 or a fragment thereof is provided, wherein the antibody or antibody fragment binds to an epitope within LAG-3.
  • an anti-LAG-3 antibody of the invention is an antibody in the form of IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE.
  • an anti-LAG-3 antibody of the invention comprises a heavy chain constant region selected from the group consisting of a heavy chain constant region such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; From the heavy chain constant region of the heavy chain constant region of, for example, IgG1, IgG2, IgG3, and IgG4, more specifically, the heavy chain constant region of IgG1, IgG2, or IgG4, such as the heavy chain constant region of human IgG1, IgG2, or IgG4.
  • the heavy chain constant region is a human IgGl or human IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is a human IgG4 heavy chain constant region. In another embodiment, the anti-LAG-3 antibody molecule has, for example, a light chain constant region selected from the kappa or lambda light chain constant region, preferably a light chain constant region of kappa (eg, human kappa).
  • the anti-LAG-3 antibody molecule comprises a heavy chain constant region of IgG4 (eg, human IgG4).
  • human IgG4 comprises a substitution at position 228 according to the EU number (eg, Ser to Pro substitution).
  • human IgG4 is mutated to AA at positions 114-115 (EU numbering) (Armour KL1, Clark MR, Hadley AG, Williamson LM, Eur J Immunol. 1999 Aug; 29(8): 2613 24, Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities).
  • the heavy chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence set forth in SEQ ID NO:46 A sequence of 96%, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the anti-LAG-3 antibody molecule comprises a kappa light chain constant region, eg, a human kappa light chain constant region.
  • the light chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 of the amino acid sequence of SEQ ID NO:47 A sequence of %, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the anti-LAG-3 antibody molecule comprises a heavy chain constant region of IgG4 (eg, a human IgG4 heavy chain constant region) and a kappa light chain constant region (eg, a human kappa light chain constant region).
  • the constant region is a mutated IgG4, eg, a mutated human IgG4 (eg, having a mutation at position 228 according to EU numbering (eg, S228P mutation)).
  • the human IgG4 heavy chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence set forth in SEQ ID NO:46, A sequence of 95%, 96%, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the human kappa light chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO:47 A sequence of 96%, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the anti-LAG-3 antibody molecule is isolated or recombinant.
  • the anti-LAG-3 antibody is a monoclonal antibody or an antibody having monospecificity.
  • the anti-LAG-3 antibody molecule can also be a humanized, chimeric, camelid, shark or antibody molecule produced in vitro.
  • the anti-LAG-3 antibody is humanized.
  • the anti-LAG-3 antibody is a human antibody.
  • at least a portion of the framework sequence of the anti-LAG-3 antibody is a human consensus framework sequence.
  • an anti-LAG-3 antibody of the invention further encompasses an antibody fragment thereof, preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv) or ( Fab') 2 , single domain antibody, double antibody (dAb) or linear antibody.
  • an antibody fragment thereof preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv) or ( Fab') 2 , single domain antibody, double antibody (dAb) or linear antibody.
  • the anti-LAG-3 antibody molecule is in the form of a bispecific or multispecific antibody molecule.
  • the bispecific antibody molecule has a first binding specificity for LAG-3 and for PD-1, TIM-3, CEACAM (eg, CEACAM-1 and/or CEACAM-5), PD-L1 Or the second binding specificity of PD-L2.
  • the bispecific antibody molecule binds to LAG-3 and PD-1.
  • the bispecific antibody molecule binds to LAG-3 and PD-L1.
  • the bispecific antibody molecule binds to LAG-3 and PD-L2. Any combination of the foregoing molecules can be produced in a multispecific antibody molecule.
  • Multispecific antibody molecules such as trispecific antibody molecules, comprising a first binding specificity for LAG-3 and a second and third binding specificity for one or more of the following molecules: PD-1, TIM- 3.
  • CEACAM eg, CEACAM-1 or CEACAM-5
  • the anti-LAG-3 antibody molecule is used in combination with a bispecific molecule comprising one or more of: PD-1, TIM-3, CEACAM (eg, CEACAM-1 or CEACAM-5), PD -L1 or PD-L2.
  • a bispecific molecule comprising one or more of: PD-1, TIM-3, CEACAM (eg, CEACAM-1 or CEACAM-5), PD -L1 or PD-L2.
  • the invention provides a nucleic acid encoding any of the above anti-LAG-3 antibodies or fragments thereof.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector.
  • a host cell comprising the nucleic acid or the vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from the group consisting of a yeast cell, a mammalian cell (eg, a CHO cell or a 293 cell), or other cell suitable for use in the preparation of an antibody or antigen-binding fragment thereof.
  • the host cell is prokaryotic, such as an E. coli cell.
  • the invention provides a method of making an anti-LAG-3 antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises expressing the antibody or fragment thereof (preferably an antigen-binding fragment) suitable for expression
  • the host cell is cultured under conditions of a nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated.
  • the method further comprises recovering an anti-LAG-3 antibody or fragment thereof, preferably an antigen binding fragment, from the host cell.
  • the invention provides an immunoconjugate comprising any of the anti-LAG-3 antibodies and other materials provided herein, such as a cytotoxic agent or label.
  • the immunoconjugate is used to prevent or treat a tumor (eg, cancer) or an infectious disease.
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • the infectious disease is a chronic infection.
  • the invention provides compositions comprising any of the anti-LAG-3 antibodies or fragments thereof, preferably antigen binding fragments thereof, or immunoconjugates thereof, preferably a composition, which is a pharmaceutical composition.
  • the composition further comprises a pharmaceutical excipient.
  • a composition eg, a pharmaceutical composition, comprises an anti-LAG-3 antibody or fragment thereof of the invention, or an immunoconjugate thereof, and one or more additional therapeutic agents (eg, chemotherapeutic agents, cytotoxicity) Agent, vaccine, other antibody, anti-infective active agent or immunomodulatory agent (for example, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule), preferably an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-PD- A combination of L2 antibodies).
  • additional therapeutic agents eg, chemotherapeutic agents, cytotoxicity
  • Agent e.g, chemotherapeutic agents, cytotoxicity
  • vaccine e.g, other antibody, anti-infective active agent or immunomodulatory agent (for example, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule), preferably an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-PD- A combination of L2 antibodies).
  • the pharmaceutical composition is for use in preventing or treating a tumor (eg, cancer) or an infection.
  • a tumor eg, cancer
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • the infectious disease is a chronic infection.
  • the invention relates to a method of preventing or treating a tumor (eg, cancer) or an infectious disease in a subject or individual, the method comprising administering to the subject an effective amount of any of the anti-LAGs described herein -3 antibody or fragment thereof, pharmaceutical composition or immunoconjugate.
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • the infectious disease is a chronic infection.
  • the invention features a use of any of the anti-LAG-3 antibodies or fragments thereof described herein for the manufacture of a medicament for treating a tumor (eg, cancer) or infection in a subject.
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • the infectious disease is a chronic infection.
  • the invention in another aspect, relates to a method of preventing or treating a tumor (eg, cancer) or an infectious disease in a subject or individual, the method comprising administering to the subject an effective amount of any of the antibodies described herein A LAG-3 antibody or fragment, pharmaceutical composition or immunoconjugate thereof, and a PD-1 axis binding antagonist or a drug or active agent comprising the PD-1 axis binding antagonist.
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • the infectious disease is a chronic infection.
  • the invention relates to any of the anti-LAG-3 antibodies or fragments thereof described herein in combination with a PD-1 axis binding antagonist for use in the preparation of a tumor (eg, cancer) or infectious disease in a subject.
  • a tumor eg, cancer
  • infectious disease is a chronic infection.
  • the PD-1 axis binding antagonist comprises, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody.
  • the treatment modality includes surgical treatment and/or radiation therapy.
  • the additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, or an immunomodulatory agent (eg, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule).
  • the subject or individual is a non-human animal. In some embodiments, the subject or individual is a mammal, preferably a human.
  • the invention relates to a method of detecting LAG-3 in a sample, the method comprising (a) contacting the sample with any of the anti-LAG-3 antibodies or fragments thereof described herein; and (b) detecting the anti-LAG- 3 Formation of a complex between an antibody or a fragment thereof and LAG-3.
  • the anti-LAG-3 antibody is detectably labeled.
  • the invention relates to a kit or article comprising any of the anti-LAG-3 antibodies or fragments thereof described herein.
  • the kit or article of manufacture comprises an anti-LAG-3 antibody or fragment thereof described herein, and an optional pharmaceutical excipient.
  • the kit or article further comprises instructions for administering a drug to treat a tumor or infection.
  • the invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein, or any combination thereof, are suitable for use in any and all of the anti-LAG-3 antibodies or fragments, methods and uses thereof of the invention described herein.
  • Figure 1 shows the binding ability of the parent antibody and cell surface hLAG-3 detected by flow cytometry.
  • Figure 2 shows the binding ability of affinity matured antibodies to cell surface hLAG-3 as detected by flow cytometry.
  • Figure 3 shows that anti-LAG-3 antibodies detected by flow cytometry block the interaction of human MHC II (HLA-DR) and LAG-3.
  • Figure 4 shows the binding ability of anti-LAG-3 antibody and activated human CD4+ T cells detected by flow cytometry.
  • Figure 5 shows the binding ability of anti-LAG-3 antibody and cell surface mouse LAG-3 detected by flow cytometry.
  • Figure 6 shows the inhibition of tumors in a CT26 xenograft model using a combination of an anti-LAG-3 antibody and an anti-PD-1 antibody.
  • Figure 7 shows the inhibition of tumors in the NOG model using an anti-LAG-3 antibody in combination with an anti-PD-1 antibody or PD-L1.
  • Figure 8 shows the IL-2 standard curve.
  • Figure 9 shows the effect of antibody or antibody combinations on the secretion of IL-2 by activated human CD4+ T cells.
  • IC50 produces a concentration of 50% inhibition
  • VH heavy chain variable region VH heavy chain variable region
  • Binding affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of a binding pair (eg, an antibody and an antigen), unless otherwise indicated.
  • the affinity of molecule X for its partner Y is generally expressed by the equilibrium dissociation constant (K D ). Affinity can be measured by conventional methods known in the art, including those known in the art and described herein.
  • lymphocyte activating gene-3 or "LAG-3” includes all isotypes, mammalian (eg, human) LAG-3, a species homolog of human LAG-3, and at least one common to LAG-3. An analog of an epitope.
  • Amino acid sequences and nucleotide sequences of LAG-3 e.g., human LAG-3) are known in the art, for example, see Triebel et al, (1990) J. Exp. Med. 171: 1393-1405.
  • the term “human LAG-3” refers to the complete amino acid sequence of human sequence LAG-3, such as human LAG-3 of Genbank Accession No. NP_002277.
  • mouse LAG-3 refers to the complete amino acid sequence of mouse sequence LAG-3, such as mouse LAG-3 of Genbank Accession No. NP_032505.
  • LAG-3 is also referred to in the prior art as, for example, CD223.
  • the human LAG-3 sequence may have, for example, a conservative mutation or a mutation in a non-conserved region different from the human LAG-3 of Genbank Accession No. NP_002277, and the LAG-3 is substantially identical to the human LAG-3 of Genbank Accession No. NP_002277.
  • Biological function for example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3, which is specifically bound by the antibodies of the present disclosure, or the biological function of human LAG-3 is to bind to MHC class II molecules .
  • anti-LAG-3 antibody refers to an antibody capable of binding LAG with sufficient affinity. -3 protein or a fragment thereof.
  • the anti-LAG-3 antibody binds to a non-LAG-3 protein to a lesser extent than about 10%, about 20%, about 30%, about 40%, about 50% of the binding of the antibody to LAG-3.
  • monoclonal antibody or “mAb” or “Mab” refers to a single copy or cloned antibody derived from, for example, a eukaryotic, prokaryotic or phage clone, and does not refer to methods of its production.
  • Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant techniques, phage display technology, synthetic techniques such as CDR grafting, or a combination of such or other techniques known in the art.
  • “Native antibody” refers to a naturally occurring immunoglobulin molecule having a different structure.
  • a native IgG antibody is an isotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains bonded with a disulfide. From N to C, each heavy chain has a variable region (VH), also known as a variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C, each light chain has a variable region (VL), also known as a variable light or light chain variable domain, followed by a constant light (CL) domain.
  • the antibody light chain can be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ).
  • a "native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • the native sequence human Fc region comprises the native sequence human IgG1 Fc region (non-A and A allotype); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region; and naturally occurring variants thereof .
  • Antibody fragment refers to a molecule that is distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single chain antibodies (eg, 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 (eg, human LAG-3) that specifically interacts with an antibody molecule.
  • This portion referred to herein as an epitope determinant
  • an epitope determinant typically comprises an element such as an amino acid side chain or a sugar side chain or a component thereof.
  • Epitope determinants can be defined according to methods known in the art or disclosed herein (e.g., by crystallography or by hydrogen-oxime exchange).
  • At least one or some portion of the antibody molecule that specifically interacts with an epitope determinant is generally located within the CDR.
  • epitopes have specific three dimensional structural characteristics.
  • epitopes have specific charge characteristics. Some epitopes are linear epitopes, while others are conformational epitopes.
  • 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 its competition assay Binding of the antigen, in other words, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the antibody to its antigen in a 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 more of the binding of the reference antibody to its antigen in a competition assay.
  • the reference antibody blocks 50%, 60%, 70%, 80%, 90% or more 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 an antibody competes with another assay such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition. Assay (see for example Stahli et al, 1983, Methods in Enzymology 9: 242-253).
  • An antibody that inhibits (eg, competitively inhibits) binding of a reference antibody to its antigen refers to an antibody that inhibits binding of 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody to its antigen. . Conversely, the reference antibody inhibits binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90% or more.
  • the binding of an antibody to its antigen can be measured by affinity (eg, equilibrium dissociation constant). Methods for determining affinity are known in the art.
  • An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of binding at least 50%, 60%, 70%, 80%, 90% or 95% 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 a sequence that is hypervariable in an antibody variable domain and that forms a structurally defined loop ("hypervariable loop") and/or contains an antigen contact residue ( The area of the "antigen contact point”).
  • the CDR is primarily responsible for binding to an epitope.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • the CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al.
  • the residues of each CDR are as follows.
  • the CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence (e.g., any of the exemplary CDRs of the invention).
  • the CDRs of the antibodies of the invention are determined by Kabat rules, rules are determined by IMGT, or rules are determined by AbM, or by a combination thereof, for example, the sequences are as shown in Table 1.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different.
  • the scope of the antibody also encompasses an antibody whose variable region sequence comprises the particular CDR sequence, but due to the application of a different protocol (eg Different assignment system rules or combinations result in different claimed CDR boundaries than the specific CDR boundaries defined by the present invention.
  • Antibodies with different specificities have different binding sites for different antigens
  • CDRs have different CDRs (under the same assignment system).
  • the CDRs differ between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding.
  • the minimum binding unit can be a sub-portion of the CDR.
  • residues of the remainder of the CDR sequences can be determined by the structure of the antibody and protein folding. Accordingly, the invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • IgA immunoglobulin 1
  • IgG 2 immunoglobulin 2
  • IgG 3 immunoglobulin 3
  • IgG 4 immunoglobulin 1
  • IgA 2 immunoglobulin 2
  • the heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Antibody in IgG form refers to the IgG form to which the heavy chain constant region of an antibody belongs.
  • the heavy chain constant regions of all antibodies of the same type are identical, and the heavy chain constant regions differ between different types of antibodies.
  • an antibody in the form of IgG4 refers to an Ig domain whose heavy chain constant region Ig domain is IgG4.
  • PD-1 axis binding antagonist refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby removing signals originating from the PD-1 signaling axis. Conducted T cell dysfunction - one result is the restoration or enhancement of T cell function (eg proliferation, cytokine production, target cell killing).
  • PD-1 axis binding antagonists include PD-1 binding antagonists (eg, anti-PD-1 antibodies), PD-L1 binding antagonists (eg, anti-PD-L1 antibodies), and PD-L2 binding antagonists. (eg anti-PD-L2 antibody).
  • PD-1 binding antagonist refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the binding of PD-1 to one or more of its binding partners (such as PD-L1, PD-L2). Interacting signal transduction.
  • the PD-1 binding antagonist is a molecule that inhibits PD-1 binding to one or more of its binding partners.
  • the PD-1 binding antagonist inhibits PD-1 binding to PD-L1 and/or PD-L2.
  • a PD-1 binding antagonist comprises an anti-PD-1 antibody that reduces, blocks, inhibits, eliminates or interferes with signal transduction from PD-1 interaction with PD-L1 and/or PD-L2, and antigen binding thereof.
  • the PD-1 binding antagonist reduces a cell surface protein-mediated negative costimulatory signal (via PD-1 mediated signaling) expressed on or via T lymphocytes, thereby enabling dysfunctional T cells Less dysfunctional (eg, enhancing effector response to antigen recognition).
  • the PD-1 binding antagonist is an anti-PD-1 antibody.
  • the PD-1 binding antagonist is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab) or AMP-224 as disclosed in WO2015/095423.
  • the anti-PD-1 antibody is "Antibody C" as disclosed in WO 2017/133540.
  • the anti-PD-1 antibody is "Antibody D" as disclosed in WO 2017/025016.
  • PD-L1 binding antagonist refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the binding of PD-L1 from one or more of its binding partners (such as PD-1, B7-1). Interacting signal transduction.
  • the PD-L1 binding antagonist is a molecule that inhibits PD-L1 binding to its binding partner.
  • the PD-L1 binding antagonist inhibits PD-L1 binding to PD-1 and/or B7-1.
  • the PD-L1 binding antagonist comprises reducing, blocking, inhibiting, eliminating or interfering with the interaction of PD-L1 from one or more of its binding partners (such as PD-1, B7-1) Signal transduced anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules.
  • the PD-L1 binding antagonist reduces a cell surface protein-mediated negative costimulatory signal (via PD-L1 mediated signaling) expressed on or via T lymphocytes, thereby enabling dysfunctional T cells Less dysfunctional (eg, enhancing effector response to antigen recognition).
  • the PD-L1 binding antagonist is an anti-PD-L1 antibody.
  • the anti-PD-L1 antibody is YW243.55.S70, MDX-1105, MPDL3280A or MEDI4736 as disclosed in WO2015/095423.
  • PD-L2 binding antagonist refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signals derived from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. divert.
  • the PD-L2 binding antagonist is a molecule that inhibits PD-L2 binding to one or more of its binding partners.
  • the PD-L2 binding antagonist inhibits PD-L2 binding to PD-1.
  • the PD-L2 antagonist comprises reducing, blocking, inhibiting, eliminating or interfering with signal transduction derived from the interaction of PD-L2 with one or more of its binding partners, such as PD-1.
  • Anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules are included in the PD-L2 binding antagonist.
  • the PD-L2 binding antagonist reduces a cell surface protein-mediated negative costimulatory signal (via PD-L2 mediated signaling) expressed on or via T lymphocytes, thereby enabling dysfunctional T cells Less dysfunctional (eg, enhancing effector response to antigen recognition).
  • the PD-L2 binding antagonist is an immunoadhesin.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcR Fc receptor
  • cytotoxic cells eg, NK cells, neutrophils, and macrophages.
  • Secretory immunoglobulins enable these cytotoxic effector cells to specifically bind to target cells carrying the antigen, followed by cytotoxicity to kill the cytotoxic form of the target cells.
  • the main cell that mediates ADCC NK cells, express only Fc ⁇ RIII, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991) summarizes FcR expression on hematopoietic cells.
  • an in vitro ADCC assay can be performed, such as that described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Effector cells that can be used in such assays include PBMC and NK cells. Alternatively/in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as those disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998).
  • cytotoxic agent or "cytotoxic factor” as used in the present invention refers to a substance which inhibits or prevents cellular function and/or causes cell death or destruction. Examples of cytotoxic agents are disclosed in WO 2015/153513, WO 2016/028672 or WO 2015/138920.
  • therapeutic agent encompasses any substance that is effective in preventing or treating a tumor (eg, cancer) and an infection (eg, a chronic infection), including a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, or An immunomodulator, such as any of the materials disclosed in WO2016/028672 or WO2015/138920, which can be used in combination with an anti-LAG-3 antibody.
  • “Chemotherapeutic agents” include chemical compounds that are useful in the treatment of cancer. Examples of chemotherapeutic agents are disclosed in WO 2015/153513 or WO 2016/028672 or WO 2015/138920.
  • cytokine is a generic term for a protein that is released by one cell population and acts as an intercellular medium on another cell.
  • cytokines are lymphokines, mononuclear factors; interleukins (IL) such as IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL. -7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including LIF and kit ligand (KL) And ⁇ -interferon.
  • IL interleukins
  • IL-1 interleukins
  • IL-2 interleukins
  • IL-3 interleukins
  • IL-5 IL-6
  • IL. -7 IL-8
  • IL-9 IL-11, IL-12, IL-15
  • tumor necrosis factor such as TNF- ⁇ or TNF- ⁇
  • KL kit ligand
  • costimulatory molecule refers to a related binding partner on a T cell that specifically binds to a costimulatory ligand, and thus is mediated by T cells, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for efficient immune responses.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activation molecules of signaling lymphocytes (SLAM proteins), NK cell activating receptors , BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS , ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA
  • activator or "agonist” includes a substance that increases certain parameters (eg, activity) of a given molecule (eg, a costimulatory molecule).
  • a given molecule eg, a costimulatory molecule
  • the term includes a substance that increases the activity of the given molecule by at least 5%, 10%, 25%, 50%, 75% or more (eg, costimulatory activity).
  • Immunoassay molecule means a group of molecules on the cell surface of CD4 T cells and CD8 T cells. These molecules can effectively act as “brakes” that down-regulate or inhibit anti-tumor immune responses.
  • Immunological checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, and LAG-3, which directly inhibit Immune Cells.
  • inhibitor or "antagonist” includes a substance that reduces certain parameters (eg, activity) of a given molecule (eg, an immunological checkpoint inhibitor protein).
  • diabody refers to an antibody fragment having two antigen binding sites comprising a heavy chain variable domain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). (VH).
  • VL light chain variable domain
  • VH light chain variable domain
  • linker that is too short to be able to pair between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites.
  • Diabodies can be bivalent or bispecific. Diabodies are more fully described, for example, in EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA ) 90:6444-6448 (1993). Tri- and tetra-antibodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).
  • a “functional Fc region” possesses an “effector function” of the native sequence Fc region.
  • effector functions include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (eg, B cell receptor; BCR), and the like.
  • Such effector functions generally require that the Fc region be associated with a binding domain (eg, an antibody variable domain) and can be assessed using a variety of assays, such as those disclosed herein.
  • Antibody effector function refers to those biological activities attributable to the Fc region of an antibody that vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) Down); and B cell activation.
  • Human effector cell refers to a leukocyte that expresses one or more FcRs and functions as an effector. In certain embodiments, the cell expresses at least Fc to effector function and to function as an ADCC effector. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from their natural source, such as blood.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells
  • neutrophils effector cells can be isolated from their natural source, such as blood.
  • an effective amount refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention that, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.
  • An effective amount can be readily determined by the attending physician as a person skilled in the art by considering various factors such as the species of the mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease; Response of an individual patient; specific antibody administered; mode of administration; bioavailability characteristics of the administered formulation; selected dosing regimen; and use of any concomitant therapy.
  • Therapeutically effective amount means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required.
  • the therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof can vary depending on a variety of factors, such as the disease state, the age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are less than a therapeutically beneficial effect.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70, relative to an untreated subject. % and still more preferably at least about 80%.
  • a measurable parameter eg, a tumor growth rate
  • the ability of a compound to inhibit measurable parameters can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit, in vitro by assays known to the skilled artisan.
  • prophylactically effective amount is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount will be less than a therapeutically effective amount since the prophylactic dose is administered to a subject prior to the earlier stage of the disease or at an earlier stage of the disease.
  • Antibodies and antigen-binding fragments thereof suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterozygous, chimeric Humanized (especially grafted with CDRs), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') 2 fragments, fragments produced by Fab expression libraries, Fd, Fv, II Sulfide-linked Fv (dsFv), single-chain antibody (eg, scFv), diabody or tetra-antibody (Holliger P. et al. (1993) Proc. Natl.
  • Nanobody nanobody
  • anti-idiotypic antibody including, for example, an anti-Id antibody against an antibody of the invention
  • epitope-binding fragment of any of the above.
  • a "Fab” fragment includes a heavy chain variable domain and a light chain variable domain, and also includes a constant domain of the light chain and a first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the name for Fab' in which a cysteine residue of a constant domain carries a free thiol group.
  • the F(ab') 2 antibody fragment was originally produced as a pair of Fab' fragments with a hinge cysteine between the Fab' fragments. Other chemical couplings of antibody fragments are also known.
  • Fc region is used herein to define a C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National. Institutes of Health, Bethesda, MD, 1991.
  • variable region refers to a domain of an antibody heavy or light chain that is involved in binding of an antibody to an antigen.
  • the variable domains of the heavy and light chains of a native antibody typically have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • antibodies that bind to the antigen can be isolated using a VH or VL domain from an antibody that binds to a particular antigen to separately screen a library of complementary VL or VH domains. See, for example, Portolano et al, J. Immunol. 150: 880-887 (1993); Clarkson et al, Nature 352: 624-628 (1991).
  • “Framework” or “F R” refers to a variable domain residue other than a complementarity determining region (CDR) residue.
  • the FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4.
  • CDR and FR sequences typically appear in the following sequences of the heavy chain variable domain (VH) (or light chain variable domain (VL)):
  • the numbering of residues in each domain of an antibody is based on the EU numbering system, which is also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • full length antibody intact antibody and “intact antibody” are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.
  • Fv is the smallest antibody fragment that contains the entire antigen binding site.
  • the double-stranded Fv species consists of one heavy chain variable domain and one light chain variable domain in a tight, non-covalently associated dimer.
  • one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can be similar to a double-stranded Fv species.
  • Dimer structure association. In this configuration, it is the three CDRs of each variable domain that define the antigen binding site on the surface of the VH-VL dimer. In summary, the six CDRs confer antigen binding specificity to the antibody.
  • variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • affinity is lower than the intact binding site.
  • host cell refers to a cell into which an exogenous nucleic acid is introduced, including progeny of such a cell.
  • Host cells include “transformants” and “transformed cells” which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny of the same function or biological activity selected or selected in the originally transformed cells are included herein.
  • Human antibody refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, which utilizes a human antibody library or other human Antibody coding sequence. This definition of a human antibody specifically excludes a humanized antibody comprising a non-human antigen-binding residue.
  • Human consensus framework refers to a framework that represents the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is selected from subtypes of variable domain sequences.
  • the subtype of this sequence is a subtype as disclosed in Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Vol. 1-3).
  • the subtype is subtype kappa I as in Kabat et al. (supra).
  • the subtype is subtype III as in Kabat et al. (supra).
  • a “humanized” antibody refers to a chimeric antibody comprising an amino acid residue from a non-human CDR and an amino acid residue from a human FR.
  • a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the CDRs (eg, CDRs) correspond to those of a non-human antibody, and all Or substantially all of the FRs correspond to those of human antibodies.
  • the humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • cancer refers to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer.
  • squamous cell carcinoma e.g., epithelial squamous cell carcinoma
  • lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma
  • peritoneal cancer e.g., peritoneal cancer.
  • hepatocellular carcinoma gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, Malignant freckle-like melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as with phagomatoses, edema (such as those associated with brain
  • cell proliferative disorder and “proliferative disorder” refer to a disorder associated with a certain degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder refers to cancer.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre
  • infectious disease refers to a disease caused by a pathogen, including, for example, a viral infection, a bacterial infection, a fungal infection, or a protozoan such as a parasitic infection.
  • chronic infection refers to an infection in which an infectious agent (eg, a pathogen such as a virus, a bacterium, a protozoan such as a parasite, a fungus, or the like) has induced an immune response in an infected host, but has not yet been as acutely infected. It is also removed or eliminated from the host as in the process.
  • an infectious agent eg, a pathogen such as a virus, a bacterium, a protozoan such as a parasite, a fungus, or the like
  • Chronic infections can be persistent, latent or slow.
  • acute infections are usually resolved by the immune system for days or weeks (eg, flu)
  • persistent infections can persist for months, years, decades, or lifetime (eg, hepatitis B) at relatively low levels.
  • latent infections are characterized by long-term asymptomatic activity, interrupted by high levels of rapid infection and elevated pathogen levels over time (eg, herpes simplex).
  • slow infection is characterized by a gradual and continuous increase in disease symptoms, such as a long-term incubation period, followed by an onset of prolonged and progressive clinical processes following the onset of clinical symptoms.
  • chronic infections may not begin with the acute phase of viral proliferation (eg, picornaviruses infection, sheep visna virus, scrapie, Creutzfeldt-Jakobdisease).
  • infectious agents capable of inducing chronic infection include viruses (eg, cytomegalovirus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type I and type II, human immunodeficiency virus types 1 and 2, Human papillomavirus, human T lymphocyte virus type 1 and 2, varicella-zoster virus, etc.), bacteria (eg, Mycobacterium tuberculosis, Listeria spp.) , Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Borrelia spp., Helicobacter pylori, etc., native Animals such as parasites (eg, Leishmania spp., Plasmodium falciparum, Schistosoma spp., Toxoplasma spp., Trypanosoma species) Trypanosoma spp.), Taenia carssiceps, etc., and fungi (e
  • Additional infectious agents include prions or misfolded proteins that affect the brain or neuronal structure by further spreading protein misfolding in these tissues, resulting in the formation of amyloid plaques (which lead to cell death, tissue damage, and eventual death).
  • diseases caused by prion infection include: Creutzfeldt-Jakob disease and its varieties, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (sFI) (fatal familial Insomnia (sFI)), kuru, scrapie, bovine spongiform encephalopathy (BSE) (also known as "mad cow” disease) (Bovine spongiformencephalopathy (BSE) in cattle (aka “mad cow") "Disease)), as well as various other animal forms of encephalopathy [eg, transmissible mink encephalopathy (TME), white-tailed deer, elk, and elk (" Mule deer) chronic wasting disease (CWD), feline spongiform ence
  • an “immunoconjugate” is an antibody that is conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and that facilitates detection of the agent to which it is conjugated or fused.
  • the label itself may be detectable (e.g., radioisotope label or fluorescent label) or, in the case of enzymatic labeling, may catalyze chemical alteration of a substrate compound or composition that is detectable.
  • the term is intended to encompass the direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to a probe or antibody, and indirectly labeling the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end labeling of a biotin-containing DNA probe such that it can be detected with a fluorescently labeled streptavidin protein.
  • mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and rats
  • rodents eg, mice and rats.
  • the individual or subject is a human.
  • an “isolated” antibody is one which has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) Determined by HPLC).
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reversed phase
  • nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that typically comprises the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • isolated nucleic acid encoding an anti-LAG-3 antibody or fragment thereof refers to one or more nucleic acid molecules encoding an antibody heavy or light chain (or a fragment thereof), including such a single vector or separate vector. Nucleic acid molecules, as well as such nucleic acid molecules that are present at one or more positions in a host cell.
  • nucleic acid refers to nucleotides of any length (deoxyribonucleotides or ribonucleotides) or analogs thereof in the form of a polymer.
  • the polynucleotide may be single stranded or double stranded, and if single stranded, may be a coding strand or a non-coding (antisense) strand.
  • Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide may be further modified after polymerization, such as by conjugation to a labeling component.
  • the nucleic acid can be a recombinant polynucleotide or a genomic, cDNA, semi-synthetic or synthetic source polynucleotide that is not found in nature or linked to another polynucleotide in an unnatural layout.
  • polypeptide if single stranded
  • polymer may be linear or branched, it may contain modified amino acids, and it may be cleaved by non-amino acids.
  • the term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
  • Polypeptides can be isolated from natural sources, produced by eukaryotic or prokaryotic hosts by recombinant techniques, and can be the product of synthetic methods.
  • the sequences are aligned for optimal comparison purposes (eg, for optimal alignment, in the first and second amino acid sequences or nucleic acid sequences) Vacancies are introduced in one or both or non-homologous sequences can be discarded for comparison purposes.
  • the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% for comparison purposes. , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at the corresponding amino acid position or nucleotide position are then compared. When the 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 molecule is identical at this position.
  • Mathematical algorithms can be used to achieve sequence comparisons and percent identity calculations between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm in the GAP program that has been integrated into the GCG software package is used (at http://www.gcg.com) Obtained), using a Blossum 62 matrix or PAM250 matrix and vacancy weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6, to determine between the two amino acid sequences Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and the vacancy weights of 40, 50, 60, 70 or 80 and The length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences.
  • a particularly preferred set of parameters (and a set of parameters that should be used unless otherwise stated) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases to, for example, identify other family member sequences or related sequences. Such searches can be performed, for example, using the NBLAST and XBLAST programs of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • vacant BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.
  • the default parameters of the corresponding programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • hybridizes under conditions of low stringency, medium stringency, high stringency or very high stringency describes hybridization and washing conditions.
  • Guidance 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, incorporated by reference. Both aqueous and non-aqueous methods are described in the references and either method can be used.
  • the specific hybridization conditions referred to herein 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, Increasing the wash temperature to 55 ° C) was washed twice in 0.2X SSC, 0.1% SDS; 2) Moderate stringency hybridization conditions were in 6X SSC at about 45 ° C, followed by 0.2X SSC, 0.1% SDS at 60 ° C.
  • SSC sodium chloride/sodium citrate
  • high stringency hybridization conditions are washed one or more times in 6X SSC at about 45 ° C, then at 0.2 ° SSC, 0.1% SDS at 65 ° C; and preferably 4) extremely high Stringent hybridization conditions were washed one or more times at 65 ° C in 0.5 M sodium phosphate, 7% SDS followed by 65 ° C in 0.2X SSC, 0.1% SDS.
  • Very high stringency conditions (4) are preferred conditions and one condition that should be used unless otherwise stated.
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredient contained therein to be effective, and does not include additional toxicity to the subject to whom the composition is administered. Ingredients.
  • pharmaceutically acceptable adjuvant refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), carrier, excipient or stabilizer, and the like, to be administered with the active substance.
  • treating refers to slowing, interrupting, arresting, ameliorating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes inhibition of the occurrence or progression of a disease or condition or a symptom of a particular disease or condition.
  • a subject with a family history of cancer is a candidate for a prophylactic regimen.
  • prevention refers to the administration of a drug prior to the onset of a symptom or symptom of a cancer, particularly in a subject at risk for cancer.
  • anti-infective active agent includes any molecule that specifically inhibits or eliminates the growth of microorganisms at the concentration of administration and the interval of administration, but is not lethal to the host, such as viruses, bacteria, fungi or protozoa, such as parasites.
  • anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents.
  • the anti-infective active agent is non-toxic to the host at the concentration of administration and the interval of administration.
  • Antibacterial anti-infective active agents or antibacterial agents can be broadly classified as either bactericidal (i.e., directly killed) or bacteriostatic (i.e., preventing division). Antibacterial anti-infective active agents can be further reclassified as narrow spectrum antibacterial agents (i.e., affecting only subtype bacterial subtypes, e.g., Gram negative, etc.) or broad spectrum antibacterial agents (i.e., affecting a wide variety).
  • Examples include amikacin, gentamicin, geldanamycin, puromycin, mupirocin, nitrofurantoin, pyrazinamide, quinupristin/dalofopine, rifampicin/isofloxacin Amide or tinidazole.
  • antiviral agent includes any substance that inhibits or eliminates the growth, pathogenesis, and/or survival of a virus. This includes, for example, acyclovir, cidofovir, zidovudine, didanosine (ddI, VIDEX), zalcitabine (ddC, HIVID), stavudine (d4T, ZERIT), Lamy Fuding (3TC, EPIVIR)), azocavir (ZIAGEN), emtricitabine (EMTRIVA), and the like.
  • antifungal agent includes any substance that inhibits or eliminates the growth, pathogenesis and/or survival of fungi. This includes, for example, natamycin, bacteriocin, felofin, nystatin, amphotericin B, kandixin, patchouli, neem seed oil, coconut Oil (Coconut Oil) and the like.
  • antigenic animal agent includes any substance that inhibits or eliminates the growth, morbidity and/or survival of a protozoan organism (eg, a parasite).
  • antiprotozoal agents include antimalarial agents such as quinine, quinidine, and the like.
  • antibacterial, antiviral, antifungal, and antiprotozoal agents are described, for example, in WO 2010/077634 and the like.
  • Anti-infective active agents are also described, for example, in WO 2014/008218, WO 2016/028672 or WO 2015/138920.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes a vector that is a self-replicating nucleic acid structure and a vector that binds to the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Subject/patient sample refers to a collection of cells or fluids obtained from a patient or subject.
  • the source of the tissue or cell sample may be a solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample or a biopsy sample or a puncture sample; 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 naturally not intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • tumor samples include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (thoracic fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulation Plasma protein, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumors sample.
  • package insert is used to refer to instructions commonly included in commercial packages of therapeutic products containing information about indications, usage, dosage, administration, combination therapies, contraindications and/or warnings relating to the use of such therapeutic products. .
  • an antibody or fragment thereof of the invention binds to LAG-3.
  • an antibody or fragment thereof of the invention binds to a mammalian LAG3, such as human LAG-3 or mouse LAG-3.
  • an antibody molecule specifically binds to an epitope on LAG-3 (eg, a linear or conformational epitope).
  • the antibody molecule binds to one or more extracellular Ig-like domains of LAG-3 (eg, a first, second, third or fourth extracellular Ig-like domain of LAG-3).
  • an anti-LAG-3 antibody or fragment thereof of the invention has one or more of the following properties:
  • the anti-LAG-3 antibody of the present invention or a fragment thereof binds LAG-3 (for example, human LAG-3) with high affinity, for example, binds to LAG-3 with the following equilibrium dissociation constant (K D ), said K D is less than about 100 nM, preferably less than or equal to about 50 nM, more preferably less than or equal to about 20 nM, more preferably less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2 nM, most preferably The K D is less than or equal to about 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM.
  • K D equilibrium dissociation constant
  • the anti-LAG-3 antibodies of the invention are in the range of 0.1-20 nM, preferably 0.5-20 nM, more preferably 0.5-10 nM, 0.5-8 nM, 0.5-5 nM, and most preferably 0.5-1 nM, 0.5-0.8. nM, 0.5-0.7nM, 0.6-0.7nM K D binding of LAG-3.
  • LAG-3 is human LAG-3.
  • the LAG-3 is mouse LAG-3.
  • antibody binding affinity is determined using a bio-optical interference assay (eg, Fortebio affinity measurement) assay.
  • the antibody of the present invention or a fragment thereof binds to a cell expressing human LAG-3, for example, at less than or equal to about 3.3 nM, 3 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, EC50 of 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM or 0.5 nM.
  • the binding is determined by flow cytometry (eg, FACS).
  • the human LAG-3 expressing cell is a human LAG-3 expressing 293 cell (eg, HEK293 cell).
  • the antibody of the present invention or a fragment thereof binds to a cell expressing mouse LAG-3, for example, with an EC50 of less than or equal to about 15000 nM, 14000 nM or 13000 nM.
  • an antibody or fragment thereof of the invention binds to a cell expressing mouse LAG-3, eg, with an EC50 of less than or equal to about 50 nM, such as an EC50 of about 40-50 nM, an EC50 of about 40-45 nM, or An EC50 of approximately 42 nM.
  • the binding is determined by flow cytometry (eg, FACS).
  • the cell expressing mouse LAG-3 is a Chinese hamster ovary (CHO) cell expressing mouse LAG-3.
  • the antibody or fragment thereof of the present invention inhibits the activity of LAG-3, for example, with an IC 50 of less than or equal to about 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM or 5 nM, preferably about 1-6 nM, 1- 5nM, 4nM, 4.5nM, 5nM, 5.1nM, 5.2nM, 5.3nM, 5.4nM, 5.5nM, 5.6nM, 5.7nM, 5.8nM, 5.9nM or 6nM the IC 50.
  • the related activity of LAG-3 is the binding of an MHC class II molecule to LAG-3.
  • the antibody or fragment of the invention is less than or equal to about 20nM, 10nM, 9nM, 8nM, 7nM, 6nM 5nM or the IC 50, preferably about 1-6nM, 1-5nM, 4nM, 4.5nM , 5 nM, 5.1 nM, 5.2 nM, 5.3 nM, 5.4 nM, 5.5 nM, 5.6 nM, 5.7 nM, 5.8 nM, 5.9 nM or 6 nM of IC 50 inhibits LAG-3 and MHC class II molecules on cells expressing MHC class II molecules Combination of.
  • the MHC class II molecule is HLA-DR.
  • the cell is a CHO cell.
  • inhibition of the associated activity of LAG-3 by the antibody or fragment thereof of the invention is measured by flow cytometry (e.g., FACS).
  • the antibody or fragment thereof of the invention binds to endogenous LAG-3 on the surface of activated CD4+ and/or CD8+ T cells, for example at less than or equal to about 35 pM, 30 pMnM, 25 pM, 20 pM, 15 pM, 14 pM or 13 pM the EC 50, preferably about 1-20pM, 5-20pM, 5-15pM, 10-15pM, 11-13pM, 10pM, 11pM, 12pM 13pM, or the EC 50.
  • the activated CD4+ T cells are activated human CD4+ T cells.
  • the above binding is determined by flow cytometry (e.g., FACS). In some embodiments, flow cytometry is performed in an Accuri C6 system.
  • the antibody or fragment thereof of the present invention inhibits one or more activities of LAG-3, for example, resulting in one or more of the following: increased antigen-dependent stimulation of CD4+ T lymphocytes; increased proliferation of T cells; activation Increased expression of antigen (eg, CD25); increased expression of cytokines (eg, interferon-gamma (IFN- ⁇ ), interleukin-2 (IL-2) or interleukin-4 (IL-4); chemokines Increased expression (eg, CCL3, CCL4, or CCL5); decreased repression activity of Treg cells; increased T cell homeostasis; increased tumor infiltrating lymphocytes; or reduced immune evasion of cancer cells.
  • cytokines eg, interferon-gamma (IFN- ⁇ ), interleukin-2 (IL-2) or interleukin-4 (IL-4)
  • chemokines Increased expression (eg, CCL3, CCL4, or CCL5)
  • decreased repression activity of Treg cells increased T cell homeost
  • the anti-LAG-3 antibody or fragment thereof of the present invention is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention has one or more of the following characteristics:
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises:
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein
  • VL contains:
  • the VH comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 23, 24 or 25.
  • VL comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28 or 29.
  • the anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 22, 23, 24 or 25, and The three complementarity determining regions LCDR of the light chain variable region of SEQ ID NO: 26, 27, 28 or 29.
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein
  • the VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4 or 17, or HCDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4 or 17; HCDR2 comprises a SEQ ID NO: amino acid sequence of 5, 6, 7 or 18, or consists of the amino acid sequence, or HCDR2 comprises one, two or more than the amino acid sequence selected from SEQ ID NO: 5, 6, 7 or 18.
  • CDR complementarity determining region
  • HCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 8, 9, 10 or 19, or HCDR3 comprises and is selected from the group consisting of SEQ ID NO: an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of 8, 9, 10 or 19;
  • VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 11, 12 or 20, or LCDR1 comprises and An amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11, 12 or 20;
  • LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13 or Consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 13;
  • LCDR3 comprises a selected from the group consisting of SEQ ID NO:
  • the amino acid sequence of 14, 15, 16 or 21 consists of or consists of the amino acid sequence
  • LCDR3 comprises one, two or three compared to the amino acid sequence selected from SEQ ID NO: 14, 15, 16 or 21.
  • the invention provides an anti-LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein
  • the invention provides an anti-LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises a complementarity determining region ( CDR) HCDR1, HCDR2 and HCDR3 and the VL comprises (CDR) LCDR1, LCDR2 and LCDR3, wherein the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprised by the antibody or antigen-binding fragment thereof is as follows (Table A):
  • Table A Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in an antibody or antigen-binding fragment thereof of the present invention
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein
  • amino acid sequence of an amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, said amino acid change does not occur in the CDR region;
  • amino acid sequence of an amino acid change (preferably an amino acid substitution, more preferably an amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR regions.
  • the invention provides an anti-LAG-3 antibody or antigen-binding fragment thereof comprising 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 an antibody or antigen-binding fragment thereof of the invention
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein
  • amino acid change comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 31, 32 or 33
  • Amino acid sequence of amino acid change preferably amino acid substitution, more preferably amino acid conservative substitution
  • the amino acid change does not occur in the CDR region of the heavy chain, and more preferably, the amino acid change does not occur in Heavy chain variable region;
  • amino acid change comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 35, 36 or 37
  • Amino acid sequence of amino acid change preferably amino acid substitution, more preferably amino acid conservative substitution
  • the amino acid change does not occur in the CDR region of the light chain, and more preferably, the amino acid change does not occur in Light chain variable region.
  • the invention provides an anti-LAG-3 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the combination of a heavy chain and a light chain comprised by the antibody or antigen-binding fragment thereof is as follows (Table C):
  • Table C Exemplary combinations of heavy and light chains in an antibody or antigen-binding fragment thereof of the invention
  • the heavy and/or light chain of an anti-LAG-3 antibody or fragment thereof of the invention further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 48).
  • the amino acid changes described herein include substitutions, insertions or deletions of amino acids.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described herein occur in regions outside the CDRs (e.g., in FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • an anti-LAG-3 antibody of the invention comprises a post-translational modification to a light chain variable region, a heavy chain variable region, a light chain or a heavy chain.
  • exemplary post-translational modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • the substitution is a conservative substitution.
  • a conservative substitution is one in which one amino acid is replaced by another amino acid in the same class, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is passed through another neutral amino acid.
  • Replacement An exemplary substitution is shown in Table D below:
  • the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Addition or deletion of a glycosylation site to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.
  • one or more amino acid substitutions can be performed to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site.
  • Such aglycosylation increases the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.
  • Antibodies with altered types of glycosylation can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased aliquots of GlcNac structure. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies.
  • Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell having an altered glycosylation system.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 ( ⁇ (1,6)-fucosyltransferase), thereby allowing expression of antibodies in the Ms704, Ms705, and Ms709 cell lines. It lacks fucose on its sugar.
  • the Ms704, Ms705 and Ms709FUT8-/- cell lines can be created by targeting the disruption of the FUT8 gene in CHO/DG44 cells using two alternative vectors (see U.S. Patent Publication No.
  • EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such cell lines exhibit low fucoal by reducing or eliminating alpha-1,6 bond-associated enzymes Saccharification.
  • EP 1,176,195 also describes cell lines having low or no enzymatic activity of adding fucose to N-acetylglucosamine binding to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662) ).
  • PCT publication WO 03/035835 describes a variant CHO cell line Lec13 cell in which the ability to attach fucose to Asn(297)-linked saccharide is reduced, thereby also resulting in expression in the host cell.
  • Low fucosylation of antibodies see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740.
  • Antibodies with modified glycosylation profiles can also be produced in eggs as set forth in PCT Publication WO 06/089231.
  • an antibody having a modified glycosylation profile can be produced in a plant cell (eg, Lemna).
  • a method of producing antibodies in a plant system is disclosed in U.S. Patent Application Serial No.
  • PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein modified glycosyltransferases (eg, ⁇ (1,4)-N-acetylglucosaminyltransferase III (GnTIII)), thereby Antibodies expressed in this engineered cell line exhibit an increased aliquot of the GlcNac structure, which results in increased ADCC activity of the antibody (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).
  • glycoprotein modified glycosyltransferases eg, ⁇ (1,4)-N-acetylglucosaminyltransferase III (GnTIII)
  • the fucose residue of the antibody can be excised using a fucosidase; for example, the fucosidase alpha-L-fucosidase removes fucosyl residues from the antibody (Tarentino et al. 1975) Biochem. 14: 5516-23).
  • the antibody or fragment of the invention is glycosylated with engineered yeast N-linked glycans or CHO N-linked glycans.
  • the antibody can be PEGylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • PEG polyethylene glycol
  • pegylation is carried out via an acylation reaction or an alkylation reaction using a reactive PEG molecule (or a similar reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy polyethylene glycols or poly Ethylene glycol-maleimide.
  • the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention, see for example EP 0154316 and EP 0401384.
  • one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce an Fc region variant to enhance the effectiveness of, for example, an antibody in treating cancer or a cell proliferative disorder.
  • the anti-LAG3 antibodies and antigen-binding fragments thereof disclosed herein also include antibodies and fragments having a modified (or blocked) Fc region to provide altered effector function. See, for example, U.S. Patent No. 5,624,821, WO2003/086310, WO2005/120571, WO2006/0057702. Such modifications can be used to enhance or inhibit various responses of the immune system, which may have beneficial effects in diagnosis and treatment.
  • Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fc. Modification of the Fc can also alter the half-life of the antibody in the therapeutic antibody, thereby enabling lower frequency administration and thus increased convenience and reduced material usage. See Presta (2005) J. Allergy Clin. Immunol. 116: 731, pp. 734-735.
  • the number of cysteine residues of an antibody can be altered to modify antibody properties.
  • a modification is made to the hinge region of CH1 to alter (e.g., increase or decrease) the number of cysteine residues in the hinge region.
  • This approach is further described in U.S. Patent No. 5,677,425.
  • the number of cysteine residues in the hinge region of CH1 can be altered to, for example, facilitate assembly of the light and heavy chains or increase or decrease the stability of the antibody.
  • the antibodies provided herein can be further modified to contain other non-protein portions known in the art and readily available.
  • Portions suitable for antibody derivatization include, but are not limited to, water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, 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 (such as glycerin), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • the polymer can have any molecular weight and can be branched or unbranched.
  • the number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on factors such as, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions. .
  • the invention encompasses fragments of an anti-LAG-3 antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabody, linear antibody, single chain antibody (eg, scFv); and multispecificity formed by antibody fragments antibody.
  • antibody molecules can include heavy chain (HC) variable domain sequences and light chain (LC) variable domain sequences.
  • the antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody).
  • the antibody molecule comprises two heavy chain variable domain sequences and two light chain variable domain sequences, thereby forming two antigen binding sites, such as Fab, Fab', F(ab')2 , Fc, Fd, Fd', Fv, single chain antibodies (eg, scFv), single domain antibodies, diabody (Dab) (bivalent and bispecific), and chimeric (eg, humanized) antibodies, which may Those antibody molecules are synthesized de novo by modifying intact antibodies or by using recombinant DNA techniques.
  • Antibodies and antibody fragments can be from any antibody class including, but not limited to, IgG, IgA, IgM, IgD, and IgE and from any antibody subclass (eg, IgGl, IgG2, IgG3, and IgG4). Preparation of antibody molecules can be monoclonal or polyclonal.
  • the antibody may also be a human antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, or an antibody produced in vitro.
  • the antibody may have, for example, a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3 or IgG4.
  • the antibody may also have, for example, a light chain selected from kappa or lambda.
  • the antibodies of the invention may also be single domain antibodies.
  • Single domain antibodies can include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack a light chain, single domain antibodies derived from conventional 4-chain antibodies, or engineered antibodies.
  • a single domain antibody can be any antibody of the prior art, or any single domain antibody of the future.
  • Single domain antibodies can be derived from any species including, but not limited to, mice, humans, camels, alpaca, fish, sharks, goats, rabbits, and cattle.
  • a single domain antibody is a naturally occurring single domain antibody, referred to as a heavy chain antibody lacking a light chain.
  • Single domain antibodies are disclosed, for example, in WO 94/04678.
  • Single domain antibodies or Nanobodies can be antibodies produced from Camelidae species such as camels, alpacas, dromedaries, llamas and guanaco. Other species than camels can produce heavy chain antibodies that naturally lack light chains; such single domain antibodies are within the scope of the invention.
  • an anti-LAG-3 antibody of the invention is a humanized antibody.
  • Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J, (2008) Frontiers in Bioscience 13: 1619-1633 ).
  • Almagro & Fransson distinguish between rational approaches and empirical approaches. A rational approach is characterized by the generation of a few engineered antibody variants and assessing their binding or any other property of interest. If the design variant does not produce the expected results, then a new round of design and combined assessment is initiated. Rational approaches include CDR grafting, Resurfacing, Superhumanization, and Human String Content Optimization.
  • an anti-LAG-3 antibody of the invention is a human antibody.
  • Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol 20: 450-459 (2008).
  • a human monoclonal antibody can be produced using a transgenic mouse carrying a human immunoglobulin gene rather than a mouse system (see, for example, Wood et al., International Application WO 91/00906; Kucherlapati et al., PCT Publication WO 91/ 10741; Lonberg et al., International Application WO 92/03918; Kay et al., International Application 92/03917; Lonberg, N. et al., 1994 Nature 368: 856-859; Green, LL et al., 1994 Nature Genet. 13-21; Morrison, SL et al, 1994 Proc. Natl. Acad. Sci.
  • an anti-LAG-3 antibody of the invention is a non-human antibody, such as a rodent (mouse or rat) antibody, a goat antibody, a primate (eg, a monkey) antibody, a camelid antibody.
  • the non-human antibody is a rodent (mouse or rat) antibody. Methods of producing rodent antibodies are known in the art.
  • An antibody of the invention can be isolated by screening a combinatorial library for antibodies having the desired activity.
  • a variety of methods are known in the art for generating phage display libraries and screening for antibodies having the desired binding characteristics in these libraries. These methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further, for example, in McCafferty et al, Nature 348:552- 554; Clackso et al, Nature 352:624-628 (1991); Marks et al, J. Mol. Biol.
  • the antibody molecule is a monospecific antibody molecule and binds to a single epitope.
  • a monospecific antibody molecule has multiple immunoglobulin variable domain sequences that each bind to the same epitope.
  • the antibody molecule is a multispecific antibody molecule, eg, comprising a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable structure of the plurality of immunoglobulin variable domain sequences The domain sequence has binding specificity for the first epitope and the second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for the second epitope.
  • the first and second epitopes are on the same antigen (eg, the same protein (or subunit of a multimeric protein)).
  • the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap.
  • the first and second epitopes are on different antigens (eg, different proteins (or different subunits of a multimeric protein)).
  • the multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain.
  • the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule or a tetraspecific antibody molecule.
  • the multispecific antibody molecule is a bispecific antibody molecule.
  • Bispecific antibodies are specific for no more than two antigens.
  • the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for the first epitope and a second immunoglobulin variable domain sequence having binding specificity for the second epitope.
  • the first and second epitopes are on the same antigen (eg, the same protein (or subunit of a multimeric protein)).
  • the first and second epitopes overlap.
  • the first and second epitopes do not overlap.
  • the first and second epitopes are on different antigens (eg, different proteins (or different subunits of a multimeric protein)).
  • the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain having binding specificity for a second epitope Variable domain sequences and light chain variable domain sequences.
  • the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope.
  • the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope.
  • the bispecific antibody molecule comprises a scFv having binding specificity for a first epitope or a fragment thereof and a scFv having binding specificity for a second epitope or a fragment thereof.
  • the first epitope is on LAG-3 and the second epitope is at PD-1, TIM-3, CEACAM (eg, CEACAM-1 and/or CEACAM-5), PD-L1 or PD- On L2.
  • the second epitope is located on PD-1.
  • an antibody of the invention can be a chimeric antibody (eg, a human constant domain/mouse variable domain).
  • a "chimeric antibody” as used herein is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first antibody and the second antibody are from different species (US Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).
  • variable domain is obtained from an antibody ("parent antibody”) from an experimental animal (such as a rodent), and the constant domain sequence is obtained from a human antibody such that the resulting chimeric antibody is in a human subject The likelihood of causing an adverse immune response is lower than that of a parent (eg, mouse) antibody.
  • the invention also encompasses anti-LAG-3 monoclonal antibodies ("immunoconjugates") conjugated to other substances, such as therapeutic modules or labels, such as cytotoxic agents or immunomodulators.
  • Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for the formation of immunoconjugates are known in the art, see for example WO05/103081 or WO2015/138920 or CN 107001470A.
  • cytotoxic agents include, but are not limited to, radioisotopes (eg, iodine (131I or 125I), hydrazine (90Y), hydrazine (177Lu), hydrazine (225Ac), hydrazine, hydrazine (211At), hydrazine (186Re), hydrazine (212Bi or 213Bi), indium (111In), antimony (99mTc), phosphorus (32P), antimony (188Rh), sulfur (35S), carbon (14C), antimony (3H), chromium (51Cr), chlorine (36Cl) , cobalt (57Co or 58Co), iron (59Fe), selenium (75Se) or gallium (67Ga).
  • radioisotopes eg, iodine (131I or 125I
  • hydrazine (90Y) hydrazine (177Lu), hydrazine (225Ac), hydr
  • cytotoxic agents also include chemotherapeutic agents or other therapeutic drugs, for example, taxol, cytochalasin B, short Bacitracin D, ethidium bromide, imidin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy Anthraquinone dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, Puromycin, maytansinoid, for example, maytansinol (see U.S.
  • Patent No. 5,208,020 CC-1065 (see U.S. Patent Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof.
  • Anti-generation Xie eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine
  • alkylating agent eg, nitrogen mustard, thiobenzoic acid nitrogen
  • Mustard eg, nitrogen mustard, thiobenzoic acid nitrogen
  • CC-1065 melphalan
  • cyclophosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C and cis -dichlorodiamine platinum (II) (DDP) cisplatin)
  • anthracyclines eg, daunorubicin (formerly daunorubicin)
  • Nucleic acid of the present invention and host cell containing the same
  • the invention provides a nucleic acid encoding any of the above anti-LAG-3 antibodies or fragments thereof.
  • the nucleic acid may encode an amino acid sequence comprising a light chain variable region and/or a heavy chain variable region of an antibody, or an amino acid sequence comprising a light chain and/or a heavy chain of an antibody.
  • an exemplary nucleic acid of the invention comprises at least 85%, 90%, 91%, 92%, 93 of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42, 43, 44 or 45. a nucleic acid sequence of %, 94%, 95%, 96%, 97%, 98% or 99% identity, or comprising a selected from the group consisting of SEQ ID NOs: 38, 39, 40, 41, 42, 43, 44, 45 Nucleic acid sequence.
  • a nucleic acid of the invention comprises a code selected from any one of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37.
  • nucleic acid having an amino acid sequence or encoding and selected from any one of SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37
  • the invention also encompasses a nucleic acid that hybridizes under stringent conditions with a nucleic acid, or a nucleic acid having one or more substitutions (eg, conservative substitutions), deletions, or insertions with a nucleic acid comprising: SEQ ID NO: 38,
  • the nucleic acid sequences of 39, 40, 41, 42, 43, 44, 45 have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% a nucleic acid of an identical nucleic acid sequence; or a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 45; comprising a coding selected from the group consisting of SEQ ID NO: 22, 23, a nucleic acid of a nucleic acid sequence of the amino acid sequence shown in any one of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37; or
  • 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
  • a variety of carrier systems can be used.
  • one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus.
  • Another type of vector utilizes RNA elements derived from RNA viruses such as Semliki forest virus, oriental equine encephalitis virus, and flavivirus.
  • the expression vector of the invention is a pTT5 expression vector.
  • cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of the host cells that have been transfected.
  • the marker can, for example, provide prototrophic, biocidal (eg, antibiotic) or heavy metal (eg, copper) resistance, etc., to the auxotrophic host.
  • the selectable marker gene can be ligated directly to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional components may also be required in order to optimally synthesize mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques.
  • protoplast fusion the cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be based on the methods described in the present specification and the prior art, depending on the particular expression vector used and Mammalian host cell changes or optimization.
  • a host cell comprising a nucleic acid encoding an antibody molecule described herein or a vector described herein.
  • Suitable host cells for cloning or expressing a nucleic acid or vector encoding the antibody include prokaryotic or eukaryotic cells as described herein.
  • antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required.
  • the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.
  • the host cell is an E. coli cell.
  • the host cell is eukaryotic.
  • the host cell is selected from the group consisting of a yeast cell, a mammalian cell (eg, a human cell), an insect cell, a plant cell, or other cell suitable for use in the preparation of an antibody or antigen-binding fragment thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including glycosylation pathways that have been "humanized” resulting in the production of antibodies having partial or complete human glycosylation patterns. Fungal and yeast strains. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al, Nat. Biotech.
  • Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines engineered to be suitable for suspension growth can be used.
  • useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293 HEK or 293 cells such as, for example, Graham et al, J. Gen Virol. 36:59 (1977) ) as described in ).
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc.
  • Suitable mammalian host cell lines include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, MDCKII cells, PerC6 cell lines (eg, PERC6 cells from Crucell), oocytes, and from transgenes.
  • Animal cells such as mammary epithelial cells. Suitable insect cells include, but are not limited to, Sf9 cells.
  • the anti-LAG3 antibodies disclosed herein can be recombinantly produced. There are several methods known in the art for producing recombinant antibodies. One example of a method for recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567.
  • a method of making an anti-LAG-3 antibody comprising culturing a host cell comprising a nucleic acid encoding the antibody, such as provided above, under conditions suitable for expression of the antibody, and The antibody is optionally recovered from the host cell (or host cell culture medium).
  • a nucleic acid encoding an antibody such as an antibody described above
  • Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).
  • the host cell comprises a vector comprising a nucleic acid encoding the amino acid sequence of the VL of the antibody and a nucleic acid encoding the amino acid sequence of the VH of the antibody. In one embodiment, the host cell comprises a first vector comprising a nucleic acid encoding an amino acid sequence of the VL of the antibody and a second vector comprising a nucleic acid encoding the amino acid sequence of the VH of the antibody.
  • the anti-LAG-3 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity 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 known methods such as ELISA, Western blot, flow cytometry, magnetic beads of antibody molecules, and the like.
  • LAG-3 binding can be determined using methods known in the art, and exemplary methods are disclosed herein.
  • biophotonic interferometry eg, Fortebio affinity measurement
  • MSD assay flow cytometry
  • competition assays can be used to identify antibodies that compete with any of the anti-LAG-3 antibodies disclosed herein for binding to LAG-3.
  • a competitive antibody binds to the same epitope (eg, a linear or conformational epitope) as the epitope to which the anti-LAG-3 antibody disclosed herein binds.
  • epitope eg, a linear or conformational epitope
  • a detailed exemplary method for locating epitopes bound by antibodies is found in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
  • the invention also provides assays for identifying an anti-LAG-3 antibody having one or more of the properties described above. Antibodies having such biological activity in vivo and/or in vitro are also provided.
  • an antibody of the invention is tested for one or more of the properties described above.
  • LAG-3 is expressed on activated CD4+ and CD8+ T cells, Treg cells, natural killer (NK) cells, and plasmacytoid dendritic cells (DC).
  • LAG-3 is expressed in tumor infiltrating lymphocytes (eg, infiltrating lymphocytes in head and neck squamous cell carcinoma (HNSCC)).
  • LAG-3 is expressed on highly repressible inducible and natural Tregs.
  • Such cells also include cell lines expressing LAG-3 and cell lines that are not normally expressing LAG-3 but have been transfected with a nucleic acid encoding LAG-3.
  • any of the above assays can be performed by replacing or supplementing the anti-LAG-3 antibody with an immunoconjugate of the invention.
  • any of the above assays can be performed using an anti-LAG-3 antibody and other therapeutic agents.
  • compositions comprising an anti-LAG-3 antibody or fragment thereof or an immunoconjugate thereof, and a composition comprising a nucleic acid encoding an anti-LAG-3 antibody or fragment thereof.
  • the composition comprises one or more antibodies that bind to LAG-3, or a fragment thereof, or an immunoconjugate thereof, or one or more antibodies that encode one or more LAG-3 binding antibodies or Fragment of nucleic acid.
  • suitable pharmaceutical excipients such as pharmaceutically acceptable carriers, excipients and the like known in the art, including buffering agents.
  • Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc.
  • excipients see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R. C. Rowe, P. J. Seskey and S. C. Owen, Pharmaceutical Press, London, Chicago.
  • compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • these compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • the anti-LAG-3 antibody of the present invention having the desired purity can be mixed with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980)).
  • Pharmaceutical formulations comprising the anti-LAG-3 antibodies described herein are prepared, preferably in the form of a lyophilized formulation or an aqueous solution.
  • Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958.
  • Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter including a histidine-acetate buffer.
  • compositions or formulations of the present invention may also comprise more than one active ingredient which is required for the particular indication being treated, preferably those having complementary activities which do not adversely affect each other.
  • active ingredients such as chemotherapeutic agents and/or PD-1 axis binding antagonists (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies).
  • the active ingredient is suitably present in combination in an amount effective for the intended use.
  • the active ingredient can be any substance known in the art that can be combined with an anti-LAG-3 antibody, including chemotherapeutic agents, antibodies, and other therapeutic agents. Examples of such active ingredients are described, for example, in WO2016/028672, WO2015/042246, WO2015/138920, and the like.
  • the anti-PD-1 antibody or anti-PD-L1 antibody or anti-PD-L2 antibody is an anti-human PD-1 antibody or an anti-human PD-L1 antibody or an anti-human PD-L2 antibody, for example, humanized Anti-human PD-1 antibody or anti-human PD-L1 antibody or anti-human PD-L2 antibody.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.
  • the invention relates to a method of modulating an immune response in a subject.
  • the method comprises administering to the subject an effective amount of an antibody molecule (eg, an anti-LAG-3 antibody) or a pharmaceutical composition or immunoconjugate disclosed herein to modulate an immune response in the subject.
  • an antibody molecule eg, a therapeutically effective amount of an anti-LAG-3 antibody molecule
  • a pharmaceutical composition or immunoconjugate disclosed herein restores, enhances, stimulates, or increases an immune response in a subject.
  • the invention in another aspect, relates to a method of preventing or treating a tumor (eg, cancer) in a subject, the method comprising administering to the subject an effective amount of an antibody molecule disclosed herein (eg, anti-LAG-3 Antibody) or a pharmaceutical composition or immunoconjugate.
  • a tumor eg, cancer
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer or the like.
  • the invention relates to a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an antibody molecule disclosed herein (eg, an anti-LAG-3 antibody) Or a pharmaceutical composition or immunoconjugate.
  • an antibody molecule disclosed herein eg, an anti-LAG-3 antibody
  • a pharmaceutical composition or immunoconjugate e.g., an immunoconjugate.
  • the infectious disease is a chronic infection.
  • the invention in another aspect, relates to a method of eliciting antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of an antibody molecule disclosed herein (eg, anti-LAG -3 antibody) or a pharmaceutical composition or immunoconjugate.
  • an antibody molecule disclosed herein eg, anti-LAG -3 antibody
  • a pharmaceutical composition or immunoconjugate e.g., a pharmaceutical composition or immunoconjugate.
  • the subject can be a mammal, for example, a primate, preferably a higher primate, for example, a human (eg, a patient having the disease described herein or at risk of having the disease described herein).
  • the subject is in need of an enhanced immune response.
  • an anti-LAG-3 antibody molecule described herein restores, enhances or stimulates an antigen-specific T cell response in a subject, eg, an interleukin-2 (IL-2) in an antigen-specific T cell response Or interferon-gamma (IFN- ⁇ ) production.
  • the immune response is an anti-tumor response.
  • the subject has or is at risk of having a disease described herein (eg, a tumor or infectious disease as described herein).
  • the subject is immunocompromised or at risk of immunocompromised.
  • the subject has received or has received chemotherapy treatment and/or radiation therapy.
  • the subject is immunocompromised due to infection or has a risk of being immunocompromised by the infection.
  • the tumors described herein include, but are not limited to, solid tumors, hematological cancers (eg, leukemia, lymphoma, myeloma), and metastatic lesions thereof.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, for example, sarcomas and cancers (eg, adenocarcinomas) of multiple organ systems, such as invading the lungs, breast, lymph, gastrointestinal or colorectal, genital and genitourinary tract (eg, renal cells) , bladder cells, bladder cells), pharynx, CNS (eg, brain cells, nerve cells, or glial cells), skin (eg, melanoma), head and neck (eg, head and neck squamous cell carcinoma (HNCC) )) and those of the pancreas.
  • sarcomas and cancers eg, adenocarcinomas
  • multiple organ systems such as invading the lungs, breast, lymph, gastrointestinal or colorectal, genital and genitourinary tract (eg, renal cells) , bladder cells, bladder cells), pharynx, CNS (eg, brain cells, nerve cells, or glial cells),
  • melanoma colon cancer, gastric cancer, rectal cancer, renal cell carcinoma, breast cancer (eg, breast cancer that does not express one, two or all of the estrogen receptor, progesterone receptor, or Her2/neu, eg, , triple negative breast cancer), liver cancer, lung cancer (eg, non-small cell lung cancer (NSCLC) (eg, NSCLC with squamous and/or non-squamous structure) or small cell liver cancer), prostate cancer, head or neck Cancer (eg, HPV + squamous cell carcinoma), small bowel cancer, and esophageal cancer.
  • NSCLC non-small cell lung cancer
  • head or neck Cancer eg, HPV + squamous cell carcinoma
  • small bowel cancer small esophageal cancer.
  • hematological cancers include, but are not limited to, leukemia (eg, myeloid leukemia, lymphoid leukemia or chronic lymphocytic leukemia (CLL)), lymphoma (eg, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma) (NHL), diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma or mantle cell lymphoma (MCL), and myeloma, for example, multiple myeloma.
  • leukemia eg, myeloid leukemia, lymphoid leukemia or chronic lymphocytic leukemia (CLL)
  • lymphoma eg, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma) (NHL), diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma or mantle cell lymphoma (MCL), and my
  • the cancer is selected from the group consisting of colorectal cancer (eg, CRC), melanoma, eg, advanced melanoma (eg, stage II-IV melanoma) or HLA-A2 positive-melanoma; pancreas Cancer, for example, advanced pancreatic cancer; breast cancer, for example, metastatic breast cancer or triple-negative breast cancer; head and neck cancer (for example, HNSCC); esophageal cancer; renal cell carcinoma (RCC), for example, renal clear cell carcinoma (ccRCC) Or metastatic renal cell carcinoma (MRCC); lung cancer (eg, NSCLC); cervical cancer; bladder cancer; or hematological malignancies, eg, leukemia (eg, lymphocytic leukemia) or lymphoma (eg, Hodgkin's lymphoma) (HL), non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymph
  • CRC
  • compositions disclosed herein are useful for treating metastatic lesions associated with the aforementioned cancers.
  • the cancer is a cancer that expresses LAG-3, particularly a metastatic cancer. In some embodiments, the cancer is a cancer that expresses PD-L1. In some embodiments, the cancer is a cancer that expresses LAG-3 as well as PD-L1.
  • the cancer described herein is colon cancer and metastatic cancer thereof.
  • the infection is acute or chronic.
  • the chronic infection is a persistent, latent infection or a slow infection.
  • the chronic infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi, and protozoa.
  • the infectious disease is caused by a viral infection.
  • pathogenic viruses include (types A, B, and C) hepatitis viruses, (types A, B, and C) influenza viruses, HIV, herpes viruses (eg, VZV, HSV-1, HAV-6) , HSV-II, CMV, Epstein Barr virus +), adenovirus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, Rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma, soft prion, poliovirus, rabies virus, JC virus and plague encephalitis virus.
  • viruses include (types A, B, and C) hepatitis viruses, (types A, B, and C) influenza viruses, HIV, herpes viruses (eg, VZV, HSV-1, HAV-6) , HSV
  • the infection is a bacterial infection.
  • pathogenic bacteria causing infection include syphilis, chlamydia, rickettsial, mycobacteria, staphylococcus, streptococcus, pneumococcus, meningococcus, and gonococcus (conococci), Klebsiella, proteobacteria, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, tetanus, Botox, Anthrax, Yersinia, Leptospira and Lyme.
  • the infection is a fungal infection
  • pathogenic fungi include Candida albicans, Candida krusei, Candida glabrata ), Candida tropicalis, Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (Mucorales) Mucor, bsidia, rhizophus, Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, coarse ball Coccidioides immitis and Histoplasma capsulatum.
  • the infection is a protozoan, such as a parasitic infection
  • parasites include Entamoeba histolytica, Balantidium coli, and F. striata. Naegleria fowleri), Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, and other days Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma Gondi) and the Japanese nematode (Nippostrongylus brasiliensis).
  • the infectious disease is hepatitis (eg, hepatitis B infection).
  • Anti-LAG-3 antibody molecules (alone or in combination with a PD-1 axis binding antagonist, such as anti-PD-1 or anti-PD-L1) can be combined with conventional treatments for hepatitis B infection for therapeutic advantages.
  • the anti-LAG-3 antibody molecule is administered in combination with a hepatitis B antigen (eg, Engerix B) or a vaccine, and optionally in combination with an aluminum-containing adjuvant.
  • the infectious disease is influenza.
  • the anti-LAG-3 antibody molecule is administered in combination with an influenza antigen or vaccine.
  • a disease suitable for prevention or treatment with an anti-LAG-3 antibody or a fragment thereof of the present invention can be further referred to WO2015/138920, WO2016/028672, WO2015/042246 and the like.
  • the invention provides the use of an anti-LAG-3 antibody or fragment thereof or immunoconjugate thereof for the manufacture or preparation of a medicament for the treatment of a related disease or condition as mentioned above.
  • an antibody or antibody fragment or immunoconjugate of the invention delays the onset of a condition and/or a condition associated with the condition.
  • the prophylactic or therapeutic methods described herein further comprise administering to the subject or individual a combination of an antibody molecule (eg, an anti-LAG-3 antibody) or a pharmaceutical composition or immunoconjugate disclosed herein, And a PD-1 axis binding antagonist or a drug or immunoconjugate comprising the PD-1 axis binding antagonist.
  • the PD-1 axis binding antagonist comprises, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody.
  • the PD-1 axis binding antagonist includes, but is not limited to, a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.
  • Alternative names for "PD-1” include CD279 and SLEB2.
  • Alternative names for "PD-L1” include B7-H1, B7-4, CD274, and B7-H.
  • Alternative names for "PD-L2” include B7-DC, Btdc, and CD273.
  • PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits PD-1 binding to its ligand binding partner.
  • the PD-1 ligand binding partner is PD-L1 and/or PD-L2.
  • the PD-L1 binding antagonist is a molecule that inhibits PD-L1 binding to its binding partner.
  • the PD-L1 binding partner is PD-1 and/or B7.1.
  • the PD-L2 binding antagonist is a molecule that inhibits PD-L2 binding to its binding partner.
  • the PD-L2 binding partner is PD-1.
  • the antagonist can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA) and CT-011 (Pidilizumab).
  • the PD-1 binding antagonist is an immunoadhesin (eg, comprising extracellular or PD-1 fused to a constant region (eg, an Fc region of an immunoglobulin sequence), PD-L1 or PD-L2 Binding part of the immunoadhesin).
  • the PD-1 binding antagonist is AMP-224.
  • the PD-L1 binding antagonist is an anti-PD-L1 antibody.
  • the anti-PD-L1 binding antagonist is selected from the group consisting of YW243.55.S70, MPDL3280A, MEDI4736, and MDX-1105.
  • MDX-1105 also known as BMS-936559
  • BMS-936559 is an anti-PD-L1 antibody described in WO2007/005874.
  • the antibody YW243.55.S70 (the heavy and light chain variable region sequences are shown in SEQ ID No. 20 and 21, respectively) is the anti-PD-L1 described in WO 2010/077634 A1.
  • MDX-1106 also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab
  • Merck 3475 also known as MK-3475, SCH-900475 or pembrolizumab
  • MK-3475 SCH-900475 or pembrolizumab
  • CT-011 also known as hBAT, hBAT-1 or pidilizumab
  • AMP-224 also known as B7-DCIg
  • PD-L2-Fc fusion soluble receptor as described in WO2010/027827 and WO2011/066342.
  • the anti-PD-1 antibody is MDX-1106.
  • Alternative names for "MDX-1106” include MDX-1106-04, ONO-4538, BMS-936558 or nivolumab.
  • the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).
  • the anti-PD-1 antibody is "Antibody C" or Antibody D as described herein.
  • an anti-LAG-3 antibody of the invention can also be used in combination with an anti-PD-L1 antibody.
  • an anti-PD-L1 antibody of the invention is an anti-human PD-L1 antibody.
  • an anti-PD-L1 antibody of the invention is an antibody in IgGl form or an antibody in IgG2 form or an antibody in IgG4 form.
  • the anti-PD-L1 antibody is a monoclonal antibody.
  • the anti-PD-L1 antibody is humanized.
  • the anti-PD-L1 antibody is a chimeric antibody.
  • at least a portion of the framework sequence of the anti-PD-L1 antibody is a human consensus framework sequence.
  • an anti-PD-L1 antibody of the invention further encompasses an antibody fragment thereof, preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv) or ( Fab') 2 , single domain antibody, double antibody (dAb) or linear antibody.
  • an antibody fragment thereof preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv) or ( Fab') 2 , single domain antibody, double antibody (dAb) or linear antibody.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises
  • an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein
  • VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 51; HCDR2 comprises an amino acid selected from the group consisting of SEQ ID NO: a sequence consisting of or consisting of the amino acid sequence; HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53;
  • CDR complementarity determining region
  • VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 54; LCDR2 comprises the amino acid sequence of SEQ ID NO: 55 or Composition of the amino acid sequence; LCDR3 comprises or consists of the amino acid sequence selected from SEQ ID NO:56.
  • CDR complementarity determining region
  • an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein
  • an amino acid comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 57 a sequence consisting of or consisting of; or
  • amino acid change comprising an amino acid change having 1 or more (preferably no more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid sequences selected from SEQ ID NO: 57 (preferably) An amino acid sequence of an amino acid substitution, more preferably a conservative substitution of an amino acid, preferably, the amino acid change does not occur in the CDR region;
  • amino acid change having one or more (preferably no more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid sequences selected from SEQ ID NO: 58 (preferably)
  • amino acid sequence of an amino acid substitution more preferably a conservative substitution of an amino acid, preferably does not occur in the CDR regions.
  • an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein
  • amino acids compared to the amino acid sequence selected from SEQ ID NO: 59
  • Amino acid sequence which changes preferably amino acid substitution, more preferably amino acid conservative substitution, preferably, said amino acid change does not occur in the CDR region of the heavy chain, more preferably, said amino acid change does not occur in the heavy chain variable region ;
  • amino acids compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 60
  • Amino acid sequence which changes preferably amino acid substitution, more preferably amino acid conservative substitution
  • said amino acid change does not occur in the CDR region of the light chain, more preferably, said amino acid change does not occur in the light chain variable region .
  • the modifications of the invention against an anti-LAG-3 antibody are equally applicable to an anti-PD-L1 antibody.
  • the anti-LAG-3 antibody or fragment thereof can also be administered in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents.
  • the treatment modality includes surgery (eg, tumor resection); radiation therapy (eg, external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the illumination region is designed), local illumination (eg, pointing to a pre-selected target) Or irradiation of organs) or focused illumination).
  • the focused illumination can be selected from stereotactic radiosurgery, segmented stereotactic radiosurgery, and intensity modulated radiation therapy.
  • the focused illumination may have a radiation source selected from the group consisting of a particle beam (proton), a cobalt-60 (photon), and a linear accelerator (X-ray), for example as described in WO 2012/177624.
  • Radiation therapy can be administered by one or a combination of several methods including, but not limited to, external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, whole body radiation therapy, radiotherapy, and permanent or transient Interstitial brachytherapy.
  • brachytherapy refers to radiation therapy delivered by spatially constrained radioactive material that is inserted into the body at or near the site of a tumor or other proliferative tissue disease.
  • the term is intended to be, without limitation, including exposure to radioisotopes (eg, At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and Lu's radioisotope).
  • Suitable sources of radiation include solids and liquids.
  • the source of radiation may be a radionuclide such as I-125, I-131, Yb-169, Ir-192 as a solid state source, I-125 as a solid source or emitting photons, beta particles, gamma radiation Or other radionuclides of other therapeutic rays.
  • the radioactive material may also be a fluid made from any radionuclide solution, for example, an I-125 or I-131 solution, or a suitable fluid containing small particles of a solid radionuclide (such as Au-198, Y-90) may be used.
  • the slurry produces a radioactive fluid.
  • the radionuclide can be contained in a gel or radioactive microspheres.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, or an immunomodulatory agent (eg, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule).
  • a chemotherapeutic agent e.g., a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, or an immunomodulatory agent (eg, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule).
  • Exemplary cytotoxic agents include anti-microtubule drugs, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinblastine alkaloids, intercalating agents, capable of interfering with signal transduction Routes of active agents, pro-apoptotic active agents, proteasome inhibitors, and irradiation (eg, local or systemic exposure (eg, gamma radiation).
  • irradiation eg, local or systemic exposure (eg, gamma radiation).
  • Exemplary other antibodies include, but are not limited to, immunological checkpoint inhibitors (eg, anti-CTLA-4, anti-TIM-3, anti-CEACAM); antibodies that stimulate immune cells (eg, agonistic GITR antibodies or CD137 antibodies); anti-cancer Antibodies (eg, rituximab ( or ),Trastuzumab Tosimo monoclonal antibody Imomozumab Alemizumab Epalizumab Bevacizumab Erlotinib Cetuximab and many more.
  • immunological checkpoint inhibitors eg, anti-CTLA-4, anti-TIM-3, anti-CEACAM
  • antibodies that stimulate immune cells eg, agonistic GITR antibodies or CD137 antibodies
  • anti-cancer Antibodies eg, rituximab ( or ),Trastuzumab Tosimo monoclonal antibody Imomozumab Alemizumab Epalizumab Bevacizumab Erlotinib Cetuxim
  • chemotherapeutic agents include, but are not limited to, anastrozole Bicalutamide Bleomycin sulfate Bai Xiaoan Baixiaoan injection Capecitabine N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin Carmustine Chlorambucil Cisplatin Clarity Cyclophosphamide or ), cytarabine, cytosine arabinoside Cytarabine liposome injection dacarbazine Dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride Citric acid citrinomycin liposome injection Dexamethasone, docetaxel Doxorubicin hydrochloride Etoposide Fludarabine phosphate 5-fluorouracil Flutamide Tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea Idabis Ifosfamide Irinotecan L-as
  • Exemplary vaccines include, but are not limited to, cancer vaccines.
  • the vaccine can be a DNA based vaccine, an RNA based vaccine or a virus transduced vaccine.
  • Cancer vaccines can be prophylactic or therapeutic.
  • the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine.
  • the peptide cancer vaccine is a multivalent long peptide, a multiple peptide, a peptide mixture, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, eg, Yamada et al, Cancer Sci, 104: 14-21) , 2013).
  • anti-infective active agents include, but are not limited to, antiviral, antifungal, antiprotozoal, antibacterial agents such as the nucleoside analog zidovudine (AST), ganciclovir, foscarnet or cidovir As described above.
  • Immunomodulators include immunological checkpoint molecular inhibitors and costimulatory molecule activators.
  • the inhibitor of the immunological checkpoint molecule is CTLA-4, TIM-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (eg, CEACAM-1 and/or CEACAM-5) and / Or an inhibitor of TGFR. Inhibition of the molecule can be carried out at the DNA, RNA or protein level.
  • an inhibitory nucleic acid eg, dsRNA, siRNA, or shRNA
  • the inhibitor of the immunological checkpoint molecule is a polypeptide that binds to an immunological checkpoint molecule, eg, a soluble ligand or antibody or antibody fragment.
  • Exemplary TIM-3 antibody molecules include, but are not limited to, MBG220, MBG227, and MBG219.
  • the immunomodulatory agent is a soluble ligand (eg, CTLA-4-Ig or TIM-3-Ig) or an antibody or antibody fragment of CTLA4.
  • an anti-LAG-3 antibody molecule (alone or in combination with a PD-1 axis binding antagonist) can be administered in combination with a CTLA-4 antibody (eg, ipilimumab).
  • CTLA-4 antibody eg, ipilimumab
  • exemplary anti-CTLA4 antibodies include tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675, 206); and ipilimumab (CTLA-4 antibody, also It is called MDX-010, CAS No. 477202-00-9).
  • the immunomodulatory agent is an activator or agonist of a costimulatory molecule.
  • the agonist of the costimulatory molecule is selected from an agonist of the following molecule (eg, an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion): OX40, CD2, CD27, CDS, ICAM-1, LFA -1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.
  • an anti-LAG-3 antibody or fragment thereof, alone or in combination with a PD-1 axis binding antagonist is associated with a costimulatory molecule comprising a co-stimulatory domain of CD28, CD27, ICOS, and GITR (eg, Used in combination with signal-related agonists.
  • GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), for example, GITR fusion proteins described in U.S. Patent No. 6,111,090, European Patent No. 090,505 B1, U.S. Patent No. 8,586,023 PCT Publication Nos. WO 2010/003118 and 2011/090754.
  • An exemplary anti-GITR antibody is TRX518.
  • an anti-LAG-3 antibody or fragment thereof, alone or in combination with a PD-1 axis binding antagonist, can also be inhibited with a tyrosine kinase inhibitor (eg, receptor tyrosine kinase (RTK)) Agent) used in combination.
  • a tyrosine kinase inhibitor eg, receptor tyrosine kinase (RTK)
  • RTK receptor tyrosine kinase
  • Exemplary tyrosine kinase inhibitors include, but are not limited to, epidermal growth factor (EGF) pathway inhibitors (eg, epidermal growth factor receptor (EGFR) inhibitors), vascular endothelial growth factor (VEGF) pathway inhibitors (eg, blood vessels) Endothelial growth factor receptor (VEGFR) inhibitors (eg, VEGFR-1 inhibitors, VEGFR-2 inhibitors, VEGFR-3 inhibitors), platelet-derived growth factor (PDGF) pathway inhibitors (eg, platelet-derived growth factor receptors) A body (PDGFR) inhibitor (eg, a PDGFR- ⁇ inhibitor), a RAF-1 inhibitor, a KIT inhibitor, and a RET inhibitor.
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • VEGFR-1 inhibitors eg, VEGFR-2 inhibitors, VEGFR-3 inhibitors
  • PDGF platelet-derived growth factor pathway inhibitors
  • a body (PDGFR) inhibitor
  • an anti-LAG-3 antibody or fragment thereof can also be associated with a PI3K inhibitor, an mTOR inhibitor, a BRAF inhibitor, a MEK inhibitor, and/or a JAK2 inhibitor And so on.
  • administration of an anti-LAG-3 antibody or fragment thereof of the invention, alone or in combination with a PD-1 axis binding antagonist is combined with administration of a tumor antigen.
  • the antigen may for example be a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen.
  • the tumor antigen comprises a protein.
  • the tumor antigen comprises a nucleic acid.
  • the tumor antigen is a tumor cell.
  • an anti-LAG-3 antibody or fragment thereof of the invention can be associated with a T cell comprising a adoptively transferred chimeric antigen receptor (CAR) (eg, cytotoxicity) Treatment with T cells or CTL) is administered in combination.
  • CAR chimeric antigen receptor
  • an anti-LAG-3 antibody or fragment thereof of the invention can be administered in combination with an antineoplastic agent.
  • an anti-LAG-3 antibody or fragment thereof of the invention can be administered in combination with an oncolytic virus.
  • an anti-LAG-3 antibody or fragment thereof of the invention can be administered in combination with a cytokine.
  • the cytokine can be administered as a fusion molecule with an anti-LAG-3 antibody molecule, or as a separate composition.
  • the anti-LAG-3 antibody is administered in combination with one, two, three or more cytokines (eg, as a fusion molecule or as a separate composition).
  • the cytokine is an interleukin selected from one, two, three or more of IL-1, IL-2, IL-12, IL-12, IL-15 or IL-21 ( IL).
  • an antibody of the invention, or a fragment thereof, alone or in combination with a PD-1 axis binding antagonist can be combined with conventional cancer therapies in the art including, but not limited to: (i) radiation therapy (for example, radiation therapy, X-ray therapy, irradiation) or using ionizing radiation to kill cancer cells and shrink tumors.
  • radiation therapy For example, radiation therapy, X-ray therapy, irradiation
  • ionizing radiation to kill cancer cells and shrink tumors.
  • Radiation therapy can be administered by external beam radiation therapy (EBRT) or by internal brachytherapy; (ii) chemotherapy, or the application of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or specific effects
  • An agent that deregulates cancer cell proteins eg, tyrosine kinase inhibitor imatinib, gefitinib; monoclonal antibody, photodynamic therapy
  • immunotherapy or enhancement Host immune response (eg, vaccine)
  • hormonal therapy or blocking hormones (eg, when the tumor is hormone sensitive),
  • angiogenesis inhibitors, or blocking angiogenesis and growth and
  • an antibody of the invention, or a fragment thereof, alone or in combination with a PD-1 axis binding antagonist can be combined with conventional methods of enhancing host immune function, including but not limited to: (i) APC enhancement , for example, (a) injecting a tumor with a DNA encoding a heterologous MHC alloantigen, or (b) a gene that increases the likelihood of recognition by an immunizing antigen (eg, immunostimulatory cytokine, GM-CSF, costimulatory molecule B7.1, B7.2) Transfected biopsies of tumor cells, (iii) adoptive cellular immunotherapy, or treatment with activated tumor-specific T cells.
  • APC enhancement for example, (a) injecting a tumor with a DNA encoding a heterologous MHC alloantigen, or (b) a gene that increases the likelihood of recognition by an immunizing antigen (eg, immunostimulatory cytokine, GM-CSF, costimulatory molecule B7.1, B7.2
  • Adoptive cellular immunotherapy includes isolating tumor-infiltrating host T lymphocytes, such as by in vitro expansion of the population by IL-2 or tumor or both; in addition, dysfunctional isolated T cells can also be used in vitro by applying the antibodies of the invention To activate, the thus activated T cells can then be re-administered to the host.
  • Such combination therapies encompasses combined administration (wherein two or more therapeutic agents are included in the same formulation or separate formulations), and administered separately, in which case other therapies, such as treatments and Administration of the antibodies of the invention occurs before, simultaneously with, and/or after the therapeutic agent.
  • Antibody molecules and/or other therapies, such as therapeutic agents or treatment modalities can be administered during an active disease or during a period of lesser or less active disease.
  • the antibody molecule can be administered prior to other treatments, simultaneously with other treatments, after treatment, or during disease remission.
  • administration of the anti-LAG-3 antibody and administration of another therapy within about one month, or within about one, two or three weeks, or about 1, 2, Occurs within 3, 4, 5, or 6 days.
  • the antibody combinations described herein can be administered separately, eg, as separate antibodies, or when ligated (eg, as a bispecific or trispecific antibody molecule).
  • any treatment can be performed by replacing or supplementing the anti-LAG-3 antibody with an immunoconjugate of the invention.
  • the antibodies of the invention can be administered by any suitable method, including parenteral, intrapulmonary and intranasal, Also, if local treatment is required, it is administered intralesionally.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on the short-term or long-term nature of the administration, it can be administered by any suitable route, for example by injection, for example intravenous or subcutaneous injection.
  • Various medication schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • an antibody of the invention for the prevention or treatment of a disease, a suitable dose of an antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity of the disease, and the Whether the antibody is administered for prophylactic purposes or for therapeutic purposes, prior treatment, clinical history of the patient and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient in one treatment or through a series of treatments.
  • the dosage regimen is adjusted to provide the optimal desired response (eg, a therapeutic response).
  • a single bolus may be administered, several separate doses may be administered over time or may be proportionally reduced or increased as indicated by the acute condition of the treatment situation.
  • parenteral compositions in dosage unit form for ease of administration and uniformity.
  • Dosage unit form as used herein refers to physically discrete units suitable as a single dose for the subject to be treated; each unit contains a predetermined amount of active compound, the predetermined amount being calculated in association with the required pharmaceutical carrier. Produce the desired therapeutic effect.
  • the specifications for the dosage unit form of the present invention are directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the incorporation of such active compounds for use in the field of sensitive treatment in individuals. limit.
  • a non-limiting range of examples of therapeutically effective or prophylactically effective amounts of antibody molecules is from 0.1 to 30 mg/kg, more preferably from 1 to 25 mg/kg, more preferably from 5 to 15 mg/kg.
  • the dosage and treatment regimen of the anti-LAG-3 antibody molecule can be determined by the skilled artisan.
  • the anti-LAG-3 antibody molecule is injected (eg, subcutaneously or intravenously) at about 1 to 40 mg/kg, eg, 1 to 30 mg/kg, eg, about 5 to 25 mg/kg, about 10 Administration is carried out at a dose of 20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20 mg/kg, 15 to 25 mg/kg or about 3 mg/kg.
  • the dosage regimen can vary, for example, from once a week to once every 2, 3 or 4 weeks.
  • the anti-LAG-3 antibody molecule is administered every other week at a dose of about 10 to 20 mg/kg.
  • the antibody molecule can be administered by intravenous infusion at a rate of more than 20 mg/min, for example, 20-40 mg/min and preferably greater than or equal to 40 mg/min, to achieve about 35 to 440 mg/m2, preferably about 70 to 310 mg/ A dose of m2 and more preferably from about 110 to 130 mg/m2. In an embodiment, an infusion rate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg. In one embodiment, the anti-LAG-3 antibody molecule is administered at a dose of about 3 to 800 mg (eg, about 3, 20, 80, 240, or 800 mg) (eg, intravenously).
  • a dose of about 3 to 800 mg eg, about 3, 20, 80, 240, or 800 mg
  • the anti-LAG-3 antibody molecule is administered alone at a dose of about 20 to 800 mg (eg, about 3, 20, 80, 240, or 800 mg). In other embodiments, the anti-LAG-3 antibody molecule is administered at a dose of about 3 to 240 mg (eg, about 3, 20, 80, or 240 mg) to a second active agent or treatment (eg, a second active agent described herein) Or therapeutic means) combined administration. In one embodiment, the anti-LAG-3 antibody molecule is administered every 2 weeks during each 8 week cycle (eg, between weeks 1, 3, 5, 7), eg, up to 96 weeks.
  • the antibody molecule can be administered by intravenous infusion at a rate of more than 20 mg/min, for example, 20-40 mg/min and preferably greater than or equal to 40 mg/min, to achieve about 35 to 440 mg/m2, preferably A dose of about 70 to 310 mg/m2 and more preferably about 110 to 130 mg/m2 is used. In an embodiment, an infusion rate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg.
  • the antibody molecule is administered by intravenous infusion at a rate of less than 10 mg/min, for example, less than or equal to 5 mg/min, to achieve from about 1 to 100 mg/m2, for example, from about 5 to 50 mg/m2, about A dose of 7 to 25 mg/m2 and more preferably about 10 mg/m2. In some embodiments, the antibody is infused over a period of about 30 minutes.
  • the anti-LAG-3 antibody molecule is administered in combination with an anti-PD-1 antibody molecule.
  • exemplary dosages that may be used include about 1 to 10 mg/kg (eg, 3 mg/kg) of an anti-PD-1 antibody molecular dose.
  • the anti-LAG-3 antibody molecule can be administered in combination at a dose of from about 20 to 800 mg, for example, about 20, 80, 240 or 800 mg.
  • the anti-LAG-3 antibody molecule is administered every 2 weeks (eg, during weeks 1, 3, 5, 7) during every 8 week cycle (eg, up to 96 weeks).
  • any of the anti-LAG-3 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of LAG-3 in a biological sample.
  • detection includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic binding of antibody molecules, ELISA assays. Method, PCR-technology (for example, RT-PCR).
  • the biological sample is a blood, serum or other liquid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is from a hyperproliferative or cancerous lesion.
  • an anti-LAG-3 antibody for use in a diagnostic or detection method.
  • a method of detecting the presence of LAG-3 in a biological sample comprises detecting the presence of a LAG-3 protein in a biological sample.
  • LAG-3 is human LAG-3.
  • the method comprises contacting a biological sample with an anti-LAG-3 antibody as described herein under conditions that permit binding of the anti-LAG-3 antibody to LAG-3, and detecting the anti-LAG-3 antibody Whether a complex is formed between LAG-3 and LAG-3. Formation of the complex indicates the presence of LAG-3.
  • the method can be an in vitro or in vivo method.
  • an anti-LAG-3 antibody is used to select a subject suitable for treatment with an anti-LAG-3 antibody, for example wherein LAG-3 is a biomarker for selecting the subject.
  • a cancer or tumor can be diagnosed using an antibody of the invention, eg, to assess (eg, monitor) the treatment or progression of a disease (eg, a hyperproliferative or cancerous disease) described herein in a subject, its diagnosis, and/or Staging.
  • a disease eg, a hyperproliferative or cancerous disease
  • Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected portions, such as enzymes or ligands, for example, By enzymatic reaction or molecular interaction.
  • directly detected labels or moieties such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels
  • indirectly detected portions such as enzymes or ligands, for example, By enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbrella Umbelliferone, Luciferiferase, for example, firefly luciferase and bacterial luciferase (U.S. Patent No.
  • fluorescein 2,3-dihydropyridazinedione, horseradish peroxidase (HR), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lytic enzyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin/avidin , spin labeling, phage labeling, stable free radicals, and more.
  • HR horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lytic enzyme carbohydrate oxidase, for example
  • the sample is obtained prior to treatment with an anti-LAG-3 antibody. In some embodiments, the sample is obtained prior to treatment with a cancer drug. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (eg, a core biopsy), a surgical specimen (eg, from a surgically resected specimen), or a fine needle aspirate.
  • FFPE formalin fixed, paraffin coated
  • LAG-3 is detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval.
  • a method of treating a tumor or infection comprising: examining a subject (eg, a sample) (eg, a sample of a subject comprising cancer cells) for detecting the presence of LAG-3, The LAG-3 value is thus determined, the LAG-3 value is compared to the control value, and if the LAG-3 value is greater than the control value, the subject is administered a therapeutically effective amount of the individual, optionally in combination with one or more other therapies.
  • an anti-LAG-3 antibody eg, an anti-LAG-3 antibody described herein
  • a PD-1 axis binding antagonist thereby treating a tumor or infection.
  • a yeast-based antibody presentation library was amplified according to the existing method (WO2009036379; WO 2010105256; W02012009568), wherein the diversity of each library reached 1 ⁇ 10 9 .
  • the first two rounds of screening used Miltenyi's MACS system for magnetically activated cell sorting.
  • FACS wash buffer phosphate buffer, containing 0.1% bovine serum albumin
  • Human LAG-3 antigen Human LAG-3 antigen (ArcoBiosystems).
  • the next round of sorting was performed using a flow cytometer: approximately 1 ⁇ 10 8 yeast cells obtained by MACS screening were washed three times with FACS buffer for human LAG containing low concentrations of biotin (100-1 nM). The -3 antigen was cultured at room temperature. After discarding the culture medium and washing the cells twice with FACS wash buffer, the cells were mixed with LC-FITC (FITC-labeled goat anti-human immunoglobulin F (ab') kappa chain antibody, Southern Biotech) (1:100 dilution).
  • LC-FITC FITC-labeled goat anti-human immunoglobulin F (ab') kappa chain antibody, Southern Biotech
  • SA-633 streptavidin-633, Molecular Probes
  • SA-PE streptavidin-phycoerythrin, Sigma
  • the yeast cells expressing the anti-human LAG-3 antibody obtained by the screening were shaken at 30 ° C for 48 hours to express an antibody against human LAG-3. After the end of the induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 min, and the supernatant was harvested.
  • the anti-human LAG-3 antibody in the supernatant was purified using Protein A, eluted with a pH 2.0 acetic acid solution, and the anti-human LAG-3 antibody was harvested with an antibody purity of >95%.
  • This screening obtained antibody ADI-26789 and antibody ADI-26869.
  • This method introduces mutations into the antibody heavy chain region by conventional mismatch PCR methods.
  • the base mismatch probability was increased to about 0.01 bp by using the 1 uM highly mutated base analogs dPTP and 8-oxo-dGTP.
  • the obtained mismatched PCR product was constructed by homologous recombination into a vector containing a heavy chain constant region.
  • the CDRH3 gene of the progeny antibody obtained by the VHmut method was constructed into a 1 ⁇ 10 8 diversity CDRH1/CDRH2 gene pool, and subjected to 3 rounds of screening. In the first round, the MACS method was used, and in the second and third rounds, the FACS method was used, and the antibody antigen conjugate was subjected to affinity pressurization to select the antibody with the highest affinity.
  • the CDR regions, the light chain variable region and the heavy chain variable region, the amino acid sequences of the light and heavy chains of the four antibodies (ADI-26789, ADI-26869, ADI-31851, ADI-31853) exemplified in the present invention, and Corresponding nucleic acid sequences and numbers are set forth in the "Table 1-4" section of this application.
  • the cDNA encoding the light chain amino acid sequence and the heavy chain amino acid sequence of each anti-LAG-3 antibody was cloned into the expression vector pTT5, respectively, according to a conventional method in the art.
  • the above expression vector containing the antibody gene of interest and the transfection reagent PEI (Polysciences) were transiently transfected into cultured human kidney blast cell 293 cells (Invitrogen) according to the protocol provided by the manufacturer, after transfection, the medium was discarded and fresh The cells were diluted to 4 ⁇ 10 6 /ml in EXPI293 medium (Gibco). The cells were cultured for 7 days at 37 ° C, 5% CO 2 , and fresh medium was added every 48 hours. After 7 days, centrifuge at 1300 rpm for 20 min. The supernatant was taken and the supernatant was purified with Protein A to give the antibody a purity of >95%.
  • control antibodies used in the examples were also expressed and purified in HEK293 cells:
  • 25F7 is a human LAG-3 antibody transiently expressed in HEK293 cells, the sequence of which is identical to the sequence of the antibody "25F7" in US20170137514A1.
  • BAP050 is an anti-human LAG-3 antibody transiently expressed in HEK293 cells, the sequence of which is identical to the sequence of the antibody "BAP050” in US Patent WO 2015/138920 A1.
  • the equilibrium dissociation constant (KD) of the above four exemplary antibodies of the present invention in combination with human LAG-3 (hLAG-3) was determined by Biote Interferometry (ForteBio) assay.
  • the ForteBio affinity assay 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). Briefly, the sensor was equilibrated in assay buffer for 30 minutes, then on-line for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto an AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to 100 nM human LAG-3 antigen (Arco Biosystems) for 5 minutes, after which the sensor was transferred to assay buffer for 5 minutes for dissociation rate measurement. Analysis of the kinetics was performed using a 1:1 binding model.
  • affinities of ADI-26789, ADI-26869, ADI-31851, ADI-31853, and control antibodies 25F7 and BAP050 are shown in Table 6 in experiments conducted as described in the above assays.
  • Example 5 Binding of the anti-LAG-3 antibody of the present invention to human LAG-3
  • Binding of the above four exemplary antibodies of the invention to human LAG-3 was measured in a flow cytometry based assay.
  • 293 cells overexpressing human LAG-3 were generated by transfecting human blastocysts 293 cells (Invitrogen) with pCHO1.0 vector (Invitrogen) carrying human LAG-3 cDNA (Sino Biological) cloned into the multiple cloning site MCS (293-hLAG-3 cells).
  • 293-hLAG-3 cells (0.2 ⁇ 10 6 cells) were mixed with different concentrations of the experimental antibodies (ADI-26789, ADI-26869 and control antibody 25F7) prepared as described above (antibody dilution method was: the highest antibody concentration was 500 nM, three-fold dilution in PBS containing 0.1% bovine serum albumin (BSA) for a total of 8 concentrations tested). Incubate on ice for 30 minutes. The cells were then washed at least twice, and a 1:100 dilution of secondary antibody (PE-labeled goat anti-human IgG antibody, Southern Biotech, final concentration of 5 ⁇ g/ml) was added and incubated on ice (protected from light) for 30 minutes. The cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on an Accuri C6 system (BD Biosciences) and a concentration-dependent curve was fitted with GraphPad according to its MFI.
  • Affinity-optimized anti-hLAG-3 antibodies ADI-31851 and ADI-31853 bind to hLAG-3 overexpressing HEK293 cells with EC50 values of 0.501 nM and 0.4332 nM, respectively, which are superior to control antibody 25F7 and BAP050 overexpressing HEK293 cells.
  • the binding capacity of hLAG-3 (EC50 values were 2.593 nM and 1.409 nM, respectively).
  • ADI-31851, ADI-31853 The ability of ADI-31851, ADI-31853 to block the binding of human LAG-3 to MHC II (HLA) on the cell surface was measured by flow cytometry.
  • HLA-DR- ⁇ and HLA-DR ⁇ 1 two-stage DNA having the sequence described below were simultaneously constructed into pCHO1.0 vector (Invitrogen) and transfected into CHO-S cells (Invitrogen, ExpiCHO TM Expression System Kit, article number: A29133 CHO cells (CHO-DR cells) whose surface overexpresses human HLA-DR are produced.
  • the antigen rhLAG3 protein (huFc) (Sino Biological) was diluted to 40 nM, 50 ⁇ l/well.
  • the antibodies prepared as described above (ADI-31851, ADI-31853 and control antibody 25F9) were subjected to 3-fold serial dilution from the highest concentration of 80 nM, a total of 8 dilution gradients, 50 ⁇ l/well, and incubated on PBS ice for 30 min. The concentration was 20 nM and the maximum final concentration of the antibody was 40 nM.
  • CHO-DR cells were adjusted to 3 x 10 5 cells/well, 100 ⁇ l/well.
  • the cells were centrifuged at 300 g for 5 min, the supernatant was discarded, and resuspended in the antigen-antibody mixture. Incubate on ice for 30 min, add PBS 100 ⁇ l/well, centrifuge at 300 g for 5 min, wash once with PBS, add 100 ⁇ l of goat anti-human IgG-PE (Southern Biotech)/well diluted 1:100, ice bath for 20 min, add PBS 100 ⁇ l/well, 300 g Centrifuge for 5 min and wash once with PBS. The cell fluorescence signal values were measured by resuspending in 100 ⁇ l of PBS and cell flow meter (BD Biosciences).
  • Human CD4+ T cells were activated to express LAG-3 protein on their surface. This study measured the binding ability of ADI-31851, ADI-31853 and activated human CD4 + T cells by flow cytometry.
  • PBMC separation Take 50 ml of fresh blood from donors, add 2.5 times PBS, gently add to FiColl (Thermo), divide into 4 tubes, 12.5 ml per tube, 400 g, centrifuge for 30 min, and stop at 0 deceleration. Pipette the middle white strip into PBS and wash twice with PBS.
  • CD4+ T cell isolation The kit was operated according to the 'Untouched CD4+T cell isolation' kit (11346D, Invitrogen) instructions. PBMC was cultured for 2 h, and the suspended cell liquid was aspirated into a 15 ml centrifuge tube, centrifuged at 200 g for 10 min, and 500 ⁇ l of the separation solution, 100 ⁇ l of AB-type serum, 100 ⁇ l of purified antibody were resuspended in the pellet, incubated at 4 ° C for 20 min, and washed once with the separation solution.
  • Bead Buffer Invitrogen
  • Bead was removed in a magnetic field, and the T cell medium was washed once, resuspended in 8 ml of medium, and cultured at 37 ° C, 6% CO 2 .
  • the wells were 150 ⁇ l/well, the other wells were 100 ⁇ l/well, and the antibodies ADI-31851 and ADI-31853 and the control antibody 25F7 prepared as described above were added to the first column of wells at a final concentration of 10 nM, and mixed, and 50 ⁇ l was taken into the next row of wells. And so on. Make 3 duplicate wells per sample.
  • Example 8 Binding of anti-LAG-3 antibody of the present invention to mouse LAG-3
  • Binding of the two exemplary antibodies ADI-31851 and ADI-31853 of the present invention to mouse LAG-3 was measured in a flow cytometry-based assay.
  • Mouse LAG-3cDNA (Sino Biological) carrying cloned into the multiple cloning site by transfecting pCHO1.0 vector (Invitrogen) into CHO-S cells (Invitrogen, ExpiCHO TM Expression System Kit , NO: A29133), is too small to generate the expression CHO cells of mouse LAG-3 (CHO-mLAG-3 cells).
  • CHO-mLAG-3 cells (0.2 ⁇ 10 6 cells) were mixed with different concentrations of the experimental antibodies (ADI-31851, ADI-31853, and control antibodies 25F7 and BAP050) prepared as described above (antibody dilution method: highest antibody) At a concentration of 500 nM, three dilutions were made in PBS containing 0.1% bovine serum albumin (BSA) for a total of 8 concentrations). Incubate on ice for 30 minutes. The cells were then washed at least twice, and a 1:100 dilution of secondary antibody (PE-labeled goat anti-human IgG antibody, Southern Biotech, final concentration of 5 ⁇ g/ml) was added and incubated on ice (protected from light) for 30 minutes. The cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on an Accuri C6 system (BD Biosciences) and a concentration-dependent curve was fitted with GraphPad according to its MFI.
  • mice Female BALB/c mice (about 8 weeks old) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The mice were domesticated for 7 days after arrival and the study was started.
  • Mouse colon cancer cell line CT26 (ATCC# CRL-2638) was purchased from ATCC and routinely subcultured for subsequent in vivo experiments in strict accordance with ATCC requirements. The cells were collected by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 5 x 10 6 /ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdomen region of BALB/c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) to establish a tumor-bearing mouse model.
  • mice were divided into four groups (8 mice per group), each of which was injected subcutaneously with the following doses of antibody:
  • mice IgG (equitech-Bio), 10 mg/kg;
  • mice meeting the experimental requirements were randomly divided into groups of 8 each. Each group of mice was dosed as above on days 7, 10, 14 and 17 with the above four groups of reagents, respectively.
  • Tumors and body weight were measured twice weekly throughout the study, and mice were euthanized when the tumor reached the endpoint (tumor volume > 2500 mm 3 ) or when the mice had >20% weight loss.
  • Tumor size from each group of mice was plotted against time. Analysis of variance (ANOVA) was used to determine statistical significance. A P value of ⁇ 0.05 was considered to be statistically significant in all analyses performed using Prism 5 statistical software (GraphPad software).
  • This study used a humanized mouse model to study the combination of anti-human LAG-3 antibody ADI-31853 and anti-PD-1 antibody “Antibody D” (IBI308, WO2017/025016) or anti-PD-L1 antibody (HZ3266-IgG1N297A). Antitumor activity.
  • anti-tumor effects of anti-LAG-3 antibodies were determined on NOG mice using A375 (ATCC) human skin cancer cells.
  • A375 A375 human skin cancer cells.
  • NOG model A375 tumor-bearing mouse model
  • the tumor volume and body weight of the mice in each group during the administration were monitored, and the frequency of administration was 2 times/week, and the administration was carried out for 2 weeks for a total of 5 times.
  • the monitoring frequency was 2 times/week, and the monitoring was continued for 4 weeks.
  • the dosage and mode of administration were as shown in Table 2.
  • the relative tumor inhibition rate (TGI%) was calculated after the end of administration.
  • the cDNA encoding the light chain amino acid sequence and the heavy chain amino acid sequence (Table 3) of the anti-PD-L1 antibody HZ3266-IgG1N297A was cloned into the expression vector pMD20-T vector (Clontech), respectively, according to a conventional method in the art, and the plasmid was obtained for transformation. dye.
  • 293F cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1.5 ⁇ 10 6 cells/ml one day before transfection. The cell density on the day of transfection was approximately 3 x 10 6 cells/ml.
  • a final volume of 1/10 of F17 medium (Gibco, A13835-01) was used as a transfection buffer, and an appropriate plasmid was added and mixed.
  • Add appropriate polyethyleneimine (PEI) Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI is 1:3 in 293F cells), mix and incubate for 10 min at room temperature to obtain a DNA/PEI mixture.
  • PEI polyethyleneimine
  • the gravity column used for purification was treated with 0.5 M NaOH overnight, and the glass bottle and the like were washed with distilled water and then dry-baked at 180 ° C for 4 hours to obtain a purification column.
  • the collected medium was centrifuged at 4500 rpm for 30 min before purification, and the cells were discarded.
  • the supernatant was then filtered using a 0.22 ⁇ l filter.
  • Each tube was filled with 1 ml of ProteinA and equilibrated with 10 ml of binding buffer (sodium phosphate 20 mM. NaCl 150 mM, pH 7.0). The filtered supernatant was added to the purification column and re-equilibrated with 15 ml of binding buffer.
  • elution buffer citric acid + sodium citrate 0.1 M, pH 3.5
  • eluate was collected
  • 80 ⁇ l of Tris-HCl was added per 1 ml of the eluate.
  • the collected antibodies were concentrated by ultrafiltration into PBS (Gibco, 70011-044), and the concentration was measured.
  • the equilibrium dissociation constant (KD) of the anti-PD-L1 antibody (HZ3266-IgG1N297A) binding to human PD-L1 was determined by bioluminescence interferometry (ForteBio).
  • the ForteBio affinity assay 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 instrument setting parameters are as follows: running steps: Baseline, Loading ⁇ 1 nm, Baseline, Association, and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the rotation speed is 400 rpm, and the temperature is 30 °C. KD values were analyzed using ForteBio analysis software. The measured K D value was 0.724 nM.
  • mice NOG mice, female, 7-8 weeks (the age of mice at the time of tumor cell inoculation), weighing 17.6-24.2 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The mice were domesticated for 7 days after arrival and the study was started.
  • Human skin cancer cells A375 (ATCC# CRL-1619) were purchased from ATCC and routinely subcultured for subsequent in vivo experiments in strict accordance with ATCC requirements. The cells were collected by centrifugation, resuspended in sterile PBS, and adjusted to a cell density of 30 ⁇ 10 6 /ml. After NOG mice have been intravenously injected with human PBMC, the right back is shaved and subcutaneously injected with 0.2 ml/A of A375 cells. Tumor cells were examined for tumor volume 7 days after inoculation, and mice with an average tumor volume in the range of 70-71 mm 3 were randomly grouped according to tumor volume.
  • mice that met the mean tumor volume requirement were randomized into groups of 8 animals each. Each group of mice was dosed as above on days 7, 10, 14 and 17 with the above six groups of reagents, respectively.
  • Tumors and body weight were measured twice weekly throughout the study, and mice were euthanized when the tumor reached the endpoint or when the mice had >20% weight loss.
  • Example 11 Activation of human CD4 + T cells by anti-LAG-3 antibody combined with anti-PD-L1 antibody
  • human CD4 + T cell activation assay was used to detect the activation of human T cells by ADI-31853. Beads stimulated activation (as described in Example 7) of human CD4 + T cells, mature DC and different concentrations of ADI-31853, 25F7 alone solution, PD-L1 antibody (HZ3266-IgG1N297A, prepared as described above) alone, And the combination was incubated for a total of 3 days in a 96-well U-bottom plate.
  • IL-2 concentration in the supernatant was measured, and the activation of T cells by ADI-31853, 25F7 alone, and with the PD-L1 antibody (HZ3266-IgG1N297A, prepared as described above) was compared.
  • Human PBMC cells (prepared as described in Example 7) were removed from the liquid nitrogen tank, thawed rapidly in a 37 ° C water bath, transferred to 20 ml human T cell medium (containing 1 DNase (Sigma)), centrifuged at 400 g. 10min. The cells were resuspended in T75 flasks with 20 ml of human T cell medium (1 DNase) and cultured in a 37 ° C, 5% CO 2 cell incubator for 24 h.
  • human T cell medium containing 1 DNase (Sigma)
  • DC cell separation discard the suspension cell solution, add 9 ml DC medium to the remaining cells, add 3 ml DC medium after 2 days of culture, and then culture for 5 days, then add rTNFa (1000 U/ml), IL-1b (5 ng/ml). IL-6 (10 ng/ml) and 1 ⁇ M PGE2 (Tocris) were cultured for 2 days as DC cells for lymphocyte mixed reaction (MLR).
  • MLR lymphocyte mixed reaction
  • T cell culture medium and DC cell culture medium are both CTS AIM V SFM, and 1000 U/ml IL-4 and 1000 U/ml GM-CSF are additionally added to the DC medium.
  • the item number and batch number are as follows:
  • Human PBMC cells (prepared as described in Example 7) were removed from the liquid nitrogen tank, thawed rapidly in a 37 ° C water bath, then transferred to human T cell medium (1 DNase) as described above, and centrifuged at 400 g for 10 min. .
  • Human T cell medium (1 DNase) as described above, and centrifuged at 400 g for 10 min. .
  • Mature DC cells were mixed with CD4+ cells at a volume of 200 ⁇ l per well, 10000 DC cells, 100000 CD4+ cells, and antibody (200 nM, 4 fold serial dilution, SEE (Toxin technology) 1 ng/ml), DC, CD4+ cells, The mixed cells were used as a negative control and mixed for 3 days.
  • the antibody concentrations used are as follows:
  • Anti-LAG-3 ADI-31853 200nM start, 4x dilution, total 10 concentrations
  • Anti-PD-L1 200nM start, 4 times dilution, a total of 10 concentrations
  • Anti-LAG-3 (200nM start, 4x dilution, total 10 concentrations) + anti-PD-L1 (12.5nM)
  • the concentration of IL-2 in the medium was measured using Cisbio Human IL-2kit.
  • test solution (reagents are all from Human IL2 1000tests kit, Cisbio)
  • the medium supernatant and the standard gradient solution (10 ⁇ l) were added to a 96-well microplate, and 10 ⁇ l of the test solution was added to each well, and centrifuged at 400 g for 1 min, protected from light, and incubated at room temperature for 2 h.
  • the OD value was read at 616 nm and 665 nm using a multi-function microplate reader, and the data was analyzed.
  • Ratio OD 665nm / OD 616nm ⁇ 10 4
  • the IL-2 standard curve was obtained.
  • the IL-2 level of human CD4+ T cells in each group was calculated by a standard curve.

Abstract

本发明涉及特异性结合LAG-3的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物。此外，本发明涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途。此外，本发明涉及这些抗体和抗体片段的治疗和诊断用途。特别地，本发明涉及这些抗体和抗体片段与其它治疗剂，例如抗PD-1或抗PD-L1抗体的联合治疗。

Description

抗LAG-3抗体及其用途
本发明涉及特异性结合LAG-3的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物。此外，本发明涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途。此外，本发明涉及这些抗体和抗体片段的治疗和诊断用途。特别地，本发明涉及这些抗体和抗体片段与其它疗法，例如治疗方式或治疗剂，例如抗PD-1或抗PD-L1抗体的联合治疗。
背景技术
淋巴细胞激活基因3(LAG-3)，也称为CD223，是由LAG3基因在人体中编码的I型跨膜蛋白。LAG-3的分子性质和生物学功能已经有充分的表征和描述，参见例如Sierro等人，Expert Opin Ther Targets(2011)15(1)：91-101。LAG-3是一种CD4样蛋白，在T细胞(特别是活化的T细胞)、自然杀伤细胞、B细胞和浆细胞样树突状细胞表面表达。已显示LAG-3是负共刺激受体，即抑制性受体。
LAG-3与MHC II类分子结合(Baixeras等(1992)J.Exp.Med.176：327-337；Huard等(1996)Eur.J.Immunol，26：1180-1186)，在抗原呈递细胞(APC)如树突状细胞、巨噬细胞和B细胞的表面处组成型表达高水平的分子家族。LAG-3功能依赖于与II类MHC的结合和通过其胞质结构域的信号传导。已经提出，LAG-3与MHC II类分子的直接结合在下调CD4+T淋巴细胞的抗原依赖性刺激中起作用(Huard等，(1994)Eur.J.Immunol.24：3216-3221)。还认为LAG3和II类MHC之间的相互作用在调节树突细胞功能中起作用(Andreae等人.J Immunol 168：3874-3880，2002)。近期的临床前研究也已经记录了LAG-3在CD8 T-细胞耗竭中的作用(Blackburn等人.Nat Immunol 10：29-37，2009)。
研究表明，慢性病毒感染后耗尽的CD8+T细胞表达多种抑制性受体(如PD-1，CD160和2B4)。LAG-3在LCMV感染后以高水平表达，并且显示阻断PD-1/PD-L1途径与阻断LAG-3显著降低慢性感染小鼠中的病毒载量(Blackburn等人，Nat Immunol(2009)10：29-37)。还显示PD-1/PD-L1途径和LAG-3阻断剂的联合抑制提供了抗肿瘤功效(Jing等人，Journal for ImmunoTherapy of Cancer(2015)3：2)。
鉴于LAG-3的上述重要作用，需要开发调节其活性的新的抗LAG-3抗体，特别是人源化或人抗体。这类抗体能够更好地用于治疗肿瘤以及其它疾病如感染。还希望获得能够与其他疗法(例如治疗剂，例如抗PD-1或抗PD-L1抗体)联合用于治疗肿瘤，特别是转移性或难治性肿瘤，或者治疗感染，例如慢性感染的新的抗LAG-3抗体。此外，还需要能够用于小鼠模型的抗鼠LAG-3抗体，以便于研究抗体的体内生物学活性。
发明概述
本文公开了与LAG-3结合的抗体分子，例如全人抗体分子或人源化抗体分子。还提供了编码所述抗体或其抗体片段的核酸、用于产生抗体分子的表达载体、宿主细胞和方法。还提供包含抗LAG-3的抗体分子的免疫缀合物、多特异性或双特异性抗体分子，以及药物组合物。本文公开的抗LAG-3抗体分子可以单独或联合其他疗法，例如治疗剂(例如抗PD-1抗体或抗PD-L1抗体)或治疗方式，用来治疗、预防和/或诊断肿瘤疾病以及感染性疾病。此外，本文还公开了用于检测LAG-3的组合物和方法以及使用抗LAG-3抗体分子治疗多种疾病(包括肿瘤和/或感染性疾病)的方法。
因此，在一些实施方案中，本发明的抗体或其片段(特异性)结合LAG-3。在一些实施方案中，本发明的抗体或其片段(特异性)结合人LAG-3或小鼠LAG-3。
在一些实施方案中，本发明的抗LAG-3抗体或其片段以高亲和力结合LAG-3(例如人LAG-3)，例如，以以下平衡解离常数(K D)与LAG-3结合，所述K D小于大约100nM，优选地，小于或等于大约50nM，更优选地小于或等于大约20nM，更优选地小于或等于大约10nM、9nM、8nM、7nM、6nM、5nM、4nM、3nM或2nM，最优选地，所述K D小于或等于大约1nM、0.9nM、0.8nM或0.7nM。 在一些实施方案中，本发明的抗LAG-3抗体以0.1-20nM，优选地0.5-20nM，更优选地0.5-10nM、0.5-8nM、0.5-5nM，最优选地0.5-1nM、0.5-0.8nM、0.5-0.7nM、0.6-0.7nM的K D结合LAG-3。在一些实施方案中，LAG-3为人LAG-3。在一些实施方案中，LAG-3为小鼠LAG-3。在一些实施方案中，抗体结合亲和力是使用生物光干涉测定法(例如Fortebio亲和测量)测定法测定的。
在一些实施方案中，本发明的抗体或其片段结合表达人LAG-3的细胞，例如，以小于或等于大约3.3nM、3nM、2nM、1.5nM、1.4nM、1.3nM、1.2nM、1.1nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM或0.5nM的EC50。在一些实施方案中，所述结合用流式细胞术(例如FACS)测定。在一些实施方案中，表达人LAG-3的细胞为表达人LAG-3的293细胞(例如HEK293细胞)。
在一些实施方案中，本发明的抗体或其片段结合表达小鼠LAG-3的细胞，例如，以小于或等于大约15000nM、14000nM或13000nM的EC50。在一些实施方案中，本发明的抗体或其片段结合表达小鼠LAG-3的细胞，例如，以小于或等于大约50nM的EC50，例如以大约40-50nM的EC50、大约40-45nM的EC50或大约42nM的EC50。在一些实施方案中，用流式细胞术(例如FACS)测定所述结合。在一些实施方案中，表达小鼠LAG-3的细胞为表达小鼠LAG-3的中国仓鼠卵巢(CHO)细胞。
在一些实施方案中，本发明的抗体或其片段抑制LAG-3的相关活性，例如以小于或等于大约20nM、10nM、9nM、8nM、7nM、6nM或5nM的IC 50，优选以大约1-6nM、1-5nM、4nM、4.5nM、5nM、5.1nM、5.2nM、5.3nM、5.4nM、5.5nM、5.6nM、5.7nM、5.8nM、5.9nM或6nM的IC 50。在一些实施方案中，LAG-3的相关活性是MHC II类分子与LAG-3的结合。在一些实施方案中，本发明的抗体或其片段以小于或等于大约20nM、10nM、9nM、8nM、7nM、6nM或5nM的IC 50，优选以大约1-6nM、1-5nM、4nM、4.5nM、5nM、5.1nM、5.2nM、5.3nM、5.4nM、5.5nM、5.6nM、5.7nM、5.8nM、5.9nM或6nM的IC 50抑制LAG-3与表达MHC II类分子的细胞上的MHC II类分子的结合。在一些实施方案中，MHC II类分子是HLA-DR。在一些实施方案中，细胞为CHO细胞。在一些实施方案中，用流式细胞术(例如FACS)测量所述本发明的抗体或其片段对LAG-3的相关活性的抑制。
在一些实施方案中，本发明的抗体或其片段结合激活的CD4+和/或CD8+T细胞的表面上的内源LAG-3，例如以小于或等于大约35pM、30pMnM、25pM、20pM、15pM、14pM或13pM的EC 50，优选大约1-20pM、5-20pM、5-15pM、10-15pM、11-13pM、10pM、11pM、12pM或13pM的EC 50。在一些实施方案中，激活的CD4+T细胞为激活的人CD4+T细胞。在一些实施方案中，用流式细胞术(例如FACS)测定上述结合。在一些实施方案中，在Accuri C6系统中进行流式细胞术。
在一些实施方案中，本发明的抗体或其片段抑制LAG-3的一种或多种活性，例如，导致以下一种或多种：CD4+T淋巴细胞的抗原依赖性刺激增加；T细胞增殖增加；活化抗原(例如，CD25)的表达增加；细胞因子(例如，干扰素-扰素增加；细胞因、白介素-2(IL-2)或白介素-4(IL-4))的表达增加；趋化因子(例如，CCL3、CCL4或CCL5)的表达增加；Treg细胞的阻抑活性减少；T细胞稳态增加；肿瘤浸润型淋巴细胞增加；或癌细胞的免疫逃避减少。
在一些实施方案中，本发明的抗LAG-3抗体或其片段能够诱发抗体依赖性细胞介导的细胞毒性(ADCC)。
在一些实施方案中，本发明的抗LAG-3抗体单独或与其他的疗法(例如治疗方式和/或治疗剂)组合能够有效治疗肿瘤(例如癌症)或感染性疾病(例如慢性感染)。优选地，本发明的抗LAG-3抗体能够与抗PD-1或抗PD-L1抗体联合治疗肿瘤，特别是转移性或难治性肿瘤。在一些实施方案中，肿瘤是癌症。在一些实施方案中，肿瘤是胃肠道肿瘤。在一些实施方案中，癌症是结肠癌。
在一些实施方案中，本发明抗LAG-3抗体或其片段的重链和/或轻链还包含信号肽序列，例如 METDTLLLWVLLLWVPGSTG(SEQ ID NO：48)。
在一些实施方案中，本发明的抗体还涵盖抗LAG-3抗体的氨基酸序列的变体，以及与上文所述的任何抗LAG-3抗体或其片段结合相同表位的抗体。
在某些实施方案中，提供结合LAG-3或其片段的抗体或抗体片段(优选的抗原结合片段)，其中所述抗体或抗体片段结合LAG-3内的表位。
在一些实施方案中，本发明的抗LAG-3抗体是IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD或IgE形式的抗体。在一些实施方案中，本发明的抗LAG-3抗体包含选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区的重链恒定区；特别地，选自例如IgG1、IgG2、IgG3和IgG4的重链恒定区的重链恒定区，更具体地IgG1、IgG2或IgG4的重链恒定区，例如人IgG1、IgG2或IgG4的重链恒定区。在一个实施方案中，重链恒定区是人IgG1或人IgG4重链恒定区。在一个实施方案中，重链恒定区是人IgG4重链恒定区。在另一个实施方案中，抗LAG-3抗体分子具有例如选自κ或λ轻链恒定区的轻链恒定区、优选地κ(例如，人κ)的轻链恒定区。
在又一个实施方案中，抗LAG-3抗体分子包含IgG4(例如，人IgG4)的重链恒定区。在一个实施方案中，人IgG4包含在根据EU编号的位置228处的置换(例如，Ser至Pro置换)。在又一个实施方案中，人IgG4在第114-115位(EU编号)被突变为AA(Armour KL1，Clark MR，Hadley AG，Williamson LM，Eur J Immunol.1999 Aug；29(8)：2613-24，Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities)。在一个实施方案中，重链恒定区包含SEQ ID NO：46所示的氨基酸序列，或与之具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的序列，或由所述序列组成。
在又一个实施方案中，抗LAG-3抗体分子包含κ轻链恒定区，例如，人κ轻链恒定区。在一个实施方案中，轻链恒定区包含SEQ ID NO：47的氨基酸序列，或与之具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的序列，或由所述序列组成。
在另一个实施方案中，抗LAG-3抗体分子包含IgG4的重链恒定区(例如，人IgG4重链恒定区)和κ轻链恒定区(例如，人κ轻链恒定区)。在一个实施方案中，恒定区是突变的IgG4，例如，突变的人IgG4(例如，具有在根据EU编号的位置228处的突变(例如，S228P突变))。在一些实施方案中，人IgG4重链恒定区包含SEQ ID NO：46所示的氨基酸序列，或与之具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的序列，或由所述序列组成。在一个实施方案中，人κ轻链恒定区包含SEQ ID NO：47的氨基酸序列，或与之具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的序列，或由所述序列组成。
在一个实施方案中，抗LAG-3抗体分子是分离的或重组的。
在一些实施方案中，抗LAG-3抗体是单克隆抗体或具有单特异性的抗体。抗LAG-3抗体分子也可以是人源化的、嵌合的、骆驼科(camelid)、鲨鱼或体外生成的抗体分子。在一些实施方案中，抗LAG-3抗体是人源化的。在一些实施方案中，抗LAG-3抗体是人抗体。在一些实施方案中，至少部分的抗LAG-3抗体的构架序列是人共有构架序列。在一个实施方案中，本发明的抗LAG-3抗体还涵盖其抗体片段，优选地选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)或(Fab’) 2、单结构域抗体、双抗体(dAb)或线性抗体。
在某些实施方案中，抗LAG-3抗体分子处于双特异性或多特异性抗体分子形式。在一个实施方案中，双特异性抗体分子具有针对LAG-3的第一结合特异性和针对PD-1、TIM-3、CEACAM(例如，CEACAM-1和/或CEACAM-5)、PD-L1或PD-L2的第二结合特异性。在一个实施方案中，双特异性抗体分子与LAG-3和PD-1结合。在另一个实施方案中，双特异性抗体分子与LAG-3和PD-L1 结合。在又一个实施方案中，双特异性抗体分子与LAG-3和PD-L2结合。可以在多特异性抗体分子中产生前述分子的任何组合。多特异性抗体分子例如三特异性抗体分子，其包含针对LAG-3的第一结合特异性和针对以下一种或多种的分子的第二及第三结合特异性：PD-1、TIM-3、CEACAM(例如、CEACAM-1或CEACAM-5)、PD-L1或PD-L2。
在其他实施方案中，抗LAG-3抗体分子与包含以下一种或多种的双特异性分子组合使用：PD-1、TIM-3、CEACAM(例如、CEACAM-1或CEACAM-5)、PD-L1或PD-L2。
在一方面，本发明提供了编码以上任何抗LAG-3抗体或其片段的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的，例如大肠杆菌细胞。
在一个实施方案中，本发明提供制备抗LAG-3抗体或其片段(优选的抗原结合片段)的方法，其中所述方法包含在适于表达编码所述抗体或其片段(优选的抗原结合片段)的核酸的条件下培养所述宿主细胞，以及任选地分离所述抗体或其片段(优选地抗原结合片段)。在某个实施方案中，所述方法还包括从宿主细胞回收抗LAG-3抗体或其片段(优选地抗原结合片段)。
在一些实施方案中，本发明提供了免疫缀合物，其包含本文中提供的任何抗LAG-3抗体和其它物质，例如细胞毒性剂或标记。在一些实施方案中，所述免疫缀合物用于预防或治疗肿瘤(例如癌症)或感染性疾病。优选地，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。优选地，感染性疾病是慢性感染。
在一些实施方案中，本发明提供包含本文所述的任何抗LAG-3抗体或其片段(优选地其抗原结合片段)或其免疫缀合物的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的抗LAG-3抗体或其片段或其免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)，优选地抗PD-1抗体、抗PD-L1抗体或抗PD-L2抗体)的组合。
在一些实施方案中，所述药物组合物用于预防或治疗肿瘤(例如癌症)或感染。优选地，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。优选地，感染性疾病是慢性感染。
在另一方面中，本发明涉及预防或治疗受试者或个体肿瘤(例如癌症)或感染性疾病的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗LAG-3抗体或其片段、药物组合物或免疫缀合物。在一个实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。在一个实施方案中，感染性疾病是慢性感染。在另一方面，本发明还涉及本文所述的任何抗LAG-3抗体或其片段制备用于在受试者中治疗肿瘤(例如癌症)或感染的药物的用途。在一个实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。在一个实施方案中，感染性疾病是慢性感染。
在另一方面中，本发明涉及预防或治疗受试者或个体肿瘤(例如癌症)或感染性疾病的方法，所述方法包括向所述受试者联合施用有效量的本文所述的任何抗LAG-3抗体或其片段、药物组合物或免疫缀合物，以及PD-1轴结合拮抗剂或包含所述PD-1轴结合拮抗剂的药物或活性剂。在一个实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。在一个实施方案中，感染性疾病是慢性感染。在另一方面，本发明还涉及本文所述的任何抗LAG-3抗体或其片段与PD-1轴结合拮抗剂组合在制备用于在受试者中治疗肿瘤(例如癌症)或感染性疾病的药物中的用途。在一个实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。在一个实施方案中，感染性疾病是慢性感染。
在一些实施方案中，PD-1轴结合拮抗剂包括例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗 体。
在进一步的一些实施方案中，在本文所述的预防或治疗方法中，还包括向所述受试者或个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方案中，治疗方式包括手术治疗和/或放射疗法。在一些实施方案中，其它治疗剂选自化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)。
在一些实施方案中，受试者或个体是非人动物。在一些实施方案中，受试者或个体是哺乳动物，优选地人。
在一方面中，本发明涉及检测样品中LAG-3的方法，所述方法包括(a)将样品与本文所述的任何抗LAG-3抗体或其片段接触；和(b)检测抗LAG-3抗体或其片段和LAG-3间的复合物的形成。在一个实施方案中，抗LAG-3抗体是被可检测地标记的。
在一些实施方案中，本发明涉及试剂盒或制品，其包含本文所述的任何抗LAG-3抗体或其片段。在一些实施方案中，所述试剂盒或制品包含本文所述的抗LAG-3抗体或其片段与任选的药用辅料。在一些实施方案中，该试剂盒或制品进一步包含关于施用药物来治疗肿瘤或感染的说明书。
本发明还涵盖本文所述的任何实施方案的任意组合。本文所述的任何实施方案或其任何组合适用于本文所述的发明的任何和所有抗LAG-3抗体或其片段、方法和用途。
附图说明
图1显示了用流式细胞术检测的亲本抗体和细胞表面hLAG-3的结合能力。
图2显示了用流式细胞术检测的亲和力成熟后的抗体和细胞表面hLAG-3的结合能力。
图3显示了用流式细胞术检测的抗LAG-3抗体阻断人MHC II(HLA-DR)和LAG-3的相互作用。
图4显示了用流式细胞术检测的抗LAG-3抗体和激活的人CD4+T细胞的结合能力。
图5显示了用流式细胞术检测的抗LAG-3抗体和细胞表面小鼠LAG-3的结合能力。
图6显示了用抗LAG-3抗体和抗PD-1抗体联合使用在CT26移植瘤模型中对肿瘤的抑制作用。
图7显示了用抗LAG-3抗体和抗PD-1抗体或PD-L1联合使用在NOG模型中对肿瘤的抑制作用。
图8显示了IL-2标准曲线。
图9显示了抗体或抗体组合对活化人CD4+T细胞分泌IL-2的影响。
发明详述
缩写
除非另外说明，否则本说明书中的缩写具有以下含义：
使用以下缩写：
ADCC     抗体依赖性细胞介导的毒性
CDC      补体依赖性细胞毒性
CDR      在免疫球蛋白可变区中的互补决定区
CHO      中国仓鼠卵巢
EC50     导致50％效力或结合的浓度
K D       平衡解离常数
ELISA    酶联免疫吸附测定
FR       抗体构架区
IC50     产生50％抑制的浓度
Ig       免疫球蛋白
Kabat    通过Elvin A.Kabat((1991)Sequences of Proteins of Immun ological Interest，第5版Public Health Service，National Institutes of Health，Bethesda，Md.)设立的免疫球蛋白比对和编号系统
mAb或Mab或MAb         单克隆抗体
PCR                   聚合酶链式反应
IFN                   干扰素
VL                    轻链可变区
VH                    重链可变区
LC                    轻链
HC                    重链
HCDR                  重链互补决定区
LCDR                  轻链互补决定区
定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
“亲和力”是指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。除非另有说明，在用于本文时，“结合亲和力”指反映结合对的成员(例如抗体与抗原)之间1∶1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可用平衡解离常数(K D)来表述。亲和力可通过本领域知道的常用方法来测量，包括现有技术已知以及本文中所描述的那些。
术语“淋巴细胞活化基因-3”或“LAG-3”包括全部同种型，哺乳动物(例如，人)LAG-3、人LAG-3的物种同源物和包含LAG-3的至少一个共同表位的类似物。LAG-3(例如，人LAG-3)的氨基酸序列和核苷酸序列是本领域已知的，例如，参见Triebel等人，(1990)J.Exp.Med.171：1393-1405。在一些实施方案中，术语“人LAG-3”指的是人序列LAG-3，如Genbank登录号为NP_002277的 人LAG-3的完整氨基酸序列。在一些实施方案中，术语“小鼠LAG-3”指的是小鼠序列LAG-3，如Genbank登录号为NP_032505的小鼠LAG-3的完整氨基酸序列。LAG-3在现有技术中也称为，例如，CD223。人LAG-3序列可以具有例如保守突变或在非保守区中的突变而不同于Genbank登录号NP_002277的人LAG-3，并且该LAG-3与Genbank登录号NP_002277的人LAG-3具有基本上相同的生物功能。例如，人LAG-3的生物功能为具有在LAG-3的胞外结构域中的表位，其被本公开的抗体特异性结合，或者人LAG-3的生物功能为与MHC II类分子结合。
本文所用的术语“抗LAG-3抗体”、“抗LAG-3”、“LAG-3抗体”或“结合LAG-3的抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合LAG-3蛋白或其片段。在一个实施方案中，抗LAG-3抗体与非LAG-3蛋白结合的程度低于所述抗体与LAG-3结合的约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％或约90％或以上，如例如通过放射性免疫测定(RIA)或生物光干涉测定法或MSD测定法测量的。
如本文所用，“单克隆抗体”或“mAb”或”Mab”指来源于例如真核生物的、原核生物的或噬菌体克隆的单一拷贝或克隆的抗体，而不指其产生的方法。单克隆抗体或其抗原结合片段可以例如通过杂交瘤技术、重组技术、噬菌体展示技术、合成技术例如CDR嫁接、或此类或其它本领域已知的技术的组合来产生。
“天然抗体”指具有不同结构的天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由以二硫化物键合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域或重链可变域，接着是三个恒定域(CH1，CH2，和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域或轻链可变域，接着是一个恒定轻(CL)域。根据其恒定域氨基酸序列，抗体轻链可归入两种类型中的一种，称作卡帕(κ)和拉姆达(λ)。“天然序列Fc区”包含与在自然界中找到的Fc区的氨基酸序列相同的氨基酸序列。天然序列人Fc区包括天然序列人IgG1 Fc区(非A和A同种异型)；天然序列人IgG2 Fc区；天然序列人IgG3 Fc区；和天然序列人IgG4 Fc区；及其天然存在变体。
“抗体片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’)2；双抗体；线性抗体；单链抗体(例如scFv)；单结构域抗体；双价或双特异性抗体或其片段；骆驼科抗体；和由抗体片段形成的双特异性抗体或多特异性抗体。
如本文所用，术语“表位”指抗原(例如，人LAG-3)中与抗体分子特异性相互作用的部分。这部分(本文中称作表位决定簇)一般包含元件如氨基酸侧链或糖侧链或是其组成部分。表位决定簇可以按照本领域已知的或本文公开的方法(例如，通过结晶学或通过氢-氘交换法)限定。抗体分子上与表位决定簇特异性相互作用的至少一个或某些部分一般位于CDR内。通常，表位具有特定的三维结构特征。通常，表位具有特定电荷特征。一些表位是线性表位，而另一些是构象表位。
与参照抗体“结合相同或重叠表位的抗体”是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合，反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。
与参照抗体竞争结合其抗原的抗体是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。众多类型的竞争性结合测定可用于确定一种抗体是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methods in Enzymology 9：242-253)。
抑制(例如竞争性抑制)参照抗体与其抗原的结合的抗体是指这样的抗体，其抑制50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体抑制50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。抗体与其抗原的结合可以亲和力(例如平衡解离常数)衡量。测定亲和力的方法是本领域已知的。
与参照抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有参照抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883，Al-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
Figure PCTCN2018124315-appb-000001
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR 序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.(1991))的编号位置。
在一个实施方案中，本发明抗体的CDR通过Kabat规则确定边界，通过IMGT确定规则，或通过AbM确定规则，或通过其组合确定规则，例如其序列如表1所示。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR(在同一指派系统下)。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat，Chothia，AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
本领域已知五个主要类别的抗体：IgA，IgD，IgE，IgG和IgM，并且这些抗体中的数个可以进一步被划分为亚类(同种型)，例如，IgG 1，IgG 2，IgG 3，IgG 4，IgA 1和IgA 2。对应于不同类别的免疫球蛋白的重链恒定结构域分别被称为α，δ，ε，γ和μ。
“IgG形式的抗体”是指抗体的重链恒定区所属于的IgG形式。所有同一型的抗体的重链恒定区都是相同的，不同型的抗体之间的重链恒定区不同。例如，IgG4形式的抗体是指其重链恒定区Ig结构域为IgG4的Ig结构域。
术语“PD-1轴结合拮抗剂”是指如下的分子，其抑制PD-1轴结合配偶与一种或多种它的结合配偶相互作用，从而去除源自PD-1信号传导轴上的信号传导的T细胞功能障碍——一项结果是恢复或增强T细胞功能(例如增殖，细胞因子生成，靶细胞杀伤)。如本文中使用的，PD-1轴结合拮抗剂包括PD-1结合拮抗剂(例如抗PD-1抗体)，PD-L1结合拮抗剂(例如抗PD-L1抗体)和PD-L2结合拮抗剂(例如抗PD-L2抗体)。
术语“PD-1结合拮抗剂”指如下的分子，其降低、阻断、抑制、消除或干扰源自PD-1与一种或多种它的结合配偶(诸如PD-L1，PD-L2)相互作用的信号转导。在一些实施方案中，PD-1结合拮抗剂是抑制PD-1结合一种或多种它的结合配偶的分子。在一个特定方面，PD-1结合拮抗剂抑制PD-1结合PD-L1和/或PD-L2。例如，PD-1结合拮抗剂包括降低、阻断、抑制、消除或干扰源自PD-1与PD-L1和/或PD-L2相互作用的信号转导的抗PD-1抗体、其抗原结合片段、免疫粘附素、融合蛋白、寡肽和其它分子。在一个实施方案中，PD-1结合拮抗剂降低由或经由T淋巴细胞上表达的细胞表面蛋白质介导的负面共刺激信号(经由PD-1介导信号传导)，从而使得功能障碍性T细胞不太功能障碍性(例如增强对抗原识别的效应器应答)。在一些实施方案中，PD-1结合拮抗剂是抗PD-1抗体。在一个具体实施方案中，PD-1结合拮抗剂是WO2015/095423中公开的MDX-1106(nivolumab)、MK-3475(pembrolizumab)、CT-011(pidilizumab)或AMP-224。在一个具体 的实施方案中，抗PD-1抗体是WO2017/133540中公开的“Antibody C”。在另一个具体的实施方案中，抗PD-1抗体是WO2017/025016中公开的“Antibody D”。
术语“PD-L1结合拮抗剂”指如下的分子，其降低、阻断、抑制、消除或干扰源自PD-L1与一种或多种它的结合配偶(诸如PD-1，B7-1)相互作用的信号转导。在一些实施方案中，PD-L1结合拮抗剂是抑制PD-L1结合它的结合配偶的分子。在一个特定方面，PD-L1结合拮抗剂抑制PD-L1结合PD-1和/或B7-1。在一些实施方案中，PD-L1结合拮抗剂包括降低、阻断、抑制、消除或干扰源自PD-L1与一种或多种它的结合配偶(诸如PD-1，B7-1)相互作用的信号转导的抗PD-L1抗体、其抗原结合片段、免疫粘附素、融合蛋白、寡肽和其它分子。在一个实施方案中，PD-L1结合拮抗剂降低由或经由T淋巴细胞上表达的细胞表面蛋白质介导的负面共刺激信号(经由PD-L1介导信号传导)，从而使得功能障碍性T细胞不太功能障碍性(例如增强对抗原识别的效应器应答)。在一些实施方案中，PD-L1结合拮抗剂是抗PD-L1抗体。在一个具体方面，抗PD-L1抗体是WO2015/095423中公开的YW243.55.S70、MDX-1105、MPDL3280A或MEDI4736。
术语“PD-L2结合拮抗剂”指如下的分子，其降低、阻断、抑制、消除或干扰源自PD-L2与一种或多种它的结合配偶(诸如PD-1)相互作用的信号转导。在一些实施方案中，PD-L2结合拮抗剂是抑制PD-L2结合一种或多种它的结合配偶的分子。在一个特定方面，PD-L2结合拮抗剂抑制PD-L2结合PD-1。在一些实施方案中，PD-L2拮抗剂包括降低、阻断、抑制、消除或干扰源自PD-L2与一种或多种它的结合配偶(诸如PD-1)相互作用的信号转导的抗PD-L2抗体、其抗原结合片段、免疫粘附素、融合蛋白、寡肽和其它分子。在一个实施方案中，PD-L2结合拮抗剂降低由或经由T淋巴细胞上表达的细胞表面蛋白质介导的负面共刺激信号(经由PD-L2介导信号传导)，从而使得功能障碍性T细胞不太功能障碍性(例如增强对抗原识别的效应器应答)。在一些实施方案中，PD-L2结合拮抗剂是免疫粘附素。
“抗体依赖性细胞介导的细胞毒性”或“ADCC”指其中结合到某些细胞毒性细胞(例如NK细胞，嗜中性粒细胞和巨噬细胞)上存在的Fc受体(FcR)上的分泌型免疫球蛋白，使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞的细胞毒性形式。介导ADCC的主要细胞，NK细胞，只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。Ravetch和Kinet，Annu.Rev.Immunol.9：457-92(1991)第464页表3总结了造血细胞上的FcR表达。为了评估目的分子的ADCC活性，可进行体外ADCC测定法，诸如美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中所记载的。可用于此类测定法的效应细胞包括PBMC和NK细胞。可选地/另外地，可在体内评估目的分子的ADCC活性，例如在动物模型中，诸如Clynes等人，PNAS(USA)95：652-656(1998)中所披露的。
术语“细胞毒性剂”或“细胞毒性因子”用在本发明中指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。细胞毒性剂例子参见WO2015/153513、WO2016/028672或WO2015/138920中所公开的那些。
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤(例如癌症)和感染(例如慢性感染)中有效的任何物质，包括化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂或免疫调节剂，例如WO2016/028672或WO2015/138920中所公开的可以与抗LAG-3抗体联合使用的任何物质。
“化疗剂”包括在治疗癌症中有用的化学化合物。化疗剂的例子参见WO2015/153513或WO2016/028672或WO2015/138920中所公开的那些。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子，单核因子；白介素(IL)，诸如IL-1、IL-1胞、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-11、IL-12、IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括LIF和kit配体(KL)和γ-干扰素。如本文中使用的，术语细胞因子包括来自天然来源 或来自重组细胞培养物的蛋白质及天然序列细胞因子的生物学活性等效物，包括通过人工合成产生的小分子实体，及其药剂学可接受的衍生物和盐。
术语“共刺激分子”指T细胞上与共刺激配体特异性结合，因而由T细胞介导共刺激反应(如但不限于增殖)的相关结合配偶体。共刺激分子是高效免疫应答要求的除抗原受体或其配体之外的细胞表面分子。共刺激分子包括但不限于：MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、信号传导淋巴细胞的活化分子(SLAM蛋白)、NK细胞活化受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CDS、ICAM-1、LFA-1(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a和与CD83特异性结合的配体。
术语“激活剂”或“激动剂”包括使所给出分子(例如，共刺激分子)的某些参数(例如，活性)增加的物质。例如，这个术语包括使得所给出的分子增加至少5％、10％、25％、50％、75％或更多的活性(例如，共刺激活性)的物质。
术语“免疫检查点分子”意指在CD4 T细胞和CD8 T细胞的细胞表面上的一组分子。这些分子可以有效地充当下调或抑制抗肿瘤免疫应答的“刹车”。免疫检查点分子包括但不限于程序性死亡1(PD-1)、细胞毒T淋巴细胞抗原4(CTLA-4)、B7H1、B7H4、OX-40、CD137、CD40和LAG-3，它们直接抑制免疫细胞。
术语“抑制剂”或“拮抗剂”包括使所给出分子(例如，免疫检查点抑制蛋白)的某些参数(例如，活性)降低的物质。例如，这个术语包括使得所给出的分子被抑制至少5％、10％、20％、30％、40％或更多的活性(例如，PD-1活性或PD-L1活性)的物质。因此，抑制作用不必是100％。
术语“双抗体”指具有两个抗原结合位点的抗体片段，所述片段在相同的多肽链(VH-VL)中包含与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用因为太短而不能在相同链上的两个结构域之间配对的接头，迫使所述结构域与另一条链的互补结构域配对从而产生两个抗原结合位点。双抗体可以是二价的或双特异性的。双抗体更充分地描述于例如EP 404,097；WO 1993/01161；Hudson等，Nat.Med.9：129-134(2003)；和Hollinger等，美国国家科学院学报(Proc.Natl.Acad.Sci.USA)90：6444-6448(1993)中。三抗体和四抗体同样描述于Hudson等，Nat.Med.9：129-134(2003)中。
“功能性Fc区”拥有天然序列Fc区的“效应器功能”。例示性的“效应器功能”包括C1q结合；CDC；Fc受体结合；ADCC；吞噬作用；细胞表面受体(例如B细胞受体；BCR)下调等。此类效应器功能一般要求Fc区与结合结构域(例如抗体可变域)联合，而且可以使用多种测定法来评估，例如本文所公开的那些。
“效应子功能”指那些可归于抗体Fc区且随抗体同种型而变化的生物学活性。抗体效应子功能的实例包括：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)下调；和B细胞活化。
“人效应细胞”指表达一种或多种FcR并行使效应器功能的白细胞。在某些实施方案中，该细胞至少表达Fc使效应器功并行使ADCC效应器功能。介导ADCC的人白细胞的例子包括外周血单个核细胞(PBMC)、天然杀伤(NK)细胞、单核细胞、细胞毒性T细胞和嗜中性粒细胞。效应细 胞可以从其天然来源分离，例如血液。
术语“有效量”指本发明的抗体或片段或缀合物或组合物的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；它的大小、年龄和一般健康；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其缀合物或组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。可选地，可以通过检验化合物抑制的能力评价组合物的这种特性，所述抑制在体外通过熟练技术人员已知的测定法。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
适用于本发明的“抗体及其抗原结合片段”包括但不限于多克隆、单克隆、单价、双特异性、异缀合物、多特异性、重组、异源、异源杂合、嵌合、人源化(特别是嫁接有CDR的)、去免疫的、或人的抗体，Fab片段、Fab′片段、F(ab′) 2片段、由Fab表达库产生的片段、Fd、Fv、二硫化物连接的Fv(dsFv)、单链抗体(例如scFv)、双抗体或四抗体(Holliger P.等(1993)Proc.Natl.Acad.Sci.U.S.A.90(14)，6444-6448)、纳米抗体(nanobody)(也称为单结构域抗体)、抗独特型(抗Id)抗体(包括例如针对本发明抗体的抗Id抗体)和上述任一种的表位结合片段。
“Fab”片段包括重链可变结构域和轻链可变结构域，并且还包括轻链的恒定结构域以及重链的第一恒定结构域(CH1)。Fab’片段因在重链CH1结构域的羧基末端增加了一些残基(包括来自抗体铰链区的一个或多个半胱氨酸)而与Fab片段不同。Fab’-SH是对其中恒定结构域的半胱氨酸残基携带一个游离硫醇基的Fab’的称谓。F(ab’) 2抗体片段最初是作为成对Fab’片段生成的，在Fab’片段之间具有铰链半胱氨酸。抗体片段的其它化学偶联也是已知的。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)。(参见，例如，Kindt等Kuby Immunology，6 th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。参见，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。
“构架”或“F R”是指除互补决定区(CDR)残基之外的可变结构域残基。可变结构域的FR通常由四个FR结构域组成：FR1，FR2，FR3和FR4。因此，CDR和FR序列通常出现在重链可变结构域(VH)(或轻链可变结构域(VL))的以下序列中：
FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
除非另有说明，抗体各个结构域中的残基的编号根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
术语“全长抗体”、“完整的抗体”和“完整抗体”在本文被可交换地用于指结构与天然抗体结构基本相似或具有包含如本文所定义的Fc区的重链的抗体。
“Fv”是包含完整抗原结合位点的最小抗体片段。在一个实施方案中，双链Fv种类由一个重链可变结构域和一个轻链可变结构域以紧密的，非共价缔合的二聚体组成。在单链Fv(scFv)种类中，一个重链可变结构域和一个轻链可变结构域可以通过柔性肽接头共价连接从而使轻链和重链可以以类似于双链Fv种类的“二聚体”结构缔合。在这种构型中，正是每个可变结构域的三个CDR作用来限定了VH-VL二聚体的表面上的抗原结合位点。总而言之，六个CDR将抗原结合特异性赋予抗体。然而，即使是单个可变结构域(或只包含对抗原特异的三个CDR的一半Fv)也具有识别和结合抗原的能力，尽管亲和性低于完整结合位点。关于scFv的综述参见例如Pluckthun于The Pharmacology of Monoclonal Antibodies，卷113，Rosenburg和Moore编辑，(Springer-Verlag，New York，1994)，第269-315页中。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
“人抗体”指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于下述抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
“人共有构架”是指这样的构架，即在选择人免疫球蛋白VL或VH构架序列中，其代表最常出现的氨基酸残基。一般而言，对人免疫球蛋白VL或VH序列的选择是从可变结构域序列的亚型中选择。一般而言，该序列的亚型是如Kabat等(Sequences of Proteins of Immunological Interest，第五版，NIH Publication 91-3242，Bethesda MD(1991)，1-3卷)中公开的亚型。在一个实施方案中，对于VL，该亚型是如Kabat等(见上文)中的亚型κI。在一个实施方案中，对于VH，该亚型是如Kabat等(见上文)中的亚型III。
“人源化”抗体是指包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体将包含基本上所有的至少一个、通常两个可变结构域，其中所有或基本上所有的CDR(例如，CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃癌(包括胃肠癌和胃肠基质癌)，胰腺癌，成胶质细胞 瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，尿道癌，肝瘤，乳腺癌，结肠癌，直肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌，前列腺癌，外阴癌，甲状腺癌，肝癌，肛门癌，阴茎癌，黑素瘤，浅表扩散性黑素瘤，恶性雀斑样痣黑素瘤，肢端黑素瘤，结节性黑素瘤，多发性骨髓瘤和B细胞淋巴瘤，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖，脑瘤和脑癌，以及头颈癌，及相关转移。
术语“细胞增殖性病症”和“增殖性病症”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，细胞增殖性病症指癌症。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”，“癌性”，“细胞增殖性病症”，“增殖性病症”和“肿瘤”在本文中提到时并不互相排斥。
术语“感染性疾病”是指病原体引发的疾病，包括例如病毒感染、细菌感染、真菌感染或者原生动物例如寄生虫感染。
术语“慢性感染”是指这样的感染，其中传染原(例如，病原体如病毒、细菌、原生动物例如寄生虫、真菌或诸如此类)已经在感染的宿主中诱导了免疫应答，但尚未如在急性感染过程中一样被从宿主中清除或消除。慢性感染可以是持续性的、潜伏性的或缓慢的。尽管急性感染通常被免疫系统在数天或数周(如流感)内解决，持续性的感染可以相对低的水平持续数月、数年、数十年或一生(例如，乙型肝炎)。相比之下，潜伏性的感染的特征是长期的无症状活动，被一段时间的迅速增加的高度感染和升高的病原体水平不时打断(例如单纯疱疹)。最后，缓慢感染的特征是疾病症状的逐渐和连续增加，诸如长期的潜伏期，随后在临床症状出现后是延长的和进展的临床过程开始。不像潜伏性的和持续性的感染，慢性感染可以不以病毒增殖的急性期开始(例如，小RNA病毒感染(picornaviruses infection)、绵羊髓鞘脱落病毒(visna virus)、瘙痒病(scrapie)、克雅氏病(Creutzfeldt-Jakobdisease))。能够诱导慢性感染的示例性传染原包括病毒(例如，巨细胞病毒、EB病毒、乙型肝炎病毒、丙型肝炎病毒、单纯疱疹病毒I型和II型、人免疫缺陷病毒1型和2型，人乳头状瘤病毒、人T淋巴细胞病毒1型和2型，水痘-带状疱疹病毒等等)，细菌(例如，结核分枝杆菌(Mycobacterium tuberculosis)，李斯特菌属物种(Listeria spp.)，肺炎克雷伯菌(Klebsiella pneumoniae)，肺炎链球菌(Streptococcus pneumoniae)，金黄色葡萄球菌(Staphylococcus aureus)，疏螺旋体属物种(Borrelia spp.)，幽门螺旋杆菌(Helicobacter pylori)等等)，原生动物例如寄生虫(例如，利什曼原虫属物种(Leishmaniaspp.)，恶性疟原虫(Plasmodium falciparum)，血吸虫属物种(Schistosoma spp.)，弓形虫属物种(Toxoplasma spp.)，锥虫属物种(Trypanosoma spp.)，Taenia carssiceps等等)，和真菌(例如，曲霉属物种(Aspergillus spp.)，白色念珠菌(Candida albicans)，粗球孢子菌(Coccidioides immitis)，夹膜组织胞浆菌(Histoplasma capsulatum)，卡氏肺囊虫(Pneumocystis carinii)等等)。另外的传染原包括朊病毒或错误折叠的蛋白质，其通过在这些组织中进一步传播蛋白错误折叠影响脑或神经元结构，导致形成淀粉样蛋白斑(其导致细胞死亡、组织损伤和最终死亡)。由朊病毒感染导致的疾病的实例包括：克雅氏病及其变种(Creutzfeldt-Jakob disease and its varieties)，Gerstmann-Straussler-Scheinker syndrome(GSS)，致命性家族性失眠症(sFI)(fatal familial insomnia(sFI))，库鲁病(kuru)，瘙痒病(scrapie)，牛的牛海绵状脑病(BSE)(又名“疯牛”病)(Bovine spongiformencephalopathy(BSE)in cattle(aka“mad cow”disease))，以及其他各种动物形式的脑病[例如，传染性水貂脑病(TME)(transmissible mink encephalopathy(TME))，白尾鹿(white-tailed deer)、麇鹿(elk)和骡鹿(mule deer)中慢性消耗性疾病(chronicwasting disease(CWD))，猫海绵状脑病(feline spongiform encephalopathy)，尼牙薮羚(nyala)、羚羊(oryx)和更大角羚(greater kudu)中的外来有蹄类脑病(EUE)(exoticungulate encephalopathy(EUE)，鸵鸟的海绵状脑病(spongiform encephalopathy of  theostrich)]。
“免疫缀合物”是与一个或多个其它物质(包括但不限于细胞毒性剂或标记)缀合的抗体。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体，以及通过与直接被标记的另一种试剂反应来间接标记探针或抗体。间接标记的实例包括使用荧光标记的二级抗体进行的一级抗体的检测和具有生物素的DNA探针的末端标记，使得其可以用荧光标记的链霉抗生素蛋白来检测。
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848：79-87(2007)。
“分离的”核酸是指这样的核酸分子，其已经与其天然环境的组分分离。分离的核酸包括包含在通常包含该核酸分子的细胞中的核酸分子，但是该核酸分子存在于染色体外或在不同于其天然染色体位置的染色体位置处。
“分离的编码抗LAG-3抗体或其片段的核酸”是指一个或多个核酸分子，其编码抗体重链或轻链(或其片段)，包括在单一载体或分开的载体中的这样的核酸分子，以及存在于宿主细胞中的一个或多个位置处的这样的核酸分子。
术语“核酸”、“核酸序列”、“核苷酸序列”或“多核苷酸序列”和“多核苷酸”互换使用。它们指聚合物形式的任何长度的核苷酸(脱氧核糖核苷酸或核糖核苷酸)或其类似物。多核苷酸可以是单链或双链，并且如果为单链，可以是编码链或非编码(反义)链。多核苷酸可以包含修饰的核苷酸，如甲基化核苷酸及核苷酸类似物。核苷酸的序列可以被非核苷酸组分打断。可以在聚合后进一步修饰多核苷酸，如通过与标记组分缀合。核酸可以是重组多核苷酸或在自然界中不存在或与另一个多核苷酸以非自然布局连接的基因组来源、cDNA来源、半合成来源或合成来源的多核苷酸。
术语“多肽”、“肽”和“蛋白质”(如果为单链)在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。多肽可以从天然来源分离，可以通过重组技术从真核或原核宿主产生并且可以是合成方法的产物。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48：444-453)算法(在http：//www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http：//www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法，((1989)CABIOS，4：11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人，(1990)J.Mol.Biol.215：403-10的NBLAST及XBLAST程序执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本发明核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本发明蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人，(1997)Nucleic Acids Res.25：3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如XBLAST和NBLAST)的默认参数。参见http：//www.ncbi.nlm.nih.gov。
如本文所用，术语“在低严格性、中等严格性、高严格性或极高严格性条件下杂交”描述了杂交和洗涤条件。进行杂交反应的指导可以在通过引用方式并入的Current Protocols in Molecular Biology，John Wiley & Sons，N.Y.(1989)，6.3.1-6.3.6中找到。参考文献中描述了含水方法和非含水方法并且可以使用任一方法。本文中提及的特异性杂交条件如下：1)低严格性杂交条件是在约45℃于6X氯化钠/柠檬酸钠(SSC)中，随后至少在50℃(对于低严格性条件，可以增加洗涤的温度至55℃)于0.2X SSC，0.1％SDS中洗涤两次；2)中等严格性杂交条件是在约45℃于6X SSC中、随后在60℃在0.2X SSC、0.1％SDS中洗涤一次或多次；3)高严格性杂交条件是在约45℃在6X SSC中、随后在65℃于0.2X SSC、0.1％SDS中洗涤一次或多次；并且优选地4)极高严格性杂交条件是在65℃于0.5M磷酸钠、7％SDS中、随后在65℃于0.2X SSC、0.1％SDS中洗涤一次或多次。极高严格性条件(4)是优选的条件和除非另外说明，否则应当使用的一个条件。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、载体、赋形剂或稳定剂等。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“抗感染活性剂”包括在施用浓度和给药间隔下特异性抑制或消除微生物生长但对宿主不致命的任何分子，所述微生物诸如病毒、细菌、真菌或原生动物，例如寄生虫。用于本文时，术语抗感染活性剂包括抗生素、抗菌剂、抗病毒剂、抗真菌剂和抗原生动物剂。在一个具体方面中，抗 感染活性剂在施用浓度和给药间隔对宿主是无毒的。
抗细菌的抗感染活性剂或抗菌剂可广泛的分类为杀菌的(即，直接杀死)或抑菌的(即，阻止分裂)。抗菌的抗感染活性剂可进一步再分类为窄谱抗菌剂(即，仅影响小类细菌亚型，例如，革兰氏阴性等)或广谱抗菌剂(即，影响广泛种类)。实例包括阿米卡星、庆大霉素、格尔德霉素、除莠霉素、莫匹罗星、呋喃妥因、吡嗪酰胺、奎奴普丁/达福普汀、利福平/异福酰胺或替硝唑等。
术语“抗病毒剂”包括抑制或消除病毒生长、致病和/或存活的任何物质。这包括例如阿昔洛韦、西多福韦、齐多夫定、去羟肌苷(ddI，VIDEX)、扎西他滨(ddC，HIVID)、司他夫定(d4T，ZERIT)、拉米夫定(3TC，EPIVIR))、阿巴卡韦(ZIAGEN)、恩曲他滨(EMTRIVA)等。
术语“抗真菌剂”包括抑制或消除真菌生长、致病和/或存活的任何物质。这包括例如那他霉素、龟裂杀菌素、非律平、制霉菌素、两性霉素B、坎底辛、绿叶刺蕊草(patchouli)、印度楝树种子油(neem seed oil)、椰子油(Coconut Oil)等。
术语“抗原生动物剂”包括抑制或消除原生动物生物体(例如寄生虫)生长、发病和/或存活的任何物质。抗原生动物剂的实例包括抗疟疾试剂例如，奎宁、奎尼丁等
示例性的抗菌剂、抗病毒剂、抗真菌剂、抗原生动物剂参见例如WO2010/077634等。抗感染活性剂还参见例如WO2014/008218、WO2016/028672或WO2015/138920。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
“受试者/患者样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。肿瘤样品的例子在本文中包括但不限于肿瘤活检、细针吸出物、支气管灌洗液、胸膜液(胸水)、痰液、尿液、手术标本、循环中的肿瘤细胞、血清、血浆、循环中的血浆蛋白质、腹水、衍生自肿瘤或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定的、石蜡包埋的肿瘤样品或冷冻的肿瘤样品。
术语“包装插页”用于指治疗产品的商业包装中通常包含的用法说明书，其含有关于涉及此类治疗产品应用的适应症，用法，剂量，施用，联合疗法，禁忌症和/或警告的信息。
本发明的抗体
因此，在一些实施方案中，本发明的抗体或其片段结合LAG-3。在一些实施方案中，本发明的抗体或其片段结合哺乳动物LAG3，例如人LAG-3或小鼠LAG-3。例如，抗体分子与LAG-3上的表位(例如，线性或构象表位)特异性结合。在一些实施方案中，抗体分子与LAG-3的一个或多个胞外Ig样结构域(例如，LAG-3的第一、第二、第三或第四胞外Ig样结构域)结合。
在一些实施方案中，本发明的抗LAG-3抗体或其片段具有以下一种或多种性质：
(1)本发明的抗LAG-3抗体或其片段以高亲和力结合LAG-3(例如人LAG-3)，例如，以以下平衡解离常数(K D)与LAG-3结合，所述K D小于大约100nM，优选地，小于或等于大约50nM，更优选地小于或等于大约20nM，更优选地小于或等于大约10nM、9nM、8nM、7nM、6nM、5nM、4nM、3nM或2nM，最优选地，所述K D小于或等于大约1nM、0.9nM、0.8nM或0.7nM。在一些实施方案中，本发明的抗LAG-3抗体以0.1-20nM，优选地0.5-20nM，更优选地0.5-10nM、0.5-8nM、0.5-5nM， 最优选地0.5-1nM、0.5-0.8nM、0.5-0.7nM、0.6-0.7nM的K D结合LAG-3。在一些实施方案中，LAG-3为人LAG-3。在一些实施方案中，LAG-3为小鼠LAG-3。在一些实施方案中，抗体结合亲和力是使用生物光干涉测定法(例如Fortebio亲和测量)测定法测定的。
(2)本发明的抗体或其片段结合表达人LAG-3的细胞，例如，以小于或等于大约3.3nM、3nM、2nM、1.5nM、1.4nM、1.3nM、1.2nM、1.1nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM或0.5nM的EC50。在一些实施方案中，所述结合用流式细胞术(例如FACS)测定。在一些实施方案中，表达人LAG-3的细胞为表达人LAG-3的293细胞(例如HEK293细胞)。
(3)本发明的抗体或其片段结合表达小鼠LAG-3的细胞，例如，以小于或等于大约15000nM、14000nM或13000nM的EC50。在一些实施方案中，本发明的抗体或其片段结合表达小鼠LAG-3的细胞，例如，以小于或等于大约50nM的EC50，例如以大约40-50nM的EC50、大约40-45nM的EC50或大约42nM的EC50。在一些实施方案中，用流式细胞术(例如FACS)测定所述结合。在一些实施方案中，表达小鼠LAG-3的细胞为表达小鼠LAG-3的中国仓鼠卵巢(CHO)细胞。
(4)本发明的抗体或其片段抑制LAG-3的相关活性，例如以小于或等于大约20nM、10nM、9nM、8nM、7nM、6nM或5nM的IC 50，优选以大约1-6nM、1-5nM、4nM、4.5nM、5nM、5.1nM、5.2nM、5.3nM、5.4nM、5.5nM、5.6nM、5.7nM、5.8nM、5.9nM或6nM的IC 50。在一些实施方案中，LAG-3的相关活性是MHC II类分子与LAG-3的结合。在一些实施方案中，本发明的抗体或其片段以小于或等于大约20nM、10nM、9nM、8nM、7nM、6nM或5nM的IC 50，优选以大约1-6nM、1-5nM、4nM、4.5nM、5nM、5.1nM、5.2nM、5.3nM、5.4nM、5.5nM、5.6nM、5.7nM、5.8nM、5.9nM或6nM的IC 50抑制LAG-3与表达MHCII类分子的细胞上的MHCII类分子的结合。在一些实施方案中，MHCII类分子是HLA-DR。在一些实施方案中，细胞为CHO细胞。在一些实施方案中，用流式细胞术(例如FACS)测量所述本发明的抗体或其片段对LAG-3的相关活性的抑制。
(5)本发明的抗体或其片段结合激活的CD4+和/或CD8+T细胞的表面上的内源LAG-3，例如以小于或等于大约35pM、30pMnM、25pM、20pM、15pM、14pM或13pM的EC 50，优选大约1-20pM、5-20pM、5-15pM、10-15pM、11-13pM、10pM、11pM、12pM或13pM的EC 50。在一些实施方案中，激活的CD4+T细胞为激活的人CD4+T细胞。在一些实施方案中，用流式细胞术(例如FACS)测定上述结合。在一些实施方案中，在Accuri C6系统中进行流式细胞术。
(6)本发明的抗体或其片段抑制LAG-3的一种或多种活性，例如，导致以下一种或多种：CD4+T淋巴细胞的抗原依赖性刺激增加；T细胞增殖增加；活化抗原(例如，CD25)的表达增加；细胞因子(例如，干扰素-γ(IFN-γ)、白介素-2(IL-2)或白介素-4(IL-4))的表达增加；趋化因子(例如，CCL3、CCL4或CCL5)的表达增加；Treg细胞的阻抑活性减少；T细胞稳态增加；肿瘤浸润型淋巴细胞增加；或癌细胞的免疫逃避减少。
(7)本发明的抗LAG-3抗体或其片段能够诱发抗体依赖性细胞介导的细胞毒性(ADCC)。
在一些实施方案中，本发明的抗LAG-3抗体或其抗原结合片段具有以下一个或多个特性：
(i)显示与本发明抗体(例如表3所列的任一抗体)对LAG-3相同或相似的结合亲和力和/或特异性；
(ii)抑制(例如，竞争性抑制)本发明抗体(例如表3所列的任一抗体)与LAG-3的结合；
(iii)与本发明抗体(例如表3所列的任一抗体)结合相同或重叠的表位；
(iv)与本发明抗体(例如表3所列的任一抗体)竞争结合LAG-3；
(v)具有本发明抗体(例如表3所列的任一抗体)的一个或多个生物学特性。
示例性的抗体
在一些实施方案中，本发明的抗LAG-3抗体或其抗原结合片段包含重链可变区(VH)，其中所述VH包含
(i)表B所列任一抗体的VH中所含的三个互补决定区域(CDR)，或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列。
在一些实施方案中，本发明的抗LAG-3抗体或其抗原结合片段包含轻链可变区(VL)，其中所述VL包含：
(i)表B所列任一抗体的VL中所含的三个互补决定区域(CDR)；或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列。
在一些实施方案中，本发明的抗LAG-3抗体或其抗原结合片段包含重链可变区VH和/或轻链可变区VL，其中
(a)所述VH包含
(i)表B所列任一抗体的VH中所含的三个互补决定区域(CDR)，或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；和/或
(b)所述VL包含：
(i)表B所列任一抗体的VL中所含的三个互补决定区域(CDR)；或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列。
在优选的实施方案中，VH包含选自SEQ ID NO：22、23、24或25所示的氨基酸序列，或由所述氨基酸序列组成。
在优选的实施方案中，VL包含选自SEQ ID NO：26、27、28或29所示的氨基酸序列，或由所述氨基酸序列组成。
在优选的实施方案中，本发明抗LAG-3抗体或其抗原结合片段包含如SEQ ID NO：22、23、24或25所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：26、27、28或29所示的轻链可变区的3个互补决定区LCDR。
在一些实施方案中，本发明的抗LAG-3抗体或其抗原结合片段包含重链可变区(VH)和/或轻链可变区(VL)，其中
(i)所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含选自SEQ ID NO：1、2、3、4或17的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与选自SEQ ID NO：1、2、3、4或17的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；HCDR2包含选自SEQ ID NO：5、6、7或18的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与选自SEQ ID NO：5、6、7或18的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；HCDR3包含选自SEQ ID NO：8、9、10或19的氨基酸序列或由所述氨基酸序列组成，或者HCDR3包含与选自SEQ ID NO：8、9、10或19的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；
和/或
(ii)其中所述VL包含互补决定区域(CDR)LCDR1、LCDR2和LCDR3，其中LCDR1包含选自SEQ ID NO：11、12或20的氨基酸序列或由所述氨基酸序列组成，或者LCDR1包含与选自SEQ ID NO：11、12或20的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；LCDR2包含选自SEQ ID NO：13的氨基酸序列或由所述氨基酸序列组成， 或者LCDR2包含与选自SEQ ID NO：13的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；LCDR3包含选自SEQ ID NO：14、15、16或21的氨基酸序列或由所述氨基酸序列组成，或者LCDR3包含与选自SEQ ID NO：14、15、16或21的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在优选的实施方案中，本发明提供抗LAG-3抗体或其抗原结合片段，其包含重链可变区(VH)和/或轻链可变区(VL)，其中
(a)所述VH包含
(i)表A所示的HCDR1、HCDR2和HCDR3的组合；或
(ii)(i)的HCDR组合的变体，所述变体在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；
和/或
(ii)所述VL包含
(i)表A所示的LCDR1、LCDR2和LCDR3的组合；或者
(ii)(i)的LCDR组合的变体，所述变体在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)。
在优选的实施方案中，本发明提供抗LAG-3抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3并且所述VL包含(CDR)LCDR1、LCDR2和LCDR3，其中所述抗体或其抗原结合片段所包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3的组合如下表(表A)所示：
表A：本发明抗体或其抗原结合片段中HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3的示例性组合
Figure PCTCN2018124315-appb-000002
在一些实施方案中，本发明的抗LAG-3抗体或其抗原结合片段包含重链可变区VH和/或轻链可变区VL，其中，
(a)重链可变区VH
(i)包含与选自SEQ ID NO：22、23、24或25的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
(ii)包含选自SEQ ID NO：22、23、24或25的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：22、23、24或25的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中；
和/或
(b)轻链可变区VL
(i)包含与选自SEQ ID NO：26、27、28或29的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：26、27、28或29的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：26、27、28或29的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。
在优选的实施方案中，本发明提供抗LAG-3抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中所述抗体或其抗原结合片段所包含的重链可变区VH和轻链可变区VL的组合如下表(表B)所示：
表B：本发明抗体或其抗原结合片段中重链可变区VH和轻链可变区VL的示例性组合
Figure PCTCN2018124315-appb-000003
在一些实施方案中，本发明的抗LAG-3抗体或其抗原结合片段包含重链和/或轻链，其中
(a)重链
(i)包含与选自SEQ ID NO：30、31、32或33的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：30、31、32或33的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：30、31、32或33的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中；
和/或
(b)轻链
(i)包含与选自SEQ ID NO：34、35、36或37的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：34、35、36或37的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：34、35、36或37的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中。
在优选的实施方案中，本发明提供抗LAG-3抗体或其抗原结合片段，其包含重链和轻链，其中所述抗体或其抗原结合片段所包含的重链和轻链的组合如下表(表C)所示：
表C：本发明抗体或其抗原结合片段中重链和轻链的示例性组合
Figure PCTCN2018124315-appb-000004
在一些实施方案中，本发明抗LAG-3抗体或其片段的重链和/或轻链还包含信号肽序列，例如METDTLLLWVLLLWVPGSTG(SEQ ID NO：48)。
在本发明的一个实施方案中，本文所述的氨基酸改变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。
任选地，本发明的抗LAG-3抗体包括对轻链可变区、重链可变区、轻链或重链的翻译后修饰。示例性的翻译后修饰包括二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合。
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。示例性的置换如下表D所示：
表D
Figure PCTCN2018124315-appb-000005
在某些实施方案中，本文中所提供的抗体经改变以增加或降低抗体糖基化的程度。对抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。
举例而言，可实施一或多种氨基酸置换以消除一或多个可变区构架糖基化位点，由此消除该位 点处的糖基化。这类无糖基化可增加抗体对抗原的亲和力。例如参见美国专利第5,714,350号及第6,350,861号。可制备具有改变类型的糖基化的抗体，例如具有减小量的岩藻糖基残基的低岩藻糖化抗体或具有增加的等分GlcNac结构的抗体。这类改变的糖基化模式已显示可增加抗体的ADCC能力。可通过例如在具有改变的糖基化体系的宿主细胞中表达抗体来实现这类糖类修饰。具有改变的糖基化体系的细胞已在本领域中阐述，且可用作为在其中表达本发明的抗体以由此产生具有改变的糖基化的抗体的宿主细胞。举例而言，细胞系Ms704、Ms705及Ms709缺乏岩藻糖基转移酶基因FUT8(α(1，6)-岩藻糖基转移酶)，从而在Ms704、Ms705及Ms709细胞系中表达的抗体在其糖类上缺乏岩藻糖。可以通过使用两种代替载体靶向破坏CHO/DG44细胞中的FUT8基因来创建Ms704、Ms705及Ms709FUT8-/-细胞系(参见美国专利公开20040110704号及Yamane-Ohnuki等人(2004)Biotechnol Bioeng 87：614-22)。EP 1,176,195描述了具有功能受破坏的编码岩藻糖基转移酶的FUT8基因的细胞系，从而在这类细胞系中表达的抗体通过减少或消除α-1，6键相关酶来展现低岩藻糖化。EP 1,176,195还描述了具有向结合抗体Fc区的N-乙酰葡糖胺添加岩藻糖的低酶活性或不具有该酶活性的细胞系，例如大鼠骨髓瘤细胞系YB2/0(ATCC CRL 1662)。PCT公开文本WO 03/035835描述了一种变体CHO细胞系Lec13细胞，其中使岩藻糖附接至Asn(297)-连接的糖类的能力降低，从而亦导致在该宿主细胞中表达的抗体的低岩藻糖化(亦参见Shields等人(2002)J.Biol.Chem.277：26733-26740)。具有经修饰的糖基化概况的抗体亦可产生于鸡蛋中，如PCT公开文本WO06/089231中所阐述。备选地，具有经修饰的糖基化概况的抗体可产生于植物细胞(例如青萍(Lemna))中。在植物系统中产生抗体的方法公开在对应于Alston及Bird LLP代理档案号：040989/314911的2006年8月11日提出申请的美国专利申请案中。PCT公开文本WO 99/54342描述了经工程化以表达糖蛋白修饰糖基转移酶(例如β(1，4)-N-乙酰葡糖胺基转移酶III(GnTIII))的细胞系，从而在该工程化细胞系中表达的抗体展现增加的等分GlcNac结构，其导致增加的抗体的ADCC活性(亦参见Umana等人(1999)Nat.Biotech.17：176-180)。备选地，可使用岩藻糖苷酶切除抗体的岩藻糖残基；举例而言，岩藻糖苷酶α-L-岩藻糖苷酶自抗体去除岩藻糖基残基(Tarentino等人(1975)Biochem.14：5516-23)。
在本发明的一个实施方案中，本发明所述抗体或片段用经工程改造的酵母N-连接的聚糖或CHO N-连接的聚糖糖基化。
本发明所涵盖的另一种对本文所述抗体或其片段的修饰是聚乙二醇化(pegylation)。可对抗体实施聚乙二醇化以例如增加抗体的生物(例如血清)半衰期。为将抗体聚乙二醇化，通常使抗体或其片段与聚乙二醇(PEG)(例如PEG的反应性酯或醛衍生物)在其中一或多个PEG基团变得附接至抗体或抗体片段的条件下发生反应。优选地，经由使用反应性PEG分子(或类似反应性水溶性聚合物)进行酰化反应或烷基化反应来实施聚乙二醇化。如本文中所使用，术语“聚乙二醇”意图涵盖已用于衍生化其他蛋白质的PEG的任一形式，例如单(C1-C10)烷氧基-或芳氧基聚乙二醇或聚乙二醇-马来酰亚胺。在某些实施方案中，要聚乙二醇化的抗体是无糖基化抗体。本领域中已知使蛋白质聚乙二醇化的方法且可将其应用于本发明的抗体，例如参见EP 0154316及EP 0401384。
在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体，以便增强例如抗体治疗癌症或细胞增殖性疾病的有效性。本文中公开的抗-LAG3抗体和其抗原结合片段也包括具有修饰的(或封闭的)Fc区以提供改变的效应子功能的抗体和片段。参见，例如，美国专利号5,624,821、WO2003/086310、WO2005/120571、WO2006/0057702。可以使用这样的修饰增强或抑制免疫系统的各种反应，这可能具有在诊断和治疗中的有益效果。Fc区的修饰包括氨基酸变化(置换、缺失和插入)、糖基化或去糖基化、和添加多个Fc。对Fc的修饰还可以改变治疗性抗体中的抗体的半衰期，从而实现更低频率的给药和因而增加的方便和减少的材料使用。参见Presta(2005)J.Allergy Clin.Immunol.116：731，734-735页。
在一个实施方案中，可以改变抗体的半胱氨酸残基数目以修饰抗体特性。例如对CH1的铰链 区实施修饰，从而改变(例如增加或降低)铰链区中的半胱氨酸残基的数目。此办法进一步阐述于美国专利第5,677,425号中。可以改变CH1的铰链区中半胱氨酸残基的数目以例如促进轻链及重链的装配或增加或降低抗体的稳定性。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。聚合物可具有任何分子量，并且可为分支或未分支的。连接到抗体的聚合物的数目可变化，并且如果连接多于一个聚合物，那么其可为相同或不同分子。一般说来，用于衍生化的聚合物的数目和/或类型可基于包括但不限于有待改善的抗体的特定特性或功能、抗体衍生物是否将在确定条件下用于疗法中等考虑因素加以确定。
在一些实施方案中，本发明涵盖抗LAG-3抗体的片段。抗体片段的实例包括但不限于Fv、Fab、Fab′、Fab’-SH，F(ab’) 2、双抗体、线性抗体、单链抗体(例如scFv)；和由抗体片段形成的多特异性抗体。
例如，抗体分子可以包括重链(HC)可变结构域序列和轻链(LC)可变结构域序列。在一个实施方案中，抗体分子包含一条重链和一条轻链(在本文中称作半抗体)或由其组成。在另一个例子中，抗体分子包含两个重链可变结构域序列和两个轻链可变结构域序列，因而形成两个抗原结合位点，如Fab、Fab’、F(ab’)2、Fc、Fd、Fd’、Fv、单链抗体(例如scFv)、单结构域抗体、双抗体(Dab)(双价和双特异性)和嵌合(例如，人源化)抗体，它们可以通过修饰完整抗体产生，或使用重组DNA技术从头合成那些抗体分子。这些功能性抗体片段保留选择性地与其相应抗原或受体结合的能力。抗体和抗体片段可以来自任何抗体类别，包括但不限于IgG、IgA、IgM、IgD和IgE并且来自任何抗体亚类(例如，IgG1、IgG2、IgG3和IgG4)。抗体分子的制备可以是单克隆或多克隆的。抗体也可以是人抗体、人源化抗体、嵌合抗体、CDR移植抗体或体外生成的抗体。抗体可以具有例如选自IgG1、IgG2、IgG3或IgG4的重链恒定区。抗体还可以具有例如选自κ或λ的轻链。
本发明的抗体也可以是单结构域抗体。单结构域抗体可以包括其互补决定区是单结构域多肽组成部分的抗体。例子包括但不限于重链抗体、天然缺少轻链的抗体、衍生自常规4-链抗体的单结构域抗体或工程化抗体。单结构域抗体可以是现有技术的任何抗体，或将来的任何单结构域抗体。单结构域抗体可以衍生自任何物种，包括但不限于小鼠、人、骆驼、羊驼、鱼类、鲨鱼、山羊、兔和牛。根据本发明的另一个方面，单结构域抗体是天然存在的单结构域抗体，称作缺少轻链的重链抗体。这类单结构域抗体例如在WO 94/04678中公开。单结构域抗体或纳米抗体可以是自骆驼科(Camelidae)物种(例如骆驼、羊驼、单峰驼、驼羊和原驼)中产生的抗体。除骆驼之外的其他物种可以产生天然缺少轻链的重链抗体；这类单结构域抗体处于本发明的范围内。
在一些实施方案中，本发明的抗LAG-3抗体是人源化抗体。用于使抗体人源化的不同方法是技术人员已知的，如由Almagro&Fransson综述的，其内容通过提述完整并入本文(Almagro JC和Fransson J，(2008)Frontiers in Bioscience 13：1619-1633)。Almagro&Fransson区分理性办法和经验办法。理性办法的特征在于生成少数工程化抗体变体并评估其结合或任何其它感兴趣的特性。如果设计的变体不产生预期的结果，那么启动新一轮的设计和结合评估。理性办法包括CDR嫁接、表面重建(Resurfacing)、超人源化(Superhumanization)和人字符串内容优化(Human StringContent Optimization)。相比之下，经验办法基于生成大的人源化变体库并使用富集技术或高通量筛选选出最佳克隆。因而，经验办法依赖于能够对大量抗体变体进行搜索的可靠的选择和/或筛选系统。体外展示技术，如噬菌体展示和核糖体展示满足这些要求并且是技术人员公知的。经验办法包括FR库、导向选择(Guided selection)、构架改组(Framework-shuffling)和Humaneering。
在一些实施方案中，本发明的抗LAG-3抗体是人抗体。可使用本领域中已知的各种技术来制备人抗体。人抗体一般描述于van Dijk和van de Winkel，Curr.Opin.Pharmacol 5：368-74(2001)以及Lonberg，Curr.Opin.Immunol 20：450-459(2008)。例如，可以使用携带人免疫球蛋白基因而非小鼠系统的转基因小鼠，产生人单克隆抗体(参见，例如，Wood等人，国际申请WO 91/00906；Kucherlapati等人，PCT公开WO 91/10741；Lonberg等人，国际申请WO 92/03918；Kay等人，国际申请92/03917；Lonberg，N.等人，1994 Nature 368：856-859；Green，L.L.等人，1994 Nature Genet.7：13-21；Morrison，S.L.等人，1994Proc.Natl.Acad.Sci.USA 81：6851-6855；Bruggeman等人，1993 Year Immunol 7：33-40；Tuaillon等人，1993 PNAS 90：3720-3724；Bruggeman等人，1991 Eur J Immunol 21：1323-1326)。
在一些实施方案中，本发明的抗LAG-3抗体是非人抗体，例如啮齿类(小鼠或大鼠)抗体、山羊抗体、灵长类(例如，猴)抗体、骆驼抗体。优选地，非人抗体是啮齿类(小鼠或大鼠)抗体。产生啮齿类抗体的方法是本领域已知的。
可通过在组合文库中筛选具有所需活性的抗体来分离本发明抗体。举例来说，本领域中已知多种用于产生噬菌体展示文库并且在这些文库中筛选具有所需结合特征的抗体的方法。这些方法于例如Hoogenboom等人，Methods in Molecular Biology 178：1-37(O′Brien等人编，Human Press，Totowa，NJ，2001)中评述，并且进一步于例如McCafferty等人，Nature 348：552-554；Clackso等人，Nature 352：624-628(1991)；Marks等人，J.Mol.Biol.222：581-597(1992)；Marks及Bradbury，Methods in Molecular Biology 248：161-175(Lo编，Human Press，Totowa，NJ，2003)；Sidhu等人，J.Mol.Biol.338(2)：299-310(2004)；Lee等人，J.Mol.Biol340(5)：1073-1093(2004)；Fellouse，Proc.Natl.Acad.Sci.USA 101(34)：12467-12472(2004)；以及Lee等人，J.Immunol.Methods284(1-2)：119-132(2004)中描述。
在一个实施方案中，抗体分子是单特异性抗体分子并且结合单一表位。例如，单特异性抗体分子具有各自结合相同表位的多个免疫球蛋白可变结构域序列。
在一个实施方案中，抗体分子是多特异性抗体分子，例如，它包含多个免疫球蛋白可变结构域序列，其中多个免疫球蛋白可变结构域序列的第一免疫球蛋白可变结构域序列具有针对第一表位的结合特异性并且多个免疫球蛋白可变结构域序列的第二免疫球蛋白可变结构域序列具有针对第二表位的结合特异性。在一个实施方案中，第一和第二表位在相同抗原(例如，相同蛋白质(或多聚体蛋白的亚基))上。在一个实施方案中，第一和第二表位重叠。在一个实施方案中，第一和第二表位不重叠。在一个实施方案中，第一和第二表位在不同抗原(例如，不同蛋白质(或多聚体蛋白的不同亚基))上。在一个实施方案中，多特异性抗体分子包含第三、第四或第五免疫球蛋白可变结构域。在一个实施方案中，多特异性抗体分子是双特异性抗体分子、三特异性抗体分子或四特异性抗体分子。
在一个实施方案中，多特异性抗体分子是双特异性抗体分子。双特异性抗体对不多于两种抗原具有特异性。双特异性抗体分子以针对第一表位具有结合特异性的第一免疫球蛋白可变结构域序列和针对第二表位具有结合特异性的第二免疫球蛋白可变结构域序列为特征。在一个实施方案中，第一和第二表位在相同抗原(例如，相同蛋白质(或多聚体蛋白的亚基))上。在一个实施方案中，第一和第二表位重叠。在一个实施方案中，第一和第二表位不重叠。在一个实施方案中，第一和第二表位在不同抗原(例如，不同蛋白质(或多聚体蛋白的不同亚基))上。在一个实施方案中，双特异性抗体分子包含针对第一表位具有结合特异性的重链可变结构域序列和轻链可变结构域序列以及针对第二表位具有结合特异性的重链可变结构域序列和轻链可变结构域序列。在一个实施方案中，双特异性抗体分子包含针对第一表位具有结合特异性的半抗体和针对第二表位具有结合特异性的半抗体。在一个实施方案中，双特异性抗体分子包含针对第一表位具有结合特异性的半抗体或其片段和针对第二表位具有结合特异性的半抗体或其片段。在一个实施方案中，双特异性抗体分子包含针对 第一表位具有结合特异性的scFv或其片段和针对第二表位具有结合特异性的scFv或其片段。在一个实施方案中，第一表位位于LAG-3上并且第二表位位于PD-1、TIM-3、CEACAM(例如，CEACAM-1和/或CEACAM-5)、PD-L1或PD-L2上。在优选的实施方案中，第二表位位于PD-1上。
在一些实施方案中，本发明的抗体可以是嵌合抗体(例如，人恒定结构域/小鼠可变结构域)。本文中使用的“嵌合抗体”是具有来自第一抗体的可变结构域和来自第二抗体的恒定结构域的抗体，其中所述第一抗体和第二抗体来自不同的物种(美国专利号4,816,567；和Morrison等人，(1984)Proc.Natl.Acad.Sci.USA 81：6851-6855)。通常，所述可变结构域得自来自实验动物(诸如啮齿动物)的抗体(“亲本抗体”)，且所述恒定结构域序列得自人抗体，使得得到的嵌合抗体在人受试者中引起不利免疫应答的可能性低于亲本(例如，小鼠)抗体。
在一些实施方案中，本发明还涵盖与其它物质，例如治疗性模块或标记物，如细胞毒性剂或免疫调节剂，缀合的抗LAG-3单克隆抗体(“免疫缀合物”)。细胞毒性剂包括任何对细胞有害的药剂。适合于形成免疫缀合物的细胞毒性剂(例如化疗剂)的例子是本领域中已知的，参见例如WO05/103081或WO2015/138920或CN 107001470A。例如，细胞毒性剂包括但不限于：放射性同位素(例如，碘(131I或125I)、钇(90Y)、镥(177Lu)、锕(225Ac)、镨、砹(211At)、铼(186Re)、铋(212Bi或213Bi)、铟(111In)、锝(99mTc)、磷(32P)、铑(188Rh)、硫(35S)、碳(14C)、氚(3H)、铬(51Cr)、氯(36Cl)、钴(57Co或58Co)、铁(59Fe)、硒(75Se)或镓(67Ga)。细胞毒性剂的实例还包括化疗剂或其它治疗药物，例如，紫杉酚、松胞菌素B、短杆菌肽D、溴化乙锭、依米丁、丝裂霉素、依托泊苷、替尼泊苷、长春新碱、长春碱、秋水仙碱、多柔比星、道诺霉素、二羟基炭疽菌素二酮、米托蒽醌、光神霉素、放线菌素D、1-脱氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普萘洛尔、嘌呤霉素、类美登素(maytansinoid)，例如，美登醇(参见美国专利号5,208,020)、CC-1065(参见美国专利号5,475,092、5,585,499、5,846,545)及其类似物或同源物。细胞毒性剂还包括例如抗代谢物(例如，甲氨蝶呤、6-巯基嘌呤、6-硫鸟嘌呤、阿糖胞苷、5-氟尿嘧啶达卡巴嗪)、烷基化剂(例如，氮芥、硫代苯丁酸氮芥、CC-1065、美法仑、卡莫司汀(BSNU)和罗莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲霉素、丝裂霉素C和顺-二氯二胺铂(II)(DDP)顺铂)、蒽环类(例如，道诺霉素(以前柔红霉素)和多柔比星)、抗生素(例如，更生霉素(dactinomycin)(前称放线菌素D)、博来霉素、光神霉素和安曲霉素(AMC))和抗有丝分裂剂(例如，长春新碱、长春碱、紫杉酚和类美登素))；生长抑制剂；酶及其片段如核酸水解酶；抗生素；毒素如小分子毒素或细菌、真菌、植物或动物起源的酶促活性毒素，包括其片段和/或变体；和已知的各种抗肿瘤或抗癌剂。
本发明的核酸以及包含其的宿主细胞
在一方面，本发明提供了编码以上任何抗LAG-3抗体或其片段的核酸。所述核酸可以编码包含抗体的轻链可变区和/或重链可变区的氨基酸序列，或包含抗体的轻链和/或重链的氨基酸序列。
例如，本发明的示例性的核酸包含与选自SEQ ID NO：38、39、40、41、42、43、44或45的核酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的核酸序列，或包含选自SEQ ID NO：38、39、40、41、42、43、44、45的核酸序列。例如，本发明的核酸包含编码选自SEQ ID NO：22、23、24、25、26、27、28、29、30、31、32、33、34、35、36或37中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO：22、23、24、25、26、27、28、29、30、31、32、33、34、35、36或37中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。
本发明还涵盖与下述核酸在严格性条件下杂交的核酸或与下述核酸具有一个或多个置换(例如保守性置换)、缺失或插入的核酸：包含与选自SEQ ID NO：38、39、40、41、42、43、44、45的核酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的 核酸序列的核酸；或包含选自SEQ ID NO：38、39、40、41、42、43、44、45的核酸序列的核酸；包含编码选自SEQ ID NO：22、23、24、25、26、27、28、29、30、31、32、33、34、35、36或37中任一项所示氨基酸序列的核酸序列的核酸；或包含编码与选自SEQ ID NO：22、23、24、25、26、27、28、29、30、31、32、33、34、35、36或37中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸序列的核酸。
在一个实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。可以使用众多载体系统。例如，一个类别的载体利用衍生自动物病毒例如牛乳头瘤病毒、多瘤病毒、腺病毒、痘苗病毒、杆状病毒、逆转录病毒(劳斯肉瘤病毒、MMTV或MOMLV)或SV40病毒的DNA元件。另一类载体利用衍生自RNA病毒如Semliki森林病毒、东方马脑炎病毒和黄病毒的RNA元件。在优选的实施方案中，本发明的表达载体是pTT5表达载体。
另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。标记物可以例如向营养缺陷型宿主提供原养型、杀生物抗性(例如，抗生素)或重金属(如铜)抗性等。可选择标记基因可以与待表达的DNA序列直接连接或通过共转化引入相同的细胞中。也可能需要额外元件以便最佳合成mRNA。这些元件可以包括剪接信号，以及转录启动子、增强子和终止信号。
一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。
在一个实施方案中，提供包含编码本文所述的抗体分子的核酸或本文所述载体的宿主细胞。用于克隆或表达编码抗体的核酸或载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，抗体可在细菌中产生，特别当不需要糖基化和Fc效应子功能时。对于抗体片段和多肽在细菌中的表达，见，例如，美国专利号5,648,237，5,789,199和5,840,523，还见Charlton，Methods in Molecular Biology，卷248(B.K.C.Lo，编辑，Humana Press，Totowa，NJ，2003)，第245-254页，其描述抗体片段在大肠杆菌中的表达)。在表达后，抗体可以从可溶级分中的细菌细胞糊状物分离，并且可以进一步纯化。在一个实施方案中，宿主细胞是大肠杆菌细胞。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如人细胞)、昆虫细胞、植物细胞或适用于制备抗体或其抗原结合片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是编码抗体的载体的合适克隆或表达宿主，包括糖基化途径已经进行“人源化”从而导致产生具有部分或完全人糖基化模式的抗体的真菌和酵母菌株。参见Gerngross，Nat.Biotech.22：1409-1414(2004)，和Li等，Nat.Biotech.24：210-215(2006)。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(293HEK或293细胞，如例如Graham等，J.Gen Virol.36：59(1977)中所描述的)等。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞(Urlaub等，Proc.Natl.Acad.Sci.USA 77：216(1980))；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。关于适合产生抗体的某些哺乳动物宿主细胞系的综述见例如Yazaki和Wu，Methods in Molecular Biology，卷248(B.K.C.Lo，ed.，Humana Press，Totowa，NJ)，第255-268页(2003)。其它有用的宿主细胞还包括但不限于Vero细胞、Hela细胞、COS细胞、CHO细胞、HEK293细胞、BHK细胞、MDCKII细胞、PerC6细胞系(例如，来自Crucell的PERC6 细胞)、卵母细胞和来自转基因动物的细胞，例如乳腺上皮细胞。合适的昆虫细胞包括但不限于Sf9细胞。
制备本发明抗体及其抗原结合片段的方法
可以重组生产本文公开的抗-LAG3抗体。存在几种本领域已知的用于生产重组抗体的方法。用于重组生产抗体的方法的一个例子公开在美国专利号4,816,567中。
在一个实施方案中，提供了制备抗LAG-3抗体的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体的核酸的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。为了重组产生抗LAG-3抗体，分离编码抗体(例如上文所描述的抗体)的核酸，并将其插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序(例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针进行)。
在一个实施方案中，宿主细胞包含含有编码抗体的VL的氨基酸序列的核酸以及编码抗体的VH的氨基酸序列的核酸的载体。在一个实施方案中，宿主细胞包含含有编码抗体的VL的氨基酸序列的核酸的第一载体和含有编码抗体的VH的氨基酸序列的核酸的第二载体。
测定法
可以通过本领域中已知的多种测定法对本文中提供的抗LAG-3抗体进行鉴定、筛选，或表征其物理/化学特性和/或生物学活性。一方面，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA、Western印迹，流式细胞术、抗体分子复合的磁珠等来进行。可使用本领域已知方法来测定LAG-3结合，本文中公开了例示性方法。在一些实施方案中，使用生物光干涉测定法(例如Fortebio亲和测量)或MSD测定法或流式细胞术。
另一方面，可使用竞争测定法来鉴定与本文中公开的任何抗LAG-3抗体竞争对LAG-3的结合的抗体。在某些实施方案中，此类竞争性抗体结合与本文中公开的任何抗LAG-3抗体所结合表位相同的表位(例如线性或构象表位)。用于定位抗体所结合表位的详细例示性方法见Morris(1996)“Epitope Mapping Protocols”，Methods in Molecular Biology vol.66(Humana Press，Totowa，NJ)。
本发明还提供了用于鉴定具有上文所述的一种或多种性质的抗LAG-3抗体的测定法。还提供在体内和/或在体外具有此类生物学活性的抗体。
在某些实施方案中，对本发明的抗体测试上文所述的一种或多种性质。
供任何上述体外测定法使用的细胞包括天然表达LAG-3或经改造而表达LAG-3的细胞或细胞系。几种细胞类型表达LAG-3。例如，LAG-3在活化的CD4+和CD8+T细胞、Treg细胞、天然杀伤(NK)细胞和浆细胞样树状细胞(DC)上表达。LAG-3在肿瘤浸润性淋巴细胞(例如，头颈鳞状细胞癌(HNSCC)中的浸润性淋巴细胞)中表达。LAG-3在高度阻抑性诱导型和天然Treg上表达。例如，在黑素瘤和结直肠癌中高度阻抑性FoxP3+nTreg和FoxP3-iTreg为LAG-3阳性(Camisaschi等人(2010)J.Immunol.184(11)：6545-6551；Scurr等人(2014)Mucosal.Immunol.7(2)：428-439)。此类细胞还包括表达LAG-3的细胞系和并非正常情况下表达LAG-3但已经用编码LAG-3的核酸转染的细胞系。
可以理解的是，能够使用本发明的免疫缀合物替换或补充抗LAG-3抗体来进行任何上述测定法。
可以理解的是，能够使用抗LAG-3抗体和其它的治疗剂来进行任何上述测定法。
药物组合物和药物制剂
本发明还包括包含抗LAG-3抗体或其片段或其免疫缀合物的组合物(包括药物组合物或药物制剂)和包含编码抗LAG-3抗体或其片段的核酸的组合物。在某些实施方案中，组合物包含一种或多种结合LAG-3的抗体或其片段或其免疫缀合物或一种或多种编码一种或多种结合LAG-3的抗体或其片段的核酸。这些组合物还可以包含合适的药用辅料，如本领域中已知的药用载体、赋形剂等，包括缓冲剂。
适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。
合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”，第五版，R.C.Rowe，P.J.Seskey和S.C.Owen，PharmaceuticalPress，London，Chicago。
若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准载体和/或赋形剂，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
可以通过将具有所需纯度的本发明的抗LAG-3抗体与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本文所述的抗LAG-3抗体的药物制剂，优选地以冻干制剂或水溶液的形式。
示例性的冻干抗体制剂描述于美国专利号6,267,958。水性抗体制剂包括美国专利号6,171,586和WO2006/044908中所述的那些，后一种制剂包括组氨酸-乙酸盐缓冲剂。
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它抗癌活性成分，例如化疗剂和/或PD-1轴结合拮抗剂(例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体)。所述活性成分以对于目的用途有效的量合适地组合存在。活性成分可以是本领域已知的能够与抗LAG-3抗体组合的任何物质，包括化疗剂、抗体以及其他的治疗剂。这些活性成分的例子参见例如WO2016/028672、WO2015/042246、WO2015/138920等等。
在一些实施方案中，抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体是抗人PD-1抗体或抗人PD-L1抗体或抗人PD-L2抗体，例如人源化的抗人PD-1抗体或抗人PD-L1抗体或抗人PD-L2抗体。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
抗体的用途
在一方面中，本发明涉及调节对象中免疫反应的方法。该方法包括向对象施用有效量的本文公开的抗体分子(例如，抗LAG-3抗体)或药物组合物或免疫缀合物，从而调节对象中的免疫反应。在一个实施方案中，本文公开的抗体分子(例如，治疗有效量的抗LAG-3抗体分子)或药物组合物或免疫缀合物恢复、增强、刺激或增加对象中的免疫反应。
在另一方面中，本发明涉及预防或治疗受试者的肿瘤(例如癌症)的方法，所述方法包括向所述受试者施用有效量的本文公开的抗体分子(例如，抗LAG-3抗体)或药物组合物或免疫缀合物。在一个实施方案中，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌等。
在另一方面中，本发明涉及预防或治疗受试者的感染性疾病的方法，所述方法包括向所述受试 者施用有效量的本文公开的抗体分子(例如，抗LAG-3抗体)或药物组合物或免疫缀合物。在一个实施方案中，感染性疾病是慢性感染。
在另一方面，本发明涉及在受试者中引起抗体依赖性细胞介导的细胞毒性的方法，所述方法包括向所述受试者施用有效量的本文公开的抗体分子(例如，抗LAG-3抗体)或药物组合物或免疫缀合物。
受试者可以是哺乳动物，例如，灵长类，优选地，高级灵长类，例如，人类(例如，患有本文所述疾病或具有患有本文所述疾病的风险的患者)。在一个实施方案中，受试者需要增强免疫反应。在一些实施方案中，本文所述的抗LAG-3抗体分子恢复、增强或刺激受试者中的抗原特异性T细胞反应，例如，抗原特异性T细胞反应中的白介素-2(IL-2)或干扰素-γ(IFN-γ)产生。在一些实施方案中，免疫反应是抗肿瘤反应。在一个实施方案中，受试者患有本文所述疾病(例如，如本文所述的肿瘤或感染性疾病)或具有患有本文所述疾病的风险。在某些实施方案中，受试者免疫受损或具有免疫受损风险。例如，受试者接受或已经接受过化疗治疗和/或放射疗法。备选地或组合下，受试者因感染而免疫受损或具有因感染而免疫受损的风险。
在一些实施方案中，本文所述的肿瘤，例如癌症，包括但不限于实体瘤、血液学癌(例如，白血病、淋巴瘤、骨髓瘤)及其转移性病灶。在一个实施方案中，癌症是实体瘤。实体瘤的例子包括恶性肿瘤，例如，多个器官系统的肉瘤和癌(例如，腺癌)，如侵袭肺、乳腺、淋巴、胃肠道或结直肠、生殖器和生殖泌尿道(例如，肾细胞、膀胱细胞、膀胱细胞)、咽、CNS(例如，脑细胞、神经细胞或神经胶质细胞)、皮肤(例如，黑素瘤)、头部和颈部(例如，头颈鳞状细胞癌(HNCC))和胰的那些。例如，黑素瘤、结肠癌、胃癌、直肠癌、肾细胞癌、乳腺癌(例如，不表达一种、两种或全部雌激素受体、孕酮受体或Her2/neu的乳腺癌，例如，三阴性乳腺癌)、肝癌、肺癌(例如，非小细胞肺癌(NSCLC)(例如，具有鳞状和/或非鳞状结构的NSCLC)或小细胞肝癌)、前列腺癌、头部或颈部癌(例如，HPV+鳞状细胞癌)、小肠癌和食道癌。血液学癌的例子包括但不限于白血病(例如，髓样白血病、淋巴样白血病或慢性淋巴细胞白血病(CLL))、淋巴瘤(例如，霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤(DLBCL)、T细胞淋巴瘤或套细胞淋巴瘤(MCL))和骨髓瘤，例如，多发性骨髓瘤。癌症可以处于早期、中期或晚期或是转移性癌。
在一些实施方案中，癌症选自结直肠癌(例如，CRC)、黑素瘤，例如，晚期黑素瘤(例如，II-IV期黑素瘤)或HLA-A2阳性-黑素瘤；胰腺癌，例如，晚期胰腺癌；乳腺癌，例如，转移性乳腺癌或三阴性乳腺癌；头颈癌(例如，HNSCC)；食管癌；肾细胞癌(RCC)，例如，肾透明细胞癌(ccRCC)或转移性肾细胞癌(MRCC)；肺癌(例如，NSCLC)；宫颈癌；膀胱癌；或血液学恶性病，例如，白血病(例如，淋巴细胞白血病)或淋巴瘤(例如，霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤(DLBCL)、套细胞淋巴瘤(MCL)或CLL，例如，复发性或顽固性慢性淋巴细胞白血病)。
本文公开的方法和组合物可用于治疗与前述癌症相关的转移性病灶。
在一些实施方案中，所述癌症是表达LAG-3的癌症，特别是转移性癌症。在一些实施方案中，所述癌症是表达PD-L1的癌症。在一些实施方案中，所述癌症是表达LAG-3以及PD-L1的癌症。
在一些实施方案中，本文所述的癌症是结肠癌及其转移性癌症。
在一些实施方案中，所述感染是急性的或慢性的。在一些实施方案中，所述慢性感染是持续性的、潜伏的感染或缓慢感染。在一些实施方案中，所述慢性感染是因为选自细菌、病毒、真菌和原生动物的病原体导致的。
在一些实施方案中，所述感染性疾病是由于病毒感染引起的。致病性病毒的一些例子包括(甲型、乙型和丙型)肝炎病毒、(甲型、乙型和丙型)流感病毒、HIV、疱疹病毒(例如，VZV、HSV-1、 HAV-6、HSV-II、CMV、Epstein Barr病毒+)、腺病毒、黄病毒、艾柯病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞体病毒、腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革病毒、乳头状瘤、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、JC病毒和虫媒脑炎病毒。
在一些实施方案中，所述感染是细菌感染。引起感染的病原菌的一些例子包括梅毒菌、衣原体、立克次体菌、分枝杆菌、葡萄球菌、链球菌、肺炎球菌、脑膜炎球菌和淋球菌(conococci)、克雷伯菌、变形菌、沙雷菌、假单胞菌、军团菌、白喉杆菌、沙门氏菌、芽孢杆菌、霍乱菌、破伤风菌、肉毒杆菌、炭疽菌、鼠疫菌、钩端螺旋体病菌和莱姆病菌。
在一些实施方案中，所述感染是真菌感染，致病真菌的一些例子包括假丝酵母(白假丝酵母(Candida albicans)、克柔假丝酵母(Candida krusei)、光滑假丝酵母(Candida glabrata)、热带假丝酵母(Candida tropicalis)等)、新型隐球酵母(Cryptococcus neoformans)、曲霉(Aspergillus)(烟曲霉(Aspergillus fumigatus)、黑曲霉(Aspergillus niger)等)、毛霉目(Mucorales)(毛霉(mucor)、犁头霉(bsidia)、根霉(rhizophus))、申克孢子丝菌(Sporothrix schenkii)、皮炎芽生菌(Blastomyces dermatitidis)、巴西副球孢子菌(Paracoccidioides brasiliensis)、粗球孢子菌(Coccidioides immitis)和荚膜组织胞浆菌(Histoplasma capsulatum)。
在一些实施方案中，所述感染是原生动物例如寄生虫感染，寄生虫的一些例子包括包括痢疾阿米巴(Entamoeba histolytica)、结肠小袋纤毛虫(Balantidium coli)、福氏耐格里变形虫(Naegleria fowleri)、棘阿米巴属物种(Acanthamoeba sp.)、蓝氏贾第鞭毛虫(Giardia lambia)、隐孢子虫属物种(Cryptosporidium sp.)、卡氏肺囊虫(Pneumocystis carinii)、间日疟原虫(Plasmodium vivax)、田鼠巴贝虫(Babesia microti)、布氏锥虫(Trypanosoma brucei)、枯氏锥虫(Trypanosoma cruzi)、杜氏利什曼原虫(Leishmania donovani)、刚地弓形虫(Toxoplasma gondi)和巴西日圆线虫(Nippostrongylus brasiliensis)。
在一个实施方案中，感染性疾病是肝炎(例如，乙型肝炎感染)。抗LAG-3抗体分子(单独或与PD-1轴结合拮抗剂，例如抗PD-1或抗PD-L1，组合)可以出于治疗优势与乙型肝炎感染的常规治疗组合。在某些实施方案中，抗LAG-3抗体分子与乙型肝炎抗原((例如，Engerix B))或疫苗组合施用，和任选地联合含铝佐剂组合施用。
在另一个实施方案中，感染性疾病是流感。在某些实施方案中，抗LAG-3抗体分子与流感抗原或疫苗组合施用。
适于用本发明的抗LAG-3的抗体或其片段预防或治疗的疾病可进一步参见WO2015/138920、WO2016/028672、WO2015/042246等。
在其他方面，本发明提供抗LAG-3抗体或其片段或其免疫缀合物在生产或制备药物中的用途，所述药物用于治疗上文提及的相关疾病或病症。
在一些实施方案中，本发明的抗体或抗体片段或免疫缀合物会延迟病症和/或与病症相关的症状的发作。
联合疗法
在一些实施方案中，本文所述的预防或治疗方法还包括向所述受试者或个体联合施用本文公开的抗体分子(例如，抗LAG-3抗体)或药物组合物或免疫缀合物，以及PD-1轴结合拮抗剂或包含所述PD-1轴结合拮抗剂的药物或免疫缀合物。在一些实施方案中，PD-1轴结合拮抗剂包括例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体。
在一些实施方案中，PD-1轴结合拮抗剂包括但不限于PD-1结合拮抗剂，PD-L1结合拮抗剂和PD-L2结合拮抗剂。″PD-1″的备选名称包括CD279和SLEB2。″PD-L1″的备选名称包括B7-H1、B7-4、CD274和B7-H。″PD-L2″的备选名称包括B7-DC、Btdc和CD273。在一些实施方案中，PD-1、PD-L1和PD-L2是人PD-1、PD-L1和PD-L2。在一些实施方案中，PD-1结合拮抗剂是抑制PD-1结合其配体结合配偶的分子。在一个具体方面，PD-1配体结合配偶是PD-L1和/或PD-L2。在另一个实施方案中，PD-L1结合拮抗剂是抑制PD-L1结合其结合配偶的分子。在一个具体方面，PD-L1结合配偶是PD-1和/或B7.1。在另一个实施方案中，PD-L2结合拮抗剂是抑制PD-L2结合其结合配偶的分子。在一个具体方面，PD-L2结合配偶是PD-1。拮抗剂可以是抗体，其抗原结合片段、免疫粘附素、融合蛋白或寡肽。在一些实施方案中，PD-1结合拮抗剂是抗PD-1抗体(例如人抗体，人源化抗体，或嵌合抗体)。在一些实施方案中，抗PD-1抗体选自下组：MDX-1106(nivolumab，OPDIVO)，Merck 3475(MK-3475，pembrolizumab，KEYTRUDA)和CT-011(Pidilizumab)。在一些实施方案中，PD-1结合拮抗剂是免疫粘附素(例如包含融合至恒定区(例如免疫球蛋白序列的Fc区)的，PD-L1或PD-L2的胞外或PD-1结合部分的免疫粘附素)。在一些实施方案中，PD-1结合拮抗剂是AMP-224。在一些实施方案中，PD-L1结合拮抗剂是抗PD-L1抗体。在一些实施方案中，抗PD-L1结合拮抗剂选自下组：YW243.55.S70，MPDL3280A，MEDI4736和MDX-1105。MDX-1105，也称作BMS-936559，是WO2007/005874中记载的抗PD-L1抗体。抗体YW243.55.S70(重和轻链可变区序列分别显示于SEQ ID No.20和21)是WO 2010/077634 A1中记载的抗PD-L1。MDX-1106，也称作MDX-1106-04，ONO-4538，BMS-936558或nivolumab，是WO2006/121168中记载的抗PD-1抗体。Merck 3475，也称作MK-3475，SCH-900475或pembrolizumab，是WO2009/114335中记载的抗PD-1抗体。CT-011，也称作hBAT，hBAT-1或pidilizumab，是WO2009/101611中记载的抗PD-1抗体。AMP-224，也称作B7-DCIg，是WO2010/027827和WO2011/066342中记载的PD-L2-Fc融合可溶性受体。在一些实施方案中，抗PD-1抗体是MDX-1106。″MDX-1106″的备选名称包括MDX-1106-04，ONO-4538，BMS-936558或nivolumab。在一些实施方案中，抗PD-1抗体是nivolumab(CAS注册号：946414-94-4)。在优选的实施方案中，抗PD-1抗体是本文所述的“Antibody C”或Antibody D。
在一些实施方案中，本发明的抗LAG-3抗体也可以与抗PD-L1抗体联合用于治疗。
在一些实施方案中，本发明的抗PD-L1抗体是抗人PD-L1抗体。在一些实施方案中，本发明的抗PD-L1抗体是IgG1形式的抗体或IgG2形式的抗体或IgG4形式的抗体。在一些实施方案中，抗PD-L1抗体是单克隆抗体。在一些实施方案中，抗PD-L1抗体是人源化的。在一些实施方案中，抗PD-L1抗体是嵌合抗体。在一些实施方案中，至少部分的抗PD-L1抗体的框架序列是人共有框架序列。在一个实施方案中，本发明的抗PD-L1抗体还涵盖其抗体片段，优选地选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)或(Fab’) 2、单结构域抗体、双抗体(dAb)或线性抗体。
在一些具体的实施方案中，本发明的抗PD-L1抗体或其抗原结合片段包含
(i)如SEQ ID NO：57所示的重链可变区的3个互补决定区HCDR，和/或
(ii)如SEQ ID NO：58所示的轻链可变区的3个互补决定区LCDR。
在一些实施方案中，本发明的抗PD-L1抗体或其抗原结合片段包含重链可变区(VH)和/或轻链可变区(VL)，其中
(i)所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：51的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含选自SEQ ID NO：52的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：53的氨基酸序列或由所述氨基酸序列组成；
和/或
(ii)其中所述VL包含互补决定区域(CDR)LCDR1、LCDR2和LCDR3，其中LCDR1包含 SEQ ID NO：54的氨基酸序列或由所述氨基酸序列组成；LCDR2包含SEQ ID NO：55的氨基酸序列或由所述氨基酸序列组成；LCDR3包含选自SEQ ID NO：56的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，本发明的抗PD-L1抗体或其抗原结合片段包含重链可变区VH和/或轻链可变区VL，其中，
(a)重链可变区VH
(i)包含与选自SEQ ID NO：57的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
(ii)包含选自SEQ ID NO：57的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：57的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中；
和/或
(b)轻链可变区VL
(i)包含与选自SEQ ID NO：58的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：58的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：58的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的抗PD-L1抗体或其抗原结合片段包含重链和/或轻链，其中
(a)重链
(i)包含与选自SEQ ID NO：59的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：59的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：59的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中；
和/或
(b)轻链
(i)包含与选自SEQ ID NO：60的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：60的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：60的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中。
在一些实施方案中，本发明针对抗LAG-3抗体的修饰同样适用于抗PD-L1抗体。
在进一步的一些实施方案中，单独或与PD-1轴结合拮抗剂组合的抗LAG-3抗体或其片段还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用。
在一些实施方案中，治疗方式包括外科手术(例如肿瘤切除术)；放射疗法(例如，外粒子束疗法，它涉及其中设计照射区域的三维适形放射疗法)、局部照射(例如，指向预选靶或器官的照射)或聚焦 照射)等。聚焦照射可以选自立体定位放射手术、分割立体定位放射手术和强度调节型放射疗法。聚焦照射可以具有选自粒子束(质子)、钴-60(光子)和直线加速器(X射线)的辐射源，例如，如WO 2012/177624中描述。
放射疗法可以通过几种方法之一或方法组合施用，所述方法包括而不限于外粒子束疗法、内部放射疗法，植入物照射、立体定位放射手术、全身放射疗法、放疗法和永久或短暂间质近距放射疗法。术语“近距放射疗法”指通过空间限制的放射性物质递送的放射疗法，所述放射性物质在肿瘤或其他增殖性组织疾病部位处或其附近插入体内。该术语意在而不限于包括暴露于放射性同位素(例如At-211、I-131、I-125、Y-90、Re-186、Re-188、Sm-153、Bi-212、P-32和Lu的放射性同位素)。合适辐射源包括固体和液体。通过非限制性举例方式，辐射源可以是放射性核素，如I-125、I-131、Yb-169、Ir-192作为固态源、I-125作为固态源或发射光子、β粒子、γ辐射或其他治疗性射线的其他放射性核素。放射性物质也可以是从任何放射性核素溶液，例如，I-125或I-131溶液制成的流体，或可以使用含有固体放射性核素(如Au-198、Y-90)小颗粒的合适流体的浆液产生放射性流体。另外，放射性核素可以包含在凝胶或放射性微球状体中。
在一些实施方案中，治疗剂选自化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)。
示例性的细胞毒性剂包括抗微管药物、拓扑异构酶抑制剂、抗代谢药、有丝分裂抑制剂、烷基化剂、蒽环类、长春碱类生物碱、嵌入剂、能够干扰信号转导途径的活性剂、促凋亡活性剂、蛋白酶体抑制剂和照射(例如，局部或全身照射(例如、γ辐射)。
示例性的其它抗体包括但不限于免疫检查点抑制剂(例如，抗CTLA-4、抗TIM-3、抗CEACAM)；刺激免疫细胞的抗体(例如，激动性GITR抗体或CD137抗体)；抗癌抗体(例如，利妥昔单抗(
Figure PCTCN2018124315-appb-000006
或
Figure PCTCN2018124315-appb-000007
)、曲妥珠单抗
Figure PCTCN2018124315-appb-000008
托西莫单抗
Figure PCTCN2018124315-appb-000009
替伊莫单抗
Figure PCTCN2018124315-appb-000010
阿来组单抗
Figure PCTCN2018124315-appb-000011
依帕珠单抗
Figure PCTCN2018124315-appb-000012
贝伐珠单抗
Figure PCTCN2018124315-appb-000013
厄洛替尼
Figure PCTCN2018124315-appb-000014
西妥昔单抗
Figure PCTCN2018124315-appb-000015
等等。
示例性的化疗剂包括但不限于阿那曲唑
Figure PCTCN2018124315-appb-000016
比卡鲁胺
Figure PCTCN2018124315-appb-000017
硫酸博来霉素
Figure PCTCN2018124315-appb-000018
白消安
Figure PCTCN2018124315-appb-000019
白消安注射剂
Figure PCTCN2018124315-appb-000020
卡培他滨
Figure PCTCN2018124315-appb-000021
N4-戊氧羰基-5-脱氧-5-氟胞苷、卡铂
Figure PCTCN2018124315-appb-000022
卡莫司汀
Figure PCTCN2018124315-appb-000023
苯丁酸氮芥
Figure PCTCN2018124315-appb-000024
顺铂
Figure PCTCN2018124315-appb-000025
克拉立滨
Figure PCTCN2018124315-appb-000026
环磷酰胺(
Figure PCTCN2018124315-appb-000027
或
Figure PCTCN2018124315-appb-000028
)、阿糖胞苷、胞嘧啶阿拉伯糖苷
Figure PCTCN2018124315-appb-000029
阿糖胞苷脂质体注射剂
Figure PCTCN2018124315-appb-000030
达卡巴嗪
Figure PCTCN2018124315-appb-000031
更生霉素(dactinomycin)(放线菌素D、Cosmegan)、盐酸道诺霉素
Figure PCTCN2018124315-appb-000032
柠檬酸道诺霉素脂质体注射剂
Figure PCTCN2018124315-appb-000033
地塞米松、多西紫杉醇
Figure PCTCN2018124315-appb-000034
盐酸多柔比星
Figure PCTCN2018124315-appb-000035
依托泊苷
Figure PCTCN2018124315-appb-000036
磷酸氟达拉滨
Figure PCTCN2018124315-appb-000037
5-氟尿嘧啶
Figure PCTCN2018124315-appb-000038
氟他胺
Figure PCTCN2018124315-appb-000039
tezacitibine、吉西他滨(双氟脱氧胞苷)、羟基脲
Figure PCTCN2018124315-appb-000040
伊达比星
Figure PCTCN2018124315-appb-000041
异环磷酰胺
Figure PCTCN2018124315-appb-000042
伊立替康
Figure PCTCN2018124315-appb-000043
L-天冬酰胺酶
Figure PCTCN2018124315-appb-000044
亚叶酸钙、美法仑
Figure PCTCN2018124315-appb-000045
6-巯基嘌呤
Figure PCTCN2018124315-appb-000046
甲氨蝶呤
Figure PCTCN2018124315-appb-000047
米托蒽醌(米托蒽醌)、米罗他(mylotarg)、紫杉醇
Figure PCTCN2018124315-appb-000048
phoenix(钇90/MX-DTPA)、喷司他丁、聚苯丙生20联用卡莫司汀植入物
Figure PCTCN2018124315-appb-000049
柠檬酸他莫昔芬
Figure PCTCN2018124315-appb-000050
替尼泊苷
Figure PCTCN2018124315-appb-000051
6-硫鸟嘌呤、塞替派、替拉扎明
Figure PCTCN2018124315-appb-000052
注射用盐酸拓扑替康
Figure PCTCN2018124315-appb-000053
长春碱
Figure PCTCN2018124315-appb-000054
长春新碱
Figure PCTCN2018124315-appb-000055
长春瑞滨(长春瑞滨)、依鲁替尼、吉利德(idelalisib)和贝伦妥单抗-维多汀(brentuximab vedotin)。
示例性的疫苗包括但不限于癌症疫苗。疫苗可以是基于DNA的疫苗、基于RNA的疫苗或基于病毒转导的疫苗。癌症疫苗可以是预防性的或治疗性的。在一些实施方案中，该癌症疫苗是肽癌症疫苗，其在一些实施方案中是个性化肽疫苗。在一些实施方案中，该肽癌症疫苗是多价长肽、多重肽、肽混合物、杂合肽，或经肽脉冲的树突细胞疫苗(参见例如Yamada等人，Cancer  Sci，104：14-21，2013)。
示例性的抗感染活性剂包括但不限于，抗病毒剂、抗真菌剂、抗原生动物剂、抗菌剂，例如核苷类似物齐多夫定(AST)、更昔洛韦、膦甲酸或cidovir，如上文所述。
免疫调节剂包括免疫检查点分子抑制剂和共刺激性分子激活剂。
在一些实施方案中，免疫检查点分子的抑制剂是CTLA-4、TIM-3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4、CEACAM(例如，CEACAM-1和/或CEACAM-5)和/或TGFR的抑制剂。对分子的抑制可以在DNA、RNA或蛋白质水平进行。在一些实施方案中，抑制性核酸(例如，dsRNA、siRNA或shRNA)可以用来抑制免疫检查点分子的表达。在其他实施方案中，免疫检查点分子的抑制剂是与免疫检查点分子结合的多肽，例如，可溶性配体或抗体或抗体片段。示例性TIM-3抗体分子包括但不限于MBG220、MBG227和MBG219。
在其他实施方案中，免疫调节剂是CTLA4的可溶性配体(例如，CTLA-4-Ig或TIM-3-Ig)或抗体或抗体片段。例如，抗LAG-3抗体分子(单独或与PD-1轴结合拮抗剂)可以与CTLA-4抗体(例如，伊匹木单抗)组合施用。示例性抗CTLA4抗体包括曲美木单抗(tremelimumab)(从Pfizer可获得的IgG2单克隆抗体，以前称作ticilimumab、CP-675，206)；和伊匹木单抗(CTLA-4抗体，也称作MDX-010、CAS No.477202-00-9)。
在一些实施方案中，免疫调节剂是共刺激分子的激活剂或激动剂。在一个实施方案中，共刺激分子的激动剂选自以下分子的激动剂(例如，激动性抗体或其抗原结合片段、或可溶性融合物)：OX40、CD2、CD27、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD30、CD40、BAFFR、HVEM、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160、B7-H3或CD83配体。在另一个实施方案中，单独或与PD-1轴结合拮抗剂组合的抗LAG-3抗体或其片段与包括CD28、CD27、ICOS和GITR的共刺激结构域的共刺激分子(例如，与正向信号相关的激动剂)组合使用。示例性GITR激动剂例如包括GITR融合蛋白和抗GITR抗体(例如，双价的抗GITR抗体)，例如，以下文献中描述的GITR融合蛋白：美国专利号6,111,090、欧洲专利号090505B1、美国专利号8,586,023、PCT公开号WO 2010/003118和2011/090754。一个示例性抗GITR抗体是TRX518。
在进一步的一些实施方案中，单独或与PD-1轴结合拮抗剂组合的抗LAG-3抗体或其片段还能与酪氨酸激酶抑制剂(例如，受体酪氨酸激酶(RTK)抑制剂)组合使用。示例性酪氨酸激酶抑制剂包括但不限于表皮生长因子(EGF)途径抑制剂(例如，表皮生长因子受体(EGFR)抑制剂)、血管内皮生长因子(VEGF)途径抑制剂(例如，血管内皮生长因子受体(VEGFR)抑制剂(例如，VEGFR-1抑制剂、VEGFR-2抑制剂、VEGFR-3抑制剂)、血小板衍生生长因子(PDGF)途径抑制剂(例如，血小板衍生生长因子受体(PDGFR)抑制剂(例如，PDGFR-β抑制剂))、RAF-1抑制剂、KIT抑制剂和RET抑制剂。
在一些实施方案中，单独或与PD-1轴结合拮抗剂组合的抗LAG-3抗体或其片段还能与PI3K抑制剂、mTOR抑制剂、BRAF抑制剂、MEK抑制剂和/或JAK2抑制剂等组合使用。
在本发明的任何方法的一些实施方案中，单独或与PD-1轴结合拮抗剂组合的本发明的抗LAG-3抗体或其片段的施用与肿瘤抗原的施用组合。抗原可以例如是肿瘤抗原、病毒抗原、细菌性抗原或来自病原体的抗原。在一些实施方案中，肿瘤抗原包含蛋白质。在一些实施方案中，肿瘤抗原包含核酸。在一些实施方案中，肿瘤抗原是肿瘤细胞。
在一些实施方案中，单独或与PD-1轴结合拮抗剂组合的本发明的抗LAG-3抗体或其片段可以与包含过继转移表达嵌合抗原受体(CAR)的T细胞(例如细胞毒性T细胞或CTL)的治疗联合施用。
在一些实施方案中，单独或与PD-1轴结合拮抗剂组合的本发明的抗LAG-3抗体或其片段可以 与抗肿瘤剂联合施用。
在一些实施方案中，单独或与PD-1轴结合拮抗剂组合的本发明的抗LAG-3抗体或其片段可以与溶瘤病毒联合施用。
在一些实施方案中，单独或与PD-1轴结合拮抗剂组合的本发明的抗LAG-3抗体或其片段可以与细胞因子联合施用。细胞因子可以作为与抗LAG-3抗体分子的融合分子施用，或作为单独的组合物施用。在一个实施方案中，抗LAG-3抗体与一种、两种、三种或更多种细胞因子(例如，作为融合分子或作为单独组合物)组合施用。在一个实施方案中，细胞因子是选自IL-1、IL-2、IL-12、IL-12、IL-15或IL-21中一者、两者、三者或更多者的白介素(IL)。
在一些实施方案中，单独或与PD-1轴结合拮抗剂组合的本发明的抗体或其片段可以与本领域常规的癌症疗法组合，常规的癌症疗法包括但不限于：(i)放射疗法(例如，放射疗法、X射线疗法、照射)或用电离辐射杀死癌细胞并缩小肿瘤。放射疗法可经体外放射治疗(EBRT)或经内部近距离放射疗法施用；(ii)化学疗法，或应用细胞毒药物，其一般影响快速分裂的细胞；(iii)靶向疗法，或特异性影响癌细胞蛋白去调节的药剂(例如，酪氨酸激酶抑制剂伊马替尼(imatinib)、吉非替尼(gefitinib)；单克隆抗体，光动力学疗法)；(iv)免疫疗法，或增强宿主免疫应答(例如，疫苗)；(v)激素疗法，或阻断激素(例如，当肿瘤是激素敏感的时候)，(vi)血管发生抑制剂，或阻断血管形成和生长，和(vii)姑息护理，或这样的治疗，其涉及改善护理质量以降低疼痛、恶心、呕吐、腹泻和出血，其中疼痛药物如吗啡(morphine)和羟考酮(oxycodone)，抗-催吐药如昂丹司琼(ondansetron)和阿瑞匹坦(aprepitant)，从而可容许更具攻击性的治疗方案。
在一些实施方案中，单独或与PD-1轴结合拮抗剂组合的本发明的抗体或其片段可以与增强宿主免疫功能的常规方法组合，所述常规方法包括但不限于：(i)APC增强，诸如(a)向肿瘤注射编码异源MHC同种异体抗原的DNA，或(b)用增加免疫抗原识别可能性的基因(例如免疫刺激细胞因子、GM-CSF、共刺激分子B7.1、B7.2)转染活检的肿瘤细胞，(iii)过继性细胞免疫疗法，或用活化的肿瘤特异性T细胞治疗。过继性细胞免疫疗法包括分离肿瘤浸润的宿主T淋巴细胞，诸如通过IL-2或肿瘤或两者刺激体外扩增该群体；此外，功能障碍的分离的T细胞也可通过体外应用本发明的抗体来活化，如此活化的T细胞可然后重新施用给宿主。
上文所述的各种组合疗法可以进一步组合以用于治疗。
更多的抗LAG-3抗体与其他治疗方式或治疗剂的组合的例子可以参见WO2015/138920、WO2016/028672、WO2015/042246等。
此类组合疗法涵盖组合施用(其中两种或更多种治疗剂包含在同一配制剂或分开的配制剂中)，和分开施用，在该情况中，可以在施用别的疗法，例如治疗方式和/或治疗剂之前，同时，和/或之后发生本发明的抗体的施用。抗体分子和/或其他疗法，例如治疗剂或治疗方式可以在活动性疾病期间或在缓解或活动度更小的疾病期间施用。抗体分子可以在其他治疗前、与其他治疗同时、治疗后或在疾病缓解期间施用。
在一个实施方案中，抗LAG-3抗体的施用和别的疗法(例如治疗方式或治疗剂)的施用彼此在约一个月内，或约一，两或三周内，或约1，2，3，4，5，或6天内发生。
在一些实施方案中，本文中描述的抗体组合可以分别施用，例如，作为单独的抗体分别施用，或连接时(例如作为双特异性或三特异性抗体分子)施用。
可以理解的是，能够使用本发明的免疫缀合物替换或补充抗LAG-3抗体来进行任何治疗。
施用途径和剂量
本发明的抗体(以及包含其的药物组合物或免疫缀合物，以及任何另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。 肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的抗体的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、所述抗体是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
在一些实施方案中，调整剂量方案以提供最佳的所需反应(例如，治疗反应)。例如，可以施用单次团注，可以随时间推移施用几个分开的剂量或可以如治疗情况的危急性所示，按比例减少或增加该剂量。特别有利的是以剂量单位形式配制肠胃外组合物以易于剂量的施用和均匀性。如本文所用的剂量单位形式指适合作为用于待治疗对象的单一剂量的物理分立的单元；每个单元含有预定量的活性化合物，所述的预定量经计算与所要求的药用载体结合时产生所需的治疗效果。用于本发明剂量单位形式的规格直接取决于(a)活性化合物的独特特征和待实现的特定治疗效果，以及(b)混合这种活性化合物用于个体中敏感性治疗的领域内所特有的限制。
在一些实施方案中，抗体分子治疗有效量或预防有效量的示例的非限制性范围是0.1-30mg/kg、更优选地1-25mg/kg、更优选地5-15mg/kg。抗LAG-3抗体分子的剂量和治疗方案可以由技术人员确定。在某些实施方案中，抗LAG-3抗体分子通过注射(例如，皮下或静脉内)以约1至40mg/kg，例如，1至30mg/kg，例如，约5至25mg/kg、约10至20mg/kg、约1至5mg/kg、1至10mg/kg、5至15mg/kg、10至20mg/kg、15至25mg/kg或约3mg/kg的剂量施用。给药方案可以从例如一周一次变动至每2、3或4周一次。在一个实施方案中，抗LAG-3抗体分子以约10至20mg/kg的剂量每隔一周施用。抗体分子可以通过静脉内输注以超过20mg/分钟，例如，20-40mg/分钟和优选地大于或等于40mg/分钟的速率施用，以达到约35至440mg/m2、优选地约70至310mg/m2和更优选地约110至130mg/m2的剂量。在实施方案中，约110至130mg/m2的输注速率实现约3mg/kg的水平。在一个实施方案中，抗LAG-3抗体分子以约3至800mg(例如，约3、20、80、240或800mg)的剂量施用(例如，静脉内施用)。在某些实施方案中，抗LAG-3抗体分子以约20至800mg(例如，约3、20、80、240或800mg)的剂量单独施用。在其他实施方案中，抗LAG-3抗体分子以约3至240mg(例如，约3、20、80或240mg)的剂量与第二活性剂或治疗方式(例如，本文所述的第二活性剂或治疗方式)组合施用。在一个实施方案中，抗LAG-3抗体分子在每个8周循环期间每2周施用(例如，在第1、3、5、7周期间)，例如，最多96周。
在一些实施方案中，抗体分子可以通过静脉内输注以超过20mg/分钟，例如，20-40mg/分钟和优选地大于或等于40mg/分钟的速率施用，以达到约35至440mg/m2、优选地约70至310mg/m2和更优选地约110至130mg/m2的剂量。在实施方案中，约110至130mg/m2的输注速率实现约3mg/kg的水平。在其他实施方案中，抗体分子通过静脉内输注以小于10mg/分钟，例如，小于或等于5mg/分钟的速率施用，以达到约1至100mg/m2，例如，约5至50mg/m2、约7至25mg/m2和更优选地，约10mg/m2的剂量。在一些实施方案中，抗体是经约30分钟时间输注。
在另一个实施方案中，抗LAG-3抗体分子与抗PD-1抗体分子组合施用。可以是使用的示例性剂量包括约1至10mg/kg(例如，3mg/kg)的抗PD-1抗体分子剂量。抗LAG-3抗体分子可以约20至800mg，例如，约20、80、240或800mg的剂量联合施用。在一个实施方案中，抗LAG-3抗体分子在每8周循环期间(例如，最多96周)每2周施用(例如，在第1、3、5、7周期间)。
用于诊断和检测的方法和组合物
在某些实施方案中，本文中提供的任何抗LAG-3抗体或其抗原结合片段可以用于检测LAG-3在生物样品中的存在。术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉 及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自过度增生性或癌性病灶。
在一个实施方案中，提供用于诊断或检测方法的抗LAG-3抗体。在另一个方面中，提供检测LAG-3在生物样品中的存在的方法。在某些实施方案中，方法包含检测LAG-3蛋白在生物样品中的存在。在某些实施方案中，LAG-3是人LAG-3。在某些实施方案中，所述方法包括将生物样品与如本文所述的抗LAG-3抗体在允许抗LAG-3抗体与LAG-3结合的条件下接触，并检测在抗LAG-3抗体和LAG-3之间是否形成复合物。复合物的形成表示存在LAG-3。该方法可以是体外或体内方法。在一个实施方案中，抗LAG-3抗体被用于选择适合利用抗LAG-3抗体的治疗的受试者，例如其中LAG-3是用于选择所述受试者的生物标记物。
在一个实施方案中，可以使用本发明抗体诊断癌症或肿瘤，例如评价(例如，监测)对象中本文所述疾病(例如，过度增生性或癌性疾病)的治疗或进展、其诊断和/或分期。
在某些实施方案中，提供标记的抗LAG-3抗体。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。示例性标记包括但不限于，放射性同位素32P、14C、125I、3H和131I，荧光团如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰(dansyl)，伞形酮(umbelliferone)，荧光素酶(luceriferase)，例如，萤火虫荧光素酶和细菌荧光素酶(美国专利号4,737,456)，荧光素，2，3-二氢酞嗪二酮，辣根过氧化物酶(HR)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶解酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶如尿酸酶和黄嘌呤氧化酶，以及利用过氧化氢氧化染料前体的酶如HR，乳过氧化物酶，或微过氧化物酶(microperoxidase)，生物素/亲和素，自旋标记，噬菌体标记，稳定的自由基，等等。
在本文中提供的任何发明的一些实施方案中，样品是在用抗LAG-3抗体治疗之前获得的。在一些实施方案中，样品是在用癌症药物治疗之前获得的。在一些实施方案中，样品是在癌症已经转移之后获得的。在一些实施方案中，样品是福尔马林固定、石蜡包膜(FFPE)的。在一些实施方案中，样品是活检(例如芯活检)，手术标本(例如来自手术切除的标本)，或细针吸出物。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测LAG-3。
在一些实施方案中，提供了一种治疗肿瘤或感染的方法，所述方法包括：对受试者(例如，样品)(例如，包含癌细胞的受试者样品)检验LAG-3的存在，因而确定LAG-3值，将LAG-3值与对照值比较，并且如果LAG-3值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的单独或与PD-1轴结合拮抗剂组合的抗LAG-3抗体(例如，本文所述的抗LAG-3抗体)，因而治疗肿瘤或感染。
本发明示例性抗LAG-3抗体的序列
Figure PCTCN2018124315-appb-000056
Figure PCTCN2018124315-appb-000057
Figure PCTCN2018124315-appb-000058
Figure PCTCN2018124315-appb-000059
Figure PCTCN2018124315-appb-000060
本发明的这些以及其它方面和实施方案在附图(附图简述紧随其后)和以下的发明详述中得到描述并且示例于以下实施例中。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
实施例
实施例1.酵母展示技术筛选抗LAG-3全人源抗体
基于酵母的抗体展示(yeast-based antibody presentation)文库，按照现有的方法(WO2009036379；WO 2010105256；W02012009568)进行扩增，其中每个库的多样性达到1×10 9。简言之，前两轮的筛选使用Miltenyi公司的MACS系统进行磁性激活细胞分选。首先，将文库的酵母细胞(～1×10 10细胞/文库)分别在FACS洗涤缓冲液中(磷酸盐缓冲液，含有0.1％牛血清蛋白)室温孵化15分钟，缓冲液中含有100nM生物素标记的人LAG-3抗原(ArcoBiosystems)。使用50ml预冷的FACS洗涤缓冲液洗一次，再用40ml相同洗涤缓冲液重悬细胞，并加入500μl链霉素微珠(Miltenyi LS)于4℃孵化15分钟。1000rpm离心5min弃去上清后用5ml FACS洗涤缓冲液重悬细胞，将细胞溶液加到Miltenyi LS柱中。加样完成后，用FACS洗涤缓冲液洗柱3次，每次3ml。从磁性区域取下Miltenyi LS柱，用5ml生长培养基洗脱，收集洗脱的酵母细胞并在37℃过夜生长。
使用流式细胞仪进行下一轮的分选：将经过MACS系统筛选获得的大约1×10 8的酵母细胞用FACS缓冲液洗三次，于含有低浓度生物素(100-1nM)标记的人LAG-3抗原中室温下培养。弃去培养液，细胞用FACS洗涤缓冲液洗两次之后，将细胞与LC-FITC(FITC标记山羊抗人免疫球蛋白F(ab’)kappa链抗体，Southern Biotech)(1∶100稀释)混合，并与SA-633(链霉亲和素-633，Molecular Probes)(1∶500稀释)或SA-PE(链霉亲和素-藻红蛋白，Sigma)(1∶50稀释)试剂混合，4℃下培养15分钟。用预冷的FACS洗涤缓冲液洗脱两次，并重悬于0.4ml缓冲液中，将细胞转移到带滤器的分离管中。使用FACS ARIA(BD Biosciences)分选细胞。
将通过筛选获得的表达抗人LAG-3抗体的酵母细胞在30℃下震荡诱导48小时以表达抗人LAG-3的抗体。诱导结束之后，1300rpm离心10min去除酵母细胞，收获上清液。使用Protein A对上清液中的抗人LAG-3抗体进行纯化，pH2.0醋酸溶液洗脱，收获抗人LAG-3抗体，抗体纯度＞95％。
本次筛选获得抗体ADI-26789及抗体ADI-26869。
实施例2抗人LAG-3抗体的亲和力优化
为了获得更高亲和力的抗人LAG-3抗体，我们通过以下方法对抗体ADI-26789及ADI-26869进行了优化。
VHmut筛选
该方法是通过常规的错配PCR的方法向抗体重链区域引入突变。PCR过程中，通过使用1uM高突变的碱基类似物dPTP和8-oxo-dGTP，从而将碱基错配概率提高至约0.01bp。
获得的错配PCR的产物通过同源重组的方法构建入含有重链恒定区的载体中。通过这种方法，在包括LAG-3抗原滴度、未标记抗原竞争以及使用母抗体竞争的筛选压力下，我们获得了库容量为1×10 7的次级库。通过FACS方法进行了3轮成功筛选。
CDRH1/CDRH2筛选
把VHmut方法获得的子代抗体的CDRH3基因构建入1×10 8多样性的CDRH1/CDRH2基因库中，并对其进行了3轮筛选。第一轮使用MACS方法，而第二、三轮使用FACS方法，对抗体抗原结合物进行亲和力加压，筛选出最高亲和力的抗体。
经过以上亲和力成熟过程，我们获得了亲和力提高的抗人LAG-3单克隆抗体ADI-31851和ADI-31853。
实施例3.HEK293细胞中的表达和纯化
本发明示例的4个抗体(ADI-26789，ADI-26869，ADI-31851，ADI-31853)的CDR区、轻链可变区和重链可变区、轻链和重链的氨基酸序列，以及对应的核酸序列以及编号在本申请的“表1-4”部分列出。
根据本领域常规方法，将编码各条抗LAG-3抗体的轻链氨基酸序列和重链氨基酸序列的cDNA分别克隆到表达载体pTT5中。
将含有目标抗体基因的上述表达载体与转染试剂PEI(Polysciences)按照生产产商提供的方案瞬时转染培养的人肾胚细胞293细胞(Invitrogen)，转染后，弃去培养基并用新鲜的EXPI293培养基(Gibco)把细胞稀释到4×10 6/ml。在37℃，5％CO 2的条件下培养细胞7天，每48小时流加新鲜培养基。7天后，1300rpm离心20min。取上清液，用Protein A纯化上清液，使抗体的纯度＞95％。
在HEK293细胞中还表达并且纯化实施例中使用的下述对照抗体：
对照抗体
25F7
BAP050
其中25F7是在HEK293细胞中瞬时表达的人LAG-3抗体，其序列与美国专利US20170137514A1中的抗体“25F7”的序列相同。BAP050是在HEK293细胞中瞬时表达的抗人LAG-3抗体，其序列与美国专利WO2015/138920A1中的抗体“BAP050”的序列相同。
实施例4：本发明抗LAG-3抗体的亲和力测定
采用生物光干涉测量(ForteBio)测定法测定本发明上述4个示例抗体结合人LAG-3(hLAG-3)的平衡解离常数(KD)。
ForteBio亲和力测定按照现有的方法(Estep，P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs，2013.5(2)：p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30分钟，然后线上检测60秒建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器(ForteBio)上进行ForteBio亲和测量。再将具有加载的抗体的传感器暴露于100nM的人LAG-3抗原(ArcoBiosystems)中作用5分钟，之后将传感器转移至分析缓冲液解离5分钟用于解离速率测量。使用1∶1结合模型进行动力学的分析。
在如以上测定法所述进行的实验中，ADI-26789、ADI-26869、ADI-31851、ADI-31853以及对照抗体25F7和BAP050亲和力如表6所示。
表6：通过生物光干涉测量本发明抗体的结合动力学
Figure PCTCN2018124315-appb-000061
Figure PCTCN2018124315-appb-000062
可见，本发明上述4个示例抗体均显示极高的亲和力，其中ADI-31851和ADI-31853具有比25F7更高、和BAP050相似的亲和力。
实施例5：本发明抗LAG-3抗体与人LAG-3的结合
在基于流式细胞术的测定法中测量本发明的上述4个示例抗体与人LAG-3的结合。
通过将携带克隆至多克隆位点MCS的人LAG-3 cDNA(Sino Biological)的pCHO1.0载体(Invitrogen)转染至人肾胚细胞293细胞(Invitrogen)，产生过表达人LAG-3的293细胞(293-hLAG-3细胞)。
将293-hLAG-3细胞(0.2×10 6个细胞)与不同浓度的如上所述制备的实验抗体(ADI-26789、ADI-26869及对照抗体25F7)混合(抗体稀释方法为：最高抗体浓度为500nM，三倍稀释在含0.1％牛血清白蛋白(BSA)的PBS中，总共测试了8个浓度)。冰上孵育30分钟。然后将细胞洗涤至少两次，加入1∶100稀释的二抗(PE标记的羊抗人IgG抗体，SouthernBiotech，终浓度为5μg/ml)，冰上(避光)孵育30分钟。将细胞洗涤至少两次并通过流式细胞术进行分析。在Accuri C6系统(BD Biosciences)上进行流式细胞术检测，并根据其MFI用GraphPad拟合浓度依赖的曲线。
在如以上测试法所述进行的实验中，ADI-26789和ADI-26869结合HEK293细胞上过表达的hLAG-3，EC50值分别为2.137nM和2.909nM，优于对照抗体25F9与HEK293细胞上过表达的hLAG-3的结合能力(对照抗体25F9的EC50值为3.339nM)(参见图1)。
亲和力优化的抗hLAG-3抗体ADI-31851和ADI-31853结合HEK293细胞上过表达的hLAG-3，EC50值分别为0.501nM和0.4332nM，优于对照抗体25F7和BAP050与HEK293细胞上过表达的hLAG-3的结合能力(EC50值分别为2.593nM和1.409nM)。(参见图2)
实施例6.本发明抗LAG-3抗体对人LAG-3配体MHC II与LAG-3相互作用的阻断
通过流式细胞术测量ADI-31851、ADI-31853阻断人LAG-3与细胞表面的MHC II(HLA)结合的能力。
将具有如下所述序列的HLA-DR-α和HLA-DRβ1两段DNA同时构建到pCHO1.0载体(Invitrogen)中，转染到CHO-S细胞(Invitrogen，ExpiCHO TM Expression System Kit，货号：A29133)中产生表面过表达人HLA-DR的CHO细胞(CHO-DR细胞)。
HLA-DRβ1
Figure PCTCN2018124315-appb-000063
HLA-DR-N
Figure PCTCN2018124315-appb-000064
Figure PCTCN2018124315-appb-000065
将抗原rhLAG3蛋白(huFc)(Sino Biological)稀释至40nM，50μl/孔。将如上所述制备的抗体(ADI-31851、ADI-31853和对照抗体25F9)从最高浓度80nM开始进行3倍梯度稀释，共8个稀释梯度，50μl/孔，于PBS冰上孵育30min，抗原终浓度20nM，抗体最高终浓度40nM。将CHO-DR细胞调节至3×10 5细胞/孔，100μl/孔。细胞于300g离心5min，弃上清，重悬于抗原抗体混合液。冰上孵育30min，加PBS 100μl/孔，300g离心5min，PBS清洗1次，加1∶100稀释的100μl山羊抗人IgG-PE(SouthernBiotech)/孔，冰浴20min，加PBS 100μl/孔，300g离心5min，PBS清洗1次。用100μl PBS重悬，细胞流式仪(BD Biosciences)检测细胞荧光信号值。
实验结果表明，ADI-31851和ADI-31853均可以有效阻断LAG-3和其配体MHC II(HLA-DR)的结合，其阻断能力和对照抗体25F9一致。具体而言，ADI-31851和ADI-31853阻断人LAG-3与MHC II(HLA-DR)的结合的IC50分别为5.85nM和4.533nM。对照抗体25F9阻断人LAG-3与MHCII(HLA-DR)的结合的IC50为5.14nM。(参见图3)。
实施例7.抗体与激活的T细胞结合实验
人CD4+T细胞被激活后其表面会表达LAG-3蛋白，本研究通过流式细胞术测量ADI-31851、ADI-31853和激活的人CD4 +T细胞的结合能力。
PBMC分离：取捐赠者新鲜血液50ml，添加2.5倍PBS，轻轻加入到FiColl(Thermo)，分4管，每管12.5ml，400g，30min离心，0减速度停止。吸取中间白色条带至PBS中，PBS洗2次。
CD4+T细胞分离：按照‘Untouched CD4+T cell isolation’试剂盒(11346D，Invitrogen)说明书，利用该试剂盒进行操作。PBMC静置培养2h，吸取悬浮的细胞液至15ml离心管中，200g离心10min，沉淀加入500μl分离液、100μl AB型血清、100μl纯化抗体重悬，4℃孵育20min，用分离液清洗一次，再加入500μl Bead Buffer(Invitrogen)孵育15min，磁场去除Bead，T细胞培养基洗一次，使用8ml培养基重悬，37℃、6％CO 2培养。
按照磁珠：CD4 +T细胞＝1∶1的比例向CD4 +T细胞中加入抗CD3/CD28磁珠(Gibco)刺激3天，调节细胞密度至1×10 6个/ml，分装第一孔150μl/孔，其他孔100μl/孔，第一列孔中分别加如上所述制备的抗体ADI-31851和ADI-31853和对照抗体25F7，终浓度10nM，混匀，吸取50μl入下一列孔，以此类推。每个样品做3个复孔。冰浴30min，400g离心5min，PBS清洗2次，添加1∶100稀释的PE-抗人Fc抗体(SouthernBiotech)50μl，冰浴30min，400g离心5min，PBS清洗2次，用60μl PBS重悬，通过流式细胞术进行分析。在Accuri C6系统(BD Biosciences)上进行流式细胞术，并根据其MFI用GraphPad拟合浓度依赖的曲线。
实验结果表明，ADI-31851和ADI-31853能和激活的人CD4 +T细胞结合，EC50值分别为0.013nM和0.011nM，结合能力优于对照抗体25F7(对照抗体的EC50值为0.036nM)(参见图4)。
实施例8：本发明抗LAG-3抗体与小鼠LAG-3的结合
在基于流式细胞术的测定法中测量本发明的2个示例抗体ADI-31851及ADI-31853与小鼠LAG-3的结合。
通过转染携带克隆至多克隆位点的小鼠LAG-3cDNA(Sino Biological)的pCHO1.0载体(Invitrogen)至CHO-S细胞(Invitrogen，ExpiCHO TM Expression System Kit，货号：A29133)，产生过表达小鼠LAG-3的CHO细胞(CHO-mLAG-3细胞)。
将CHO-mLAG-3细胞(0.2×10 6个细胞)与不同浓度的如上所述制备的实验抗体(ADI-31851、ADI-31853及对照抗体25F7和BAP050)混合(抗体稀释方法为：最高抗体浓度 为500nM，三倍稀释在含0.1％牛血清白蛋白(BSA)的PBS中，总共测试了8个浓度)。冰上孵育30分钟。然后将细胞洗涤至少两次，加入1∶100稀释的二抗(PE标记的羊抗人IgG抗体，SouthernBiotech，终浓度为5μg/ml)，冰上(避光)孵育30分钟。将细胞洗涤至少两次并通过流式细胞术进行分析。在Accuri C6系统(BD Biosciences)上进行流式细胞术检测，并根据其MFI用GraphPad拟合浓度依赖的曲线。
在如以上测试法所述进行的实验中，亲和力优化的抗hLAG-3抗体ADI-31851和ADI-31853能结合CHO细胞上过表达的小鼠LAG-3，其中ADI31853结合能力较好(EC50＝42.33nM)，ADI-31851结合能力较弱。而对照抗体25F7和BAP050均不能和CHO细胞上过表达的小鼠LAG-3结合。(参见图5)
实施例9.本发明抗LAG-3抗体的抗肿瘤活性
本研究利用CT26移植瘤小鼠模型研究了抗人LAG-3抗体ADI-31853单独使用或和抗鼠PD-1抗体“Antibody C”(WO2017/133540)联合使用的抗肿瘤活性。
小鼠：
雌性BALB/c小鼠(约8周大)购自北京维通利华实验动物技术有限公司。小鼠在到达后驯化7天，随后开始研究。
细胞
小鼠结肠癌细胞CT26(ATCC# CRL-2638)购自ATCC，并严格按照ATCC要求进行常规传代培养用于后续体内实验。离心收集细胞，在无菌PBS中重悬细胞并调整细胞密度为5×10 6个/ml。在第0天取0.2ml细胞悬液皮下接种至BALB/c小鼠(北京维通利华实验动物技术有限公司)右侧腹部区域中来建立荷瘤小鼠模型。
给药
将小鼠分为四组(每组8只小鼠)，每组分别皮下注射如下剂量的抗体：
(1)小鼠IgG(equitech-Bio)，10mg/kg；
(2)PD-1(Antibody C)，1mg/kg；
(3)LAG-3(ADI-31853)，10mg/kg；
(4)LAG-3(ADI-31853)，10mg/kg PBS+PD-1(Antibody C)，1mg/kg。
试剂注射
接种后第7天，将符合实验要求的小鼠随机分组，每组8只。分别用如上四组试剂在第7天、第10天、第14天和第17天为每组小鼠按如上剂量给药。
分析
在整个研究期间每周测量两次肿瘤和体重，当肿瘤达到端点时(肿瘤体积＞2500mm 3)或当小鼠具有＞20％体重减轻时，使小鼠安乐死。采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。将来自每组的小鼠的肿瘤尺寸与时间作图。使用方差分析(ANOVA)来确定统计显著性。＜0.05的P值被视为在所有分析中具有统计显著性，所述分析是用Prism 5统计软件(GraphPad软件)执行。
实验结果见图6，可见本申请的抗LAG-3单克隆抗体ADI-31853和抗PD-1单克隆抗体“Antibody C”联合使用时与IgG对照(equitech-Bio)及这两个抗体分别使用相比，能显著抑制肿瘤的生长。
实施例10.本发明抗LAG-3抗体与其他抗体联用的抗肿瘤活性
本研究利用人源化小鼠模型研究了抗人LAG-3抗体ADI-31853和抗PD-1抗体“Antibody D”(IBI308，WO2017/025016)或抗PD-L1抗体(HZ3266-IgG1N297A)联合使用的抗肿瘤活性。
本研究采用A375(ATCC)人的皮肤癌细胞在NOG小鼠上测定抗LAG-3抗体的抗肿瘤 作用。预先静脉注射人的PBMC(2×10 6个细胞/小鼠)(AllCells)，然后采用皮下接种的方式建立A375荷瘤小鼠模型(NOG模型)，成瘤后分组，给予不同抗体的治疗，监测给药期间各组小鼠肿瘤体积和体重变化，给药频率为2次/周，给药2周，共给药5次。监测频率均为2次/周，连续监测4周，给药剂量和方式如表2。给药结束后计算相对肿瘤抑制率(TGI％)。
抗PD-L1抗体(HZ3266-IgG1N297A)
根据本领域常规方法，将编码抗PD-L1抗体HZ3266-IgG1N297A的轻链氨基酸序列和重链氨基酸序列(表3)的cDNA分别克隆到表达载体pMD20-T载体(Clontech)，获取质粒用于转染。
根据所需转染体积传代293F细胞(Invitrogen)，转染前一天将细胞密度调整至1.5×10 6个细胞/ml。转染当天细胞密度约为3×10 6个细胞/ml。取终体积1/10的F17培养基(Gibco，A13835-01)作为转染缓冲液，加入适当的质粒，混匀。加合适的聚乙烯亚胺(PEI)(Polysciences，23966)到质粒中(质粒与PEI的比例在293F细胞中为1∶3)，混匀后室温孵育10min，获得DNA/PEI混合物。用DNA/PEI混合物重悬细胞后，36.5℃，8％的CO 2。24h后补加转染体积2％的FEED(Sigma)，于36.5℃，120rpm，8％的CO 2条件下培养。连续培养至第6天或者细胞活力≤60％时，收集细胞上清进行纯化。
将纯化使用的重力柱使用0.5M NaOH过夜处理，玻璃瓶等用蒸馏水洗净后在180℃4h干烤，获得纯化柱。纯化前将收集的培养基4500rpm离心30min，弃掉细胞。再将上清使用0.22μl的滤器过滤。每管装填1ml ProteinA，并使用10ml结合缓冲液(磷酸钠20mM.NaCl 150mM，PH7.0)平衡。将过滤后的上清加入纯化柱后使用15ml结合缓冲液再平衡。加5ml洗脱缓冲液(柠檬酸+柠檬酸钠0.1M，PH3.5)，收集洗脱液，每1ml的洗脱液加入80μl Tris-HCl。将收集的抗体超滤浓缩交换到PBS(Gibco，70011-044)中，并检测浓度。
采用生物光干涉测定法(ForteBio)测定抗PD-L1抗体(HZ3266-IgG1N297A)结合人PD-L1的平衡解离常数(KD)。ForteBio亲和力测定按照现有的方法(Estep，P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs，2013.5(2)：第270-8页)进行。
实验开始前半个小时，根据样品数量，取合适数量的AMQ(Pall，1506091)(用于样品检测)或AHQ(Pall，1502051)(用于阳性对照检测)传感器浸泡于SD buffer(PBS 1×，BSA 0.1％，Tween-20 0.05％)中。
取100μl的SD缓冲液、抗体、抗原(包括人PD-L1均自Acrobiosystems购买)分别加入到96孔黑色聚苯乙烯半量微孔板(Greiner，675076)中。根据样品位置布板，选择传感器位置。仪器设置参数如下：运行步骤：Baseline、Loading～1nm、Baseline、Association和Dissociation；各个步骤运行时间取决于样品结合和解离速度，转速为400rpm，温度为30℃。使用ForteBio分析软件分析KD值。测得的K D值为0.724nM。
小鼠：NOG小鼠，雌性，7-8周(肿瘤细胞接种时的小鼠周龄)，体重17.6-24.2g，购自北京维通利华实验动物技术有限公司。小鼠在到达后驯化7天，随后开始研究。
细胞：人的皮肤癌细胞A375(ATCC# CRL-1619)购自ATCC，并严格按照ATCC要求进行常规传代培养用于后续体内实验。离心收集细胞，在无菌PBS中重悬细胞并调整细胞密度为30×10 6个/ml。NOG小鼠已经静脉注射了人的PBMC后，右侧背部剃毛，皮下注射A375细胞0.2ml/只。肿瘤细胞接种7天后检测各只小鼠瘤体积，挑选出瘤平均体积在70-71mm 3范围内的小鼠按瘤体积随机分组。
给药：每组分别皮下注射如下剂量的抗体：
(1)人IgG(equitech-Bio)，20mg/kg；
(2)PD-1(Antibody D，IBI308)，10mg/kg；
(3)LAG-3(ADI-31853)，10mg/kg；
(4)LAG-3(ADI-31853)，10mg/kg+PD-1(Antibody D，IBI308)，10mg/kg；
(5)PD-L1(HZ3266-IgG1N297A)，10mg/kg；
(6)LAG-3(ADI-31853)，10mg/kg+PD-L1(HZ3266-IgG1N297A)，10mg/kg。
接种后第7天，将符合瘤平均体积要求的小鼠随机分组，每组8只。分别用如上六组试剂在第7天、第10天、第14天和第17天为每组小鼠按如上剂量给药。
分析：在整个研究期间每周测量两次肿瘤和体重，当肿瘤达到端点时或当小鼠具有＞20％体重减轻时，使小鼠安乐死。采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。将来自每组的小鼠的肿瘤尺寸与时间作图。使用方差分析(ANOVA)来确定统计显著性。＜0.05的P值被视为在所有分析中具有统计显著性。
实验结果见图7A或B，可见本申请的抗LAG-3单克隆抗体ADI-31853(31853)和抗PD-1单克隆抗体“Antibody D”(IBI308)或抗PD-L1单克隆抗体HZ3266-IgG1N297A(HZ3266)联合使用时与IgG对照(equitech-Bio)(hIgG)及这两个抗体分别使用相比，能显著抑制肿瘤的生长。
实施例11.抗LAG-3抗体与抗PD-L1抗体组合对人CD4 +T细胞活化作用研究
本实验采用人CD4 +T细胞激活法检测ADI-31853对人T细胞的激活作用。将Beads刺激活化(如实施例7所述)的人CD4 +T细胞、成熟DC和不同浓度的ADI-31853、25F7单独溶液、PD-L1抗体(HZ3266-IgG1N297A，如上所述制备)单独溶液、以及其组合在96孔U底板中共孵育3天。最后检测上清中的IL-2浓度，比较ADI-31853、25F7单独，以及与PD-L1抗体(HZ3266-IgG1N297A，如上所述制备)对T细胞的激活作用。
实验步骤
准备DC
从液氮罐中取出人PBMC细胞(如实施例7所述制备)，在37℃水浴锅中迅速解冻，转移至20ml人T细胞培养基(含1‰DNA酶(Sigma))中，400g离心10min。用20ml人T细胞培养基(1‰DNA酶)重悬至T75培养瓶中，37℃、5％CO2细胞培养箱中培养24h。
DC细胞分离：弃悬浮细胞液，剩下细胞添加9ml DC培养基，培养2天后添加3ml DC培养基，再培养第5天，然后添加rTNFa(1000U/ml)，IL-1b(5ng/ml)，IL-6(10ng/ml)和1μM PGE2(Tocris)培养2天，作为淋巴细胞混合反应(MLR)的DC细胞。
其中，T细胞培养基及DC细胞培养基均为CTS AIM V SFM，DC培养基中还额外加入了1000U/ml IL-4和1000U/ml GM-CSF。货号及批号如下：
Figure PCTCN2018124315-appb-000066
人CD4 +T细胞纯化
从液氮罐中取出人PBMC细胞(如实施例7所述制备)，在37℃水浴锅中迅速解冻，然后转移至如上所述人T细胞培养基(1‰DNA酶)中，400g离心10min。采用如实施例7所述的Untouched CD4+T cell isolation试剂盒分离纯化人CD4 +T细胞，并用Dynabeads TM Human T-Activator CD3/CD28 for T Cell Expansion and Activation激活人CD4 +T细胞，37℃、 5％CO 2细胞培养箱中培养三天。
上样
将成熟的DC细胞与CD4+细胞混合，每孔体积200μl，DC细胞10000个，CD4+细胞100000个，加入抗体(200nM，4倍系列稀释，SEE(Toxin technology)1ng/ml)，DC、CD4+细胞、混合细胞作为阴性对照，混合培养3天。
所利用的抗体浓度如下：
抗LAG-3 ADI-31853：200nM起始，4倍稀释，共计10个浓度
抗PD-L1：200nM起始，4倍稀释，共计10个浓度
25F7：200nM起始，4倍稀释，共计10个浓度
抗LAG-3(200nM起始，4倍稀释，共计10个浓度)+抗PD-L1(12.5nM)
25F7(200nM起始，4倍稀释，共计10个浓度)+抗PD-L1(12.5nM)
IL-2检测
采用Cisbio Human IL-2kit检测培养基中IL-2的浓度。
(1)检测液配制(试剂均来自Human IL2 1000tests试剂盒，Cisbio)
取30μl Human IL-2 d2 antibody(来源)至570μl检测缓冲液(来源)中，取30μl Human IL-2 cryptate antibody(来源)至570μl检测缓冲液中，二者1∶1混合。
(2)标准品溶液梯度稀释
表7.IL-2标准品溶液的梯度稀释
Figure PCTCN2018124315-appb-000067
(3)检测
取培养基上清和标准品梯度溶液(10μl)加入96孔微孔板，每孔加入10μl检测液，400g离心1min，避光，室温孵育2h。
采用多功能酶标仪，在616nm和665nm读取OD值，分析数据。
算法公式为：Ratio＝OD 665nm/OD 616nm×10 4
ΔF％＝(Ratio 标准/样品-Ratio 标准0)/Ratio 标准0
实验结果
以标准品梯度浓度的ΔRatio为横坐标，IL-2水平为纵坐标，得IL-2标准曲线，如图8所示，以标准曲线，计算各组中人CD4+T细胞的IL-2水平。如图9所示，与对照(25F7)或抗LAG-3抗体ADI31853单独相比，本发明的抗LAG-3抗体(ADI-31853)与抗PD-L1抗体(HZ3266)组合更加显著激活人CD4 +T细胞。

Claims (28)

  1. 结合LAG-3的抗体或其抗原结合片段，所述抗体包含
    (i)如SEQ ID NO：22所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：26所示的轻链可变区的3个互补决定区LCDR，或者
    (ii)如SEQ ID NO：23所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：27所示的轻链可变区的3个互补决定区LCDR，或者
    (iii)如SEQ ID NO：24所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：28所示的轻链可变区的3个互补决定区LCDR，或者
    (iv)如SEQ ID NO：25所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：29所示的轻链可变区的3个互补决定区LCDR。
  2. 结合LAG-3的抗体或其抗原结合片段，所述抗体包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中
    HCDR1包含SEQ ID NO：1、2、3、4或17所示的氨基酸序列，HCDR2包含SEQ ID NO：5、6、7或18所示的氨基酸序列，HCDR3包含SEQ ID NO：8、9、10或19所示的氨基酸序列，LCDR1包含SEQ ID NO：11、12或20所示的氨基酸序列，LCDR2包含SEQ ID NO：13所示的氨基酸序列，且LCDR3包含SEQ ID NO：14、15、16或21所示的氨基酸序列。
  3. 结合LAG-3的抗体或其抗原结合片段，所述抗体包含重链可变区和/或轻链可变区，其中
    所述重链可变区包含：
    (i)表B所列任一抗体的VH中所含的三个互补决定区域(HCDR)；或
    (ii)表A所示的HCDR1、HCDR2和HCDR3的组合；
    和/或
    所述轻链可变区包含：
    (i)表B所列任一抗体的VL中所含的三个互补决定区域(LCDR)；或
    (ii)表A所示的LCDR1、LCDR2和LCDR3的组合。
  4. 权利要求1至3中任一项的抗体或其抗原结合片段，所述抗体包含轻链可变区和/或重链可变区，其中，重链可变区包含与选自SEQ ID NO：22、23、24或25的氨基酸序列具有至少90％同一性的氨基酸序列或由其组成；和/或
    轻链可变区包含与选自SEQ ID NO：26、27、28或29的氨基酸序列具有至少90％同一性的氨基酸序列或由其组成。
  5. 权利要求1至3中任一项的抗体或其抗原结合片段，所述抗体包含
    含有SEQ ID NO：22、23、24或25所示的氨基酸序列的重链可变区，和/或含有SEQ ID NO：26、27、28或29所示的氨基酸序列的轻链可变区。
  6. 权利要求1至5中任一项的抗体或其抗原结合片段，所述抗体包含
    (a)重链
    (i)包含与选自SEQ ID NO：30、31、32或33的氨基酸序列具有至少85％同一性的氨基酸序列或由其组成；或者
    (ii)包含选自SEQ ID NO：30、31、32或33的氨基酸序列或由其组成；
    和/或
    (b)轻链
    (i)包含与选自SEQ ID NO：34、35、36或37的氨基酸序列具有至少85％同一性的氨基酸序列或由其组成；或者
    (ii)包含选自SEQ ID NO：34、35、36或37的氨基酸序列或由其组成。
  7. 权利要求1至6中任一项的结合LAG-3的抗体或其抗原结合片段，其具有以下一个或多个特性：
    (1)以高亲和力结合LAG-3(例如人LAG-3)，例如，以以下平衡解离常数(K D)与LAG-3结合，所述K D小于大约8nM；
    (2)结合表达人LAG-3的细胞，例如，以小于或等于大约1nM的EC50；
    (3)结合表达小鼠LAG-3的细胞，例如，以小于或等于大约50nM的EC50，例如以大约40-50nM的EC50；
    (4)抑制LAG-3的相关活性，例如以小于或等于大约5nM的IC 50抑制LAG-3与表达MHC II类分子的细胞上的MHC II类分子的结合；
    (5)结合激活的CD4+和/或CD8+T细胞的表面上的内源LAG-3，例如以小于或等于大约35pM；
    (6)抑制LAG-3的一种或多种活性，例如，导致以下一种或多种：CD4+T淋巴细胞的抗原依赖性刺激增加；T细胞增殖增加；活化抗原(例如，CD25)的表达增加；细胞因子(例如，干扰素-γ(IFN-γ)、白介素-2(IL-2)或白介素-4(IL-4))的表达增加；趋化因子(例如，CCL3、CCL4或CCL5)的表达增加；Treg细胞的阻抑活性减少；T细胞稳态增加；肿瘤浸润型淋巴细胞增加；或癌细胞的免疫逃避减少；
    (7)能够诱发抗体依赖性细胞介导的细胞毒性(ADCC)；
    (8)显示与表3所列的任一抗体对LAG-3相同或相似的结合亲和力和/或特异性；
    (9)抑制(例如，竞争性抑制)表3所列的任一抗体与LAG-3的结合；
    (10)与表3所示的任一抗体结合相同或重叠的表位；
    (11)与表3所示的任一抗体竞争结合LAG-3；
    (12)具有表3所列的任一抗体分子的一个或多个生物学特性。
  8. 权利要求1至7中任一项的抗LAG-3抗体或其抗原结合片段，其中所述抗体是IgG4形式的抗体或其抗原结合片段，任选地所述抗LAG-3抗体或其抗原结合片段包含κ轻链恒定区，例如人κ轻链恒定区。
  9. 权利要求1至8中任一项的抗LAG-3抗体或其抗原结合片段，其中所述抗体是单克隆抗体。
  10. 权利要求1至9中任一项的抗LAG-3抗体或其抗原结合片段，其中所述抗体是人源化的抗体或人抗体。
  11. 权利要求1至10中任一项的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体例如scFv、(Fab’) 2片段、单结构域抗体、双抗体(dAb)或线性抗体。
  12. 权利要求1至11中任一项的抗体或其抗原结合片段，其中所述抗体是双特异性抗体或多特异性抗体，优选地，所述双特异性抗体结合LAG-3和PD-1，结合LAG-3和PD-L1，或结合LAG-3和PD-L2；优选地，所述多特异性抗体包含针对LAG-3的第一结合特异性和针对以下一种或多种的分子的第二及第三结合特异性：PD-1(例如人PD-1)、TIM-3、CEACAM(例如、CEACAM-1或CEACAM-5)、PD-L1(例如人PD-L1)或PD-L2(例如人PD-L2)。
  13. 分离的核酸，其编码权利要求1至12中任一项的抗LAG-3抗体或其抗原结合片段。
  14. 包含权利要求13的核酸的载体，优选地所述载体是表达载体，例如pTT5载体。
  15. 包含权利要求13的核酸或权利要求14的载体的宿主细胞，优选地，所述宿主细胞是原核的或真核的，更优选的选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞，最优选地，所述宿主细胞是293细胞或CHO细胞。
  16. 制备抗LAG-3抗体或其抗原结合片段的方法，所述方法包括在适于表达编码权利要求1至12中任一项的抗LAG-3抗体或其抗原结合片段的核酸的条件下培养权利要求15的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述抗LAG-3抗体或其抗原结合片段。
  17. 免疫缀合物，其包含权利要求1至12中任一项的抗LAG-3抗体或其抗原结合片段和其它物质，例如细胞毒性剂。
  18. 药物组合物，其包含权利要求1至12中任一项的抗LAG-3抗体或其抗原结合片段或者权利要求17的免疫缀合物，以及任选地药用辅料。
  19. 药物组合物，其包含权利要求1至12中任一项的抗LAG-3抗体或其抗原结合片段或者权利要求17的免疫缀合物，以及其它治疗剂，以及任选地药用辅料；优选地，所述其它治疗剂选自化疗剂、其它抗体、细胞毒性剂、疫苗、抗感染活性剂或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)，优选地抗PD-1抗体、抗PD-L1抗体或抗PD-L2抗体，优选地，抗人PD-1抗体或抗人PD-L1抗体，例如人源化的抗人PD-1抗体或人源化的抗人PD-L1抗体。
  20. 权利要求19所述的药物组合物，其中所述抗PD-L1抗体包含
    (i)如SEQ ID NO：57所示的重链可变区的3个互补决定区HCDR，和/或
    (ii)如SEQ ID NO：58所示的轻链可变区的3个互补决定区LCDR。
  21. 权利要求19所述的药物组合物，其中所述抗PD-L1抗体重链可变区(VH)和/或轻链可变区(VL)，其中
    (i)所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：51的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含选自SEQ ID NO：52的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：53的氨基酸序列或由所述氨基酸序列组成；
    和/或
    (ii)其中所述VL包含互补决定区域(CDR)LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO：54的氨基酸序列或由所述氨基酸序列组成；LCDR2包含SEQ ID NO：55的氨基酸序列或由所述氨基酸序列组成；LCDR3包含选自SEQ ID NO：56的氨基酸序列或由所述氨基酸序列组成。
  22. 有效量的权利要求1至12中任一项的抗LAG-3抗体或其抗原结合片段、或权利要求17的免疫缀合物、或权利要求18至21中任一项的药物组合物在制备药物中的用途，所述药物用于在受试者中预防或治疗受试者或个体肿瘤或感染性疾病。
  23. 有效量的权利要求1至12中任一项的抗LAG-3抗体或其抗原结合片段、或权利要求17的免疫缀合物、或权利要求18或19的药物组合物，以及PD-1轴结合拮抗剂或包含所述PD-1轴结合拮抗剂的药物或活性剂在制备药物中的用途，所述药物用于在受试者中预防或治疗受试者或个体肿瘤或感染性疾病，优选地，所述PD-1轴结合拮抗剂是抗PD-1抗体或抗PD-L1抗体，例如抗人PD-1抗体或抗人PD-L1抗体，例如人源化的抗人PD-1抗体或人源化的抗人PD-L1抗体。
  24. 权利要求22或23的用途，其中所述肿瘤是癌症，例如胃肠道肿瘤，例如胃肠道癌症，例如结肠癌；或者所述感染性疾病是慢性感染。
  25. 权利要求22至24中任一项所述的用途，其还包括向所述受试者联合施用一种或多种其它疗法，所述疗法例如包括治疗方式和/或其它治疗剂，优选地，所述治疗方式包括手术治疗和/或放射疗法，或者所述治疗剂选自化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)，优选地，所述其他治疗剂抗PD-1抗体、抗PD-L1抗体或抗PD-L2抗体，优选地，抗人PD-1抗体或抗人PD-L1抗体，例如人源化的抗人PD-1抗体或人源化的抗人PD-L1抗体。
  26. 权利要求23至25中任一项所述的用途，其中所述抗PD-L1抗体包含
    (i)如SEQ ID NO：57所示的重链可变区的3个互补决定区HCDR，和/或
    (ii)如SEQ ID NO：58所示的轻链可变区的3个互补决定区LCDR。
  27. 权利要求23至25中任一项所述的用途，其中所述抗PD-L1抗体重链可变区(VH)和/或轻链可变区(VL)，其中
    (i)所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：51的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含选自SEQ ID NO：52的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：53的氨基酸序列或由所述氨基酸序列组成；
    和/或
    (ii)其中所述VL包含互补决定区域(CDR)LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO：54的氨基酸序列或由所述氨基酸序列组成；LCDR2包含SEQ ID NO：55的氨基酸序列或由所述氨基酸序列组成；LCDR3包含选自SEQ ID NO：56的氨基酸序列或由所述氨基酸序列组成。
  28. 检测样品中LAG-3的方法，所述方法包括
    (a)将样品与权利要求1至12中任一项的任何抗LAG-3抗体或其抗原结合片段接触；和
    (b)检测抗LAG-3抗体或其抗原结合片段和LAG-3间的复合物的形成；任选地，抗LAG-3抗体是被可检测地标记的。
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