WO2019129136A1 - 抗pd-l1抗体及其用途 - Google Patents

抗pd-l1抗体及其用途 Download PDF

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Publication number
WO2019129136A1
WO2019129136A1 PCT/CN2018/124314 CN2018124314W WO2019129136A1 WO 2019129136 A1 WO2019129136 A1 WO 2019129136A1 CN 2018124314 W CN2018124314 W CN 2018124314W WO 2019129136 A1 WO2019129136 A1 WO 2019129136A1
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Prior art keywords
antibody
amino acid
acid sequence
seq
chain variable
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PCT/CN2018/124314
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English (en)
French (fr)
Inventor
缪小牛
胡化静
刘军建
陈炳良
李莉
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Innovent Biologics Suzhou Co Ltd
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Innovent Biologics Suzhou Co Ltd
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Priority claimed from CN201811567281.3A external-priority patent/CN109970857B/zh
Priority to JP2020525995A priority Critical patent/JP7534954B2/ja
Application filed by Innovent Biologics Suzhou Co Ltd filed Critical Innovent Biologics Suzhou Co Ltd
Priority to KR1020207013300A priority patent/KR102384845B1/ko
Priority to BR112020007725-9A priority patent/BR112020007725A2/pt
Priority to CA3076447A priority patent/CA3076447A1/en
Priority to US16/648,033 priority patent/US11512131B2/en
Priority to NZ762662A priority patent/NZ762662B2/en
Priority to AU2018395084A priority patent/AU2018395084B2/en
Priority to EP18894248.6A priority patent/EP3733704A4/en
Priority to SG11202002530RA priority patent/SG11202002530RA/en
Publication of WO2019129136A1 publication Critical patent/WO2019129136A1/zh
Priority to IL273411A priority patent/IL273411A/en
Anticipated expiration legal-status Critical
Priority to JP2024125864A priority patent/JP2024164038A/ja
Ceased legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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

Definitions

  • the present invention relates to novel antibodies and antibody fragments that specifically bind to PD-L1 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 therapy of these antibodies and antibody fragments with other therapies, such as therapeutic modalities or therapeutic agents.
  • Programmed Death Ligand 1 is a protein involved in suppressing immune system responses during chronic infection, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 regulates the immune response by binding to an inhibitory receptor called programmed death 1 (PD-1) expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function through interaction with another receptor, B7.1 (also known as B7-1 or CD80). The formation of the PD-L1/PD-1 and PD-L1/B7.1 complex negatively regulates T cell receptor signaling, resulting in subsequent down-regulation of T cell activation and inhibition of anti-tumor immune activity.
  • PD-1 programmed death 1
  • PD-L1 is overexpressed in many cancers, including a wide variety of solid tumors, such as bladder tumors, breast tumors, colon tumors, lung tumors, melanoma, ovarian tumors, salivary tumors, gastric tumors, and thyroid tumors.
  • solid tumors such as bladder tumors, breast tumors, colon tumors, lung tumors, melanoma, ovarian tumors, salivary tumors, gastric tumors, and thyroid tumors.
  • Overexpression of PD-L1 in tumor cells promotes tumor invasion and is often associated with poor prognosis.
  • antibody molecules that bind to PD-L1. 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 antibody molecule against PD-L1, and pharmaceutical compositions are also provided.
  • the anti-PD-L1 antibody molecules disclosed herein can be used to treat, prevent, and/or diagnose neoplastic diseases as well as infectious diseases, either alone or in combination with other therapies, such as therapeutic agents or therapeutic modalities. Further, disclosed herein are compositions and methods for detecting PD-L1, and methods of preventing or treating a variety of diseases, including tumors and/or infectious diseases, using anti-PD-L1 antibody molecules.
  • an antibody of the invention or a fragment thereof (specific) binds to PD-L1. In some embodiments, an antibody of the invention or a fragment thereof (specific) binds to human PD-L1.
  • an anti-PD-L1 antibody or fragment thereof of the invention binds PD-L1 (eg, human PD-L1) with high affinity, eg, binds to PD-L1 with the following equilibrium dissociation constant (K D ),
  • K D is less than about 50 nM, preferably less than or equal to about 20 nM, more preferably less than or equal to about 15 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.
  • an anti-PD-L1 antibody of the invention is 0.1-10 nM, preferably 0.5-10 nM, more preferably 0.6-10 nM, 0.7-8 nM, 0.7-5 nM, and most preferably 0.5-1.5 nM, 0.7- 1.5 dM, 0.7-1 nM K D binds to PD-L1.
  • PD-L1 is human PD-L1.
  • 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 PD-L1, eg, at less than or equal to about 4 nM, 3.5 nM, 3 nM, 2.9 nM, 2.8 nM, 2.7 nM, 2.6 nM, 2.5 nM EC50 of 2.4 nM, 2.3 nM, 2.2 nM, 2.1 nM, 2 nM, 1.9 nM, 1.8 nM, 1.7 nM or 1.6 nM.
  • the binding is determined by flow cytometry (eg, FACS).
  • the cell expressing human PD-L1 is a CHO cell expressing human PD-L1.
  • an antibody or fragment thereof of the invention blocks the activity of PD-L1, for example, with an EC of less than or equal to about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM. 50, preferably about 0.1-1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM 1nM or the EC 50.
  • the related activity of PD-L1 is the binding of PD-L1 to PD-1.
  • the antibody or fragment of the invention in the MOA assay of less than or equal to about 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM or 0.7nM of EC 50, preferably from about 0.1 -1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM EC 50 of 1nM or inhibiting PD-L1 PD-1 binding with.
  • the cell is a CHO cell.
  • an antibody or fragment thereof of the invention increases T cell function. In some embodiments, an antibody or fragment thereof of the invention increases T cell proliferation. In some embodiments, an antibody or fragment thereof of the invention increases IFN-[gamma] secretion. In some embodiments, an antibody or fragment thereof of the invention increases IL-2 secretion. In some embodiments, an antibody or fragment thereof of the invention increases IFN-[gamma] secretion and IL-2 secretion. In some embodiments, the increase is determined in a mixed lymphocyte reaction (MLR). In some embodiments, the ability of an antibody or fragment thereof of the invention to activate T cells is superior to known anti-PD-L1 antibodies, such as Tecentriq.
  • MLR mixed lymphocyte reaction
  • an antibody of the invention or a fragment thereof has a lower viscosity than a known anti-PD-Ll antibody (eg, Tecentriq) and thus has better drug availability.
  • the antibody or fragment thereof of the invention in a Zenix column assay, has a residence time (RT) of less than about 10 minutes, about 9 minutes, or about 8 minutes, preferably, a residence time of about 7 minutes. Between -9 minutes, preferably between about 7-8.5 minutes, about 7.5-8.5 minutes, about 7-8 minutes, or about 7.5-8 minutes, such as about 7.5 minutes, 7.6 minutes, 7.7 minutes, 7.8 minutes, 7.9 minutes, 8 minutes, 8.1 minutes, 8.2 minutes, 8.3 minutes, 8.4 minutes, 8.5 minutes.
  • RT residence time
  • an antibody or fragment thereof of the invention inhibits one or more activities of PD-L1, for example, resulting in one or more of: increased tumor infiltrating lymphocytes, T cell receptor mediated proliferation Increase, or the immune evasion of cancer cells is reduced.
  • an anti-PD-L1 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-PD-Ll antibody of the invention is effective to treat a tumor (eg, a cancer) or an infection (eg, a chronic infection).
  • a tumor eg, a cancer
  • an infection eg, a chronic infection
  • the tumor is a tumor immune escape.
  • 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-PD-L1 antibody or fragment thereof of the invention further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68).
  • the antibodies of the invention also encompass variants of the amino acid sequence of an anti-PD-L1 antibody, as well as antibodies that bind to the same epitope as any of the anti-PD-L1 antibodies or fragments thereof described above.
  • an anti-PD-L1 antibody of the invention further comprises a human or murine constant region.
  • an anti-PD-L1 antibody of the invention is an antibody in the form of IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE.
  • an anti-PD-L1 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 or IgG4, such as the heavy chain constant region of human IgG1 or IgG4.
  • the heavy chain constant region is a human IgGl or human IgG4 heavy chain constant region.
  • the anti-PD-L1 antibody of the invention comprises a murine constant region selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3.
  • an anti-PD-L1 antibody molecule of the invention 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-PD-L1 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 anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG1 (eg, human IgG1).
  • human IgGl comprises a substitution at position 297 according to the EU number (eg, Asn to Ala substitution). In one embodiment, human IgGl comprises a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both substitutions (eg, Asp to Ala at position 265 according to EU numbering) Replacement and/or Pro to Ala replacement at position 329 according to EU numbering). In one embodiment, human IgGl comprises a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or both substitutions (eg, Leu to Ala at position 234 according to EU numbering) Replacement and/or replacement of Leu to Ala at position 235 according to EU numbering).
  • EU number eg, Asn to Ala substitution
  • human IgGl comprises a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both substitutions (eg, Asp to Ala at position 265 according to
  • the 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: 64, 65 or 66 A sequence of %, 95%, 96%, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the anti-PD-L1 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:67 A sequence of %, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG1 (eg, a heavy chain constant region of human IgG1) and a kappa light chain constant region (eg, a human kappa light chain constant region).
  • human IgGl comprises a substitution at position 297 according to the EU number (eg, Asn to Ala substitution).
  • the human IgG1 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: 64 or 65 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:67 A sequence of 96%, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the anti-PD-L1 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) and/or having a position at positions 114-115 ( EU number) mutation to AA 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:66. 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:67 A sequence of 96%, 97%, 98%, 99% or more identity, or consists of the sequence.
  • the anti-PD-L1 antibody molecule is isolated or recombinant.
  • the anti-PD-L1 antibody is a monoclonal antibody or an antibody having monospecificity.
  • the anti-PD-L1 antibody molecule can also be a humanized, chimeric, human antibody molecule.
  • the anti-PD-L1 antibody is a chimeric antibody.
  • the anti-PD-L1 antibody is a humanized antibody.
  • the anti-PD-L1 antibody is a human 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 molecule is in the form of a bispecific or multispecific antibody molecule.
  • the bispecific antibody molecule has a first binding specificity for PD-L1 and a second binding specificity for LAG-3.
  • the bispecific antibody molecule binds to PD-L1 and LAG-3.
  • a multispecific antibody molecule can have any combination of binding specificities for PD-L1 with other targets.
  • the invention provides a nucleic acid encoding any of the above anti-PD-L1 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 (e.g., CHO cell or 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-PD-L1 antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises, at an expression suitable for expression of the antibody or fragment thereof (preferably an antigen-binding fragment)
  • 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-PD-L1 antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.
  • the invention provides an immunoconjugate comprising any of the anti-PD-L1 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 tumor immune escape.
  • 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-PD-L1 antibodies or fragments thereof (preferably antigen-binding fragments thereof) described herein, or immunoconjugates thereof, preferably the compositions are pharmaceutical compositions.
  • the composition further comprises a pharmaceutical excipient.
  • a composition eg, a pharmaceutical composition, comprises an anti-PD-L1 antibody of the invention, or a fragment thereof, or an immunoconjugate thereof, and one or more additional therapeutic agents (eg, chemotherapeutic agents, other antibodies)
  • additional therapeutic agents eg, chemotherapeutic agents, other antibodies
  • a combination of a cytotoxic agent, a vaccine, 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 pharmaceutical composition is for use in preventing or treating a tumor (eg, cancer) or an infection.
  • the tumor is a tumor immune escape.
  • 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 anti-PD described herein -L1 antibody or fragment thereof, pharmaceutical composition or immunoconjugate.
  • the tumor is a tumor immune escape.
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • the infectious disease is a chronic infection.
  • the invention relates to the use of any of the anti-PD-L1 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 tumor immune escape.
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • the infectious disease is a chronic infection.
  • 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, an anti-infective active agent, other antibodies, 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, such as a mammal, preferably a human.
  • the invention relates to a method of detecting PD-L1 in a sample, the method comprising (a) contacting a sample with any of the anti-PD-L1 antibodies or fragments thereof described herein; and (b) detecting anti-PD- Formation of a complex between the L1 antibody or a fragment thereof and PD-L1.
  • the anti-PD-L1 antibody is detectably labeled.
  • the invention relates to a kit or article comprising any of the anti-PD-L1 antibodies or fragments thereof described herein.
  • the kit or article of manufacture comprises an anti-PD-L1 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-PD-L1 antibodies or fragments, methods and uses thereof of the invention described herein.
  • Figure 1 shows the binding of the anti-PD-L1 antibody of the present invention and CHO-PDL1 cells detected by FACS.
  • Figure 2 shows the binding of the anti-PD-L1 antibody of the present invention and CHO-PDL1 cells detected by FACS.
  • Figure 3 shows the blocking activity of the antibody of the present invention against PD-1/PD-L1 interaction detected by the MOA method.
  • Figures 4A and 4B show activation of T cells (relative expression of IL-2) by antibodies of the invention as detected by MLR assay.
  • FIGS 5A and 5B show the activation of T cells (relative expression of IFN- ⁇ ) by the antibody of the present invention detected by the MLR assay.
  • Figure 6 shows the inhibitory effect of the antibody of the present invention on tumors.
  • Figure 7 shows the inhibition of tumors by the antibody of the present invention in combination with an anti-LAG-3 antibody.
  • 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.
  • programmed cell death 1 ligand 1 refers to Any native PD-L1 of any vertebrate origin, including any mammalian species, such as primates (eg, humans) and rodents (eg, mice and rats).
  • the terms encompass “full length”, unprocessed PD-L1, and any form of PD-L1 produced by processing in a cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
  • the basic structure of PD-L1 includes four domains: an extracellular Ig-like V-domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. Additional information regarding the human PD-L1 gene (including genomic DNA sequences) can be found under NCBI Gene ID No. 29126. Additional information regarding the mouse PD-L1 gene (including genomic DNA sequences) can be found under NCBI Gene ID No. 60533. The amino acid sequence of an exemplary full length human PD-L1 protein can be found, for example, under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7, while an exemplary full length mouse PD can be found, for example, under NCBI Accession No. NP_068693 or Uniprot Accession No. Q9EP73. L1 protein sequence.
  • anti-PD-L1 antibody refers to an antibody capable of binding PD with sufficient affinity. -L1 protein or a fragment thereof.
  • the anti-PD-L1 antibody binds to a non-PD-L1 protein to a lesser extent than about 10%, about 20%, about 30%, about 40%, about 50% of the binding of the antibody to PD-L1.
  • 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 IgGl 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 PD-L1) 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 by 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 bordered by Chothia rules or Kabat rules, for example, the sequences are 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 IgGl form refers to an Ig domain whose heavy chain constant region Ig domain is IgG1.
  • 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 WO2015/153513, WO2016/028672, WO2015/138920, WO2016/007235.
  • 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 WO2016/007235 or WO2010/077634 or US60/696426, which can be used in combination with an anti-PD-L1 antibody.
  • a tumor eg, cancer
  • an infection eg, a chronic infection
  • An immunomodulator such as any of the materials disclosed in WO2016/007235 or WO2010/077634 or US60/696426, which can be used in combination with an anti-PD-L1 antibody.
  • “Chemotherapeutic agents” include chemical compounds that are useful in the treatment of cancer. Examples of chemotherapeutic agents are disclosed in WO2016/007235, WO2010/077634, US60/696426 or WO2016/061142, US61/264061 or WO2016/007235.
  • 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-6 interleukins
  • IL-8 interleukins
  • IL-9 tumor necrosis factor
  • TNF- ⁇ or TNF- ⁇ tumor necrosis factor
  • other polypeptide factors including LIF and kit ligand (KL) and ⁇ -interferon.
  • the term cytokine includes
  • 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).
  • the term includes a substance that inhibits the given molecule by at least 5%, 10%, 20%, 30%, 40% or more of activity (eg, LAG-3 activity). Therefore, the inhibition does not have to be 100%.
  • 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 FcyRIII and functions 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
  • 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% or 90%.
  • 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 the CDR residues of the complementarity determining region.
  • 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
  • cancers suitable for treatment by the antibodies of the invention include non-small cell lung cancer, squamous cell carcinoma, small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glial Tumor, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid Cancer, liver cancer, leukemia and head and neck cancer, including those metastatic forms of cancer.
  • 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.
  • tumor immune escape refers to tumors escaping immune recognition and clearance.
  • tumor immunity is “treated” and the tumor is recognized and attacked by the immune system.
  • tumor recognition include tumor binding, tumor shrinkage, and tumor clarity.
  • 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-PD-L1 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 a 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 about 45 C in 6X sodium chloride/sodium citrate (SSC) followed by at least 50 C (for low stringency conditions, washing can be increased) The temperature was up to 55 C) washed twice in 0.2X SSC, 0.1% SDS; 2) medium stringency hybridization conditions were washed once 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 one or more washes in 6X SSC at about 45 ° C followed by 0.2X SSC, 0.1% SDS at 65 ° C; and preferably 4) very high stringency hybridization The 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)), excipient, carrier or stabilizer, and the like, which are 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, WO 2015/138920 or WO 2016/061142.
  • 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 of the invention or a fragment thereof binds to PD-L1.
  • an antibody or fragment thereof of the invention binds to a mammalian PD-L1, such as human PD-L1.
  • an antibody molecule specifically binds to an epitope (eg, a linear or conformational epitope) on PD-L1.
  • the antibody molecule binds to one or more extracellular domains of PD-L1.
  • an anti-PD-L1 antibody or fragment thereof of the invention has one or more of the following properties:
  • the anti-PD-L1 antibody of the present invention or a fragment thereof binds PD-L1 (for example, human PD-L1) with high affinity, for example, binds to PD-L1 with the following equilibrium dissociation constant (K D ), said K D is less than about 50 nM, preferably less than or equal to about 20 M, more preferably less than or equal to about 15 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 of less than or equal to about 1.5nM, 1.4nM, 1.3nM, 1.2nM, 1.1nM, 1nM, 0.9nM or 0.8nM.
  • K D equilibrium dissociation constant
  • an anti-PD-L1 antibody of the invention is 0.1-10 nM, preferably 0.5-10 nM, more preferably 0.6-10 nM, 0.7-8 nM, 0.7-5 nM, and most preferably 0.5-1.5 nM, 0.7- 1.5 dM, 0.7-1 nM K D binds to PD-L1.
  • PD-L1 is human PD-L1.
  • 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 PD-L1, for example, at less than or equal to about 4 nM, 3.5 nM, 3 nM, 2.9 nM, 2.8 nM, 2.7 nM, 2.6 nM, 2.5 nM, 2.4 nM.
  • the binding is determined by flow cytometry (eg, FACS).
  • the cell expressing human PD-L1 is a CHO cell expressing human PD-L1.
  • the antibody or fragment thereof of the present invention blocks the relative activity of PD-L1, for example, with an EC 50 of less than or equal to about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM or 0.7 nM, preferably about 0.1-1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM 1nM or the EC 50.
  • the related activity of PD-L1 is the binding of PD-L1 to PD-1.
  • the antibody or fragment of the invention in the MOA assay of less than or equal to about 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM or 0.7nM of EC 50, preferably from about 0.1 -1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM EC 50 of 1nM or inhibiting PD-L1 PD-1 binding with.
  • the cell is a CHO cell.
  • the antibody or fragment thereof of the present invention enhances T cell function, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.
  • the antibody or fragment thereof of the present invention enhances T cell proliferation, for example, in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.
  • the antibody or fragment thereof of the present invention enhances IFN- ⁇ secretion, for example, in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.
  • the antibody or fragment thereof of the present invention enhances IL-2 secretion, for example, in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.
  • the antibody of the present invention or a fragment thereof has a lower viscosity than a known anti-PD-L1 antibody (e.g., Tecentriq), and thus has better drug-forming properties.
  • the antibody or fragment thereof of the invention in a Zenix column assay, has a residence time (RT) of less than about 10 minutes, about 9 minutes, or about 8 minutes, preferably, a residence time of about 7 minutes. Between -9 minutes, preferably between about 7-8.5 minutes, about 7.5-8.5 minutes, about 7-8 minutes, or about 7.5-8 minutes, such as about 7.5 minutes, 7.6 minutes, 7.7 minutes, 7.8 minutes, 7.9 minutes, 8 minutes, 8.1 minutes, 8.2 minutes, 8.3 minutes, 8.4 minutes, 8.5 minutes.
  • RT residence time
  • the antibody or fragment thereof of the present invention inhibits one or more activities of PD-L1, for example, resulting in one or more of: an increase in tumor infiltrating lymphocytes, an increase in T cell receptor-mediated proliferation, or The immune escape of cancer cells is reduced.
  • the anti-PD-L1 antibody of the present invention or a fragment thereof is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention has one or more of the following properties:
  • an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein said VH comprises
  • an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises:
  • an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and 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: 26, 27, 28, 29, 30 or 31.
  • VL comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36 or 37.
  • 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 selected from SEQ ID NO: 1, 2, 3 or 4, 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 or 4; HCDR2 comprising an SEQ ID NO: 5 Or the amino acid sequence of 6, 7, 8, or 9, or HCDR2 comprises one, two or more than the amino acid sequence selected from SEQ ID NO: 5, 6, 7, 8, or 9.
  • CDR complementarity determining region
  • HCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 10, 11, 12 or 13 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 10, 11, 12 or 13;
  • VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 14, 15 or 16, 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: 14, 15 or 16; LCDR2 comprising a selected from the group consisting of SEQ ID NOs: 17, 18, 19 Or the amino acid sequence of 20 or consists of the amino acid sequence, or LCDR2 comprises one, two or three changes compared to the amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20 (preferably amino acid substitution, preferably Amino acid sequence of conservative substitution); LCDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 25, or LCDR3 comprises and is selected from the group consisting of SEQ ID NO: 21, 22, 23 The amino acid sequence of 24 or 25 has an amino acid sequence having one, two
  • the invention provides an anti-PD-L1 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-PD-L1 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-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
  • amino acid change comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30 or 31; 2, 1) amino acid sequence of 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 change comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36 or 37; 2.
  • amino acid sequence of an amino acid change preferably amino acid substitution, more preferably amino acid conservative substitution, preferably, the amino acid change does not occur in the CDR region.
  • the invention provides an anti-PD-L1 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-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no) than an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42, 43, 44 or 45
  • An amino acid sequence of more than 5, 4, 3, 2, 1) amino acid changes preferably amino acid substitutions, more preferably amino acid conservative substitutions, preferably, the amino acid changes do not occur in the CDR regions of the heavy chain, more preferably The amino acid change does not occur in the heavy chain variable region;
  • amino acid sequence of amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) of 3, 2, 1), preferably, said amino acid change does not occur in the CDR region of the light chain, more preferably, said amino acid The change does not occur in the light chain variable region.
  • the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the combination of the heavy chain and the 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-PD-L1 antibody or fragment thereof of the invention further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68).
  • 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-PD-L1 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 were 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.
  • Anti-PD-L1 antibodies eg, humanized antibodies or chimeric antibodies
  • antigen-binding fragments thereof disclosed herein also include antibodies and fragments having modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent No. 5,624,821, WO2003/086310, WO2005/120571, WO2006/0057702.
  • modifications can be used to enhance or inhibit various responses of the immune system, possibly with 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-PD-L1 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), single domain antibody; Multispecific antibodies formed.
  • 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).
  • an antibody molecule comprises two heavy chain variable domain sequences and two light chain variable domain sequences, thereby forming two antigen binding sites.
  • Fab fragment antigen binding
  • Fab' fragment antigen binding
  • Fc single chain antibody
  • Fd single domain antibody
  • Dab diabody
  • inlay Antibodies eg, humanized
  • These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors.
  • 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-PD-L1 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 its 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-PD-L1 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. 7: 13. -21; Morrison, SL et al, 1994 Proc. Natl. Acad. Sci.
  • an anti-PD-L1 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 is a chimeric antibody.
  • Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA. 81:6851-6855, 1984.
  • the chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region.
  • the chimeric antibody is a "class switching" antibody, wherein the class or subclass has been altered compared to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • the chimeric antibody is a humanized antibody.
  • non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains, wherein, for example, a CDR (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence.
  • the humanized antibody optionally will also comprise at least a portion of the human constant region.
  • some of the FR residues in the humanized antibody are replaced by corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), eg, to restore or improve antibody specificity or affinity.
  • 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 PD-L1 and the second epitope is on LAG-3, OX40, TIM-3, CEACAM (eg, CEACAM-1 and/or CEACAM-5) or PD-L2 .
  • the invention also encompasses anti-PD-L1 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 WO2015/153513 or WO2015/138920.
  • 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, such as paclitaxel, cytochalasin B, gramicidin D, Ethidium bromide, ipecaine, mitomycin, epipodophyllotoxin, epipodophyllotoxin, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, Dihydroxy anthraquinone dione, mitoxantrone, glomerin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol And puromycin and their analogs or homologs.
  • chemotherapeutic agents or other therapeutic drugs such as paclitaxel, cytochalasin B, gramicidin D, Ethidium bromide, ipecaine, mitomycin, epipodophyllotoxin, epipodophyllotoxin, vincris
  • Cytotoxic agents also include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, Aminomethamine (decarbazine)), an alkylating agent (eg, nitrogen mustard, thioepachlorambucil, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, Busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthranil (eg, daunorubicin) (formerly known as daunorubicin) and doxorubicin), antibiotics (eg, actinomycin D (formerly known as actinomycin), bleomycin, phosfomycin, and amphotericin (AMC) ), and anti-mitotic agents
  • Nucleic acid of the present invention and host cell containing the same
  • the invention provides a nucleic acid encoding any of the above anti-PD-L1 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 present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 26 to 51, or encoding a nucleic acid selected from any one selected from the group consisting of SEQ ID NOs: 26 to 51.
  • the invention also encompasses a nucleic acid that hybridizes under stringent conditions with a nucleic acid as described below or a nucleic acid having one or more substitutions (eg, conservative substitutions), deletions, or insertions with a nucleic acid comprising: encoding from SEQ ID NO: 26 to a nucleic acid of a nucleic acid sequence of the amino acid sequence of any one of 51; or comprising at least 85%, 90%, 91%, 92% of the amino acid sequence selected from any one of SEQ ID NOS: 26 to 51; A nucleic acid of a nucleic acid sequence of an amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • 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, for example, in bacteria, particularly when glycosylation and Fc effector functions are not required.
  • For expression of antibody fragments and polypeptides in bacteria see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, also to Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003).
  • 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. 24: 210-215 (2006).
  • 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; human embryonic kidney line (293 HEK or 293 cells, eg eg Graham et al, J. Gen Virol. 36: 59 (1977) (described in) and so on.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 216 (1980)); and myeloma cell lines such as Y0. , NS0 and Sp2/0.
  • mammalian host cell lines suitable for producing antibodies see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).
  • Other useful host cells 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 transgenic animals.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • the anti-PD-L1 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-PD-L1 antibody comprises 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-PD-L1 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.
  • PD-L1 binding can be assayed using methods known in the art, and exemplary methods are disclosed herein.
  • biophotonic interferometry eg, Fortebio affinity measurement
  • MSD assay flow cytometry
  • a competition assay can be used to identify antibodies that compete with any of the anti-PD-L1 antibodies disclosed herein for binding to PD-L1.
  • a competitive antibody binds to the same epitope (eg, a linear or conformational epitope) as the epitope to which any of the anti-PD-L1 antibodies disclosed herein bind.
  • 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 anti-PD-L1 antibodies 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.
  • Cells for use in any of the above in vitro assays include cells or cell lines that naturally express PD-L1 or that are engineered to express PD-L1.
  • any of the above assays can be performed by replacing or supplementing the anti-PD-Ll antibody with an immunoconjugate of the invention.
  • any of the above assays can be performed using an anti-PD-Ll antibody and other therapeutic agents.
  • the present invention also encompasses a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising an anti-PD-L1 antibody or a fragment thereof or an immunoconjugate thereof, and a composition comprising a nucleic acid encoding an anti-PD-L1 antibody or a fragment thereof.
  • the composition comprises one or more antibodies that bind to PD-L1, or a fragment thereof, or an immunoconjugate thereof, or one or more antibodies that encode one or more antibodies that bind to PD-L1 or Fragment of nucleic acid.
  • These compositions may also contain suitable pharmaceutical excipients such as pharmaceutically acceptable carriers, excipients and the like known in the art, including buffers.
  • 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.
  • 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-PD-L1 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-PD-L1 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 ingredient such as chemotherapeutic agents and/or cytotoxic agents.
  • 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 to be capable of combination with an anti-PD-L1 antibody, including chemotherapeutic agents, other antibodies, and other therapeutic agents. Examples of such active ingredients are described, for example, in WO2010/077634, WO2016/061142, US61/264061, US60/696426, WO2016/007235 and the like.
  • the active ingredient is an anti-LAG-3 antibody, eg, a human anti-LAG-3 antibody, preferably, the anti-LAG-3 antibody is humanized
  • 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-PD-L1 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-PD-Ll 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-PD-L1 Antibody) or a pharmaceutical composition or immunoconjugate.
  • a tumor eg, cancer
  • the tumor is a tumor immune escape.
  • the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.
  • 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-PD-L1 antibody) Or a pharmaceutical composition or immunoconjugate.
  • an antibody molecule disclosed herein eg, an anti-PD-L1 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-PD -L1 antibody) or a pharmaceutical composition or immunoconjugate.
  • an antibody molecule disclosed herein eg, anti-PD -L1 antibody
  • a pharmaceutical composition or immunoconjugate e.g., anti-PD -L1 antibody
  • 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-PD-L1 antibody molecule described herein increases T cell proliferation.
  • an anti-PD-L1 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, soft tissue tumors, and metastatic lesions.
  • solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems (including adenocarcinoma and squamous cell carcinoma), such as invasive liver, lung, breast, lymph, gastrointestinal (eg, colon), reproduction Those of the urinary tract (eg, kidney, bladder epithelial cells), prostate, and pharynx.
  • Adenocarcinomas include malignant tumors such as most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma in lung cancer, small bowel cancer, and esophageal cancer.
  • Squamous cell carcinomas include malignant tumors such as those in the lungs, esophagus, skin, head and neck area, mouth, anus, and cervix.
  • the cancer is melanoma, eg, advanced melanoma.
  • the cancer is a gastrointestinal cancer, such as colon cancer. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention.
  • Non-limiting examples of preferred cancers for treatment include lymphomas (eg, diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma), breast cancer (eg, metastatic breast cancer), Lung cancer (eg, non-small cell lung cancer (NSCLC), eg, stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myeloma (eg, multiple myeloma), leukemia (eg, , chronic myeloid leukemia), skin cancer (eg, melanoma (eg, stage III or IV melanoma) or Merkel cell carcinoma), head and neck cancer (eg, head and neck squamous cell carcinoma (HNSCC)), spinal cord development Adverse syndrome, bladder cancer (eg, transitional cell carcinoma), renal cancer (eg, renal cell carcinoma, eg, clear cell renal cell carcinoma, eg, advanced
  • cancers examples include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach-esophage cancer, stomach cancer, Testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer , thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, children Solid tumor, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodys
  • metastatic cancer e.g., metastatic carcinoma expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144)
  • metastatic cancer e.g., metastatic carcinoma expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144)
  • PD-L1 metastatic carcinoma expressing PD-L1
  • Tumor immune escape can also be treated using the antibody molecules described herein.
  • the tumor is a cancer that expresses elevated levels of 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 infection, a 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 pathogen is a bacterium.
  • the bacterium is selected from the group consisting of Mycobacterium spp., Salmonella spp., Listeria spp, and Streptococcus spp. ), Haemophilus (spp.), Neisseria spp., Klebsiella spp., Borrelia spp., fragile Bacterioides fragillis, Treponema spp., and Helicobacter pylori.
  • the pathogen is a virus.
  • the virus is selected from the group consisting of an infectious virus, such as hepatitis B virus or hepatitis C virus (hepatitis-B, -C), Herpes simplex virus-I (herpes simplex virus-I, -II), human immunodeficiency virus-I, -II, cytomegalovirus, Eppstein Barr virus, human papillomavirus, human T Human T lymphotrophic viruses (I, -II), varicallazoster.
  • infectious virus such as hepatitis B virus or hepatitis C virus (hepatitis-B, -C), Herpes simplex virus-I (herpes simplex virus-I, -II), human immunodeficiency virus-I, -II, cytomegalovirus, Eppstein Barr virus, human papillomavirus, human T Human T lymphotrophic viruses (I, -II), varicallazoster.
  • the pathogen is a fungus.
  • the condition caused by the fungus is selected from the group consisting of: aspergilosis, blastomycosis, candidiasis albicans, coccidioiodmycosis immitis, histoplasmosis ( Histoplasmosis), paracoccidioiomycosis, microsporidiosis.
  • the pathogen is a protozoan, such as a parasite.
  • the condition caused by the protozoan is selected from the group consisting of: leishmaniasis, plasmodiosis (ie, malaria), cryptosporidiosis, toxoplasma Toxoplasmosis, trypanosomiasis, and helminth infection, including those caused by trematodes (such as schistosomiasis), cestodes (such as echinococcosis), and nematodes (nemotodes) (eg, diseases caused by trchinosis, ascariasis, filariosis, and strongylodiosis).
  • the infection is a hepatitis infection, such as a hepatitis B or hepatitis C infection.
  • Anti-PD-L1 antibody molecules can be combined with conventional treatments for hepatitis B infection or hepatitis C infection for therapeutic advantages.
  • the anti-PD-L1 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.
  • a hepatitis B antigen eg, Engerix B
  • a vaccine optionally in combination with an aluminum-containing adjuvant.
  • the infectious disease is influenza.
  • the anti-PD-L1 antibody molecule is administered in combination with an influenza antigen or vaccine.
  • a disease suitable for the prevention or treatment of an antibody against PD-L1 of the present invention or a fragment thereof can be further described in WO2010/077634, WO2016/061142, US60/696426, WO2016/007235 or US61/264061.
  • the invention provides the use of an anti-PD-Ll antibody or fragment thereof or immunoconjugate thereof for the manufacture or preparation of a medicament for the prevention or 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-PD-L1 antibody or fragment thereof) or a pharmaceutical composition or immunoconjugate disclosed herein. And one or more other therapies, such as therapeutic modalities and/or other therapeutic agents.
  • an antibody molecule eg, an anti-PD-L1 antibody or fragment thereof
  • a pharmaceutical composition or immunoconjugate disclosed herein e.g., a pharmaceutical composition or immunoconjugate disclosed herein.
  • 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, or anti-LAG-3); antibodies that stimulate immune cells (eg, agonistic GITR antibodies or CD137) Antibody); an anti-cancer antibody (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, or anti-LAG-3
  • antibodies that stimulate immune cells eg, agonistic GITR antibodies or CD137
  • an anti-cancer antibody eg, rituximab ( or ),Trastuzumab Tosimo monoclonal antibody Imomozumab Alemizumab Epalizumab Be
  • 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 PD-1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM (eg, CEACAM-1, -3, and/or -5) Inhibitors of VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR ⁇ . Inhibition of the molecule can be carried out at the DNA, RNA or protein level. In some embodiments, an inhibitory nucleic acid (eg, dsRNA, siRNA, or shRNA) can be used to inhibit expression of an immunological checkpoint molecule.
  • 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 (eg, PD-1-Ig or CTLA-4Ig), or an antibody or antigen-binding fragment thereof; , with PD-1, PD-L2, CEACAM (eg, CEACAM-1, -3, and/or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 An antibody or fragment thereof that binds to and/or TGFR ⁇ or a combination thereof.
  • an immunological checkpoint molecule eg, a soluble ligand (eg, PD-1-Ig or CTLA-4Ig), or an antibody or antigen-binding fragment thereof; , with PD-1, PD-L2, CEACAM (eg, CEACAM-1, -3, and/or -5), CTLA-4, TIM-3, LAG-3,
  • the immunomodulatory agent is an inhibitor of CEACAM (eg, CEACAM-1, -3, and/or -5) (eg, human CEACAM (eg, CEACAM-1, -3, and/or -5)) .
  • the immunomodulatory agent is an inhibitor of LAG-3 (eg, human LAG-3).
  • the inhibitor of LAG-3 is an antibody molecule directed against LAG-3, for example, a human anti-LAG-3 antibody, preferably, the anti-LAG-3 antibody is humanized.
  • the immunomodulatory agent is an inhibitor of TIM-3 (eg, human TIM-3).
  • the inhibitor of TIM-3 is an antibody molecule directed against TIM-3.
  • 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, CD28, 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 body.
  • the immunomodulatory agent is a GITR agonist. In one embodiment, the GITR agonist is an antibody molecule directed against GITR. In other embodiments, the immunomodulatory agent is an OX40 agonist. In one embodiment, the OX40 agonist is an antibody molecule directed against OX40.
  • an anti-PD-L1 antibody or fragment thereof of the invention can also be used in combination with a tyrosine kinase inhibitor (eg, a receptor tyrosine kinase (RTK) inhibitor).
  • 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
  • an anti-PD-L1 antibody or fragment thereof of the invention can also be used in combination with a PI3K inhibitor, an mTOR inhibitor, a BRAF inhibitor, a MEK inhibitor, and/or a JAK2 inhibitor, and the like.
  • administration of an anti-PD-L1 antibody or fragment thereof of the invention is combined with administration of an 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-PD-L1 antibody or fragment thereof of the invention can be administered in combination with treatment comprising a T cell (eg, a cytotoxic T cell or CTL) that adopts a adoptive transfer of a chimeric antigen receptor (CAR).
  • a T cell eg, a cytotoxic T cell or CTL
  • CAR chimeric antigen receptor
  • an anti-PD-L1 antibody of the invention can be administered in combination with an anti-neoplastic agent.
  • an anti-PD-L1 antibody of the invention can be administered in combination with an oncolytic virus.
  • the oncolytic virus is capable of selectively replicating in cancer cells and triggers cancer cell death or delays its growth. In some cases, oncolytic viruses have no or minimal effect on non-cancerous cells.
  • Oncolytic viruses include, but are not limited to, oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sindbis virus, oncolytic influenza virus, or oncolytic RNA Virus (eg, oncolytic reovirus, oncolytic Newcastle disease virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)).
  • the oncolytic virus is a virus described in US 2010/0178684 A1, for example, a recombinant oncolytic virus.
  • an anti-PD-L1 antibody of the invention can be administered in combination with a cytokine.
  • an antibody of the invention or a fragment thereof can be combined with cancer therapy conventional in the art, including but not limited to: (i) radiation therapy (eg, radiation therapy, X-ray therapy, irradiation) or Kill cancer cells with ionizing radiation and shrink the tumor.
  • radiation therapy eg, radiation therapy, X-ray therapy, irradiation
  • Kill cancer cells with ionizing radiation and shrink the tumor e.g., radiation therapy, X-ray therapy, irradiation
  • 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 can be combined with conventional methods of enhancing host immune function, including but not limited to: (i) APC enhancement, such as (a) injection of a heterologous MHC into a tumor. DNA of an allogeneic antigen, or (b) a tumor cell transfected with a gene that increases the likelihood of recognition of an immune antigen (eg, immunostimulatory cytokine, GM-CSF, costimulatory molecule B7.1, B7.2), Iii) Adoptive cellular immunotherapy or treatment with activated tumor-specific T cells.
  • APC enhancement such as (a) injection of a heterologous MHC into a tumor. DNA of an allogeneic antigen, or (b) a tumor cell transfected with a gene that increases the likelihood of recognition of an immune antigen (eg, immunostimulatory cytokine, GM-CSF, costimulatory molecule B7.1, B7.2), Iii) Adoptive cellular immunotherapy or treatment with activated tumor-specific
  • 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-PD-L1 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-PD-Ll antibody with the immunoconjugate of the invention.
  • an anti-PD-L1 antibody of the invention can be used in combination with an anti-LAG-3 antibody.
  • an anti-LAG-3 antibody of the invention is an anti-human LAG-3 antibody.
  • an anti-LAG3 antibody of the invention is an antibody in IgGl form or an antibody in IgG2 form or an antibody in IgG4 form.
  • the anti-LAG-3 antibody is a monoclonal antibody.
  • the anti-LAG-3 antibody is humanized.
  • the anti-LAG-3 antibody is a chimeric 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.
  • an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention 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
  • 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: 69; 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: 71;
  • 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: 72; LCDR2 comprises the amino acid sequence of SEQ ID NO: 73 or Composition of the amino acid sequence; LCDR3 comprises or consists of the amino acid sequence selected from SEQ ID NO:74.
  • CDR complementarity determining region
  • 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 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: 75 (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: 76 (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-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein
  • amino acids compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 77 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:78
  • 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-PD-L1 antibody are equally applicable to an anti-LAG-3 antibody.
  • 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.
  • the dosage and treatment regimen of the anti-PD-L1 antibody molecule can be determined by the skilled artisan.
  • the anti-PD-L1 antibody molecule is injected (eg, subcutaneously or intravenously) at about 1 to 30 mg/kg, for example, about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to A dose of 5 mg/kg, or about 3 mg/kg is administered.
  • the dosage regimen can vary, for example, from once a week to once every 2, 3 or 4 weeks.
  • the anti-PD-L1 antibody molecule is administered every other week at a dose of about 10 to 20 mg/kg.
  • the anti-PD-L1 antibody molecule is less than or equal to about 5 mg/kg; less than or equal to about 4 mg/kg; less than or equal to about 3 mg/kg; less than or equal to about 2 mg/kg; less than or equal to about A dose of 1 mg/kg is administered alone or in combination (e.g., in combination with an anti-LAG-3 antibody molecule) every other week.
  • the anti-PD-L1 antibody molecule is administered at a dose of about 1 to 5 mg/kg every other week, at a dose of about 1 to 4 mg/kg every other week, at a dose of about 1 to 3 mg/kg every other week or A dose of about 1 to 2 mg/kg is administered every other week.
  • the anti-LAG-3 antibody molecule is administered at a dose of about 1 to 5 mg/kg every other week, at a dose of about 1 to 4 mg/kg every other week, at a dose of about 1 to 3 mg/kg every other week or A dose of about 1 to 2 mg/kg is administered alone or in combination (e.g., in combination with an anti-LAG-3 antibody molecule) every other week.
  • any of the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of PD-L1 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-PD-L1 antibody for use in a diagnostic or detection method is provided.
  • a method of detecting the presence of PD-L1 in a biological sample is provided.
  • the methods comprise detecting the presence of a PD-L1 protein in a biological sample.
  • PD-L1 is human PD-L1.
  • the method comprises contacting a biological sample with an anti-PD-L1 antibody as described herein under conditions that permit binding of the anti-PD-L1 antibody to PD-L1, and detecting the anti-PD-L1 antibody Whether a complex is formed between PD and L1. Formation of the complex indicates the presence of PD-L1.
  • the method can be an in vitro or in vivo method.
  • an anti-PD-L1 antibody is used to select a subject suitable for treatment with an anti-PD-L1 antibody, for example wherein PD-L1 is a biomarker for selection of 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 labeled anti-PD-L1 antibody is provided.
  • 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.
  • 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-PD-L1 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
  • PD-L1 is detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after a 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 PD-L1, The PD-L1 value is thus determined, the PD-L1 value is compared to a control value, and if the PD-L1 value is greater than the control value, the subject is administered a therapeutically effective amount of an anti-antibody, optionally in combination with one or more other therapies.
  • a PD-L1 antibody eg, an anti-PD-L1 antibody described herein
  • Hybridoma technology is achieved by fusing two cells while maintaining the main features of both. These two cells are antigen-immunized mouse spleen cells and mouse myeloma cells, respectively.
  • the main feature of mouse spleen cells (B lymphocytes) immunized by specific antigens is its antibody secretion function, but it cannot be continuously cultured in vitro, and mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, The so-called immortality.
  • the selective medium only the hybrid cells in which the B cells are fused with the myeloma cells can have the ability to continue to culture, and form a cell clone having both the secretory function of the antibody and the immortality of the cells.
  • mice were immunized with hPD-L1 protein, and then spleen cells and myeloma cells of mice were fused to obtain hybridoma cells capable of expressing positive antibodies.
  • mice were sacrificed by cervical dislocation, and the body surface was sterilized with 75% alcohol for 5 min, and then placed on the anatomical plate of the mouse in the ultra-clean table, and the left side was placed, and the limbs were fixed with a 7-gauge needle.
  • the spleen was removed by aseptic opening of the abdominal cavity, washed with a basal medium (configuration method as follows), and the connective tissue attached thereto was carefully removed.
  • the spleen was then transferred to another dish containing the basal medium. Press the spleen with a elbow needle, insert a small needle into the spleen, and squeeze with the scorpion to fully release the spleen cells to make a spleen cell suspension.
  • the cell suspension was filtered through a 100 ⁇ M cell sieve, washed once with 30 ml of basal medium, and centrifuged at 1200 rpm for 6 min.
  • Lysis of red blood cells The supernatant was removed and the cells were resuspended in 10 ml RBC lysis buffer (GIBCO). Then add 20 ml of RBC lysis buffer. The suspension was allowed to stand for 5 min and centrifuged at 1100 rpm for 6 min. After removing the supernatant, the cells were resuspended in 10 ml of basal medium, and then 30 ml of basal medium was added, and centrifuged at 1100 rpm for 6 min. After removing the supernatant, the cells were resuspended in 20 ml of basal medium and counted.
  • Electrofusion Mouse myeloma cells SP2/0 cells (ATCC) were resuspended in 20 ml of basal medium and counted. SP2/0 and spleen cells were mixed at a ratio of 1:2 to 1:1 and centrifuged at 100 rpm for 6 min. After removing the supernatant, the mixed cells were resuspended in 10 ml of fusion buffer (BTXpress). Further, 15 ml of the fusion buffer was added, and the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. After repeating the above steps, the cells were re-selected with an appropriate amount of fusion buffer, and the mixed cell density was adjusted to 1 ⁇ 10 7 cells/ml.
  • the parameters of the electrofusion amplifier are set as follows. 2 ml of the cell suspension was added to each electrofusion dish for electrofusion.
  • the cells were allowed to stand in an electric fusion dish for 5 min at room temperature. The cells were transferred to a centrifuge tube, and the cells were diluted to 1 to 2 x 104 cells/ml using a screening medium (configuration method as follows). 100 ⁇ l of the cell suspension was added to each well of a 96-well plate. The screening medium was changed on the 7th day after the fusion. Screening was performed 10 days after culture (or longer, depending on the state of cell growth). Hybridoma cells expressing a specific anti-PD-L1 antibody were screened by FACS (FACS ARIA (BD Biosciences)).
  • Subcloning step A 96-well plate was prepared, and 200 ⁇ l of the basal medium as described above was added to each of the 2nd to 8th columns. The cells of the positive wells selected by the above fusion were made into cell suspensions and added to the first column. 100 ⁇ l of the cell suspension of column 1 was added to the second column, and after thorough mixing, 100 ⁇ l was added to the next column. The above procedure was repeated until the last column volume became 300 ⁇ l; the 96-well plate was allowed to stand for 15 min, and the count was observed under a microscope. The corresponding volume of 100 cells was added to 20 ml of the basal medium as described above, and the plates were mixed and mixed at 200 ⁇ l per well. After one week, the microscope was observed under the microscope, and the monoclonal wells were judged and labeled, and the positive wells were picked out.
  • Cryopreservation of cells Observe the state of the cells, and wait for the cells to grow well. When the viability is >90%, centrifuge at 1000 rpm for 5 min to remove the supernatant. Resuspend the cells to 1 ⁇ 107 cells/ml with cryopreservation solution (45.5% FBS, 44.5% RPMI-1640, 10% DMSO), dispense into a cryotube, place in a programmed cooling box, and freeze at -80 °C. .
  • cryopreservation solution 45.5% FBS, 44.5% RPMI-1640, 10% DMSO
  • the present invention utilizes molecular biology techniques to obtain antibody sequences in anti-PD-L1 positive hybridoma cells, and uses the same to construct human and mouse chimeric antibodies.
  • RNA extraction fresh cells, centrifuged at 300 g for 5 min, the supernatant was removed, 500 ⁇ l of LY buffer (Biomiga) was added to the pellet (20 ⁇ l of ⁇ -mercaptoethanol was added per 1 ml before use), and mixed until clear. It was added to a DNA removal tube, centrifuged at 13,000 rpm for 2 min, and the flow-through was collected. 100% ethanol was added to the flow through solution at a ratio of 1/2, and mixed 5 times to clarify.
  • LY buffer Biomiga
  • RNA concentration was determined.
  • the reaction system I is configured as follows:
  • Reaction System I After incubation at 65 ° C for 5 min, it was quickly cooled on ice. The following reverse transcription system was added to Reaction System I in a total amount of 20 ⁇ l:
  • reverse transcription translation was carried out under the following conditions: 42 ° C for 60 min ⁇ 95 ° C for 5 min, and then cooled on ice to obtain cDNA.
  • the heavy and light chain variable regions were amplified by PCR, and the PCR reaction system was as follows:
  • PCR product obtained by the above PCR reaction was added, 0.5 ⁇ l of pMD20-T vector (Clontech), 5 ⁇ l of Ligation Mighty Mix (Takara), gently mixed, and reacted at 37 ° C for 2 hours to obtain a ligation product.
  • the TOP10 competent state (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was taken out at -80 °C, and thawed on ice. 5 ⁇ l of the ligation product obtained above was added to the melted TOP10 competent state, and the mixture was incubated on ice for 30 min. After heat shock at 42 °C for 90 s, it was rapidly cooled on ice for 2 min, and 900 ⁇ l of LB medium (Biotech (Shanghai) Co., Ltd.) was added to the EP tube, and cultured at 37 ° C for 1 h at 220 rpm shaker.
  • LB medium Biotech (Shanghai) Co., Ltd.
  • the murine anti-PD-L1 antibody VH and VL regions produced by the hybridoma of Example 1 which had been sequenced were PCR-amplified: the upstream and downstream primer sequences are shown in Table 5 and Table 6.
  • Primer Mix 1 was obtained for subsequent PCR amplification of VH.
  • VL primers (Primer Mix 2) of mouse anti-PD-L1 antibody:
  • Primer Mix 2 was obtained for subsequent PCR amplification of VL.
  • the PCR system is as follows:
  • Primer Mix 1 was applied for VH chain amplification; Primer Mix 2 was applied for VL chain amplification.
  • the PCR amplification product was recovered by gel cutting.
  • the homology recombination system is as follows:
  • the reaction was carried out at 37 ° C for 30 min to obtain a recombinant product.
  • the recombinant product was transformed into TOP10 competent state, and monoclonal sequencing was picked.
  • the clone containing the plasmid with the correct insertion direction was selected as a positive clone, and the positive clone was preserved.
  • a plasmid containing an anti-PD-L1 antibody was extracted from the positive clone obtained above.
  • 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 Protein A 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 CDRs, the light chain variable region and the heavy chain variable region, the amino acid sequences of the light chain and the heavy chain of the four chimeric antibodies obtained by the present invention, and the sequence numbers are shown in Tables 1-3 above.
  • the control antibody used in the present invention is Roche's PD-L1 antibody Atezolizumab (hereinafter referred to as ATE or Ate, trade name Tecentriq), and its CDR, light chain variable region and heavy chain variable region, and the amino acid sequences of the light chain and the heavy chain are also See Table 1-3 above.
  • the equilibrium dissociation constant (KD) of the antibody of the present invention in combination with 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 KD values of the chimeric antibodies 3-266.1, 4-79.2, 4-26.6, and 4-48.5 were 9.80E-10M, 7.56E-09M, 3.85E-09M, and 1.23E-09M, respectively. compared group, in this study the antibody has a K D value of a similar or better.
  • the CHO-S cells were transfected into the CHO-S cells by the pCHO1.0 vector (Invitrogen) carrying the human PD-L1 cDNA (Sino Biological) cloned into the multiple cloning site (MCS) (Invitrogen, ExpiCHO TM Expression System Kit, Cat. No. A29133 ), CHO cells (CHO-PDL1) overexpressing human PD-L1 were produced.
  • pCHO1.0 vector Invitrogen
  • MCS multiple cloning site
  • CHO-PDL1 cells were counted and diluted to 1 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to a U-bottom 96-well plate. 400 g for 5 min, centrifuge, and remove the cell culture medium. Samples (chimeric antibodies 3-266.1, 4-79.2, 4-26.6, 4-48.5, and positive control antibody Ate, respectively) (antibody dilution method: maximum antibody concentration 500 nM, triple dilution in 0.1% cattle) Serum albumin (BSA) in PBS, a total of 8 concentrations were tested.) U-plates were added and the cells were resuspended, 100 ⁇ l/well, and allowed to stand on ice for 30 min.
  • BSA Serum albumin
  • the supernatant was removed by 400 g for 5 min, and the cells were washed 1 time with PBS. 400 g of PBS was removed for 5 min, 100 ⁇ l of anti-mouse Fab FITC-labeled secondary antibody (Jackson Immuno Research) (1:500 dilution in PBS) was added to each well, and 100 ⁇ l of anti-human Fab FITC-labeled was added to the cells to which the positive control antibody was added. Secondary antibody (Jackson Immuno Research). Incubate on ice for 30 min in the dark. The supernatant was removed by 400 g for 5 min, and the cells were washed 1 time with PBS. The cells were resuspended in 100 ⁇ l of 1 ⁇ PBS and detected by FACS.
  • antibodies 3-266.1, 4-79.2, 4-26.6, and 4-48.5 all bind to human PD-L1 overexpressed on CHO cells with EC50 of 2.139 nM, 2.598 nM, 1.985 nM, and 1.995 nM, respectively.
  • the binding ability is better, and the binding ability of some antibodies is more than twice that of the control antibody.
  • the chimeric antibody obtained in Example 1 was humanized.
  • the antibody humanization process was performed using the Macromtek SmrtMol Humanize proprietary software program. First enter the sequence into the software, the system will generate a three-dimensional model of the sequence, and then humanized through the following steps:
  • the CDRs, light chain variable region and heavy chain variable region of the four humanized antibodies (HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-IgG4PAK and HZ4485-IgG1N297A) obtained by the present invention, the amino acid sequences of the light and heavy chains, please See Tables 1-3 as described above.
  • the equilibrium dissociation constant (KD) of humanized antibodies of different Fc isoforms of the invention in combination with human PD-L1 was determined using the ForteBio assay.
  • the ForteBio affinity determination method was the same as in Example 2.
  • the affinities of the antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-IgG4PAAK, HZ4485-IgG1N297A are shown in Table 8:
  • humanized antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, K D values were HZ4485-IgG1N297A 7.24E-10M, 9.35E-10M, 1.32E-09M, 3.17 E-09M, compared with the control group, the present study of humanized antibodies having a K D value of a similar or better.
  • the binding of a gradient-diluted humanized antibody of the present invention to a CHO-stabilized cell line overexpressing human PD-L1 was detected by flow cytometry.
  • the test method was the same as in Example 3 except that the antibodies used were humanized antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, HZ4485-IgG1N297A, and the antibody dilution method was as follows: the highest antibody concentration was 500 nM, and the three-fold dilution was 0.1.
  • humanized antibodies HZ3266-IgG1, HZ3266-IgG1N297A, HZ3266-G4PAAK, HZ4485-IgG1N297A bind to human PD-L1 overexpressing CHO cells with EC50 of 1.813 nM, 1.784 nM, 1.862 nM, 1.561 nM, respectively. Compared with the control antibody ATE, it has stronger binding ability.
  • Anti-PD-1/PD-L1 antibodies can block the inhibition of downstream NFAT signaling pathway by blocking the binding of PD-1 and PD-L1.
  • MOA detection system PD-1/PD-L1 Blockade Bioassay, Cell Propagation Model, Catalog J1252
  • Promega was used to detect the activation of NFAT signal by detecting the expression of the fluorescent reporter gene according to the method provided in the specification.
  • the inhibitory effect of the antibody on PD-1/PD-L1 binding was examined.
  • CHOK1-PDL1 cells (from the above MOA detection system) were placed one day before the activity test: the cells were passaged 1-2 days before the plating of CHOK1-PDL1. The culture supernatant was discarded, and the cells were washed once with PBS (Gibco). Add appropriate amount of Trypsin (Gibco) and digest at 37 ° C, 5% CO 2 for 3-5 min. Add 4 times Trypsin volume of medium, transfer the cells to a 50 ml centrifuge tube and count. Take the required volume of cells, 230g, and centrifuge for 10min. 1640 medium (Gibco) was added and the cells were resuspended to 4 x 105 cells/mL.
  • the cells were added to a 96-well white cell culture plate (Nunclon) at 100 ⁇ l/well.
  • the side holes were added with PBS, 200 ⁇ l/well.
  • the cells were cultured overnight in a 37 ° C / 5% CO 2 incubator.
  • Jurkat-PD1 cells from the MOA detection system described above were treated: cell passage was performed two days prior to activity assay. After counting, take the required volume of cells, 170 g, and centrifuge for 5 min. The cells were resuspended to 1.25 x 10 6 cells/ml with assay buffer (1640 medium (Gibco) + 1% FBS).
  • Bio-GloTM buffer from the MOA detection system described above
  • Bio-GloTM substrate from the above MOA detection system
  • Bio-GloTM reagent from the MOA detection system described above
  • the antibody was incubated with mature DC cells derived from different donors and CD4+ T cells in vitro, and the relative expression of IL2 and IFN- ⁇ in the system was detected to reflect the activation of T cells by different antibodies. .
  • 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.
  • DC cell isolation PBMC cells isolated as described above were added with 5 ml T cell culture medium (the preparation method is as follows), cultured at 37 ° C, 6% CO 2 , adherent culture for 2 h, and the suspension cell suspension was aspirated for CD4+ cell separation, leaving the cells Add 3 ml of DC medium (preparation method is shown in the following table), add 3 ml of DC medium after 2 days of culture, and then culture for 5 days, then add rTNFa (R&D Systems) (1000 U/ml), IL-1b (R&D Systems) (5 ng/ Ml), IL-6 (R&D Systems) (10 ng/ml) and 1 ⁇ M PGE2 (Tocris) were cultured for 2 days as DC cells for lymphocyte mixed reaction (MLR).
  • T cell culture medium the preparation method is as follows
  • CD4+ T cell isolation The kit was operated according to the 'Untouched CD4+ T cell isolation' kit instructions (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.
  • kit instructions 11346D, Invitrogen
  • 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 .
  • MLR experiment The mature DC cells obtained above were mixed with CD4+ cells at a volume of 200 ⁇ l per well, 10000 DC cells, and 100000 CD4+ cells, and the antibody of the present invention was added (concentration: 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16).
  • DC cells prepared above represented as DC in the table below
  • CD4+ T cells represented as CD4 in the table below
  • DC cells mixed with CD4+ cells represented as Cell in the table below
  • IgG1 disclosed in Table 3 was used as a negative control
  • DC+CD4+ T cells + anti-CD3/CD28 magnetic beads shown as Beads in the table below
  • the IL2 and IFN- ⁇ concentrations were measured (relative expression levels in DeltaF%).
  • the antibodies of the present invention can effectively activate T cells in vitro, and the partial activation effect is superior to the positive control antibody.
  • This experiment examined the drug-forming properties of antibodies by recording the residence time of an exemplary humanized antibody of the invention in a Zenix column (Sepax Technologies, Inc.). The shorter the residence time, the lower the viscosity of the reacted antibody and the better the drug-forming property.
  • Chromatographic conditions detection wavelength: 214 nm, column temperature: 25 ° C, flow rate: 0.35 ml / min, injection amount: 10 ⁇ l.
  • Sample preparation Take 100 ⁇ l of sample (antibody to be detected HZ3266-IgG1, HZ3266-IgG4PAAK, HZ3266-IgG1N297A, HZ4485-IgG1N297A; ATE as positive control, antibody concentration: 1 mg/ml), centrifuge at 13000 rpm for 5 min, and take 80 ⁇ l of supernatant in liquid phase. In the inner cannula, it is placed in the liquid sample tray for injection detection.
  • the anti-tumor effect of the PD-L1 antibody of the present invention was measured in hPD-L1 transgenic mice using MC38 cells (MC38-hPDL1) expressing human PD-L1 (Nanjing Yinhe Co., Ltd.).
  • mice Female C57B1/6 background human PDL-1 transgenic mice (about 8 weeks old) were purchased from Shanghai Southern Experimental Animal Technology Co., Ltd. The mice were domesticated for 7 days after arrival and the study was started.
  • MC38 cells (MC38-hPDL1) expressing human PD-L1 were purchased from Nanjing Yinhe Biomedical Co., Ltd. and routinely subcultured for subsequent in vivo experiments in strict accordance with the instructions. The cells were collected by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 5 x 106 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdomen region of human PD-L1 transgenic mice to establish a MC38-hPDL1 tumor-bearing mouse model.
  • mice with tumor volume ranging from 87.4 mm 3 to 228.4 mm 3 were selected and grouped according to tumor volume (8 mice per group, one group administered with IgG1, one)
  • the administration was carried out on days 6, 10, 14, 17, 21, 24, 28, 31 and 35, respectively, in which the frequency of administration was 2 times/week, and the dosage and manner of administration were as shown in Table 12. Tumor volume and body weight changes were monitored during the administration period, and the monitoring frequency was 2 times/week for 5 weeks. Body weight and tumor volume were determined before each dose.
  • the antibody of the present invention showed a remarkable antitumor effect after one week of administration (Fig. 6), and by the 35th day, one mouse of the antibody group of the present invention completely ablatedly disappeared. Body weight results showed that different antibody dose groups had no effect on the weight of tumor-bearing mice.
  • the antibody of the present invention has a significant inhibitory effect on tumors.
  • Example 11 Combination of an anti-PD-L1 antibody of the present invention and an anti-human LAG-3 antibody
  • This study investigated the antitumor activity of the anti-PD-L1 antibody (HZ3266-IgG1N297A) of the present invention in combination with an anti-human LAG-3 antibody (ADI-31853) using a humanized mouse model.
  • A375 human skin cancer cells were used to measure the anti-tumor effect of anti-PD-L1 antibodies on NCG mice.
  • Human PBMC AllCells (2 ⁇ 10 6 cells/mouse) was pre-injected intravenously, and then A375 tumor-bearing mouse model was established by subcutaneous vaccination, grouped into tumors, treated with different antibodies, and monitored during drug administration. The tumor volume and body weight of each group were changed, the frequency of administration was 2 times/week, and the administration was 2 weeks, and a total of 5 doses were administered. The monitoring frequency was 2 times/week, and the monitoring was continued for 4 weeks.
  • the dosage and mode of administration were as follows.
  • 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-LAG-3 antibody ADI-31853 was separately cloned into the expression vector pTT5 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%.
  • the equilibrium dissociation constant (KD) of ADI31853 bound to human LAG-3 (hLAG-3) was determined using a 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.
  • NOG mice female, 7-8 weeks (the age of mice at the time of tumor cell inoculation), weighing 17.6-24.2 g, 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.
  • 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 (AllCells), the right back is shaved and subcutaneously injected with A375 cells 0.2 ml/mouse. 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 with an average tumor volume meeting the above requirements were randomly divided into groups of 8 animals 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 or when the mice had >20% weight loss.

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Abstract

本发明提供了特异性结合PD-L1的抗体和抗体片段、含有所述抗体或抗体片段的组合物，以及编码所述抗体或其抗体片段的核酸、相应的宿主细胞及相关用途。此外，还提供了这些抗体和抗体片段与其它疗法的联合治疗。

Description

抗PD-L1抗体及其用途
本发明涉及特异性结合PD-L1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的联合治疗。
背景技术
程序性死亡配体1(PD-L1)是涉及在慢性感染、妊娠、组织同种异体移植、自身免疫性疾病和癌症期间抑制免疫系统应答的蛋白质。PD-L1通过结合到表达于T细胞、B细胞和单核细胞的表面上的被称为程序性死亡1(PD-1)的抑制性受体来调节免疫应答。PD-L1还通过与另一种受体B7.1(也称为B7-1或CD80)的相互作用负调节T细胞功能。PD-L1/PD-1和PD-L1/B7.1复合物的形成负调节T细胞受体信号传导，导致随后的T细胞活化的下调和抗肿瘤免疫活性的抑制。PD-L1在许多癌症中过表达，所述癌症包括多种多样的实体瘤，诸如膀胱肿瘤、乳腺肿瘤、结肠肿瘤、肺肿瘤、黑色素瘤、卵巢肿瘤、唾液肿瘤、胃肿瘤和甲状腺肿瘤。肿瘤细胞中的PD-L1过表达可促进肿瘤侵袭，并且常常与不良预后相关。
因此，本领域仍然需要新的与PD-L1的结合更好且成药性好的抗PD-L1抗体。
发明概述
本文公开了与PD-L1结合的抗体分子。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PD-L1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PD-L1抗体分子可以单独或联合其他疗法，例如治疗剂或治疗方式，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PD-L1的组合物和方法，以及使用抗PD-L1抗体分子预防或治疗多种疾病(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PD-L1。在一些实施方案中，本发明的抗体或其片段(特异性)结合人PD-L1。
在一些实施方案中，本发明的抗PD-L1抗体或其片段以高亲和力结合PD-L1(例如人PD-L1)，例如，以以下平衡解离常数(K D)与PD-L1结合，所述K D小于大约50nM，优选地，小于或等于大约20nM，更优选地小于或等于大约15nM，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1.5nM、1.4nM、1.3nM、1.2nM、1.1nM、1nM、0.9nM或0.8nM。在一些实施方案中，本发明的抗PD-L1抗体以0.1-10nM，优选地0.5-10nM，更优选地0.6-10nM、0.7-8nM、0.7-5nM，最优选地0.5-1.5nM、0.7-1.5nM、0.7-1nM的K D结合PD-L1。在一些实施方案中，PD-L1为人PD-L1。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PD-L1的细胞，例如，以小于或等于大约4nM、3.5nM、3nM、2.9nM、2.8nM、2.7nM、2.6nM、2.5nM、2.4nM、2.3nM、2.2nM、2.1nM、2nM、1.9nM、1.8nM、1.7nM或1.6nM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PD-L1的细胞为表达人PD-L1的CHO细胞。
在一些实施方案中，本发明的抗体或其片段阻断PD-L1的相关活性，例如以小于或等于大约10nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM或0.7nM的EC 50，优选以大约0.1-1nM、0.5-1nM、0.6-1nM、0.6nM、0.7nM、0.8nM、0.9nM或1nM的EC 50。在一些实施方案中，PD-L1的相关活性是PD-L1与PD-1的结合。在一些实施方案中，本发明的抗体或其片段在MOA测定中以小于或等于大约10nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM 或0.7nM的EC 50，优选以大约0.1-1nM、0.5-1nM、0.6-1nM、0.6nM、0.7nM、0.8nM、0.9nM或1nM的EC 50抑制PD-L1与PD-1的结合。在一些实施方案中，细胞为CHO细胞。
在一些实施方案中，本发明的抗体或其片段提高T细胞功能。在一些实施方案中，本发明的抗体或其片段提高T细胞增殖。在一些实施方案中，本发明的抗体或其片段提高IFN-γ分泌。在一些实施方案中，本发明的抗体或其片段提高IL-2分泌。在一些实施方案中，本发明的抗体或其片段提高IFN-γ分泌和IL-2分泌。在一些实施方案中，所述提高是在混合淋巴细胞反应(MLR)中测定的。在一些实施方案中，本发明的抗体或其片段激活T细胞的能力优于已知的抗PD-L1抗体，例如Tecentriq。
在一些实施方案中，本发明的抗体或其片段相比已知的抗PD-L1抗体(例如Tecentriq)具有更低的粘性，因此具有更好的成药性。在一些实施方案中，本发明的抗体或其片段在Zenix柱检测法中，驻留时间(RT)低于大约10分钟、大约9分钟或大约8分钟，优选地，驻留时间在大约7分钟-9分钟之间，优选地在大约7-8.5分钟，大约7.5-8.5分钟，大约7-8分钟，或大约7.5-8分钟之间，例如大约7.5分钟、7.6分钟、7.7分钟、7.8分钟、7.9分钟、8分钟、8.1分钟、8.2分钟、8.3分钟、8.4分钟、8.5分钟。
在一些实施方案中，本发明的抗体或其片段抑制PD-L1的一种或多种活性，例如，导致以下一者或多者：肿瘤浸润型淋巴细胞增加、T细胞受体介导的增殖增加、或癌细胞的免疫逃避减少。
在一些实施方案中，本发明的抗PD-L1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PD-L1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(例如慢性感染)。在一些实施方案中，肿瘤是肿瘤免疫逃逸。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PD-L1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：68)。
在一些实施方案中，本发明的抗体还涵盖抗PD-L1抗体的氨基酸序列的变体，以及与上文所述的任何抗PD-L1抗体或其片段结合相同表位的抗体。
在一些实施方案中，本发明的抗PD-L1抗体还包含人或鼠恒定区。在一些实施方案中，本发明的抗PD-L1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PD-L1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或IgG4的重链恒定区，例如人IgG1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。在一些实施方案中，本发明的抗PD-L1抗体所包含的鼠恒定区选自IgG1、IgG2A、IgG2B、IgG3。
在另一个实施方案中，本发明的抗PD-L1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PD-L1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)。在又一个实施方案中，抗PD-L1抗体分子包含IgG1(例如，人IgG1)的重链恒定区。在一个实施方案中，人IgG1包含在根据EU编号的位置297处的置换(例如，Asn至Ala置换)。在一个实施方案中， 人IgG1包括在根据EU编号的位置265处的置换、在根据EU编号的位置329处的置换或这两种置换(例如，在根据EU编号的位置265处的Asp至Ala置换和/或在根据EU编号的位置329处的Pro至Ala置换)。在一个实施方案中，人IgG1包括在根据EU编号的位置234处的置换、在根据EU编号的位置235处的置换或这两种置换(例如，在根据EU编号的位置234处的Leu至Ala置换和/或在根据EU编号的位置235处的Leu至Ala置换)。在一个实施方案中，重链恒定区包含SEQ ID NO：64、65或66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PD-L1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：67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PD-L1抗体分子包括IgG1的重链恒定区(例如，人IgG1的重链恒定区)和κ轻链恒定区(例如，人κ轻链恒定区)。在一个实施方案中，人IgG1包含在根据EU编号的位置297处的置换(例如，Asn至Ala置换)。在一些实施方案中，人IgG1重链恒定区包含SEQ ID NO：64或65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：67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PD-L1抗体分子包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)和/或具有在第114-115位(EU编号)突变为AA的突变)。在一些实施方案中，人IgG4重链恒定区包含SEQ ID NO：66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：67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PD-L1抗体分子是分离的或重组的。
在一些实施方案中，抗PD-L1抗体是单克隆抗体或具有单特异性的抗体。抗PD-L1抗体分子也可以是人源化的、嵌合的、人的抗体分子。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，抗PD-L1抗体是人源化抗体。在一些实施方案中，抗PD-L1抗体是人抗体。在一些实施方案中，至少部分的抗PD-L1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。
在某些实施方案中，抗PD-L1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PD-L1的第一结合特异性和针对LAG-3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PD-L1和LAG-3结合。多特异性抗体分子可以具有任何针对PD-L1与其他靶标的结合特异性的组合。
在一方面，本发明提供了编码以上任何抗PD-L1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PD-L1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PD-L1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PD-L1抗体和其它物质，例如细胞毒性剂或标记物。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。在一些实施方案中，肿瘤是肿瘤免疫逃逸。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PD-L1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PD-L1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、其他抗体、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))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。在一些实施方案中，肿瘤是肿瘤免疫逃逸。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PD-L1抗体或其片段、药物组合物或免疫缀合物。在一些实施方案中，肿瘤是肿瘤免疫逃逸。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PD-L1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。在一些实施方案中，肿瘤是肿瘤免疫逃逸。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、抗感染活性剂、其它抗体或免疫调节剂(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，例如哺乳动物，优选地人。
在一方面中，本发明涉及检测样品中PD-L1的方法，所述方法包括(a)将样品与本文所述的任何抗PD-L1抗体或其片段接触；和(b)检测抗PD-L1抗体或其片段和PD-L1间的复合物的形成。在一个实施方案中，抗PD-L1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PD-L1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PD-L1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PD-L1抗体或其片段、方法和用途。
附图说明
图1显示了FACS检测的本发明抗PD-L1抗体和CHO-PDL1细胞的结合情况。
图2显示了用FACS检测的本发明抗PD-L1抗体和CHO-PDL1细胞的结合情况。
图3显示了用MOA法检测的本发明抗体对PD-1/PD-L1相互作用的阻断活性。
图4A和4B显示了用MLR实验检测的本发明抗体对T细胞的激活作用(IL-2相对表达量)。
图5A和5B显示了用MLR实验检测的本发明抗体对T细胞的激活作用(IFN-γ相对表达量)。
图6显示了本发明抗体对肿瘤的抑制作用。
图7显示了本发明抗体与抗LAG-3抗体联合对肿瘤的抑制作用。
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
FACS   流式细胞术
MOA    作用机制
MLR    淋巴细胞混合反应
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。
如本文所用的术语“程序性细胞死亡1配体1”、“PD-L1”、“程序性死亡配体1”、“分化簇274”、“CD274”或“B7同系物1”是指来自任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。可在NCBI Gene ID No.29126下找到关于人PD-L1基因(包括基因组DNA序列)的另外信息。可在NCBI Gene ID No.60533下找到关于小鼠PD-L1基因(包括基因组DNA序列)的另外信息。示例性全长人PD-L1蛋白的氨基酸序列可例如在NCBI登录号NP_001254653或UniProt登录号Q9NZQ7下找到，而可例如在NCBI登录号NP_068693或Uniprot登录号Q9EP73下找到示例性全长小鼠PD-L1蛋白序列。
本文所用的术语“抗PD-L1抗体”、“抗PD-L1”、“PD-L1抗体”或“结合PD-L1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PD-L1蛋白或其片段。在一个实施方案中，抗PD-L1抗体与非PD-L1蛋白结合的程度低于所述抗体与PD-L1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IgG1Fc区(非A和A同种异型)；天然序列人IgG2Fc区；天然序列人IgG3Fc区；和天然序列人IgG4Fc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PD-L1)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。表位决定簇可以用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 PCTCN2018124314-appb-000001
Figure PCTCN2018124314-appb-000002
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Chothia规则或Kabat规则确定边界，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，IgG1形式的抗体是指其重链恒定区Ig结构域为IgG1的Ig结构域。
“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、WO2015/138920、WO2016/007235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WO2016/007235或WO2010/077634或US60/696426中所公开的可以与抗PD-L1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WO2016/007235、WO2010/077634、US60/696426或WO2016/061142、US61/264061或WO2016/007235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-1α、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”包括使得所给出分子(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抑制至少5％、10％、20％、30％、40％或更多的活性(例如，LAG-3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FcγRIII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％或90％。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 thed.，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。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括非小细胞肺癌、鳞状细胞癌、小细胞肺癌、腹膜癌、肝细胞癌、胃肠癌、胰腺癌、神经胶质瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌、白血病和头颈癌，包括那些癌症的转移性形式。
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“肿瘤免疫逃逸”是指肿瘤逃避免疫识别和清除。因此，作为治疗概念，当所述逃避减弱时，肿瘤免疫得到“治疗”，肿瘤被免疫系统识别并攻击。肿瘤识别的实例包括肿瘤结合、肿瘤收缩和肿瘤清楚。
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PD-L1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45C于6X氯化钠/柠檬酸钠(SSC)中，随后至少在50C(对于低严格性条件，可以增加洗涤的温度至55C)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))、赋形剂、载体或稳定剂等。
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、WO2015/138920或 WO2016/061142。
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PD-L1。在一些实施方案中，本发明的抗体或其片段结合哺乳动物PD-L1，例如人PD-L1。例如，抗体分子与PD-L1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PD-L1的一个或多个胞外结构域结合。
在一些实施方案中，本发明的抗PD-L1抗体或其片段具有以下一种或多种性质：
(1)本发明的抗PD-L1抗体或其片段以高亲和力结合PD-L1(例如人PD-L1)，例如，以以下平衡解离常数(K D)与PD-L1结合，所述K D小于大约50nM，优选地，小于或等于大约20M，更优选地小于或等于大约15nM，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1.5nM、1.4nM、1.3nM、1.2nM、1.1nM、1nM、0.9nM或0.8nM。在一些实施方案中，本发明的抗PD-L1抗体以0.1-10nM，优选地0.5-10nM，更优选地0.6-10nM、0.7-8nM、0.7-5nM，最优选地0.5-1.5nM、0.7-1.5nM、0.7-1nM的K D结合PD-L1。在一些实施方案中，PD-L1为人PD-L1。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PD-L1的细胞，例如，以小于或等于大约4nM、3.5nM、3nM、2.9nM、2.8nM、2.7nM、2.6nM、2.5nM、2.4nM、2.3nM、2.2nM、2.1nM、2nM、1.9nM、1.8nM、1.7nM或1.6nM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PD-L1的细胞为表达人PD-L1的CHO细胞。
(3)本发明的抗体或其片段阻断PD-L1的相关活性，例如以小于或等于大约10nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM或0.7nM的EC 50，优选以大约0.1-1nM、0.5-1nM、0.6-1nM、0.6nM、0.7nM、0.8nM、0.9nM或1nM的EC 50。在一些实施方案中，PD-L1的相关活性是PD-L1与PD-1的结合。在一些实施方案中，本发明的抗体或其片段在MOA测定中以小于或等于大约10nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM或0.7nM的EC 50，优选以大约0.1-1nM、0.5-1nM、0.6-1nM、0.6nM、0.7nM、0.8nM、0.9nM或1nM的EC 50抑制PD-L1与PD-1的结合。在一些实施方案中，细胞为CHO细胞。
(4)本发明的抗体或其片段提高T细胞功能，例如优于已知的抗PD-L1抗体，例如Tecentriq。
(5)本发明的抗体或其片段提高T细胞增殖，例如在MLR中，例如优于已知的抗PD-L1 抗体，例如Tecentriq。
(6)本发明的抗体或其片段提高IFN-γ分泌，例如在MLR中，例如优于已知的抗PD-L1抗体，例如Tecentriq。
(7)本发明的抗体或其片段提高IL-2分泌，例如在MLR中，例如优于已知的抗PD-L1抗体，例如Tecentriq。
(8)本发明的抗体或其片段相比已知的抗PD-L1抗体(例如Tecentriq)具有更低的粘性，因此具有更好的成药性。在一些实施方案中，本发明的抗体或其片段在Zenix柱检测法中，驻留时间(RT)低于大约10分钟、大约9分钟或大约8分钟，优选地，驻留时间在大约7分钟-9分钟之间，优选地在大约7-8.5分钟，大约7.5-8.5分钟，大约7-8分钟，或大约7.5-8分钟之间，例如大约7.5分钟、7.6分钟、7.7分钟、7.8分钟、7.9分钟、8分钟、8.1分钟、8.2分钟、8.3分钟、8.4分钟、8.5分钟。
(9)本发明的抗体或其片段抑制PD-L1的一种或多种活性，例如，导致以下一者或多者：肿瘤浸润型淋巴细胞增加、T细胞受体介导的增殖增加、或癌细胞的免疫逃避减少。
(10)本发明的抗PD-L1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PD-L1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PD-L1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PD-L1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PD-L1；
(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PD-L1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PD-L1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PD-L1抗体或其抗原结合片段包含重链可变区VH和轻链可变区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：26、27、28、29、30或31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：32、33、34、35、36或37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PD-L1抗体或其抗原结合片段包含
(i)如SEQ ID NO：26或30所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：32或36所示的轻链可变区的3个互补决定区LCDR，或者
(ii)如SEQ ID NO：27所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：33所示的轻链可变区的3个互补决定区LCDR，或者
(iii)如SEQ ID NO：28所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：34所示的轻链可变区的3个互补决定区LCDR，或者
(iv)如SEQ ID NO：29或31所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：35或37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包含选自SEQ ID NO：1、2、3或4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或4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、8或9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、8或9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：10、11、12或13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：10、11、12或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；
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：14、15或16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：14、15或16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：17、18、19或20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：17、18、19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；LCDR3包含选自SEQ ID NO：21、22、23、24或25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：21、22、23、24或25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PD-L1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PD-L1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 PCTCN2018124314-appb-000003
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：26、27、28、29、30或31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：26、27、28、29、30或31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、29、30或31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：32、33、34、35、36或37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：32、33、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：32、33、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PD-L1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
组合 VH，其包含下述SEQ ID NO所示的氨基 VL，其包含下述SEQ ID NO所示的氨基
酸序列或由其组成 酸序列或由其组成
(1) SEQ ID NO：26 SEQ ID NO：32
(2) SEQ ID NO：27 SEQ ID NO：33
(3) SEQ ID NO：28 SEQ ID NO：34
(4) SEQ ID NO：29 SEQ ID NO：35
(5) SEQ ID NO：30 SEQ ID NO：36
(6) SEQ ID NO：31 SEQ ID NO：37
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：38、39、40、41、42、43、44或45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：38、39、40、41、42、43、44或45的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：38、39、40、41、42、43、44或45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：46、47、48、49、50或51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：46、47、48、49、50或51的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：46、47、48、49、50或51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PD-L1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 PCTCN2018124314-appb-000004
在一些实施方案中，本发明抗PD-L1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：68)。
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PD-L1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 PCTCN2018124314-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。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WO 06/089231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PD-L1抗体(例如，人源化抗体或嵌合抗体)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，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PD-L1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)、单结构域抗体；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PD-L1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 inBioscience13：1619-1633)。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PD-L1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，1994Nature 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PD-L1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。
在某些实施方案中，本发明的抗体为嵌合抗体。某些嵌合抗体描述于例如美国专利号4,816,567；以及Morrison等Proc.Natl.Acad.Sci.USA.81：6851-6855，1984中。在一个实施方案中，嵌合抗体包含非人可变区(例如来源于小鼠、大鼠、仓鼠、兔或非人灵长类诸如猴的可变区)和人恒定区。在又一个实施方案中，嵌合抗体为“类别转换”抗体，其中类别或亚类与亲本抗体的类别或亚类相比已经改变。嵌合抗体包括其抗原结合片段。
在某些实施方案中，嵌合抗体为人源化抗体。通常，非人抗体被人源化以降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。一般说来，人源化抗体包含一个或多个可变域，其中例如CDR(或其部分)来源于非人抗体，并且FR(或其部分)来源于人抗体序列。人源化抗体任选还将包含至少一部分人恒定区。在一些实施方案中，人源化抗体中的一些FR残基被来自非人抗体(例如CDR残基所来源的抗体)的相应残基置换，例如以恢复或改善抗体特异性或亲和力。
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，Nature348：552-554；Clackso等人，Nature 352：624-628(1991)；Marks等人，J.Mol.Biol.222：581-597(1992)；Marks及Bradbury，Methods in Molecular Biology248：161-175(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PD-L1上并且第二表位位于LAG-3、OX40、TIM-3、CEACAM(例如，CEACAM-1和/或CEACAM-5)或PD-L2上。
在一些实施方案中，本发明还涵盖与其他物质，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PD-L1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WO2015/153513或WO2015/138920等。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，例如紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙啶、吐根碱、丝裂霉素、表鬼臼毒吡喃葡糖苷、表鬼臼毒噻吩糖苷、长春新碱、长春碱、秋水仙素、阿霉素、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光辉霉素、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、普萘洛尔和嘌呤霉素和它们的类似物或同系物。细胞毒性剂还包括，例如：抗代谢物(例如，氨甲喋呤、6-巯基嘌呤、6-硫代鸟嘌呤、阿糖胞苷、5-氟尿嘧啶、氨烯咪胺(decarbazine))，烷化剂(例如，氮芥、苯丁酸氮芥(thioepachlorambucil)、苯丙氨酸氮芥、卡莫司汀(BSNU)和洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露糖醇、链唑霉素、丝裂霉素C和顺-二氯二胺合铂(II)(DDP)顺铂)，氨茴霉素类(例如，柔红菌素(以前称为道诺霉素)和阿霉素)，抗生素(例如，放线菌素D(以前称为放线菌素)、博来霉素、光辉霉素和安曲霉素(AMC))，和抗有丝分裂剂(例如，长春新碱和长春碱)。
可以与抗PD-L1抗体缀合/偶联的物质还参见例如WO2010/077634、US60/696426、WO2016/007235或WO2016/061142等。
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PD-L1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编码选自SEQ ID NO：26至51中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO：26至51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：包含编码选自SEQ ID NO：26至51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：26至51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。抗体例如可在细菌中产生，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PD-L1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PD-L1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PD-L1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PD-L1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PD-L1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PD-L1抗体竞争对PD-L1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PD-L1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PD-L1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PD-L1或经改造而表达PD-L1的细胞或细胞系。
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PD-L1抗体来进行任何上述测定法。
可以理解的是，能够使用抗PD-L1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PD-L1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PD-L1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PD-L1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PD-L1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PD-L1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PD-L1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/或细胞毒性剂。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PD-L1抗体组合的任何物质，包括化疗剂、其它抗体以及其他的治疗剂。这些活性成分的例子参见例如WO2010/077634、WO2016/061142、US61/264061、US60/696426、WO2016/007235等等。
在一些实施方案中，所述活性成分是抗LAG-3抗体，例如结合人的抗LAG-3抗体，优选地，抗LAG-3抗体是人源化的
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PD-L1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PD-L1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PD-L1抗体)或药物组合物或免疫缀合物。在一些实施方案中，肿瘤是肿瘤免疫逃逸。优选地，肿瘤是胃肠道肿瘤(例如癌症)，例如结肠癌。
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PD-L1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PD-L1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PD-L1抗体分子提高T细胞增殖。在一些实施方案中，本文所述的抗PD-L1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、软组织肿瘤和转移性病灶。
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(括腺癌和鳞状细胞癌)，如侵袭肝、肺、乳腺、淋巴、胃肠道的(例如，结肠)、生殖泌尿道(例如，肾、膀胱上皮细胞)、前列腺和咽的那些癌。腺癌包括恶性肿瘤如大部分结肠癌、直肠癌、肾细胞癌、肝癌、肺癌中的非小细胞癌、小肠癌和食道癌。鳞状细胞癌包括恶性肿瘤，如在肺、食道、皮肤、头颈区域、口腔、肛门和子宫颈的那些癌。在一个实施方案中，癌症是黑素瘤，例如，晚期黑素瘤。在一个实施方案中，癌症是胃肠道癌症，例如结肠癌。前述癌的转移性病灶也可以使用本发明的方法和组合物治疗或预防。
用于治疗的优选癌症的非限制性例子包括淋巴瘤(例如，弥漫性大B细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤)、乳腺癌(例如，转移性乳腺癌)、肺癌(例如，非小细胞肺癌(NSCLC)，例如，IV期或复发性非小细胞肺癌、NSCLC腺癌、或NSCLC鳞状细胞癌)、骨髓瘤(例如，多发性骨髓瘤)、白血病(例如，慢性髓性白血病)、皮肤癌(例如，黑素瘤(例如，III期或IV期黑素瘤)或Merkel细胞癌)、头颈癌(例如，头颈鳞状细胞癌(HNSCC))、脊髓发育不良综合征、膀胱癌(例如，移行细胞癌)、肾癌(例如，肾细胞癌，例如，透明细胞肾细胞癌，例如，晚期或转移性透明细胞肾细胞癌)和结肠癌。另外，难治性或复发性恶性肿瘤可以使用本文所述的抗体分子治疗。
可以治疗的其他癌症的例子包括骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肛门癌、胃-食管癌、胃癌、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、Merkel细胞癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病，包括急性髓样白血病、慢性髓样白血病、急性淋巴母细胞性白血病、慢性淋巴细胞白血病、儿童实体瘤、淋巴细胞淋巴瘤、膀胱癌、多发性骨髓瘤、骨髓异常增生综合征、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管生成、脊枢椎肿瘤、脑干胶质瘤、垂体腺瘤、Kaposi肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱导的癌症，包括石棉诱导的那些癌症(例如，间皮瘤)和所述癌症的组合。
可以使用本文所述的抗体分子实现对转移性癌(例如，表达PD-L1的转移性癌(Iwai等人(2005)Int.Immunol.17：133-144))的治疗。
还可以使用本文所述的抗体分子对肿瘤免疫逃逸进行治疗。
在一个实施方案中，肿瘤是表达升高水平的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感染、潜伏的感染或缓慢感染。在一些实施方案中，所述慢性感染是由选自细菌、病毒、真菌和原生动物的病原体导致的。
在一些实施方案中，所述病原体是细菌。在一个实施方案中，所述细菌选自：分枝杆菌属物种(Mycobacterium spp.)、沙门氏菌属物种(Salmonellaspp.)、李斯特菌属物种(Listeria spp，)、链球菌属物种(Streptococcus spp.)、嗜血杆菌属物种(Haemophilus，spp.)、奈瑟菌属物种(Neisseria  spp.)、克雷伯氏菌属物种(Klebsiella spp.)、疏螺旋体属物种(Borrelia spp.)、脆弱拟杆菌(Bacterioides fragillis)、密螺旋体属物种(Treponema spp.)和幽门螺旋杆菌(Helicobacter pylori)。
在一些实施方案中，所述病原体是病毒。在一个实施方案中，所述病毒选自：感染病毒，例如乙型肝炎病毒或丙型肝炎病毒(hepatitis-B，-C)，单纯性疱疹病毒-I、-II(herpes simplex virus-I，-II)、人免疫缺陷病毒-I、-II(humanimmunodeficiency virus-I，-II)、巨细胞病毒(cytomegalovirus)、EB病毒(Eppstein Barr virus)、人乳头状瘤病毒(human papillomavirus)、人类T淋巴细胞白血病病毒-I或-II(human T lymphotrophic viruses，-I，-II)、水痘带状疱疹病毒(varicallazoster)。
在一些实施方案中，所述病原体是真菌。在一个实施方案中，真菌引起的病症选自：曲霉病(aspergilosis)、芽生菌病(blastomycosis)、白色念珠菌病(candidiasis albicans)、球孢子菌病(coccidioiodmycosis immitis)、组织胞浆菌病(histoplasmosis)、类球孢子菌病(paracoccidioiomycosis)、微孢子虫病(microsporidiosis)。
在一些实施方案中，所述病原体是原生动物，例如寄生虫。在一个实施方案中，所述原生生物导致的病症选自：利什曼病(leishmaniasis)、疟原虫病(plasmodiosis)(即，疟疾(malaria))、隐孢子虫病(cryptosporidiosis)、弓形体病(toxoplasmosis)、锥虫病(trypanosomiasis)和蠕虫感染(helminth infection)，包括那些由吸虫(trematodes)(例如血吸虫病(schistosomiasis))、绦虫(cestodes)(例如包虫病(echinococcosis))和线虫(nemotodes)(如旋毛虫病(trchinosis)、蛔虫病(ascariasis)、丝虫病(filariosis)和类圆线虫病(strongylodiosis))导致的病症。
在另一个实施方案中，感染是肝炎感染，例如，乙型肝炎或丙型肝炎感染。抗PD-L1抗体分子可以出于治疗优势与乙型肝炎感染或丙型肝炎感染的常规治疗组合。在某些实施方案中，抗PD-L1抗体分子与乙型肝炎抗原((例如，Engerix B))或疫苗，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PD-L1抗体分子与流感抗原或疫苗组合施用。
适于用本发明的抗PD-L1的抗体或其片段预防或治疗的疾病可进一步参见WO2010/077634、WO2016/061142、US60/696426、WO2016/007235或US61/264061。
在其他方面，本发明提供抗PD-L1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，所述药物用于预防或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PD-L1抗体或其片段)或药物组合物或免疫缀合物，以及一种或多种其它疗法，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或抗LAG-3)；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 PCTCN2018124314-appb-000006
或
Figure PCTCN2018124314-appb-000007
)、曲妥珠单抗
Figure PCTCN2018124314-appb-000008
托西莫单抗
Figure PCTCN2018124314-appb-000009
替伊莫单抗
Figure PCTCN2018124314-appb-000010
阿来组单抗
Figure PCTCN2018124314-appb-000011
依帕珠单抗
Figure PCTCN2018124314-appb-000012
贝伐珠单抗
Figure PCTCN2018124314-appb-000013
厄洛替尼
Figure PCTCN2018124314-appb-000014
西妥昔单抗
Figure PCTCN2018124314-appb-000015
等等。
示例性的化疗剂包括但不限于阿那曲唑
Figure PCTCN2018124314-appb-000016
比卡鲁胺
Figure PCTCN2018124314-appb-000017
硫酸博来霉素
Figure PCTCN2018124314-appb-000018
白消安
Figure PCTCN2018124314-appb-000019
白消安注射剂
Figure PCTCN2018124314-appb-000020
卡培他滨
Figure PCTCN2018124314-appb-000021
N4-戊氧羰基-5-脱氧-5-氟胞苷、卡铂
Figure PCTCN2018124314-appb-000022
卡莫司汀
Figure PCTCN2018124314-appb-000023
苯丁酸氮芥
Figure PCTCN2018124314-appb-000024
顺铂
Figure PCTCN2018124314-appb-000025
克拉立滨
Figure PCTCN2018124314-appb-000026
环磷酰胺(
Figure PCTCN2018124314-appb-000027
或
Figure PCTCN2018124314-appb-000028
)、阿糖胞苷、胞嘧啶阿拉伯糖苷
Figure PCTCN2018124314-appb-000029
阿糖胞苷脂质体注射剂
Figure PCTCN2018124314-appb-000030
达卡巴嗪
Figure PCTCN2018124314-appb-000031
更生霉素(dactinomycin)(放线菌素D、Cosmegan)、盐酸道诺霉素
Figure PCTCN2018124314-appb-000032
柠檬酸道诺霉素脂质体注射剂
Figure PCTCN2018124314-appb-000033
地塞米松、多西紫杉醇
Figure PCTCN2018124314-appb-000034
盐酸多柔比星
Figure PCTCN2018124314-appb-000035
Figure PCTCN2018124314-appb-000036
依托泊苷
Figure PCTCN2018124314-appb-000037
磷酸氟达拉滨
Figure PCTCN2018124314-appb-000038
5-氟尿嘧啶
Figure PCTCN2018124314-appb-000039
氟他胺
Figure PCTCN2018124314-appb-000040
tezacitibine、吉西他滨(双氟脱氧胞苷)、羟基脲
Figure PCTCN2018124314-appb-000041
伊达比星
Figure PCTCN2018124314-appb-000042
异环磷酰胺
Figure PCTCN2018124314-appb-000043
伊立替康
Figure PCTCN2018124314-appb-000044
L-天冬酰胺酶
Figure PCTCN2018124314-appb-000045
亚叶酸钙、美法仑
Figure PCTCN2018124314-appb-000046
6-巯基嘌呤
Figure PCTCN2018124314-appb-000047
甲氨蝶呤
Figure PCTCN2018124314-appb-000048
米托蒽醌(米托蒽醌)、米罗他(mylotarg)、紫杉醇
Figure PCTCN2018124314-appb-000049
phoenix(钇90/MX-DTPA)、喷司他丁、聚苯丙生20联用卡莫司汀植入物
Figure PCTCN2018124314-appb-000050
柠檬酸他莫昔芬
Figure PCTCN2018124314-appb-000051
替尼泊苷
Figure PCTCN2018124314-appb-000052
6-硫鸟嘌呤、塞替派、替拉扎明
Figure PCTCN2018124314-appb-000053
注射用盐酸拓扑替康
Figure PCTCN2018124314-appb-000054
长春碱
Figure PCTCN2018124314-appb-000055
长春新碱
Figure PCTCN2018124314-appb-000056
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PD-1、PD-L2、CTLA-4、TIM-3、LAG-3、CEACAM(例如，CEACAM-1、-3和/或-5)、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4和/或TGFR β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(例如，PD-1-Ig或CTLA-4Ig)，或抗体或其抗原结合片段；例如，与PD-1、PD-L2、CEACAM(例如，CEACAM-1、-3和/或-5)、CTLA-4、TIM-3、LAG-3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4和/或TGFR β或其组合结合的抗体或其片段。在其他实施方案中，免疫调节剂是CEACAM(例如，CEACAM-1、-3和/或-5)(例如，人CEACAM(例如，CEACAM-1、-3和/或-5))的抑制剂。在其他实施方案中，免疫调节剂是LAG-3(例如，人LAG-3)的抑制剂。在一个实施方案中，LAG-3的抑制剂是针对LAG-3的抗体分子，例如结合人的抗LAG-3抗体，优选地，抗LAG-3抗体是人源化的。在其他实施方案中，免疫调节剂是TIM-3(例如，人TIM-3)的抑制剂。在一个实施方案中，TIM-3的抑制剂是针对TIM-3的抗体分子。
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、CD28、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。在其他实施方案中，免疫调节剂是GITR激动剂。在一个实施方案中，GITR激动剂是针对GITR的抗体分子。在其他实施方案中，免疫调节剂是OX40激动剂。在一个实施方案中，OX40激动剂是针对OX40的抗体分子。
在进一步的一些实施方案中，本发明的抗PD-L1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，本发明的的抗PD-L1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，本发明的抗PD-L1抗体或其片段的施用与抗原的施用组合。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，本发明的抗PD-L1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，本发明的抗PD-L1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，本发明的抗PD-L1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，溶瘤病毒能够在癌细胞中选择性复制并且触发癌细胞死亡或延缓其生长。在一些情况下，溶瘤病毒对非癌细胞无影响或影响最小。溶瘤病毒包括但不限于溶瘤腺病毒、溶瘤单纯疱疹病毒、溶瘤逆转录病毒、溶瘤细小病毒、溶瘤痘苗病毒、溶瘤辛德比斯病毒、溶瘤流感病毒、或溶瘤RNA病毒(例如，溶瘤呼肠孤病毒、溶瘤新城疫病毒(NDV)、溶瘤麻疹病毒或溶瘤水泡性口炎病毒(VSV))。在一些实施方案中，溶瘤病毒是US 2010/0178684 A1中描述的病毒，例如，重组溶瘤病毒。
在一些实施方案中，本发明的抗PD-L1抗体或其片段可以与细胞因子联合施用。
在一些实施方案中，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，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PD-L1抗体与其他治疗方式或治疗剂的组合的例子可以参见WO2016/061142、WO2010/077634、US60/696426、US61/264061或WO2016/007235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PD-L1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PD-L1抗体来进行任何治疗。
与抗LAG-3抗体组合的联合疗法
在一些实施方案中，本发明的抗PD-L1抗体可以与抗LAG-3抗体联合用于治疗。
在一些实施方案中，本发明的抗LAG-3抗体是抗人LAG-3抗体。在一些实施方案中，本发明的抗LAG3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LAG-3抗体是单克隆抗体。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抗体是嵌合抗体。在一些实施方案中，至少部分的抗LAG-3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。
在一些具体的实施方案中，本发明的抗LAG-3抗体或其抗原结合片段包含
(i)如SEQ ID NO：75所示的重链可变区的3个互补决定区HCDR，和/或
(ii)如SEQ ID NO：76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SEQ ID NO：69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：70的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：71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：72的氨基酸序列或由所述氨基酸序列组成；LCDR2包含SEQ ID NO：73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：74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：75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：75的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：75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：76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：76的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：76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：77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：77的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：77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：78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：78的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：78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PD-L1抗体的修饰同样适用于抗LAG-3抗体。
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。
可以由技术人员确定抗PD-L1抗体分子的剂量和治疗方案。在某些实施方案中，抗PD-L1抗体分子通过注射(例如，皮下或静脉内)以约1至30mg/kg，例如，约5至25mg/kg、约10至20mg/kg、约1至5mg/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PD-L1抗体分子以约10至20mg/kg的剂量每隔一周施用。在一个实施方案中，将抗PD-L1抗体分子以小于或等于约5mg/kg；小于或等于约4mg/kg；小于或等于约3mg/kg；小于或等于约2mg/kg；小于或等于约1mg/kg的剂量每隔一周单独或组合(例如，与抗LAG-3抗体分子组合)施用。在一个实施方案中，将抗PD-L1抗体分子以大约1至5mg/kg的剂量每隔一周、大约1至4mg/kg的剂量每隔一周、大约1至3mg/kg的剂量每隔一周或大约1至2mg/kg的剂量每隔一周施用。在一个实施方案中，将抗LAG-3抗体分子以大约1至5mg/kg的剂量每隔一周、大约1至4mg/kg的剂量每隔一周、大约1至3mg/kg的剂量每隔一周或大约1至2mg/kg的剂量每隔一周单独或组合(例如，与抗LAG-3抗体分子组合)施用。
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PD-L1抗体或其抗原结合片段可以用于检测PD-L1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PD-L1抗体。在另一个方面中，提供检测PD-L1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PD-L1蛋白在生物样品中的存在。在某些实施方案中，PD-L1是人PD-L1。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PD-L1抗体在允许抗PD-L1抗体与PD-L1结合的条件下接触，并检测在抗PD-L1抗体和PD-L1之间是否形成复合物。复合物的形成表示存在PD-L1。该方法可以是体外或体内方法。在一个实施方案中，抗PD-L1抗体被用于选择适合利用抗PD-L1抗体的治疗的受试者，例如其中PD-L1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PD-L1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PD-L1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PD-L1。
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PD-L1的存在，因而确定PD-L1值，将PD-L1值与对照值比较，并且如果PD-L1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抗PD-L1抗体(例如，本文所述的抗PD-L1抗体)，因而治疗肿瘤或感染。
本发明示例性抗PD-L1抗体的序列
Figure PCTCN2018124314-appb-000057
Figure PCTCN2018124314-appb-000058
Figure PCTCN2018124314-appb-000059
Figure PCTCN2018124314-appb-000060
Figure PCTCN2018124314-appb-000061
Figure PCTCN2018124314-appb-000062
Figure PCTCN2018124314-appb-000063
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.杂交瘤细胞的制备
杂交瘤技术是通过融合两种细胞而同时保持两者的主要特征。这两种细胞分别是经抗原免疫的小鼠脾细胞和小鼠骨髓瘤细胞。被特异性抗原免疫的小鼠脾细胞(B淋巴细胞)的主要特征是它的抗体分泌功能，但不能在体外连续培养，小鼠骨髓瘤细胞则可在培养条件下无限分裂、增殖，即具有所谓永生性。在选择培养基的作用下，只有B细胞与骨髓瘤细胞融合的杂交细胞才能具有持续培养的能力，形成同时具备抗体分泌功能和保持细胞永生性两种特征的细胞克隆。本实验通过hPD-L1蛋白免疫小鼠，再获取小鼠的脾细胞和骨髓瘤细胞融合，获得能够表达阳性抗体的杂交瘤细胞。
杂交瘤融合
实验动物及免疫信息
Figure PCTCN2018124314-appb-000064
电融合皿准备：用70％乙醇彻底浸泡电融合皿，并于超净台中吹干备用。
分离脾细胞：颈脱位将小鼠处死，用75％酒精消毒体表5min，随即放入超净台内小鼠解剖板上，左侧卧位，用7号针头固定四肢。无菌打开腹腔取出脾脏，用基础培养基(配置方法如下表)洗涤，并仔细去掉周围附着的结缔组织。随后将脾脏转移到另一个盛有基础培养基的平皿中。以弯头针头压住脾脏，用小针头在脾脏上插孔，并用镊子挤压，使脾细胞充分释放，制成脾细胞悬液。细胞悬液经100μM细胞筛网过滤后用30ml基础培养基洗一遍，1200rpm离心6min。
Figure PCTCN2018124314-appb-000065
裂解红细胞：去除上清，用10ml RBC裂解缓冲液(GIBCO)重悬细胞。然后再加入20ml RBC裂解缓冲液。悬液静置5min后1100rpm离心6min。去上清后用10ml基础培养基重悬细胞，然后再加入30ml基础培养基，1100rpm离心6min。去除上清后，细胞重悬于20ml基础培养基中 并计数。
电融合：用20ml基础培养基重悬小鼠骨髓瘤细胞SP2/0细胞(ATCC)并计数。将SP2/0和脾细胞以1∶2～1∶1的比例混合，100rpm离心6min。去除上清后将混合的细胞重悬于10ml融合缓冲液(BTXpress)中。再加入15ml融合缓冲液，1000rpm离心5min，去除上清。重复上述步骤一遍后，用适量融合缓冲液重选细胞，调整混合细胞密度至1×10 7个细胞/ml。电融合仪的参数设置如下。每个电融合皿中加入2ml细胞悬液进行电融合。
Condition Mouse(SP2/0-ECF-F)
Alignment： 60v，30sec
Membrane breaking： 1500V，30μs，3X
Post-fusion pulse： 60V，3sec
电融合后铺板：细胞于电融合皿中室温静置5min。将细胞转移入离心管中，用筛选培养基(配置方法如下表)稀释细胞至1～2×104个细胞/ml。96孔板中每孔加入100μl细胞悬液。融合后第7天更换筛选培养基。培养第10天(或更久，根据细胞生长状态)后进行筛选。通过FACS(FACS ARIA(BD Biosciences))检测筛选出表达特异性抗PD-L1抗体的杂交瘤细胞。
Figure PCTCN2018124314-appb-000066
阳性杂交瘤细胞亚克隆
亚克隆步骤：准备一块96孔板，第2至第8列每孔加入200μl如上所述的基础培养基。将上述融合筛选出的阳性孔的细胞制成细胞悬液并加入第1列。将第1列细胞悬液取100μl加入第2列，充分混匀后取100μl加入下一列。重复上述步骤，直至最后一列体积变为300μl；静置96孔板15min，显微镜下观察计数。取100个细胞对应的体积加入20ml如上所述的基础培养基中，并混匀铺板，每孔200μl。一周后显微镜下观察，判断并标记出单克隆孔，待测挑出阳性孔。
细胞冻存：观察细胞状态，等细胞生长良好，活力＞90％时，1000rpm离心5min，去除上清。用冻存液(45.5％FBS，44.5％RPMI-1640，10％DMSO)重悬细胞至1×107个细胞/ml，分装至冻存管，放入程序降温盒中，-80℃冻存。
实施例2.嵌合抗体的生产和纯化
本发明利用分子生物学技术，获得抗PD-L1阳性杂交瘤细胞中的抗体序列，并利用其构建人鼠嵌合抗体。
杂交瘤测序
RNA抽提：新鲜细胞，300g离心5min，去除上清，沉淀中加入500μl LY缓冲液(Biomiga)(在使用前每1ml加入20μl β巯基乙醇)，混匀至澄清。加入到DNA去除管中，13000rpm离心2min，收集流穿液。按1/2的比例向流穿液中加入100％乙醇，混匀5次至澄清。将澄清的溶液加入到RNA收集管中，13000rpm离心1min去除液体，加入500μl RB(Recovery Buffer，回收缓冲 液)(Takara)，13000rpm离心30s，再加入500μl RNA洗涤缓冲液(Biomiga)(用之前加入乙醇)，离心30s，重复一遍上述过程后，离心彻底挥发去除乙醇后，向收集柱中加入30μl DEPC水，12000g离心2min，收集洗脱液。测定RNA浓度。
反转录获得cDNA：
配置反应体系I如下：
Figure PCTCN2018124314-appb-000067
*来自PrimeScript II 1 st Strand cDNA Synthesis Kit，购自Takara。
65℃温育5min后，迅速置冰上冷却。向反应体系I中加入下列反转录体系，总量为20μl：
Figure PCTCN2018124314-appb-000068
*来自PrimeScript II 1 st Strand cDNA Synthesis Kit，购自Takara。
缓慢混匀后按下列条件进行反转录翻译：42℃60min→95℃5min，然后放冰上冷却，获得cDNA。
将cDNA连接T载体：
PCR分别扩增重链和轻链可变区，PCR反应体系如下：
Figure PCTCN2018124314-appb-000069
Figure PCTCN2018124314-appb-000070
取4.5μl上述PCR反应获得的PCR产物，加入0.5μl pMD20-T载体(Clontech)，5μl Ligation Mighty Mix(Takara)，轻轻混匀，于37℃反应2h，获得连接产物。
转化细胞：
-80℃取出TOP10感受态(天根生化科技(北京)有限公司)，冰上融化，取上文获得的连接产物5μl加入到融化的TOP10感受态中，混匀后冰上孵育30min。42℃热激90s后迅速冰上冷却2min，向EP管中补加900μl LB培养基(生工生物工程(上海)股份有限公司)，37℃，220rpm摇床培养1h。3000g离心2min，吸除800μl上清，用剩余的培养基将菌体重悬并涂布在氨苄青霉素抗性的平板上。于37℃培养过夜，挑克隆测序。
构建嵌合抗体
PCR扩增已经测序的实施例1中杂交瘤产生的鼠抗PD-L1抗体VH及VL区：上下游引物序列见表5及表6。
表5.小鼠抗PD-L1抗体的重链可变区(VH)引物(Primer Mix 1)
Figure PCTCN2018124314-appb-000071
Figure PCTCN2018124314-appb-000072
按上述比例混合后，获得Primer Mix 1用于后续VH的PCR扩增。
表6.小鼠抗PD-L1抗体的轻链可变区(VL)引物(Primer Mix 2)：
Figure PCTCN2018124314-appb-000073
Figure PCTCN2018124314-appb-000074
按上述比例混合后，获得Primer Mix 2用于后续VL的PCR扩增。
PCR体系如下：
Figure PCTCN2018124314-appb-000075
*对于VH链扩增，应用Primer Mix 1；对于VL链扩增，应用Primer Mix 2。
切胶回收PCR扩增产物。
同源重组反应：
同源重组体系如下：
Figure PCTCN2018124314-appb-000076
37℃反应30min，获得重组产物。重组产物转化TOP10感受态，并挑取单克隆测序，选择包含插入方向正确的质粒的克隆作为阳性克隆，保存阳性克隆。
嵌合抗体的表达和纯化
从上文获得的阳性克隆中提取包含抗PD-L1抗体的质粒。
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 Protein A，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。
本发明获得的4个嵌合抗体的CDR、轻链可变区和重链可变区，轻链和重链的氨基酸序列，以及序列编号请参见上文表1-3。
本发明所用的对照抗体为罗氏的PD-L1抗体Atezolizumab(以下简称ATE或Ate，商品名Tecentriq)，其CDR、轻链可变区和重链可变区，轻链和重链的氨基酸序列也参见上文表1-3。
实施例2生物光干涉测定法测定本发明的嵌合抗体与抗原的结合动力学
采用生物光干涉测定法(ForteBio)测定本发明抗体结合人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×，BSA0.1％，Tween-20 0.05％)中。
取100μl的SD缓冲液、抗体、抗原(包括人PD-L1、小鼠PD-L1及食蟹猴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。
在以上测定法所述的实验中，抗体3-266.1、4-79.2、4-26.6、4-48.5的亲和力如表7所示：
表7.ForteBio检测抗原抗体结合的亲和力常数(平衡解离常数)
Figure PCTCN2018124314-appb-000077
在以上试验中，嵌合抗体3-266.1、4-79.2、4-26.6、4-48.5的KD值分别为9.80E-10M、7.56E-09M、3.85E-09M、1.23E-09M，与对照组相比，本研究中的抗体具有相似或更优的K D值。
实施例3嵌合抗体和过表达PD-L1的细胞的结合实验
本研究利用流式细胞仪检测了梯度稀释的本发明的嵌合抗体与表面过表达人PD-L1的CHO稳定细胞株的结合情况。
通过将带有克隆到多克隆位点(MCS)的人PD-L1 cDNA(Sino Biological)的pCHO1.0载体(Invitrogen)转染到CHO-S细胞(Invitrogen，ExpiCHO TM Expression System Kit，货号：A29133)，产生过表达人PD-L1的CHO细胞(CHO-PDL1)。
将CHO-PDL1细胞计数，并稀释至1×10 6个细胞/ml，向U型底96孔板中加入100μl/孔。400g 5min，离心，去除细胞培养基。将样品(分别是嵌合抗体3-266.1、4-79.2、4-26.6、4-48.5，以及阳性对照抗体Ate)(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)加入U型板并重悬细胞，100μl/孔，冰上静置30min。400g 5min去除上清，PBS洗细胞1遍。400g 5min去除PBS，每孔加入100μl抗鼠Fab的FITC标记的二抗(Jackson Immuno Research)(1∶500稀释于PBS中)，在加入阳性对照抗体的细胞中加入100μl抗人Fab的FITC标记的二抗(Jackson Immuno Research)。冰上避光孵育30min。400g 5min去除上清，PBS洗细胞1遍。用100μl 1×PBS重悬细胞，FACS检测。
在以上测定法所述的实验中，抗体3-266.1、4-79.2、4-26.6、4-48.5和CHO-PDL1细胞的结合情况如图1所示。
在以上试验中，抗体3-266.1、4-79.2、4-26.6、4-48.5均结合CHO细胞上过表达的人PD-L1，EC50分别为2.139nM、2.598nM、1.985nM、1.995nM，与对照抗体ATE相比，其结合力更优，部分抗体的结合能力是对照抗体的两倍多。
实施例4嵌合抗体的人源化
将实施例1得到的嵌合抗体进行人源化。抗体人源化过程使用Macromoltek公司的SmrtMolHumanize专有软件程序进行。首先将序列输入软件，系统会生成序列的三维模型，并经过以下步骤进行人源化：
①确定CDR环结构；
②在人种系序列数据库为重链和轻链的每个V/J区域找到最接近的同源序列；
③筛选与重链轻链最匹配的人种系以及最低量的回复突变；
④将嵌合抗体的CDR区构建至人的骨架区上；
⑤使用序列和结构特征，确定骨架区中起到维持CDR功能的氨基酸位置；
⑥在确定为重要的序列位置进行回复突变(返回到输入氨基酸类型)；
⑦生成人源化序列的三维模型；
⑧手工检查序列和结构，以确定可能会导致错误折叠或降低稳定性的风险位点；
⑨优化风险位点的氨基酸。
本发明获得的4个人源化抗体(HZ3266-IgG1N297A，HZ3266-IgG1、HZ3266-IgG4PAK和HZ4485-IgG1N297A)的CDR、轻链可变区和重链可变区，轻链和重链的氨基酸序列请参见如上文所述的表1-3。
实施例5 ForteBio测定人源化抗体与抗原的结合动力学
采用ForteBio测定法测定本发明不同Fc亚型的人源化抗体结合人PD-L1的平衡解离常数(KD)。ForteBio亲和力测定方法同实施例2。在以上测定法所述的实验中，抗体HZ3266-IgG1N297A、HZ3266-IgG1、HZ3266-IgG4PAAK、HZ4485-IgG1N297A的亲和力如表8所示：
表8.ForteBio检测抗原抗体结合的亲和力常数
Figure PCTCN2018124314-appb-000078
在以上试验中，本文所述的人源化抗体HZ3266-IgG1N297A、HZ3266-IgG1、HZ3266-G4PAAK、HZ4485-IgG1N297A的K D值分别为7.24E-10M、9.35E-10M、1.32E-09M、3.17E-09M，与对照组相比，本研究中的人源化抗体具有相似或更优的K D值。
实施例6人源化抗体和过表达PD-L1的细胞的结合实验
本研究利用流式细胞仪检测了梯度稀释的本发明的人源化抗体与表面过表达人PD-L1的CHO稳定细胞株(CHO-PDL1)的结合情况。试验方法同实施例3，例外是使用的抗体是人源化抗体HZ3266-IgG1N297A、HZ3266-IgG1、HZ3266-G4PAAK、HZ4485-IgG1N297A，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个浓度的人源化抗体HZ3266-IgG1N297A、HZ3266-IgG1、HZ3266-G4PAAK、HZ4485-IgG1N297A和CHO-PDL1细胞的结合情况如图2所示。
在以上试验中，人源化抗体HZ3266-IgG1、HZ3266-IgG1N297A、HZ3266-G4PAAK、HZ4485-IgG1N297A结合CHO细胞上过表达的人PD-L1，EC50分别为1.813nM、1.784nM、1.862nM、1.561nM，与对照抗体ATE相比，具有更强的结合能力。
实施例7 MOA方法检测抗体的生物学活性
抗PD-1/PD-L1抗体能够通过阻断PD-1和PD-L1的结合，从而解除对下游NFAT信号通路的抑制作用。本研究使用Promega公司提供的MOA检测系统(PD-1/PD-L1 Blockade Bioassay，Cell Propagation Model，Catalog J1252)，根据说明书提供的方法，通过检测荧光报告基因的表达反应出NFAT信号的激活情况，从而检测抗体对PD-1/PD-L1结合的抑制作用。
活性检测前一天铺CHOK1-PDL1细胞(来自上述MOA检测系统)：铺CHOK1-PDL1前1-2天传代。弃培养上清，PBS(Gibco)洗一遍细胞。加入适量Trypsin(Gibco)于37℃、5％CO 2消化3～5min。加入4倍Trypsin体积的培养基，转移细胞至50ml离心管并计数。取所需体积细胞，230g，离心10min。加入1640培养基(Gibco)，重悬细胞至4×105个细胞/mL。将细胞加入96孔白色细胞培养板(Nunclon)，100μl/孔。边孔加入PBS，200μl/孔。细胞于37℃/5％CO2培养箱中培养过夜。
处理Jurkat-PD1细胞(来自上述MOA检测系统)：活性检测前两天进行细胞传代。计数后取所需体积细胞，170g，离心5min。用测定缓冲液(1640培养基(Gibco)+1％FBS)重悬细胞至1.25×10 6个细胞/ml。
加入样品和Jurkat-PD1细胞至检测板(来自上述MOA检测系统)：弃95μl/孔CHOK1-PDL1细胞上清。加入40μl样品(本发明制备的人源化抗体HZ3266-IgG1、HZ3266-IgG1N297A、HZ4485-IgG1N297A)及阳性对照(Ate)、阴性对照(IgG1)(抗体稀释方法为：最高抗体浓度为100nM，三倍稀释在测定缓冲液中，总共测试了8个浓度)。加入40μl Jurkat-PD1细胞。于37℃/5％CO 2培养箱中培养6小时。
检测：提前将Bio-GloTM缓冲液(来自上述MOA检测系统)融化，加入Bio-GloTM底物(来自上述MOA检测系统)，混匀。6小时后，加入Bio-GloTM试剂(来自上述MOA检测系统)，80μl/孔。室温放置5～10min。读数。
在以上试验中，实验结果如图3所示，抗体HZ3266-IgG1、HZ3266-IgG1N297A、HZ4485-IgG1N297A均可以有效阻断PD1/PD-L1的相互作用。
实施例8混合淋巴细胞实验
本研究将抗体和体外培养的、来源于不同供体的成熟DC细胞及CD4+ T细胞共同孵育，通过检测体系中IL2和IFN-γ的相对表达量，从而反应出不同抗体对T细胞的激活作用。
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。
DC细胞分离：取如上所述分离的PBMC细胞添加5ml T细胞培养基(配制方法如下表)，37℃、6％CO 2、贴壁培养2h，吸取悬浮细胞液做CD4+细胞分离，剩下细胞添加3ml DC培养基(配制方法如下表)，培养2天后添加3ml DC培养基，再培养第5天，然后添加rTNFa(R&D Systems)(1000U/ml)，IL-1b(R&D Systems)(5ng/ml)，IL-6(R&D Systems)(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。
Figure PCTCN2018124314-appb-000079
CD4+T细胞分离：按照‘Untouched CD4+ T cell isolation’kit instructions(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。
MLR实验：将上述获得的成熟后的DC细胞与CD4+细胞混合，每孔体积200μl，DC细胞10000个，CD4+细胞100000个，加入本发明的抗体(浓度：100nM、20nM、4nM、0.8nM、0.16nM、0.032nM)，以上文制备的DC细胞(下表中表示为DC)、CD4+T细胞(下表中表示为CD4)、DC细胞与CD4+细胞混合(下表中表示为Cell)和本文表3中公开的IgG1作为阴性对照，DC+CD4+T细胞+抗-CD3/CD28磁珠(QIAGEN)(下表中表示为Beads)作为阳性对照，混合培养5天，利用cisbio试剂盒(Human IL2 Kit 1000 Test、Human IFN gamma 1000test)检测IL2和IFN-γ浓度(相对表达量以DeltaF％计)。
实验结果如表9、10、11和12及图4、5所示。其中表中的数据单位为DeltaF％。
表9.供体1的IL2相对表达量
Figure PCTCN2018124314-appb-000080
*阴性对照，未加入任何抗体或磁珠。
表10.供体2的IL2相对表达量
Figure PCTCN2018124314-appb-000081
*阴性对照，未加入任何抗体或磁珠。
表11.供体1的IFN-γ相对表达量
Figure PCTCN2018124314-appb-000082
*阴性对照，未加入任何抗体或磁珠。
表12.供体2的IFN-γ相对表达量
Figure PCTCN2018124314-appb-000083
*阴性对照，未加入任何抗体或磁珠。
因此，本发明的抗体均可以在体外有效激活T细胞，且部分激活效果优于阳性对照抗体。
实施例9 Zenix柱检测抗体成药性
本实验通过记录本发明的示例性人源化抗体在Zenix柱(Sepax Technologies，Inc)中的停留时间，检测了抗体的成药性。停留时间越短，反应该抗体的粘性较低，成药性较好。
色谱条件：检测波长：214nm，柱温：25℃，流速：0.35ml/min，进样量：10μl。
样品准备：取100μl样品(待检测抗体HZ3266-IgG1、HZ3266-IgG4PAAK、HZ3266-IgG1N297A、HZ4485-IgG1N297A；ATE作为阳性对照，抗体浓度：1mg/ml)，13000rpm离心5min，取80μl上清于液相内插管中，置于液相样品盘中待进样检测。
实验结果如表13所示，HZ3266的不同亚型和HZ4485在柱上的停留时间均短于对照抗体，表明HZ3266和HZ4485的成药性较好。
表13.Zenix柱检测抗体成药性
Figure PCTCN2018124314-appb-000084
实施例10.抗肿瘤药效试验
本实验采用表达人PD-L1的MC38细胞(MC38-hPDL1)(南京银河公司)在hPD-L1转基因小鼠测定本发明的PD-L1抗体的抗肿瘤作用。
人PD-L1转基因小鼠：
雌性C57Bl/6背景的人PDL-1转基因小鼠(约8周大)购自上海南方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：
表达人PD-L1的MC38细胞(MC38-hPDL1)购自南京银河生物医药有限公司，并严格按照说明书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5×106个/ml。在第0天取0.2ml细胞悬液皮下接种至人PD-L1转基因小鼠右侧腹部区域中来建立MC38-hPDL1荷瘤小鼠模型。
给药：
肿瘤细胞接种6天后检测各只小鼠瘤体积，挑选出瘤体积在87.4mm 3～228.4mm 3范围内的小鼠按瘤体积平均分组(每组8只小鼠，一组给药IgG1，一组给药本发明抗体HZ3266-IgG1N297A)。分别在接种后第6、10、14、17、21、24、28、31和35天时给药，其中给药频率为2次/周，给药剂量和方式如表12所示。在给药期间监测各组小鼠瘤体积和体重变化，监测频率均为2次/周，连续监测5周。在每次给药前测定体重和肿瘤体积。肿瘤体积测定：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。采用电子天平测定体重，每周2次。
表14.实验设计表
Figure PCTCN2018124314-appb-000085
本发明抗体在给药一周后即显示出显著的抗肿瘤作用(图6)，到第35天时，本发明抗体组有1只小鼠肿瘤完全消退。体重结果显示不同抗体剂量组对荷瘤小鼠体重无影响。
因此，本发明抗体对肿瘤有明显的抑制效果。
实施例11.本发明抗PD-L1抗体与抗人LAG-3抗体的联合
本研究利用人源化小鼠模型研究了本发明的抗PD-L1抗体(HZ3266-IgG1N297A)和抗人LAG-3抗体(ADI-31853)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NCG小鼠上测定抗PD-L1抗体的抗肿瘤作用。 预先静脉注射人的PBMC(AllCells)(2×10 6个细胞/小鼠)，然后采用皮下接种的方式建立A375荷瘤小鼠模型，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，给药剂量和方式如下。给药结束后计算相对肿瘤抑制率(TGI％)。
抗人LAG-3抗体ADI-31853
根据本领域常规方法，将编码抗LAG-3抗体ADI-31853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％。
采用生物光干涉测量(ForteBio)测定法测定结合人LAG-3(hLAG-3)的ADI31853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-31853亲和力如下：
Figure PCTCN2018124314-appb-000086
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(AllCells)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)LAG-3(ADI-31853)，10mg/kg；
(3)PD-L1(HZ3266-IgG1N297A)，10mg/kg；
(4)LAG-3(ADI-31853)，10mg/kg+PD-L1(HZ3266-IgG1N297A)，10mg/kg。
接种后第7天，将瘤平均体积符合上述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7，可见抗LAG-3单克隆抗体ADI-31853(31853)和抗PD-L1单克隆抗体(HZ3266-IgG1N297A)(HZ3266)联合使用时与人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。

Claims (28)

  1. 结合PD-L1的抗体或其抗原结合片段，所述抗体包含
    (i)如SEQ ID NO：26或30所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：32或36所示的轻链可变区的3个互补决定区LCDR，或者
    (ii)如SEQ ID NO：27所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：33所示的轻链可变区的3个互补决定区LCDR，或者
    (iii)如SEQ ID NO：28所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：34所示的轻链可变区的3个互补决定区LCDR，或者
    (iv)如SEQ ID NO：29或31所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：35或37所示的轻链可变区的3个互补决定区LCDR。
  2. 结合PD-L1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或4所示的氨基酸序列，HCDR2包含SEQ ID NO：5、6、7、8或9所示的氨基酸序列，HCDR3包含SEQ ID NO：10、11、12或13所示的氨基酸序列，LCDR1包含SEQ ID NO：14、15或16所示的氨基酸序列，LCDR2包含SEQ ID NO：17、18、19或20所示的氨基酸序列，且LCDR3包含SEQ ID NO：21、22、23、24或25所示的氨基酸序列。
  3. 结合PD-L1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，
    (i)重链可变区包含与选自SEQ ID NO：26或30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：32或36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；
    (ii)重链可变区包含与SEQ ID NO：27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SEQ ID NO：33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；
    (iii)重链可变区包含与SEQ ID NO：28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SEQ ID NO：34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；
    (iv)重链可变区包含与选自SEQ ID NO：29或31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：35或37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，所述抗体包含轻链可变区和/或重链可变区，其中，
    (i)重链可变区包含SEQ ID NO：26或30的氨基酸序列；和/或
    轻链可变区包含SEQ ID NO：32或36的氨基酸序列成；
    (ii)重链可变区包含SEQ ID NO：27的氨基酸序列；和/或
    轻链可变区包含SEQ ID NO：33的氨基酸序列；
    (iii)重链可变区包含SEQ ID NO：28的氨基酸序列；和/或
    轻链可变区包含SEQ ID NO：34的氨基酸序列；
    (iv)重链可变区包含SEQ ID NO：29或31的氨基酸序列；和/或
    轻链可变区包含SEQ ID NO：35或37的氨基酸序列。
  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：38、42、43或44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：38、42、43或44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：46或50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：46或50的氨基酸序列或由其组成；或者
    (a)重链
    (i)包含与SEQ ID NO：39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SEQ ID NO：39的氨基酸序列或由其组成；
    和/或
    (b)轻链
    (i)包含与SEQ ID NO：47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SEQ ID NO：47的氨基酸序列或由其组成；或者
    (a)重链
    (i)包含与SEQ ID NO：40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SEQ ID NO：40的氨基酸序列或由其组成；
    和/或
    (b)轻链
    (i)包含与SEQ ID NO：48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SEQ ID NO：48的氨基酸序列或由其组成；或者
    (a)重链
    (i)包含与选自SEQ ID NO：41或45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：41或45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：49或51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：49或51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PD-L1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PD-L1(例如人PD-L1)，例如，以以下平衡解离常数(K D)与PD-L1结合，所述K D小于大约2nM；
    (2)本发明的抗体或其片段结合表达人PD-L1的细胞，例如，以小于或等于大约2nM的EC50；
    (3)本发明的抗体或其片段阻断PD-L1的相关活性，例如以小于或等于大约0.7nM的EC 50；
    (4)本发明的抗体或其片段提高T细胞功能，例如优于已知的抗PD-L1抗体，例如Tecentriq；
    (5)本发明的抗体或其片段提高T细胞增殖，例如在MLR中，例如优于已知的抗PD-L1抗体，例如Tecentriq；
    (6)本发明的抗体或其片段提高IFN-γ分泌，例如在MLR中，例如优于已知的抗PD-L1抗体，例如Tecentriq；
    (7)本发明的抗体或其片段提高IL-2分泌，例如在MLR中，例如优于已知的抗PD-L1抗体，例如Tecentriq；
    (8)在Zenix柱检测法中，驻留时间(RT)低于大约10分钟；
    (9)抑制PD-L1的一种或多种活性，例如，导致以下一者或多者：肿瘤浸润型淋巴细胞增加、T细胞受体介导的增殖增加、或癌细胞的免疫逃避减少；
    (10)能够诱发抗体依赖性细胞介导的细胞毒性(ADCC)；
    (11)显示与表3所列的任一抗体对PD-L1相同或相似的结合亲和力和/或特异性；
    (12)抑制(例如，竞争性抑制)表3所列的任一抗体与PD-L1的结合；
    (13)与表3所示的任一抗体结合相同或重叠的表位；
    (14)与表3所示的任一抗体竞争结合PD-L1；
    (15)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PD-L1抗体或其抗原结合片段，其中所述抗体是IgG1形式的或IgG4形式的抗体或抗原结合片段，任选地所述抗PD-L1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PD-L1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PD-L1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或嵌合抗体。
  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或多特异性抗体，优选地，所述双特异性抗体分子与PD-L1和LAG-3结合。
  13. 分离的核酸，其编码权利要求1至12中任一项的抗PD-L1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PD-L1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PD-L1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PD-L1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PD-L1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PD-L1抗体或其抗原结合片段或权利要求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PD-L1抗体或其抗原结合片段或权利要求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、其他抗体(例如抗LAG-3抗体，例如结合人的抗LAG-3抗体，优选地，抗LAG-3抗体是人源化的)、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)。
  20. 权利要求19所述的药物组合物，其中所述抗LAG-3抗体包含
    (i)如SEQ ID NO：75所示的重链可变区的3个互补决定区HCDR，和/或
    (ii)如SEQ ID NO：76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LAG-3抗体包含重链可变区(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：69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：70的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：71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：72的氨基酸序列或由所述氨基酸序列组成；LCDR2包含SEQ ID NO：73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：74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PD-L1抗体或其抗原结合片段、或权利要求17的免疫缀合物在制备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的药物的用途。
  23. 有效量的权利要求1至12中任一项的抗PD-L1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的药物组合物以及抗LAG-3抗体(例如结合人的抗LAG-3抗体，优选地，抗LAG-3抗体是人源化的)在制备药物中的用途，其中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。
  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，其中所述药物还能够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、抗感染活性剂、其它抗 体(例如抗LAG-3抗体，例如结合人的抗LAG-3抗体，优选地，抗LAG-3抗体是人源化的)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)。
  26. 权利要求23至25中任一项所述的用途，其中所述抗LAG-3抗体包含
    (i)如SEQ ID NO：75所示的重链可变区的3个互补决定区HCDR，和/或
    (ii)如SEQ ID NO：76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LAG-3抗体包含重链可变区(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：69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：70的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：71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：72的氨基酸序列或由所述氨基酸序列组成；LCDR2包含SEQ ID NO：73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：74的氨基酸序列或由所述氨基酸序列组成。
  28. 检测样品中PD-L1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PD-L1抗体或其抗原结合片段接触；和
    (b)检测抗PD-L1抗体或其抗原结合片段和PD-L1间的复合物的形成；任选地，抗PD-L1抗体是被可检测地标记的。
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BR112020007725-9A BR112020007725A2 (pt) 2017-12-27 2018-12-27 anticorpo anti-pd-l1 e usos do mesmo
CA3076447A CA3076447A1 (en) 2017-12-27 2018-12-27 Anti-pd-l1 antibody and uses thereof
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US11359021B2 (en) 2018-01-10 2022-06-14 Jiangsu Hengrui Medicine Co., Ltd. PD-L1 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
EP4056592A4 (en) * 2019-11-08 2024-03-20 Jiangsu Simcere Pharmaceutical Co., Ltd. ANTIBODY AGAINST HUMAN PROGRAMMED CELL DEATH LIGAND 1 (PD-L1) AND USE THEREOF
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