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WO2025098364A1 - 靶向人源pd-l1的全人源抗体 - Google Patents

靶向人源pd-l1的全人源抗体 Download PDF

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Publication number
WO2025098364A1
WO2025098364A1 PCT/CN2024/130101 CN2024130101W WO2025098364A1 WO 2025098364 A1 WO2025098364 A1 WO 2025098364A1 CN 2024130101 W CN2024130101 W CN 2024130101W WO 2025098364 A1 WO2025098364 A1 WO 2025098364A1
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Prior art keywords
seq
antibody
antigen
binding portion
amino acid
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French (fr)
Inventor
李应宇
辛阳
林倩文
陈黎
梁雨
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Nanjing Probio Biotech Co Ltd
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Nanjing Probio Biotech Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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 application relates to a fully human antagonistic antibody targeting human PD-L1.
  • Canceration is the result of normal somatic cells losing their normal cell regulatory functions and gene mutations accumulating to a certain extent.
  • the immune system can only launch an effective attack after recognizing cancer cells.
  • Tumor-associated antigen a cell surface protein that is highly expressed or characteristically expressed in tumor cells
  • TAA Tumor-associated antigen
  • Antibodies targeting TAA can guide NK cells or T cells to tumor cells with high TAA expression, thereby specifically killing tumor cells.
  • cancer immunotherapy drugs on the market that rely on this mechanism, such as rituximab and trastuzumab, which have achieved good clinical results.
  • cancer cells have developed many mechanisms to evade immune regulation. For example, cancer cells disguise themselves as normal somatic cells or transmit inhibitory signals to surrounding immune cells. In particular, cancer cells highly express the PD-L1 protein, which binds to PD-1 on the surface of immune cells, causing dysfunction and decline of immune cells.
  • PD-1 is a membrane receptor protein that is mainly present on the surface of immune cells, such as T cells and B cells.
  • PD-L1 is one of the ligand proteins of PD-1 and is a type I transmembrane protein of 40KDa.
  • Normal human proteins reduce the immune system's response to normal human cells by expressing PD-L1 and binding to PD-1 on immune cells, thereby maintaining a certain degree of self-tolerance.
  • Solid tumors and hemangiomas as mentioned above, often upregulate the expression of PD-L1 and use the PD-1 and PD-L1 pathways to inhibit immune cell function, thereby avoiding immune surveillance.
  • PD-L1 can also be expressed on immune cells in the tumor microenvironment (TME), including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), T cells, B cells, macrophages, bone marrow-derived mast cells, and some non-immune cells, making the TME tumor-friendly.
  • TME tumor microenvironment
  • TAMs tumor-associated macrophages
  • MDSCs myeloid-derived suppressor cells
  • DCs dendritic cells
  • T cells B cells
  • macrophages bone marrow-derived mast cells
  • non-immune cells some non-immune cells
  • PD-1 and PD-L1 inhibitors have been widely used in tumor immunotherapy, which can inhibit the binding between PD-1 and PD-L1, thereby relieving immunosuppression and activating immune cells to attack tumor cells, thus achieving anti-tumor effects.
  • These immunotherapy drugs have shown significant efficacy in the treatment of a variety of malignant tumors, including melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, head and neck squamous cell carcinoma, etc., and are widely used in clinical practice.
  • the inventors of this application have screened several fully human PD-L1 antibodies through phage display technology.
  • the amino acid sequence of fully human antibodies is 100% human, which has the advantages of lower immunogenicity and better safety.
  • the PD-L1 antibody of the present application has comparable or higher PD-1 binding ability, comparable or higher PD-L1-PD-1 blocking activity, comparable or higher safety, and/or comparable or higher in vivo anti-tumor effect compared to prior art antibodies, such as Avelumab or Tecentriq.
  • the present application relates to an isolated monoclonal antibody (e.g., a chimeric or humanized antibody), or an antigen-binding portion thereof, which can specifically bind to PD-L1 (e.g., human, monkey, or mouse PD-L1), and can comprise i) a heavy chain variable region, which can contain VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 can respectively comprise (1) GRTALTYA (SEQ ID NO: 1), INWSGSMT (SEQ ID NO: 2), and AATRTAITMPPQRDGVDY (SEQ ID NO: 3); or (2) GSSFSLAV (SEQ ID NO: 8), ISSAAGT (SEQ ID NO: 9) , and WRGAVPGDPYGR (SEQ ID NO: 10); and/or ii) a light chain variable region, which light chain variable region may contain VL CDR1, VL CDR2 and VL CDR3,
  • PD-L1
  • the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 may respectively comprise (1) GRTALTYA (SEQ ID NO: 1), INWSGSMT (SEQ ID NO: 2), AATRTAITMPPQRDGVDY (SEQ ID NO: 3), QDISNY (SEQ ID NO: 4), DAS, and QQYDNLPRT (SEQ ID NO: 5); (2) GSSFSLAV (SEQ ID NO: 8), ISSAAGT (SEQ ID NO: 9), WRGAVPGDPYGR (SEQ ID NO: 10), QSVSNNY (SEQ ID NO: 11), GAS, and QQYGNSPGT (SEQ ID NO: 12); or (3) GSSFSLAV (SEQ ID NO: 8), ISSAAGT (SEQ ID NO: 9), WRGAVPGDPYGR (SEQ ID NO:
  • the heavy chain variable region of the antibody or its antigen-binding portion of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NOs: 6, 13, or 17.
  • the light chain variable region of the antibody or its antigen-binding portion of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NOs: 7, 14, or 18.
  • the heavy chain variable region and light chain variable region of the antibody or its antigen-binding portion of the present application may respectively comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with (1) SEQ ID NOs: 6 and 7; (2) SEQ ID NOs: 13 and 14; or (3) SEQ ID NOs: 17 and 18.
  • the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 may respectively comprise the amino acid sequences of GRTALTYA (SEQ ID NO: 1), INWSGSMT (SEQ ID NO: 2), AATRTAITMPPQRDGVDY (SEQ ID NO: 3), QDISNY (SEQ ID NO: 4), DAS, and QQYDNLPRT (SEQ ID NO: 5).
  • GRTALTYA SEQ ID NO: 1
  • INWSGSMT SEQ ID NO: 2
  • AATRTAITMPPQRDGVDY SEQ ID NO: 3
  • QDISNY SEQ ID NO: 4
  • DAS and QQYDNLPRT
  • the heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6.
  • the light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:7.
  • the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 may respectively comprise the amino acid sequences of GSSFSLAV (SEQ ID NO: 8), ISSAAGT (SEQ ID NO: 9), WRGAVPGDPYGR (SEQ ID NO: 10), QSVSNNY (SEQ ID NO: 11), GAS, and QQYGNSPGT (SEQ ID NO: 12).
  • the heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13.
  • the light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14.
  • the present application also provides an isolated monoclonal antibody or an antigen-binding portion thereof, which can specifically bind to PD-L1 and can include i) a heavy chain variable region, wherein the heavy chain variable region can contain VH CDR1, VH CDR2 and VH CDR3, and ii) a light chain variable region, which light chain variable region may contain VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 may include VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 contained in the selected heavy chain variable region and light chain variable region, and the selected heavy chain variable region and light chain variable region may respectively include the amino acid sequences shown in (1) SEQ ID NOs: 6 and 7; (2) SEQ ID NOs: 13 and 14; or (3) SEQ ID NOs: 17 and 18.
  • the isolated monoclonal antibody or its antigen-binding portion thereof of the present application may comprise a heavy chain constant region and/or a light chain constant region, wherein the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.
  • the heavy chain constant region may be an IgG, IgD, IgA, IgM or IgE heavy chain constant region, preferably a heavy chain constant region that has FcR and/or complement system protein binding ability naturally or after modification, or a functional fragment thereof, such as a hinge region, CH 2 and CH 3 fragment comprising a heavy chain constant region.
  • the heavy chain constant region may be an IgG1 heavy chain constant region, such as a human IgG1 heavy chain constant region, comprising, for example, an amino acid sequence shown in Uniprot number P01857-1.
  • the light chain constant region may be a ⁇ or ⁇ light chain constant region.
  • the light chain constant region may be a human ⁇ light chain constant region, comprising, for example, an amino acid sequence shown in Uniprot number P01834.
  • the antibody of the present application comprises two heavy chains and two light chains in some embodiments, or is composed of two heavy chains and two light chains, wherein each heavy chain comprises the above-mentioned heavy chain constant region sequence, heavy chain variable region sequence and/or CDR sequence, and each light chain comprises the above-mentioned light chain constant region sequence, light chain variable region sequence and/or CDR sequence.
  • the antibody of the present application or its antigen-binding portion thereof can be Fab, F(ab') 2 fragment, Fv, scFv or (scFv) 2 , etc.
  • the antibodies or antigen-binding portions thereof of the present application can be, for example, human, or chimeric.
  • the antibodies or antigen-binding portions thereof of the present application may be antagonistic.
  • the application also provides an immunoconjugate containing the antibody or its antigen binding portion of the application, which can be connected to a therapeutic agent such as a cytotoxic molecule or an anticancer agent.
  • the application also provides a bispecific molecule containing the antibody or its antigen binding portion of the application, which can be connected to a second functional group, for example, a second antibody, and the second functional group has a binding specificity different from the antibody or its binding portion of the application.
  • the antibody or its antigen binding portion of the application can be a part of a chimeric antigen receptor (CAR) or a genetically modified T cell receptor (TCR).
  • the application also provides immune cells containing the above-mentioned CAR and/or TCR, including T cells, NK cells, etc.
  • the antibody or its antigen binding portion of the application can also be encoded by an oncolytic virus or carried by an oncolytic virus.
  • the present application also includes nucleic acid molecules encoding antibodies or antigen-binding portions thereof, immunoconjugates, bispecific molecules, CARs, or TCRs of the present application.
  • the present application may also provide an expression vector and a host cell.
  • the expression vector may contain the nucleic acid molecules of the present application.
  • the host cell may contain the expression vector of the present application, or the nucleic acid molecules of the present application may be integrated into its genome.
  • the present application also provides a method for preparing the antibody or antigen-binding portion thereof, immunoconjugate, bispecific molecule, CAR or TCR of the present application using the host cell of the present application, comprising: (i) expressing in the host cell
  • the invention relates to a method for preparing an antibody or an antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, a CAR, or a TCR, and (ii) isolating the antibody or an antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, a CAR, or a TCR from a host cell or a culture thereof.
  • the present application also provides a composition comprising the antibody or antigen-binding portion thereof, immunoconjugate, bispecific molecule, immune cell, oncolytic virus, nucleic acid molecule, expression vector or host cell of the present application.
  • the composition may be a pharmaceutical composition and may further comprise a pharmaceutically acceptable carrier.
  • the present application provides a method for treating or alleviating a PD-L1-related disease in a subject, comprising administering a therapeutically effective amount of the present application's pharmaceutical composition to the subject.
  • PD-L1-related diseases can be tumors, including solid tumors. Solid tumors include, but are not limited to, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, or head and neck squamous cell carcinoma.
  • the pharmaceutical composition of the present application can be administered together with at least one other antibody.
  • the pharmaceutical composition of the present application can be administered together with a cytokine (eg, IL-2 and/or IL-21) or a co-stimulatory antibody (eg, CD137 antibody and/or GITR antibody).
  • a cytokine eg, IL-2 and/or IL-21
  • a co-stimulatory antibody eg, CD137 antibody and/or GITR antibody
  • the antibody or antigen-binding portion thereof of the present application can be administered together with a chemotherapeutic agent, which can be a cytotoxic agent.
  • the present application also provides a method for relieving or alleviating immunosuppression in a subject, comprising administering an effective amount of the pharmaceutical composition of the present application to the subject.
  • the method is used to relieve or alleviating immunosuppression in a tumor microenvironment, comprising administering an effective amount of the pharmaceutical composition of the present application to the tumor site.
  • the present application also provides a method for enhancing an immune response in a subject, comprising administering an effective amount of the pharmaceutical composition of the present application to the subject.
  • the present application also relates to the use of the composition of the present application, in particular the pharmaceutical composition, in the preparation of a drug for treating or alleviating PD-L1-related tumors, relieving or alleviating immunosuppression, or enhancing immune response.
  • the antibody or antigen-binding portion thereof of the present application can also be used for, for example, in vitro antigen detection, etc., comprising contacting the sample to be detected with the antibody or antigen-binding portion thereof of the present application.
  • FIG1 shows the results of ELISA testing of the binding affinity between the PD-L1 antibody of the present application and the human PD-L1 protein. fruit.
  • FIG. 2 shows the results of FACS detection of the binding affinity of the PD-L1 antibody of the present application to cells expressing human PD-L1.
  • FIG3 shows the results of FACS detection of the binding ability of the PD-L1 antibody of the present application to cells expressing monkey PD-L1.
  • FIG. 4 shows the results of FACS detection of the binding affinity of the PD-L1 antibody of the present application to cells expressing mouse PD-L1.
  • FIG5 shows the detection results of the binding affinity of the PD-L1 antibodies AHP30005 (A), AHP30019 (B), and AHP31319 (C), and Avelumab (D) of the present application to human PD-L1 protein.
  • FIG6 shows the inhibitory effect of Avelumab on the binding of the PD-L1 antibody of the present application to the human PD-L1 protein.
  • FIG. 7 shows the inhibitory effect of atezolizumab (Tecentriq) on the binding of the PD-L1 antibody of the present application to the human PD-L1 protein.
  • FIG8 shows the in vitro blocking activity of the PD-L1 antibody of the present application on the interaction between PD-1 and PD-L1.
  • PD-L1 refers to programmed death receptor-ligand 1.
  • the term includes variants, homologs, orthologs, and paralogs.
  • an antibody specific for human PD-L1 may, in some cases, cross-react with a PD-L1 protein of another species, such as monkey.
  • human PD-L1 refers to a PD-L1 protein having a human amino acid sequence, such as a PD-L1 protein having an amino acid sequence of NCBI index number NP_001300958.1 (Nasr S et al., (2023) BMC Cancer 23(1): 817).
  • monkey PD-L1 refers to a PD-L1 protein having a monkey amino acid sequence, such as a PD-L1 protein having an amino acid sequence of NCBI index number XP_033093166.1.
  • antibody as used herein is intended to include IgG, IgA, IgD, IgE and IgM full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) thereof.
  • a full-length antibody is one comprising at least two heavy (H) chains and two The heavy and light chains are connected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH or VH) and a heavy chain constant region.
  • the heavy chain constant region is composed of three domains, namely CH1 , CH2 and CH3 .
  • Each light chain is composed of a light chain variable region (abbreviated as VL or VL) and a light chain constant region.
  • the light chain constant region is composed of one domain , CL .
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conservative framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs conservative framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including binding to a variety of immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • a "functional fragment" of an antibody constant region refers to a fragment that retains certain desired functions in the constant region, such as a fragment of the heavy chain constant region that retains FcR/complement system component binding activity, such as an Fc fragment.
  • antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-L1 protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody.
  • binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL , VH , CL and CH1 ; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of VH and CH1 ; (iv) a FV fragment consisting of a single-arm VL and VH of an antibody; (v) a dAb fragment consisting of VH (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity determining region (CDR); and (vii) dAb- VL , a fragment comprising a single variable domain and a heavy chain constant domain.
  • a Fab fragment a monovalent fragment consisting of VL , VH , CL and CH1 ;
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by a recombinant method via a synthetic linker that makes the two become a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule.
  • These single-chain antibodies are also intended to be included in the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.
  • isolated antibody refers to an antibody that is substantially free of other antibodies with different antigenic specificities.
  • an isolated antibody that specifically binds to a PD-L1 protein is substantially free of antibodies that specifically bind to antigens other than the PD-L1 protein.
  • an isolated antibody that specifically binds to a human PD-L1 protein may have cross-binding properties to other antigens, such as PD-L1 proteins of other species.
  • the isolated antibody is substantially free of other cellular materials and/or chemicals.
  • monoclonal antibody or “monoclonal antibody” or “monoclonal antibody composition” refers to an antibody molecule preparation of single molecular composition.
  • a monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.
  • human antibody or “fully human antibody” refers to an antibody whose variable region framework and CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from human germline immunoglobulin sequences.
  • the human antibodies of the present application may contain amino acid residues that are not encoded by human germline immunoglobulin sequences, such as mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo. However, the term “human antibody” or “fully human antibody” does not include antibodies in which CDR sequences derived from other mammalian species are inserted into human framework sequences.
  • chimeric antibody refers to an antibody that combines genetic material from one species with genetic material from another species.
  • the chimeric antibody in the present application refers to an antibody obtained by combining non-human genetic material with human genetic material.
  • an antibody that recognizes an antigen and "an antibody specific for an antigen” are used interchangeably herein with the term “an antibody that specifically binds to an antigen.”
  • Specificity determination methods include, but are not limited to, SPR, Western blotting, ELISA, RIA, ECL, IRMA testing, and peptide scanning.
  • the term "does not substantially bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the KD for binding to the protein or cell is 1.0 ⁇ 10-6 M or more, more preferably 1.0 ⁇ 10-5 M or more, more preferably 1.0 ⁇ 10-4 M or more, 1.0 ⁇ 10-3 M or more, and more preferably 1.0 ⁇ 10-2 M or more.
  • EC50 also known as half maximal effect concentration, refers to the antibody concentration that elicits 50% of the maximal effect.
  • IC50 refers to the half inhibitory concentration, which is the concentration of a drug or inhibitor required to inhibit a specified biological process by half.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows and horses are preferred.
  • therapeutically effective amount refers to an amount of the antibody of the present application sufficient to prevent or alleviate the symptoms associated with a disease or disorder (e.g., cancer).
  • the therapeutically effective amount is related to the disease being treated, and those skilled in the art can easily determine the actual effective amount.
  • Sequence identity herein refers to the percentage of nucleotides/amino acids in a sequence that are identical to the nucleotides/amino acid residues in a reference sequence after sequence alignment, and if necessary, spaces are introduced in the sequence comparison to achieve the maximum percentage of sequence identity between the two sequences.
  • Those skilled in the art can perform pairwise sequence comparisons or multiple sequence alignments to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences by a variety of methods, such as using computer software, such as ClustalOmega, T-coffee, Kalign, and MAFFT, etc.
  • the PD-L1 antibody of the present application compared with prior art antibodies such as avelumab or atezolizumab, has i) equivalent or better (human, monkey, mouse) PD-L1 binding ability and binding specificity, ii) equivalent or higher PD-L1-PD-1 blocking activity, iii) equivalent or lower immunogenicity, and thus equivalent or higher safety, and/or iv) equivalent or higher in vivo anti-tumor effect.
  • Preferred antibodies of the present application are monoclonal antibodies.
  • the antibody or antigen-binding portion thereof may be, for example, human or chimeric.
  • Exemplary antibodies, or antigen-binding portions thereof, of the present application are those whose structural and chemical properties are described below.
  • the heavy chain variable region and light chain variable region sequences or sequence numbers of the antibodies or antigen-binding portions thereof of the present application are listed in Table 1.
  • the heavy chain variable region CDR and the light chain variable region CDR are determined by the IMGT numbering system, and the CDR sequences or sequence numbers determined thereby are listed in Table 1.
  • the heavy chain variable region CDR and the light chain variable region CDR of the antibody or antigen-binding portion thereof of the present application can also be determined by the Chothia, Kabat, AbM, or Contact numbering system based on the full-length sequence of the variable region.
  • the antibody of the present application may have a heavy chain constant region, such as a natural or modified one with FcR and/or complement system protein binding ability, particularly those with high FcR and/or complement system protein binding ability.
  • the heavy chain constant region may be an IgG1 constant region, such as a human IgG1 constant region comprising an amino acid sequence as shown in Uniprot number P01857-1.
  • the light chain constant region may be a kappa constant region, such as a human kappa constant region, which may comprise an amino acid sequence as shown in Uniprot number P01834.
  • VH and/or VL sequences (or CDR sequences) of other PD-L1 antibodies that bind to human PD-L1 can be "mixed and paired" with the VH and/or VL sequences (or CDR sequences) of the antibodies of the present application.
  • VH and VL (or CDRs therein) are mixed and paired
  • the VH sequence in a specific VH / VL pairing can be replaced by a structurally similar VH sequence.
  • the VL sequence in a specific VH / VL pairing is replaced by a structurally similar VL sequence.
  • the antibody or antigen-binding portion thereof of the present application comprises:
  • the antibody or antigen-binding portion thereof of the present application comprises:
  • the antibody or antigen-binding portion thereof of the present application includes the heavy chain variable region CDR2 of a PD-L1 antibody and the CDRs of other antibodies that bind to human PD-L1, such as the heavy chain variable region CDR1 and/or CDR3, and/or the light chain variable region CDR1, CDR2 and/or CDR3 of another PD-L1 antibody.
  • the CDR3 domain independent of CDR1 and/or CDR2, can independently determine the binding specificity of an antibody to the same antigen, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on the CDR3 sequence. See, e.g., Klimka et al., British J.
  • the antibody or antigen-binding portion thereof of the present application comprises CDR2 of the heavy chain variable region of the PD-L1 antibody and at least CDR3 of the heavy chain and/or light chain variable region of the PD-L1 antibody, or CDR3 of the heavy chain and/or light chain variable region of another PD-L1 antibody, wherein the antibody or antigen-binding portion thereof can specifically bind to human PD-L1.
  • these antibodies or antigen-binding portions thereof (a) compete for binding to PD-L1; (b) retain Functional properties; (c) binding to the same epitope; and/or (d) having a binding affinity similar to that of the PD-L1 antibody or its antigen binding portion of the present application.
  • the antibody or its antigen binding portion may also include the light chain variable region CDR2 of the PD-L1 antibody or its antigen binding portion of the present application, or the light chain variable region CDR2 of another PD-L1 antibody, wherein the antibody or its antigen binding portion specifically binds to human PD-L1.
  • the antibody of the present application may include the heavy chain/light chain variable region CDR1 of the PD-L1 antibody or its antigen binding portion of the present application, or the heavy chain and/or light chain variable region CDR1 of another PD-L1 antibody, wherein the antibody or its antigen binding portion specifically binds to human PD-L1.
  • the antibody or antigen-binding portion thereof of the present application comprises a heavy chain and/or light chain variable region sequence or CDR1, CDR2 and CDR3 sequence that has one or more conservative modifications with the PD-L1 antibody or antigen-binding portion thereof of the present application. It is known in the art that some conservative sequence modifications do not eliminate antigen binding. See, for example, Brummell et al., (1993) Biochem 32: 1180-8.
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region and the light chain variable region respectively comprising CDR1, CDR2 and CDR3, wherein:
  • the heavy chain variable region CDR1 comprises the sequence listed in Table 1, and/or conservative modifications thereof; and/or
  • the heavy chain variable region CDR2 comprises the sequence listed in Table 1, and/or conservative modifications thereof; and/or
  • the heavy chain variable region CDR3 comprises the sequence listed in Table 1, and/or conservative modifications thereof; and/or
  • the light chain variable region CDR1, and/or CDR2, and/or CDR3 comprise the sequences listed in Table 1, and/or conservative modifications thereof;
  • the antibody or antigen-binding portion thereof specifically binds to human PD-L1.
  • the antibodies of the present application have one or more of the following functional characteristics, such as high affinity and high specific binding to human PD-L1, and high blocking activity for PD-L1-PD-1.
  • the antibody or antigen-binding portion thereof can be, for example, human, or chimeric.
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid replacement, addition and deletion. Modifications can be introduced into the present application antibody or its antigen-binding portion by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacement is the replacement of amino acid residues with amino acid residues having similar side chains. Amino acid residue groups with similar side chains are known in the art.
  • amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • one or more amino acid residues in the CDR region of the antibody or antigen-binding portion thereof of the present application can be replaced with other amino acid residues of the same side chain group, and the resulting antibody can be tested for retention of function (i.e., the function described above) using the functional assays described herein.
  • the antibody or antigen-binding portion thereof of the present application can be used as the PD-L1 antibody or antigen-binding portion thereof of the present application.
  • the antibody can be genetically modified by modifying one or more residues in one or both variable regions (i.e., VH and/or VL ) (e.g., in one or more CDR regions and/or one or more framework regions) to improve binding affinity and/or increase similarity to antibodies naturally produced by certain species.
  • the antibody can be genetically modified by modifying residues in the constant region, for example, to alter the effector function of the antibody.
  • Variable region modifications can be mutating amino acid residues in the VH and/or VL CDR1, CDR2 and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the target antibody.
  • Point mutations or PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated in in vitro or in vivo assays known in the art.
  • conservative modifications known in the art are introduced.
  • Mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. In addition, no more than one, two, three, four or five residues in the CDR regions are typically changed.
  • the present application provides an isolated PD-L1 monoclonal antibody or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, which comprises: (a) a VH CDR1 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (b) a VH CDR2 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (c) a VH CDR3 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (d) a VL CDR1 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (e) a VL CDR2 region comprising a sequence of the present application, or an
  • Genetically modified antibodies of the present application include those in which genetic modifications are made in the framework residues of VH and/or VL , for example to change the antibody properties.
  • the framework modification includes mutating one or more residues in the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the immunogenicity that may result from the antibody. This method is also referred to as "deimmunization", which is described in more detail in U.S. Patent Application No. 20030153043.
  • the antibody of the present application can be genetically modified to include genetic modification in the Fc region, usually to change one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and/or antibody-dependent cellular toxicity.
  • the antibody of the present application can be chemically modified (for example, one or more chemical functional groups can be added to the antibody), or modified to change its glycosylation to change one or more functional properties of the antibody.
  • the hinge region of CH1 is modified to change, for example, increase or decrease the number of cysteine residues in the hinge region. This method is further described in U.S. Pat. No. 5,677,425.
  • the cysteine residues in the hinge region of CH1 are changed to, for example, promote the assembly of heavy and light chains or increase/decrease the stability of the antibody.
  • the Fc hinge region of the antibody is mutated to increase or decrease the bioavailability of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2 - CH3 junction region of the Fc-hinge fragment, such that the antibody has reduced SpA binding relative to native Fc-hinge domain SpA binding. This approach is described in more detail in U.S. Pat. No. 6,165,745.
  • the glycosylation of the antibody is modified.
  • a deglycosylated antibody i.e., the antibody lacks glycosylation
  • Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen.
  • Such glycosylation modification can be achieved, for example, by changing one or more glycosylation sites in the antibody sequence.
  • one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that position.
  • Such deglycosylation can increase the affinity of the antibody for an antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.
  • antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucose residues, or antibodies with increased bisecting GlcNac structures.
  • Altered glycosylation forms have been shown to increase the ADCC activity of antibodies.
  • Such glycosylation modifications can be performed, for example, by expressing antibodies in host cells with altered glycosylation systems.
  • Cells with altered glycosylation systems are known in the art, including but not limited to Slc35c1 gene knockout cell lines, FUT8 knockout cell lines, variant CHO cell lines Lec13, rat fusion tumor cell lines YB2/0, cell lines containing small interfering RNA specifically for the FUT8 gene, and cell lines co-expressing ⁇ -1,4-N-acetylglucosaminyltransferase III and Golgi ⁇ -mannosidase II. They can be used as host cells for expressing the recombinant antibodies of the present application to prepare antibodies with altered glycosylation.
  • Antibodies can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody.
  • PEG polyethylene glycol
  • the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment.
  • PEGylation is performed by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer).
  • polyethylene glycol as used herein includes any form of PEG used to derivatize other proteins, such as mono (C 1 -C 10 ) alkoxy- or aryloxypolyethylene glycol or polyethylene glycol maleimide.
  • the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present application. See, for example, EPO 154 316 and EP 0 401 384.
  • the antibodies or antigen-binding portions thereof of the present application can be characterized by their various physical properties in order to detect and/or distinguish their species.
  • an antibody or its antigen binding portion may contain one or more glycosylation sites in the light chain or heavy chain variable region. These glycosylation sites may cause increased antibody immunogenicity or cause an altered antibody pK value due to altered antigen binding. Glycosylation is known to occur in motifs containing N-X-S/T sequences. In some cases, it is preferred that the PD-L1 antibody or its antigen binding portion does not contain variable region glycosylation. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region or by mutating residues in the glycosylation region.
  • the antibody or antigen binding portion thereof does not contain an asparagine isomeric site.
  • Deamidation of asparagine may occur in NG or DG sequences, creating an isoaspartic acid residue which introduces a kink into the polypeptide chain and reduces its stability (isoaspartic acid effect).
  • Each antibody or its antigen-binding portion will have a unique isoelectric point (pI), which generally falls within the pH range of 6-9.5.
  • the pI of IgG1 antibodies generally falls within the pH range of 7-9.5, while the pI of IgG4 antibodies generally falls within the pH range of 6-8. It is speculated that antibodies with pIs outside the normal range may have some unfolded structures and be unstable under in vivo conditions. Therefore, it is preferred that the pI value of the PD-L1 antibody falls within the normal range. This can be achieved by selecting antibodies with pIs within the normal range or by mutating uncharged surface residues.
  • the monoclonal antibodies of the present application can be prepared using phage display technology.
  • Phage display technology is to insert the gene of exogenous encoded polypeptide or protein (such as antibody in the form of scFv) into the appropriate position of the structural gene of phage coat protein through genetic engineering technology, and correctly express it in the reading frame, so that the exogenous polypeptide or protein (such as scFv) forms a fusion protein on the capsid protein of the phage, and is presented on the phage surface with the reassembly of the progeny phage. Then use the target molecule (such as PD-L1) and adopt a suitable panning method to wash away the phage that does not specifically bind to the target molecule.
  • the target molecule such as PD-L1
  • antibodies or antigen-binding portions thereof of the present application can also be produced in host cell transfectomas using, for example, recombinant DNA technology combined with gene transfection methods (e.g., Morrison, S. (1985) Science 229: 1202).
  • DNA encoding partial or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors so that the genes are operably linked to transcription and translation regulatory sequences.
  • the term "operably linked” means that the antibody gene is linked to the vector so that the transcription and translation control sequences within the vector exercise their established functions of regulating antibody gene transcription and translation.
  • regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control transcription or translation of antibody genes.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and/or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, such as the adenovirus major late promoter (AdMLP) and polyoma virus.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • AdMLP adenovirus major late promoter
  • non-viral regulatory sequences may be used, such as the ubiquitin promoter or the ⁇ -globin promoter.
  • regulatory elements are composed of sequences from different sources, such as the SR ⁇ promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia type I virus.
  • the expression vector and expression control sequence are selected to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors.
  • the variable region is inserted into the expression vector of the heavy chain constant region and the light chain constant region that have encoded the desired subtype and constructs the full-length antibody gene, so that VH is operably connected to the C H in the vector, and VL is operably connected to the C L in the vector.
  • the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell.
  • the antibody chain gene can be cloned into a vector, so that the signal peptide is connected to the amino terminus of the antibody chain gene in the reading frame.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (that is, a signal peptide from a non-immunoglobulin).
  • the recombinant expression vector of the present application may carry other sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell.
  • the selectable marker gene can be used to select the host cell into which the vector has been introduced.
  • the selectable marker gene usually confers drug resistance to the host cell into which the vector has been introduced, such as G418, hygromycin, or methotrexate resistance.
  • Preferred selectable marker genes include dihydrofolate reductase (DHFR) genes (for methotrexate selection/amplification of dhfr host cells) and neo genes (for G418 selection).
  • DHFR dihydrofolate reductase
  • expression vectors encoding heavy and light chains are transfected into host cells by standard techniques. Multiple forms of the term "transfection” include a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, etc.
  • transfection include a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, etc.
  • the present application antibody or its antigen-binding portion in prokaryotic or eukaryotic host cells
  • it is preferred that the antibody is expressed in eukaryotic cells, most preferably in mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded and immunologically active antibodies than prokaryotic cells.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the present application include Slc35C1 gene knockout cell lines, FUT8 knockout cell lines, variant CHO cell lines Lec13, rat hybridoma cell lines YB2/0, cell lines containing small interfering RNA specifically for the FUT8 gene, co-expressing ⁇ -1,4-N-acetylglucosaminyltransferase III and Golgi ⁇ -mannosidase II, Chinese hamster ovary (CHO cells) (including dhfr-CHO cells administered with a DHFR selectable marker), NSO myeloma cells, COS cells and SP2 cells.
  • Slc35C1 gene knockout cell lines include Slc35C1 gene knockout cell lines, FUT8 knockout cell lines, variant CHO cell lines Lec13, rat hybridoma cell lines YB2/0, cell lines containing small interfering RNA specifically for the FUT8 gene, co-
  • the antibody When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is prepared by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell, or preferably sufficient to allow the antibody to be secreted into the culture medium in which the host cell is grown.
  • the antibody or its antigen-binding portion can be recovered from the culture medium using a protein purification method.
  • the application provides nucleic acid molecules encoding the heavy chain/light chain variable region or CDR of the antibody or antigen-binding portion thereof of the application.
  • the nucleic acid can be present in whole cells, in cell lysates, or in partially purified or substantially pure form.
  • the nucleic acid is "isolated” or “substantially pure” when purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins by standard techniques.
  • the nucleic acid of the application can be, for example, DNA or RNA, and may or may not contain intron sequences.
  • the nucleic acid is a cDNA molecule.
  • the nucleic acid of the present application can be obtained using standard molecular biology techniques.
  • cDNAs encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques.
  • nucleic acids encoding such antibodies can be collected from gene banks.
  • Preferred nucleic acid molecules of the present application include those encoding the VH and VL sequences or CDRs of the PD-L1 monoclonal antibody.
  • these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VH or VL is combined with the DNA fragment encoding another protein.
  • Another DNA fragment such as an antibody constant region or a flexible linker, is operably linked.
  • the term "operably linked" means that two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments are in the reading frame.
  • the isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably connecting the VH encoding DNA to another DNA molecule encoding the heavy chain constant region ( CH1 , CH2 and CH3 ).
  • the sequences of human heavy chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgG1 constant region.
  • the DNA encoding the VH region can be operably connected to another DNA molecule encoding only the heavy chain CH1 constant region.
  • the isolated DNA encoding the VL region can be converted into a full-length light chain gene by operably connecting the VL encoding DNA to another DNA molecule encoding the light chain constant region CL .
  • the sequences of human light chain constant region genes are known in the art, and DNA fragments including these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa and lambda constant region.
  • the DNA fragments encoding VH and VL can be operably linked to another fragment encoding a flexible linker so that the VH and VL sequences can be expressed as a continuous single-chain protein, in which the VH and VL regions are connected by the flexible linker.
  • the antibody or its antigen-binding portion thereof of the present application can be coupled with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC).
  • Suitable therapeutic agents include cytotoxic molecules, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents.
  • the antibody and therapeutic agent can be cross-linked by a joint, which can be cut, such as a peptide joint, a disulfide joint or a hydrazone joint.
  • the joint is a peptide joint, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu.
  • ADCs can be prepared as described in U.S. Patents 7,087,600, 6,989,452, and 7,129,261, PCT Publications WO 02/096910, WO 07/038,658, WO 07/051,081, WO 07/059,404, WO 08/083,312, and WO 08/103,693, U.S.
  • ADCs have a basis for application only in the case of antibody internalization.
  • Internalizing antibodies can be coupled to cytotoxic molecules so that the cytotoxic molecules specifically cause damage to cells that internalize the antibodies.
  • the cytotoxic molecules can enter target cells through antibody internalization.
  • the cytotoxic molecule can be any small molecule compound or protein molecule that causes damage to target cells, such as microtubule polymerization inhibitors, DNA damaging agents, etc.
  • the present application relates to a bispecific molecule comprising an antibody of the present application or an antigen-binding portion thereof connected to at least one other functional molecule such as another peptide or protein (e.g., another antibody or receptor ligand) to generate a bispecific molecule that binds to at least two different binding sites or targeting molecules.
  • a bispecific molecule includes molecules with three or more specificities.
  • Bispecific molecules can occur in a variety of forms and sizes. At one end of the size spectrum, bispecific molecules retain The traditional antibody format is maintained, except that it has two binding arms and each arm has a different specificity, instead of having two binding arms with the same specificity. At the other extreme is a bispecific molecule composed of two single-chain antibody fragments (scFv) connected by a peptide chain, called a Bs(scFv) 2 construct. Bispecific molecules of intermediate size include two different F(ab) fragments connected by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic modification, somatic cell hybridization or chemical methods.
  • the present application also provides a chimeric antigen receptor comprising a PD-L1 single-chain antibody scFv, wherein the scFv comprises the heavy chain and light chain CDRs, or the heavy chain and light chain variable regions described in the present application.
  • the PD-L1 chimeric antigen receptor can comprise (a) an extracellular antigen binding domain containing a PD-L1 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.
  • the present application also provides an immune cell, such as a T cell or a NK cell, which comprises the chimeric antigen receptor of the present application.
  • an immune cell such as a T cell or a NK cell, which comprises the chimeric antigen receptor of the present application.
  • Oncolytic viruses preferentially infect and kill cancer cells.
  • the antibodies or antigen-binding portions thereof of the present application can be used together with oncolytic viruses.
  • oncolytic viruses encoding antibodies or antigen-binding portions thereof of the present application can be introduced into the human body.
  • the present application provides a composition comprising an antibody or an antigen-binding portion thereof, a nucleic acid molecule, an expression vector, a host cell, an immunoconjugate, a chimeric antigen receptor, an immune cell, a bispecific antibody, and/or an oncolytic virus of the present application.
  • the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.
  • the composition may optionally include one or more other pharmaceutically effective ingredients, such as another anti-tumor antibody, or an immune enhancing antibody, or a non-antibody anti-tumor agent, or an immune enhancer.
  • the composition of the present application may be used in combination with, for example, another anticancer agent or another immune enhancer.
  • the pharmaceutical composition may contain any number of excipients.
  • Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or push injection).
  • the active ingredient can be wrapped in a material to protect it from the influence of acid and other natural conditions that may inactivate it.
  • Enteral administration refers to a mode different from the intestinal tract and local external use, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, supradinaline and intrasternal injection and push injection.
  • the antibody of the present application can be administered by a non-parenteral route, such as external, epidermal or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or local external use.
  • compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes or other ordered structures suitable for high drug concentrations.
  • the amount of active ingredient that is combined with the carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. The amount varies depending on the formula and is essentially the amount of the composition that produces a therapeutic effect. In percentage terms, this amount is about 0.01 to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier.
  • the dosage regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response).
  • a bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the severity of the therapeutic situation.
  • parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • a dosage unit form refers to physically discrete units suitable for single administration to a subject to be treated; each unit contains a predetermined amount of an active ingredient calculated to produce the desired therapeutic effect together with a pharmaceutical carrier.
  • the antibody may be administered as a sustained release formulation, in which case the required frequency of administration is reduced.
  • the dosage may be about 0.001-100 mg/kg host body weight.
  • An exemplary treatment regimen involves administration once per week.
  • a “therapeutically effective amount” of the pharmaceutical composition of the present application causes a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods.
  • a “therapeutically effective amount” inhibits tumor growth by at least about 20%, at least about 40%, even at least about 60%, and more particularly at least about 80%, compared to an untreated subject.
  • a therapeutic antibody in a therapeutically effective amount can reduce tumor size, or alleviate symptoms in a subject, who can be a human or another mammal.
  • the pharmaceutical composition can be a sustained release agent, including implants, and microcapsule delivery systems.
  • Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • the pharmaceutical composition can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal delivery devices (U.S. Pat. No. 4,486,194); (4) push injection devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196).
  • needle-free subcutaneous injection devices e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556
  • the components of the compositions of the present application may be formulated to ensure appropriate in vivo distribution.
  • the antibodies may be formulated in liposomes, which may additionally contain targeting functional groups to enhance selective delivery to specific cells or organs.
  • the application also relates to in vivo gene therapy, wherein nucleic acid molecules encoding the application antibody or its antigen-binding portion thereof, immunoconjugate, or bispecific molecule, etc. are directly introduced into the subject.
  • nucleic acid sequence encoding the application antibody or antigen-binding portion thereof is introduced into the target cell via a nucleic acid construct with or without a suitable delivery vector such as an adeno-associated viral vector through local injection.
  • suitable delivery vector such as an adeno-associated viral vector through local injection.
  • Other alternative viral vectors include but are not limited to retrovirus, adenovirus, herpes simplex virus, and papilloma virus vectors.
  • the in vivo physical transfer of viral vectors can be achieved by local injection of the desired nucleic acid construct or other suitable delivery vectors containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or microparticle bombardment (gene gun).
  • compositions of the present application have various in vitro and in vitro applications, involving, for example, the treatment of cancer, or more generally, for immune enhancement in patients with diseases such as cancer.
  • the pharmaceutical compositions can be administered to human subjects, for example, to inhibit tumor growth in vivo.
  • the present application provides a method for inhibiting tumor cell growth in a subject, comprising administering the pharmaceutical composition of the present application to the subject, thereby inhibiting tumor growth in the subject.
  • tumors that can be treated by the antibody of the present application include, but are not limited to, solid tumors.
  • Solid tumors include, but are not limited to, melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, or head and neck squamous cell carcinoma, whether primary or metastatic.
  • refractory or recurrent malignant tumors may also be treated with the pharmaceutical composition of the present application.
  • the present application provides a combination therapy in which the pharmaceutical composition of the present application is administered together with one or more other antibodies or non-antibody therapeutic agents, which can effectively inhibit tumor growth in a subject.
  • the present application provides a method for inhibiting tumor growth in a subject, comprising administering the pharmaceutical composition of the present application and one or more other antibodies, such as PD-1 antibodies, to the subject.
  • the subject is a human.
  • the present application provides a method for treating cancer, wherein the pharmaceutical composition of the present application is administered together with a chemotherapeutic agent, which may be a cytotoxic agent.
  • Other therapies that may be combined with the pharmaceutical composition of the present application include, but are not limited to, administration of an immunogenic agent, administration of interleukin 2 (IL-2), radiotherapy, surgery, or hormone removal.
  • composition of the present application can also be used to relieve or alleviate immunosuppression in a subject.
  • the present application also provides a method for relieving or relieving immunosuppression in a subject, comprising administering an effective amount of the pharmaceutical composition of the present application to the subject.
  • the method is used to relieve or alleviate immunosuppression in a tumor microenvironment, comprising administering an effective amount of the pharmaceutical composition of the present application to the tumor site.
  • composition of the present application can also be used to enhance immune response.
  • the present application provides a method for enhancing immune response in a subject, comprising administering an effective amount of the pharmaceutical composition of the present application to the subject.
  • combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.
  • Example 1 Obtaining positive phage clones expressing anti-human PD-L1 antibodies
  • the natural full-human phage display library was used to target the biotin-labeled human PD-L1 protein (Biopsy, PD1-H82E5), the cell line stably expressing human PD-L1 (Nanjing Pengbo, RD00868), and The CHO-K1 cell line (Nanjing Pengbo) was subjected to three rounds of panning to obtain the phage library panning eluate.
  • the neutralized phage panning eluate was added to the prepared TG1 bacterial solution, mixed and infected at 37°C for 45 minutes. After sufficient infection, the bacterial solution was gradient diluted, spread on an agar plate with relevant resistance, and inverted at 37°C for overnight culture.
  • a 96-well sterile deep-well plate containing 0.5mL 2YT medium (with 0.2% w/v glucose and 0.1mg/mL ampicillin antibiotics) was prepared, and the monoclonal colonies cultured in the plate were picked up with a sterile pipette tip and placed in the corresponding well plate, and cultured overnight at 37°C 220rpm (16-18 hours).
  • helper phage was added, and after oscillation and mixing, the culture was allowed to stand at 37°C for infection for 45 minutes. 0.25 mL of 2YT medium (containing 0.1 mg/mL ampicillin and kanamycin with a final concentration of 0.05 mg/mL after addition) was added, and cultured overnight at 30°C with shaking at 220 rpm (16-18 hours). The overnight expressed bacterial solution was centrifuged at 4000 rpm for 10 minutes to obtain the phage display expression supernatant, which was used for indirect ELISA binding detection of human PD-L1 protein (GenScript, Z03425) and FACS binding detection of cells expressing PD-L1 to obtain positive phage clones expressing fully human antibodies.
  • 2YT medium containing 0.1 mg/mL ampicillin and kanamycin with a final concentration of 0.05 mg/mL after addition
  • the overnight expressed bacterial solution was centrifuged at 4000 rpm for 10 minutes to obtain the phage display expression supernatant, which was
  • the plate was washed three times with 0.05% PBST and incubated at room temperature for 45 minutes with 100 ⁇ l/well of goat anti-M13 phage antibody conjugated to horseradish peroxidase (Sino-Bio).
  • the plate was washed six times with 0.05% PBST, and then TMB colorimetric solution (GenScript) was added and incubated at room temperature in the dark for 10 minutes.
  • the reaction was stopped by adding 50 ⁇ l of 1 M HCl stop solution (Sigma) and the plate was read at 450 nm using a microplate reader.
  • PD-L1 + cells Piero, RD00868
  • PD-L1 - CHO-K1 cells that stably express human PD-L1 were collected and washed three times with PBS.
  • 1.0 ⁇ 10 5 PD-L1 + cells or CHO-K1 cells, 50 ⁇ l of the supernatant to be tested, and 50 ⁇ l of 3.5 ⁇ g/mL biotin-labeled anti-phage antibody were added to a 96-well plate and incubated at 4°C for 1 hour.
  • the cells were then washed three times with PBS, 100 ⁇ l of iFluor-labeled streptavidin protein (Jackson, 016-600-084) was added, and incubated at 4°C for 45 minutes. Finally, the cells were washed three times with PBS and the signal was read with FACS Calibur (BD).
  • iFluor-labeled streptavidin protein Jackson, 016-600-084
  • Example 1 According to the results of ELISA and FACS detection in Example 1, the corresponding bacterial solution of the positive clones was selected for culture, phagemid extraction, PCR and monoclonal Sanger sequencing, and finally 3 positive clones were obtained.
  • the antibody ID numbers and antibody sequences/sequence numbers corresponding to the 3 clones are shown in Table 1.
  • the codon-optimized variable region DNA fragment was synthesized and inserted into the pcDNA3.4-Fc (HuIgG1) expression vector to form an expression plasmid.
  • the above plasmids were transfected into ExpiCHO-S cells and cultured in a shake flask at 37°C for 7 days, and the supernatant was collected for antibody purification.
  • the harvested cell culture supernatant was then diluted 1:1 with 2 ⁇ the above buffer and sterilized by filtration.
  • the purified antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system with a 10% precast gel (GenScript).
  • the gel was stained with Estain 2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained bands.
  • the heavy chain of the expressed antibody comprises a heavy chain variable region and a heavy chain constant region (Uniprot No. P01857-1), and the light chain comprises a light chain variable region and a light chain constant region (Uniprot No. P01834).
  • Indirect ELISA was used to evaluate the binding ability of the purified antibodies of the present application to human PD-L1-His recombinant protein.
  • the ELISA plate was coated with 100 ⁇ l/well of 1 ⁇ g/mL recombinant human PD-L1 recombinant protein (GenScript, Z03425) in carbonate buffer (CBS Pengbo homemade) at 4°C overnight.
  • the ELISA plate was washed with PBST (containing 0.05% Tween) and blocked with PBS containing 3% skim milk at 37°C for 1 hour.
  • the blocking solution was then discarded, and 100 ⁇ l of 3-fold gradient dilutions of the purified antibody of the present application, Avelumab (Nanjing Pengbo Synthesis), or IgG1 isotype control antibody was added to the plate, with a starting concentration of 15 ⁇ g/mL (about 100 nM), for a total of 11 test concentration gradients.
  • the plate was incubated at 37°C for 1 hour, washed 3 times with 0.05% PBST, and incubated at 37°C for 0.5 hours with 100 ⁇ l/well of mouse anti-human IgG Fc fragment conjugated with horseradish peroxidase (GenScript, A01854).
  • the plate was washed six times with 0.05% PBST, then TMB colorimetric solution (GenScript) was added and incubated at room temperature in the dark for 15 min, after which 50 ⁇ l of 1 M HCl stop solution (Sigma) was added to terminate the reaction.
  • the plate was read at 450 nm using a microplate reader.
  • the ELISA results of the binding of the three antibodies of the present application to the PD-L1 protein are shown in Figure 1, and the EC 50 values of each antibody are shown in Table 2. It can be seen that compared with avelumab, the PD-L1 antibody of the present application has a higher affinity to heavy metal ions than that of avelumab. The binding abilities of the two groups of human PD-L1 antigen proteins were comparable.
  • Stable cell lines expressing human, monkey and mouse PD-L1 proteins (Pengbo, RD00868; Pengbo, M00573; Pengbo, M00567), and PD-L1 - CHO-K1 cells were collected and washed three times with PBS.
  • 1 ⁇ 10 5 test cells and three-fold gradient dilutions of purified antibodies or controls were added to a 96-well plate, with an initial antibody concentration of 45 ⁇ g/mL (about 300 nM), a total volume of 100 ⁇ l in the well, and incubated at 4°C for 1 hour.
  • the cells were then washed three times with PBS, and 100 ⁇ l of iFluor-labeled goat anti-human IgG Fc ⁇ specific fragment antibody (Jackson, 115-545-071) was added and incubated at 4°C for 45 minutes. Finally, the cells were washed three times with PBS and the signal was read using a FACS analyzer (BD Calibur).
  • iFluor-labeled goat anti-human IgG Fc ⁇ specific fragment antibody Jackson, 115-545-071
  • Figures 2 to 4 show the results of binding tests of antibodies to stable cell lines expressing human, monkey and mouse PD-L1 proteins, respectively, and the EC 50 values are shown in Table 3. It can be seen that all antibodies of the present application bind to cells expressing human, monkey and mouse PD-L1, but not to PD-L1 negative cells, indicating that the binding of these three antibodies of the present application to human, monkey and mouse PD-L1 cells is specific.
  • the surface plasmon resonance (SPR) biosensor Biacore 8K (Situofan) was used to measure the binding affinity between the antibody of the present application and the recombinant human PD-L1 protein.
  • the antibody was immobilized on the sensor chip by the Fc capture method, and the human PD-L1 protein (GenScript, Z03425) was used as the analyte.
  • the binding was measured by continuous injection of seven different concentrations of human PD-L1 (concentration range 0.78125-200 nM) in the flow cell at a flow rate of 30 ⁇ l/min for 120 seconds, followed by injection of buffer flow dissociation for 360 seconds to measure the dissociation of the antibody and antigen protein.
  • the dissociation (kd) and association (ka) rate constants were obtained using the Biacore 8K evaluation software, and the equilibrium dissociation constant (K D ) was calculated from the ratio of kd to ka.
  • Indirect ELISA was used to detect whether the binding epitope of the antibody of the present application on the human PD-L1-His protein (GenScript, Z03425) was the same as that of avelumab and atezolizumab (Nanjing Pengbo Synthesis).
  • the ELISA plate was coated with 100 ⁇ l/well of 1 ⁇ g/mL recombinant human PD-L1 (His) protein (GenScript, Z03425) in CBS (Pengbo homemade) at 4°C overnight.
  • the plate was washed with PBST (containing 0.05% Tween), and the plate was blocked with 300 ⁇ l/well PBS containing 3% skim milk at 37°C for 1 hour.
  • the blocking solution was then discarded, and 50 ⁇ l of 3-fold gradient dilutions of the present application antibody or control were added to the plate, with a starting concentration of 15 ⁇ g/mL (about 100 nM), a total of 11 test concentration gradients, and incubated at 37°C for 1 hour.
  • biotinylated avelumab or atezolizumab was added to the plate and incubated at 37°C for 1 hour.
  • the plate was washed three times with PBST and incubated with 100 ⁇ l/well of avidin antibody conjugated to horseradish peroxidase (GenScript, M00091) at 37°C for 0.5 h.
  • the plate was washed six times with PBST, then TMB colorimetric solution (GenScript) was added and incubated at room temperature in the dark for 10 min.
  • the reaction was terminated by adding 50 ⁇ l of 1 M HCl stop solution (Sigma).
  • the plate was read at 450 nm using a microplate reader.
  • Figure 6 shows the detection results after adding biotinylated avelumab
  • Figure 7 shows the detection results after adding biotinylated atezolizumab.
  • the inhibition IC 50 of avelumab and atezolizumab on the binding of each antibody to human PD-L1 is shown in Table 5.
  • avelumab can effectively inhibit the binding of AHP30005 and AHP31319 to human PD-L1 protein, while having little effect on the binding of AHP30019 to human PD-L1 protein.
  • Atezolizumab can effectively inhibit the binding of AHP30005 to human PD-L1 protein, and also affects the binding of AHP31319 to human PD-L1 protein, but has almost no effect on the binding of AHP30019 to human PD-L1 protein. This indicates that the binding epitope of AHP30005 on PD-L1 is likely to overlap with that of atezolizumab, and the binding epitope of AHP31319 on PD-L1 is also likely to overlap with that of atezolizumab, while the binding epitope of AHP30019 should not overlap with that of atezolizumab.
  • Example 7 In vitro blocking activity of monoclonal antibodies against PD-1 and PD-L1
  • GS-J2B/PD-1 cell line, Pengbo, RD00871 a cell surface protein capable of activating TCR in an antigen-independent manner
  • GS-C3/PD-L1 cell line, Pengbo, RD00703 a cell surface protein capable of activating TCR in an antigen-independent manner
  • the PD-1 and PD-L1 blocking bioassay was performed to detect the blocking effect of the antibodies of the present application on PD-1 and PD-L1 by observing cell-dependent bioluminescence.
  • PD-L1 on GS-C3/PD-L1 cells interacts with PD-1 on Jurkat cells, inhibiting TCR signal transduction of Jurkat cells and NFAT-RE-mediated luciferase activity; and after the addition of PD-L1 antibodies that block the interaction between PD-1 and PD-L1, the inhibitory signal is released, thereby causing the activation of the TCR signaling pathway and the luminescence of NFAT-RE-mediated luciferase.
  • the GS-C3/PD-L1 cells were removed from the culture medium and washed with 2 mL of DPBS. 2 mL of trypsin was added and digested at 37°C for 5 minutes. 8 mL of cell culture medium was added to terminate the digestion, the cell suspension was collected, and the cell suspension was centrifuged at 800 rpm for 5 minutes. The cells were gently resuspended with 1 mL of F12K cell culture medium (Thermo Fisher, 21127022) to prepare a single cell suspension, and the cell suspension was diluted to ⁇ 1.25 ⁇ 10 5 /mL.
  • F12K cell culture medium Thermo Fisher, 21127022
  • 40 ⁇ l of the cell suspension was transferred to a 384-well white flat-bottomed assay plate and incubated overnight (16-20 hours) in a 37°C, 5% CO 2 incubator.
  • the Jurkat cells were removed from the culture medium and washed with 2 mL of DPBS. 2 mL of trypsin was added and digested at 37°C for 5 minutes, 8 mL of cell culture medium was added to terminate the digestion, the cell suspension was collected, and the cell suspension was centrifuged at 800 rpm for 5 minutes. The cells were gently resuspended with 1000 ⁇ l of complete culture medium to prepare a single cell suspension, and the cell suspension was diluted to ⁇ 7.5 ⁇ 10 5 /mL.
  • amino acid and nucleotide sequence information mentioned in this article is as follows.

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Abstract

一种靶向人源PD-L1的全人源抗体，包括全人源的拮抗型PD-L1抗体、或其抗原结合部分。还涉及编码该抗体或其抗原结合部分的核酸分子，用于表达该抗体或其抗原结合部分的表达载体、宿主细胞和方法，以及使用该抗体或其抗原结合部分、核酸分子、表达载体和/或宿主细胞的治疗方法。

Description

靶向人源PD-L1的全人源抗体
相关申请的交叉援引
本申请要求申请号为CN 202311466254.8、申请日为2023年11月06日的中国专利申请的优先权，该申请通过引用将其全部内容并入本文。
发明领域
本申请涉及一种靶向人源PD-L1的全人源拮抗型抗体。
背景技术
癌变是正常体细胞失去正常细胞调节功能、基因突变累计到一定程度的结果。免疫系统只有在识别癌细胞后，才能进行有效的进攻。
肿瘤相关抗原(TAA)，一种在肿瘤细胞中高表达或特征性表达的细胞表面蛋白，是免疫系统识别癌细胞的突破点之一。以TAA为目标的靶向治疗是当前癌症治疗中的重要途径之一。靶向TAA的抗体，可以将NK细胞或者T细胞引导至TAA高表达肿瘤细胞，进而对肿瘤细胞进行特异性的杀伤。市场上已经有数种依赖此机制的癌症免疫治疗药物，如利妥昔单抗和曲妥珠单抗，在临床上取得了较好的疗效。
然而，靶向治疗并非在所有癌症患者中都有效果。研究表明，癌细胞发展出了很多逃避免疫监管的机制。比如，癌细胞会将自己伪装成正常的体细胞，或者向周边免疫细胞传递抑制性信号等。特别地，癌细胞会高表达PD-L1蛋白，与免疫细胞表面的PD-1结合，引起免疫细胞的功能障碍和衰退等。
PD-1是一种膜受体蛋白，主要存在于免疫细胞表面，如T细胞和B细胞等。PD-L1是PD-1的配体蛋白之一，是大小为40KDa的I型跨膜蛋白。正常人体蛋白通过表达PD-L1并与免疫细胞上的PD-1结合，从而降低免疫系统对正常人体细胞的反应，保持一定程度的自体耐受度。实体瘤和血管瘤，如上所述，往往会上调PD-L1的表达量，利用PD-1与PD-L1通路来抑制免疫细胞功能，从而避开免疫监视。PD-L1还可在肿瘤微环境(TME)的免疫细胞，包括肿瘤相关巨噬细胞(TAM)、髓源性抑制细胞(MDSC)、树突状细胞(DC)、T细胞、B细胞、巨噬细胞、骨髓来源的肥大细胞，以及一些非免疫细胞上表达，使得TME呈现肿瘤友好性。研究表明，PD-1与PD-L1通路会降低CD8+T淋巴细胞及CD4+T淋巴细胞的活性，并抑制二者的增殖，进而抑制TME中T淋巴细胞发挥作用，降低对肿瘤的免疫杀伤功能。
PD-1和PD-L1抑制剂已被广泛用于肿瘤免疫治疗，通过抑制PD-1和PD-L1之间的结合，从而解除免疫抑制，激活免疫细胞对肿瘤细胞的攻击，达到抗肿瘤的效果。这些免疫治疗药物已经在多种恶性肿瘤的治疗中显示出显著的疗效，包括黑色素瘤、非小细胞肺癌、肾细胞癌、膀胱癌、头颈部鳞状细胞癌等，并被广泛应用于临床实践中。
发明内容
本申请的发明人，通过噬菌体展示技术，筛选到了几种全人源的PD-L1抗体。全人源抗体的氨基酸序列100％来自人类，具有免疫原性更低、安全性更好的优势。
本申请的PD-L1抗体，相比于现有技术抗体，如阿维鲁单抗(Avelumab)或阿替利珠单抗(Tecentriq)，具有相当或更高的PD-1结合力、相当或更高的PD-L1-PD-1阻断活性、相当或更高的安全性、和/或相当或更高的体内抗肿瘤效果。
因而，在第一个方面，本申请涉及一种分离的单克隆抗体(例如，嵌合、或人源抗体)、或其抗原结合部分，其能够与PD-L1(例如，人、猴、小鼠PD-L1)特异结合，可以包含i)重链可变区，该重链可变区可以含有VH CDR1、VH CDR2和VH CDR3，其中，VH CDR1、VH CDR2和VH CDR3可以分别包含(1)GRTALTYA(SEQ ID NO：1)、INWSGSMT(SEQ ID NO：2)、和AATRTAITMPPQRDGVDY(SEQ ID NO：3)；或(2)GSSFSLAV(SEQ ID NO：8)、ISSAAGT(SEQ ID NO：9)、和WRGAVPGDPYGR(SEQ ID NO：10)的氨基酸序列；和/或ii)轻链可变区，该轻链可变区可以含有VL CDR1、VL CDR2和VL CDR3，其中，VL CDR1、VL CDR2和VL CDR3可以分别包含(1)QDISNY(SEQ ID NO：4)、DAS、和QQYDNLPRT(SEQ ID NO：5)；(2)QSVSNNY(SEQ ID NO：11)、GAS、和QQYGNSPGT(SEQ ID NO：12)；或(3)QSLVHSDGNTY(SEQ ID NO：15)、EVS和MQGTHWPWT(SEQ ID NO：16)的氨基酸序列。还提供上述抗体或抗原结合部分的变体，其与上述抗体或其抗原结合部分相比，在各CDR中包含最多约3个氨基酸残基取代，例如1个、2个、或3个氨基酸残基取代。
本申请的分离的单克隆抗体或其抗原结合部分可以包含重链可变区和轻链可变区，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3可以分别包含(1)GRTALTYA(SEQ ID NO：1)、INWSGSMT(SEQ ID NO：2)、AATRTAITMPPQRDGVDY(SEQ ID NO：3)、QDISNY(SEQ ID NO：4)、DAS、和QQYDNLPRT(SEQ ID NO：5)；(2)GSSFSLAV(SEQ ID NO：8)、ISSAAGT(SEQ ID NO：9)、WRGAVPGDPYGR(SEQ ID NO：10)、QSVSNNY(SEQ ID NO：11)、GAS、和QQYGNSPGT(SEQ ID NO：12)；或(3)GSSFSLAV(SEQ ID NO：8)、ISSAAGT(SEQ ID NO：9)、WRGAVPGDPYGR(SEQ ID NO：10)、QSLVHSDGNTY(SEQ ID NO：15)、EVS和MQGTHWPWT(SEQ ID NO：16) 的氨基酸序列。在一些实施方式中，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3可以分别包含GRTALTYA(SEQ ID NO：1)、INWSGSMT(SEQ ID NO：2)、AATRTAITMPPQRDGVDY(SEQ ID NO：3)、QDISNY(SEQ ID NO：4)、DAS、和QQYDNLPRT(SEQ ID NO：5)的氨基酸序列。还提供上述抗体或抗原结合部分的变体，其与上述抗体或其抗原结合部分相比，在各CDR中包含最多约3个氨基酸残基取代，例如1个、2个、或3个氨基酸残基取代。
本申请抗体或其抗原结合部分的重链可变区可以包含与SEQ ID NOs：6、13、或17具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
本申请抗体或其抗原结合部分的轻链可变区可以包含与SEQ ID NOs：7、14、或18具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
本申请抗体或其抗原结合部分的重链可变区和轻链可变区可以分别包含与(1)SEQ ID NOs：6和7；(2)SEQ ID NOs：13和14；或(3)SEQ ID NOs：17和18具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
在一些实施方式中，本申请的分离的单克隆抗体或其抗原结合部分可以包含重链可变区和轻链可变区，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3可以分别包含GRTALTYA(SEQ ID NO：1)、INWSGSMT(SEQ ID NO：2)、AATRTAITMPPQRDGVDY(SEQ ID NO：3)、QDISNY(SEQ ID NO：4)、DAS、和QQYDNLPRT(SEQ ID NO：5)的氨基酸序列。该重链可变区可以包含与SEQ ID NO：6具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。该轻链可变区可以包含与SEQ ID NO：7具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
在一些实施方式中，本申请的分离的单克隆抗体或其抗原结合部分可以包含重链可变区和轻链可变区，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3可以分别包含GSSFSLAV(SEQ ID NO：8)、ISSAAGT(SEQ ID NO：9)、WRGAVPGDPYGR(SEQ ID NO：10)、QSVSNNY(SEQ ID NO：11)、GAS、和QQYGNSPGT(SEQ ID NO：12)的氨基酸序列。该重链可变区可以包含与SEQ ID NO：13具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。该轻链可变区可以包含与SEQ ID NO：14具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
本申请还提供一种分离的单克隆抗体或其抗原结合部分，其能够与PD-L1特异结合，可以包含i)重链可变区，该重链可变区可以含有VH CDR1、VH CDR2 和VH CDR3，和ii)轻链可变区，该轻链可变区可以含有VL CDR1、VL CDR2和VL CDR3，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3可以包含选定重链可变区和轻链可变区所含的VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3，选定重链可变区和轻链可变区可以分别包含(1)SEQ ID NOs：6和7；(2)SEQ ID NOs：13和14；或(3)SEQ ID NOs：17和18所示的氨基酸序列。
本申请的分离的单克隆抗体或其抗原结合部分可以包含重链恒定区和/或轻链恒定区，其中重链恒定区的N端与重链可变区的C端连接，轻链恒定区的N端与轻链可变区的C端连接。重链恒定区可以是IgG、IgD、IgA、IgM或IgE重链恒定区，优选为天然地或经改造后具有FcR和/或补体系统蛋白结合力的重链恒定区，或其功能片段，例如包含重链恒定区的铰链区、CH2和CH3的片段。在一个实施方式中，重链恒定区可以为IgG1重链恒定区，例如人IgG1重链恒定区，包含例如Uniprot编号P01857-1所示的氨基酸序列。轻链恒定区可以为κ或λ轻链恒定区。在一些实施方式中，轻链恒定区可以为人κ轻链恒定区，包含例如Uniprot编号P01834所示的氨基酸序列。
本申请的抗体在一些实施方式中包含两条重链和两条轻链，或由两条重链和两条轻链构成，其中各重链包含上述的重链恒定区序列、重链可变区序列和/或CDR序列，且各轻链包含上述的轻链恒定区序列、轻链可变区序列和/或CDR序列。在一些实施方式中，本申请的抗体或其抗原结合部分可以为Fab、F(ab′)2片段、Fv、scFv或(scFv)2等。
本申请的抗体或其抗原结合部分可以是例如人源、或嵌合的。
本申请的抗体或其抗原结合部分可以是拮抗型的。
本申请还提供含有本申请抗体或其抗原结合部分的免疫偶联物，该抗体或其抗原结合部分可以与治疗剂例如细胞毒性分子或抗癌剂相连接。本申请还提供含有本申请抗体或其抗原结合部分的双特异性分子，该抗体或其抗原结合部分可以连接有第二功能基团，例如，第二抗体，该第二功能基团具有不同于本申请抗体或其结合部分的结合特异性。在另一方面，本申请的抗体或其抗原结合部分可以是嵌合抗原受体(CAR)或基因改造T细胞受体(TCR)的一部分。本申请还提供含有上述CAR和/或TCR的免疫细胞，包括T细胞、NK细胞等。本申请的抗体或其抗原结合部分还可以由溶瘤病毒编码或由溶瘤病毒承载。
本申请还包括编码本申请抗体或其抗原结合部分、免疫偶联物、双特异性分子、CAR、或TCR的核酸分子。本申请还可以提供一种表达载体和一种宿主细胞。表达载体可以包含本申请的核酸分子。宿主细胞可以包含本申请的表达载体，或在其基因组中整合有本申请的核酸分子。
本申请还提供使用本申请的宿主细胞来制备本申请抗体或其抗原结合部分、免疫偶联物、双特异性分子、CAR或TCR的方法，包括：(i)在宿主细胞中表达 抗体或其抗原结合部分、免疫偶联物、双特异性分子、CAR、或TCR，以及(ii)从宿主细胞或其培养物中分离抗体或其抗原结合部分、免疫偶联物、双特异性分子、CAR、或TCR。
本申请还提供一种组合物，其包含本申请的抗体或其抗原结合部分、免疫偶联物、双特异性分子、免疫细胞、溶瘤病毒、核酸分子、表达载体或宿主细胞。在一些实施方式中，该组合物可以为药物组合物，还可以包含药学上可接受的载体。
另一方面，本申请提供一种在受试者中治疗或缓解PD-L1相关疾病的方法，包括向受试者施用治疗有效量的本申请药物组合物。PD-L1相关疾病可以为肿瘤，包括实体瘤。实体瘤包括，但不限于，黑色素瘤、非小细胞肺癌、肾细胞癌、膀胱癌、或头颈部鳞状细胞癌。在一些实施方式中，本申请的药物组合物可以与至少一种其他抗体一起施用。在另一个实施方式中，本申请的药物组合物可以与细胞因子(例如IL-2和/或IL-21)或共刺激抗体(例如CD137抗体和/或GITR抗体)一起施用。在另一个实施方式中，本申请的抗体或其抗原结合部分可以与化疗剂一起施用，该化疗剂可以是细胞毒性剂。
本申请还提供一种在受试者中解除或缓解免疫抑制的方法，包括向受试者施用有效量的本申请药物组合物。在一些实施方式中，该方法用于解除或缓解肿瘤微环境中的免疫抑制，包括向肿瘤位点施用有效量的本申请药物组合物。
本申请还提供一种在受试者中增强免疫应答的方法，包括向受试者施用有效量的本申请药物组合物。
本申请也涉及本申请的组合物，特别是药物组合物，在制备一种用于治疗或缓解PD-L1相关肿瘤、解除或缓解免疫抑制、或增强免疫应答的药物中的用途。
本申请的抗体或其抗原结合部分还可以用于例如体外抗原检测等，包括使得待检测样本与本申请的抗体或其抗原结合部分接触。
应当注意的是，在本申请中，特别是在权利要求中，术语例如“包含”、“包括”等可以具有中国专利法所赋予的意义；而术语例如“基本由......组成”具有中国专利法所赋予的意义，例如允许没有明确表述的元素的存在，但将现有技术中存在的元素、或影响本发明的基本或新的特性的元素排除在外。
基于以下具体描述和实施例，当前公开内容的其他特征和优势之处将会更加明晰，具体描述和实施例不应解读为限制性的。在本申请中引用的所有文献、Genbank记录、专利和已公开专利申请的内容通过引用的方式明确地包含在本文中。
附图说明
以下以示例方式给出但不意在将本发明限制于所述具体实施方式的具体描述，可以结合附图更好地进行理解。
图1示出经ELISA检测本申请PD-L1抗体与人源PD-L1蛋白的结合力的结 果。
图2示出经FACS检测本申请PD-L1抗体与表达人源PD-L1的细胞的结合力的结果。
图3示出经FACS检测本申请PD-L1抗体与表达猴源PD-L1的细胞的结合力的结果。
图4示出经FACS检测本申请PD-L1抗体与表达小鼠源PD-L1的细胞的结合力的结果。
图5示出本申请PD-L1抗体AHP30005(A)、AHP30019(B)、和AHP31319(C)，以及阿维鲁单抗(Avelumab)(D)与人源PD-L1蛋白的结合亲和力的检测结果。
图6示出阿维鲁单抗(Avelumab)对本申请PD-L1抗体与人源PD-L1蛋白结合的抑制作用。
图7示出阿替利珠单抗(Tecentriq)对本申请PD-L1抗体与人源PD-L1蛋白结合的抑制作用。
图8示出本申请PD-L1抗体对PD-1与PD-L1相互作用的体外阻断活性。
具体实施方式
本文中用到的术语，除非特别指出，均具有字典、教科书、技术工具书中的普通含义，或本领域技术人员通常所理解的意思。以下对于一些术语的描述，仅出于便于理解本申请的目的，而不意在对这些术语进行特别的限定，除非特别指出。
如本文和所附权利要求书中所使用的，单数形式“一个”、“一种”、和“该”包括所指对象的复数形式，除非上下文另有明确规定。
术语“或”是指列举的可选择要素中的单个要素，除非上下文明确地另外指出。
术语“包含”或“包括”是指将所述的要素、整数或步骤包括在内，但是不排除任意其他要素、整数或步骤的加入。在文中，当使用术语“包含”或“包括”时，除非另有指明，也涵盖由所述及的要素、整数或步骤的组合。
术语“PD-L1”是指程序性死亡受体-配体1。该术语包括变体、同源物、直向同源物和平行同源物。例如，对人PD-L1特异的抗体可以在某些情况下与另一物种例如猴的PD-L1蛋白交叉反应。
术语“人PD-L1”是指具有人氨基酸序列的PD-L1蛋白，例如具有NCBI索引号NP_001300958.1的氨基酸序列的PD-L1蛋白(Nasr S et al.，(2023)BMC Cancer 23(1)：817)。术语“猴PD-L1”是指具有猴氨基酸序列的PD-L1蛋白，例如具有NCBI索引号XP_033093166.1的氨基酸序列的PD-L1蛋白。
本文中的术语“抗体”意在包括IgG、IgA、IgD、IgE和IgM全长抗体及其任何抗原结合片段(即，抗原结合部分)。全长抗体是包含至少两条重(H)链和两 条轻(L)链的糖蛋白，重链和轻链由二硫键连接。各重链由重链可变区(简称VH或VH)和重链恒定区构成。重链恒定区由三个结构域构成，即CH1、CH2和CH3。各轻链由轻链可变区(简称VL或VL)和轻链恒定区构成。轻链恒定区由一个结构域CL构成。VH和VL区还可以划分为称作互补决定区(CDR)的高变区，其由较为保守的骨架区(FR)区分隔开。各VH和VL由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链和轻链的可变区包含与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括与多种免疫系统细胞(例如，效应细胞)和传统补体系统的第一组分(C1q)的结合。抗体恒定区的“功能片段”是指恒定区中保留有某些所需功能的片段，例如重链恒定区中保留有FcR/补体系统组分结合活性的片段，如Fc片段。
本文中的术语，抗体的“抗原结合部分”(或简称为抗体部分)，是指抗体的保持有特异结合抗原(例如，PD-L1蛋白)能力的一个或多个片段。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施。包含在抗体的“抗原结合部分”中的结合片段的例子包括(i)Fab片段，由VL、VH、CL和CH1构成的单价片段；(ii)F(ab′)2片段，包含铰链区二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1构成的Fd片段；(iv)由抗体单臂VL和VH构成的FV片段；(v)由VH构成的dAb片段(Ward et al.，(1989)Nature 341：544-546)；(vi)分离的互补决定区(CDR)；以及(vii)dAb-VL，一种包含单可变结构域和重链恒定结构域的片段。此外，尽管Fv片段的两个结构域VL和VH由不同的基因编码，它们可以通过重组法经由使两者成为单蛋白链的合成接头而连接，其中VL和VH区配对形成单价分子。这些单链抗体也意在包括在术语涵义中。这些抗体片段可以通过本领域技术人员已知的常用技术而得到，且片段可以通过与完整抗体相同的方式进行功能筛选。
本文所用的术语“分离的抗体”是指基本不含具有不同抗原特异性的其他抗体的抗体。例如，与PD-L1蛋白特异结合的分离抗体基本不含特异结合PD-L1蛋白之外抗原的抗体。但是，特异结合人PD-L1蛋白的分离抗体可能对其他抗原例如其他物种的PD-L1蛋白具有交叉结合性。此外，分离的抗体基本不含其他细胞材料和/或化学物质。
术语“单克隆抗体”或“单抗”或“单克隆抗体组成”是指单一分子组成的抗体分子制品。单克隆抗体组成呈现出对于特定表位的单一结合特异性和亲和力。
术语“人源抗体”或“全人源抗体”是指可变区骨架和CDR区得自人种系免疫球蛋白序列的抗体。此外，如果抗体包含恒定区，其也得自人种系免疫球蛋白序列。本申请的人源抗体可以包含不由人种系免疫球蛋白序列编码的氨基酸残基，例如通过体外随机突变或点突变或通过体内体细胞突变而导入的突变。然而，术语“人源抗体”或“全人源抗体”不包括在人骨架序列中插入得自其他哺乳动物物种的CDR序列的抗体。
术语“嵌合抗体”是指组合有一个物种的遗传物质与另一物种遗传物质的抗体。特别地，本申请中的嵌合抗体是指通过组合非人源遗传物质与人源遗传物质而得来的抗体。
术语“识别抗原的抗体”以及“对抗原特异的抗体”在本文中与术语“特异结合抗原的抗体”交替使用。
在本文中，术语“特异地识别”、或“特异地结合”靶标例如人PD-L1，是指一种抗体或抗原结合片段能够区分这种靶生物分子与一种或多种参照分子，且与靶生物分子的结合亲和力或结合活性比其他参照分子高出例如1倍、5倍、10倍等。特异性测定方法包括但不限于SPR、蛋白质印迹法、ELISA、RIA、ECL、IRMA测试以及肽扫描。
术语“基本不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的KD为1.0×10-6M以上，更优选1.0×10-5M以上，更优选1.0×10-4M以上、1.0×10-3M以上，更优选1.0×10-2M以上。
术语“EC50”，又叫半最大效应浓度，是指引起50％最大效应的抗体浓度。
术语“IC50”，是指半抑制浓度，即对指定的生物过程抑制一半时所需的药物或抑制剂的浓度。
术语“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳类和非哺乳类，例如非人灵长类、羊、狗、猫、牛、马、鸡、两栖类、和爬行类，尽管优选哺乳动物，例如非人灵长类、羊、狗、猫、牛和马。
术语“治疗有效量”是指足以防止或减缓与疾病或病症(例如癌症)相关的症状的本申请抗体量。治疗有效量与被治疗的疾病相关，其中本领域技术人员可以方便地判别出实际的有效量。
本文中的“序列同一性”是指在进行序列比对后，一条序列中与参照序列中核苷酸/氨基酸残基相同的核苷酸/氨基酸百分比，如果需要的话，在序列对比中引入空格来达到两条序列间最大的序列一致性百分比。本领域技术人员可以通过多种方法，例如使用计算机软件，来进行两两序列对比或多序列比对，以确定两条或多条核酸或氨基酸序列之间的序列一致性百分比，此类计算机软件为例如ClustalOmega、T-coffee、Kalign和MAFFT等。
本申请的PD-L1抗体，与现有技术抗体如阿维鲁单抗或阿替利珠单抗相比，具有i)相当或更好的(人、猴、鼠)PD-L1结合力和结合特异性，ii)相当或更高的PD-L1-PD-1阻断活性，iii)相当或更低的免疫原性、因而相当或更高的安全性，和/或iv)相当或更高的体内抗肿瘤效果。
优选的本申请抗体是单克隆抗体。此外，抗体或其抗原结合部分可以是例如人源的、或嵌合的。
本申请的示例性抗体或其抗原结合部分是结构和化学特性在以下描述的那些。
本申请抗体或其抗原结合部分的重链可变区和轻链可变区序列或序列号列于 表1。重链可变区CDR和轻链可变区CDR通过IMGT编号系统确定，且由此确定的CDR序列或序列号列于表1中。本申请抗体或其抗原结合部分的重链可变区CDR和轻链可变区CDR，还可以基于可变区全长序列，通过Chothia、Kabat、AbM、或Contact编号系统而确定。
本申请抗体可以具有重链恒定区，例如天然的或改造的具有FcR和/或补体系统蛋白结合力，特别是高FcR和/或补体系统蛋白结合力的那些。在一些实施方式中，重链恒定区可以是IgG1恒定区，例如包含如Uniprot编号P01857-1所示氨基酸序列的人IgG1恒定区。轻链恒定区可以为κ恒定区，例如人κ恒定区，其可以包含Uniprot编号P01834所示的氨基酸序列。
与人PD-L1结合的其他PD-L1抗体的VH和/或VL序列(或CDR序列)可以与本申请抗体的VH和/或VL序列(或CDR序列)“混合并配对”。优选地，当VH和VL(或其中的CDR)混合并配对时，特定VH/VL配对中的VH序列可以由结构近似的VH序列取代。相似地，优选特定VH/VL配对中的VL序列由结构近似的VL序列取代。
因此，在一个实施方式中，本申请的抗体或其抗原结合部分包括：
(a)包含列于表1中氨基酸序列的重链可变区；以及
(b)包含列于表1中氨基酸序列的轻链可变区，或者另一PD-L1抗体的VL，其中该抗体特异结合人PD-L1。
在另一实施方式中，本申请的抗体或其抗原结合部分包括：
(a)列于表1中的重链可变区的CDR1、CDR2和CDR3；以及
(b)列于表1中的轻链可变区的CDR1、CDR2和CDR3，或者另一PD-L1抗体的CDR，其中该抗体特异结合人PD-L1。
在另一实施方式中，本申请的抗体或其抗原结合部分包括PD-L1抗体的重链可变区CDR2以及其他结合人PD-L1的抗体的CDR，例如重链可变区CDR1和/或CDR3，和/或另一PD-L1抗体的轻链可变区CDR1、CDR2和/或CDR3。
此外，领域内公知的是，CDR3结构域，独立于CDR1和/或CDR2，可单独确定抗体对同种抗原的结合特异性，且可以预测到基于该CDR3序列可生成具有相同结合特异性的多种抗体。参见，例如Klimka et al.，British J.of Cancer 83(2)：252-260(2000)；Beiboer et al.，J.Mol.Biol.296：833-849(2000)；Rader et al.，Proc.Natl.Acad.Sci.U.S.A.95：8910-8915(1998)；Barbas et al.，J.Am.Chem.Soc.116：2161-2162(1994)；Barbas et al.，Proc.Natl.Acad.Sci.U.S.A.92：2529-2533(1995)；Ditzel et al.，J.Immunol.157：739-749(1996)。
在另一实施方式中，本申请的抗体或其抗原结合部分包含PD-L1抗体的重链可变区的CDR2以及至少PD-L1抗体的重链和/或轻链可变区的CDR3，或另一PD-L1抗体的重链和/或轻链可变区的CDR3，其中该抗体或其抗原结合部分能够特异结合人PD-L1。优选这些抗体或其抗原结合部分(a)竞争结合PD-L1；(b)保留 功能特性；(c)结合相同表位；和/或(d)具有与本申请PD-L1抗体或其抗原结合部分相似的结合亲和力。在另一实施方式中，抗体或其抗原结合部分还可以包含本申请PD-L1抗体或其抗原结合部分的轻链可变区CDR2，或者另一PD-L1抗体的轻链可变区CDR2，其中该抗体或其抗原结合部分特异结合人PD-L1。在另一实施方式中，本申请的抗体可以包括本申请PD-L1抗体或其抗原结合部分的重链/轻链可变区CDR1，或另一PD-L1抗体的重链和/或轻链可变区CDR1，其中该抗体或其抗原结合部分特异结合人PD-L1。
在另一实施方式中，本申请的抗体或其抗原结合部分包含与本申请PD-L1抗体或其抗原结合部分存在一个或多个保守修饰的重链和/或轻链可变区序列或CDR1、CDR2和CDR3序列。本领域知道，一些保守序列修改不会使抗原结合性消失。参见，例如，Brummell et al.，(1993)Biochem 32：1180-8。
因此，在一个实施方式中，抗体或其抗原结合部分包含重链可变区和/或轻链可变区，重链可变区和轻链可变区分别包含CDR1、CDR2和CDR3，其中：
(a)重链可变区CDR1包含表1列出的序列，和/或其保守修改；和/或
(b)重链可变区CDR2包含表1列出的序列，和/或其保守修改；和/或
(c)重链可变区CDR3包含表1列出的序列，和/或其保守修改；和/或
(d)轻链可变区CDR1、和/或CDR2、和/或CDR3包含表1列出的序列，和/或其保守修改；且
(e)该抗体或其抗原结合部分特异结合人PD-L1。
本申请的抗体具有一个或多个以下功能特征，例如对人PD-L1的高亲和力和高特异结合性，以及对于PD-L1-PD-1的高阻断活性。
在多个实施方式中，抗体或其抗原结合部分可以是例如人源、或嵌合的。
本文所用的术语“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请抗体或其抗原结合部分中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，本申请抗体或其抗原结合部分的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换，且得到的抗体可以使用本文所述的功能检测对其进行保留功能(即，上述的功能)的测试。
本申请的抗体或其抗原结合部分可以以具备本申请PD-L1抗体或其抗原结合 部分的一个或多个VH/VL序列的抗体作为起始材料，制备成基因修饰的抗体。抗体可以通过修饰一个或两个可变区(即，VH和/或VL)内(例如，在一个或多个CDR区和/或一个或多个骨架区)的一个或多个残基来进行基因修饰，以改善结合亲和力和/或增加与某些物种天然产生的抗体的相似性。例如，或者抗体可以通过修饰恒定区中的残基进行基因修饰，例如改变抗体的效应功能。
可变区修饰可以是将VH和/或VL CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，从而改进目标抗体的一种或多种结合特性(例如，亲和力)。可以进行点突变或PCR介导的突变来引入突变，且其对于抗体结合或其他功能特性的影响可以在本领域所知的体外或体内检测中进行评价。优选地，引入本领域所知的保守修饰。突变可以是氨基酸替换、添加或缺失，但优选为替换。此外，通常改变CDR区内的不多于一个、两个、三个、四个或五个的残基。
此外，在另一实施方式中，本申请提供分离的PD-L1单克隆抗体或其抗原结合部分，包含重链可变区和轻链可变区，其包含：(a)VH CDR1区，包含本申请的序列，或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列；(b)VH CDR2区，包含本申请的序列，或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列；(c)VH CDR3区，包含本申请的序列，或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列；(d)VL CDR1区，包含本申请的序列，或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列；(e)VL CDR2区，包含本申请的序列，或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列；和(f)VL CDR3区，包含本申请的序列，或一个、两个、三个、四个或五个氨基酸替换、缺失或添加的氨基酸序列。
本申请的基因改造抗体包括在VH和/或VL的骨架残基中做出基因修饰以例如改变抗体特性的那些。骨架修饰包括对骨架区的、或者甚至一个或多个CDR区的一个或多个残基进行突变，以去除T细胞表位，从而减少抗体的可能导致的免疫原性。该方法也称为“去免疫化”，在美国专利申请20030153043中有更加详细的描述。
此外，作为骨架或CDR区内修饰之外的另一种选择，本申请的抗体可以基因改造成在Fc区包括基因修饰，通常来改变抗体的一个或多个功能特性，例如血清半衰期、补体结合、Fc受体结合、和/或抗体依赖的细胞毒性。此外，本申请的抗体可以进行化学修饰(例如，可以向抗体附加一个或多个化学功能基团)，或者修饰成改变其糖基化，来改变抗体的一个或多个功能特性。
在一个实施方式中，CH1的铰链区进行修饰，改变，例如增加或减少铰链区的半胱氨酸残基的数量。该方法在美国专利5,677,425中进一步描述。改变CH1铰链区的半胱氨酸残基，来例如促进重链轻链的组装或增加/降低抗体的稳定性。
在另一个实施方式中，对抗体的Fc铰链区进行突变，以增加或降低抗体的生 物半衰期。更加具体地，将一个或多个氨基酸突变引入Fc铰链片段的CH2-CH3连接区，从而抗体相对于天然Fc-铰链结构域SpA结合而言，具有减弱的SpA结合力。该方法在美国专利6,165,745中有更详细的描述。
在另一实施方式中，修饰抗体的糖基化。例如，可以制备去糖基化的抗体(即，抗体缺少糖基化)。可以改变糖基化，来例如增加抗体对抗原的亲和性。这样的糖化修饰可以通过例如改变抗体序列中的一个或多个糖基化位点来达成。例如，可以做出一个或多个氨基酸替换，以消除一个或多个可变区骨架糖基化位点，从而消除该位置的糖基化。这样的去糖基化可以增加抗体对抗原的亲和性。参见，例如美国专利5,714,350和6,350,861。
此外，可以制备具有改变的糖基化类型的抗体，例如岩藻糖残基量减少的低岩藻糖基抗体，或者具有增加的平分型GlcNac结构的抗体。改变的糖基化形式被证明能增加抗体的ADCC活性。这样的糖化修饰可以通过例如在糖基化系统改变的宿主细胞中表达抗体而进行。具有改变的糖基化系统的细胞在领域中已知，包括但不限于，Slc35c1基因剔除细胞系、FUT8剔除细胞系、变异CHO细胞系Lec13、大鼠融合瘤细胞系YB2/0、包含特异性地针对FUT8基因的小干扰RNA的细胞系、共表达β-1,4-N-乙酰基葡糖胺基转移酶III和高尔基体α-甘露糖苷酶II的细胞系。它们可以用作表达本申请重组抗体的宿主细胞，以制备具有改变的糖基化的抗体
本文抗体的另一修饰是聚乙二醇化(PEG化)。抗体可以PEG化，例如来增加抗体的生物(例如，血清)半衰期。为使抗体PEG化，抗体或其片段通常与聚乙二醇(PEG)，例如PEG的反应性酯或醛类衍生物，在使一个或多个PEG基团附于抗体或抗体片段的条件下反应。优选地，PEG化通过与反应性PEG分子(或类似的有反应性的水溶性聚合物)的酰化反应或烷化反应进行。本文中所用的术语“聚乙二醇”包括任何形式的用于衍生其他蛋白的PEG，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。在某些实施方式中，需要PEG化的抗体是去糖基化的抗体。PEG化蛋白的方法在领域内已知，且可以应用到本申请的抗体。参见，例如EPO 154 316和EP 0 401 384。
本申请的抗体或其抗原结合部分可以用它们的多种物理特性进行表征，以检测和/或区别其分类。
例如，抗体或其抗原结合部分可以在轻链或重链可变区包含一个或多个糖基化位点。这些糖基化位点可能引起增加的抗体免疫原性，或由于改变的抗原结合而引起改变的抗体pK值。糖基化已知发生在含有N-X-S/T序列的基序中。在一些情况下，优选PD-L1抗体或其抗原结合部分不包含可变区糖基化。这可以通过选择不在可变区包含糖基化基序的抗体或通过突变糖基化区域的残基来实现。
在优选实施方式中，抗体或其抗原结合部分不包含天冬酰胺异构位点。天冬酰胺的脱酰胺可能出现在N-G或D-G序列，创建出异天冬氨酸残基，其向多肽链中引入扭结并降低其稳定性(异天冬氨酸效果)。
各抗体或其抗原结合部分将具有独特的等电点(pI)，基本落在6-9.5的pH范围内。IgG1抗体的pI通常落在7-9.5的pH范围内，而IgG4抗体的pI基本落在6-8的pH范围内。推测pI在正常范围外的抗体可能在体内条件下具有一些展开结构且不稳定。因此，优选PD-L1抗体的pI值落在正常范围内。这可以通过选择pI在正常范围内的抗体或通过突变不带电的表面残基来实现。
本申请的单克隆抗体可以使用噬菌体展示技术进行制备。噬菌体展示技术是将外源编码多肽或蛋白(例如scFv形式的抗体)的基因通过基因工程技术插入到噬菌体外壳蛋白结构基因的适当位置，在阅读框中正确表达，使外源多肽或蛋白(例如scFv)在噬菌体的衣壳蛋白上形成融合蛋白，随子代噬菌体的重新组装呈现在噬菌体表面。然后利用靶分子(例如PD-L1)，采用合适的淘洗方法，洗去未特异性结合靶分子的噬菌体。再用酸碱或者竞争的分子洗脱下结合的噬菌体，中和后的噬菌体感染大肠杆菌扩增，经过3-5轮的富集，逐步提高可以特异性识别靶分子的噬菌体比例，最终获得识别靶分子的多肽或者蛋白。
制备单克隆抗体的其他方法包括体细胞杂交(杂交瘤)、B淋巴细胞的病毒或致癌性转化等。制备嵌合抗体的方式也在领域内熟知。本申请的抗体或其抗原结合部分还可以使用例如重组DNA技术结合基因转染方法，在宿主细胞转染瘤中生成(例如Morrison，S.(1985)Science 229：1202)。在一个实施方式中，将由标准分子生物技术得到的编码部分或全长轻链和重链的DNA插入一个或多个表达载体中，从而基因与转录和翻译调控序列可操作地连接。在该情况下，术语“可操作地连接”是指抗体基因连接到载体中，从而载体内的转录和翻译控制序列行使它们既定的调控抗体基因转录和翻译的功能。
术语“调控序列”包括控制抗体基因转录或翻译的启动子、增强子和其他表达控制元件(例如，多腺苷酸化信号)。优选的用于哺乳动物宿主细胞表达的调控序列包括引导在哺乳动物细胞中的高水平蛋白表达的病毒元件，例如得自巨细胞病毒(CMV)、猿猴病毒40(SV40)、腺病毒的启动子和/或增强子，如腺病毒主要晚期启动子(AdMLP)和多瘤病毒。或者，可以使用非病毒调控序列，例如泛素启动子或β-珠蛋白启动子。另外，调控元件由不同来源的序列构成，例如SRα启动子系统，其包含来自SV40早期启动子的序列和人T细胞白血病I型病毒的长末端重复。表达载体和表达控制序列选为与所使用的表达宿主细胞相容。
抗体轻链基因和抗体重链基因可以插入到同一或不同的表达载体中。在优选实施方式中，可变区通过插入到已经编码所需亚型的重链恒定区和轻链恒定区的表达载体中而构建全长抗体基因，从而VH与载体中的CH可操作地连接，VL与载体中的CL可操作地连接。或者，重组表达载体可以编码促进抗体链从宿主细胞分泌的信号肽。抗体链基因可以克隆到载体中，从而信号肽在阅读框内连接到抗体链基因的氨基端。信号肽可以是免疫球蛋白信号肽或异源信号肽(即，来自非免疫球蛋白的信号肽)。
除抗体链基因和调控序列外，本申请的重组表达载体可以携带其他序列，例如调控载体在宿主细胞中复制的序列(例如，复制起始点)和可选择标记物基因。可选择标记物基因可用于选择已导入载体的宿主细胞。例如，通常可选择标记物基因赋予已导入载体的宿主细胞以药物抗性，例如G418、潮霉素、或氨甲喋呤抗性。优选的可选择标记物基因包括二氢叶酸还原酶(DHFR)基因(用于dhfr宿主细胞的氨甲喋呤选择/扩增)和neo基因(用于G418选择)。
对于轻链和重链的表达，编码重链和轻链的表达载体通过标准技术转染到宿主细胞中。多个形式的术语“转染”包括多种常用于将外源DNA导入原核或真核宿主细胞的技术，例如，电穿孔、磷酸钙沉淀、DEAE-右旋糖转染等。尽管在原核或真核宿主细胞中表达本申请抗体或其抗原结合部分在理论上是可行的，优选抗体在真核细胞中表达，最优选在哺乳动物宿主细胞中表达，因为真核细胞，特别是哺乳动物细胞，比原核细胞更可能组装并分泌适当折叠且有免疫活性的抗体。
优选的用于表达本申请重组抗体的哺乳动物宿主细胞包括Slc35C1基因剔除细胞系、FUT8剔除细胞系、变异CHO细胞系Lec13、大鼠融合瘤细胞系YB2/0、包含特异性地针对FUT8基因的小干扰RNA的细胞系、共表达β-1,4-N-乙酰基葡糖胺基转移酶III和高尔基体α-甘露糖苷酶II、中国仓鼠卵巢(CHO细胞)(包括与DHFR可选择标记物一起施用的dhfr-CHO细胞)、NSO骨髓瘤细胞、COS细胞和SP2细胞。当编码抗体基因的重组表达载体导入哺乳动物宿主细胞时，通过将宿主细胞培养足以使得宿主细胞中抗体表达、或优选地足以使得使抗体分泌到宿主细胞生长的培养基中的一段时间，从而制备抗体。抗体或其抗原结合部分可以使用蛋白纯化方法从培养基中回收。
在另一方面，本申请提供编码本申请抗体或其抗原结合部分的重链/轻链可变区或CDR的核酸分子。核酸可以存在整细胞中，在细胞裂解液中，或处于部分纯化或基本纯的形式。当通过标准技术从其他细胞组分或其他污染物例如其他细胞核酸或蛋白中纯化出来后，核酸是“分离的”或“基本纯的”。本申请的核酸可以为例如DNA或RNA，且可能包含或可能不包含内含子序列。在优选实施方式中，核酸是cDNA分子。
本申请的核酸可以使用标准的分子生物学技术获得。对于由杂交瘤(例如，由携带人免疫球蛋白基因的转基因小鼠制备的杂交瘤)表达的抗体，编码杂交瘤制备的抗体的轻链和重链的cDNA可以通过标准PCR扩增或cDNA克隆技术获得。对于(例如使用噬菌体展示技术)从免疫球蛋白基因库获得的抗体，编码这类抗体的核酸可以从基因库中收集。
优选的本申请核酸分子包括编码PD-L1单克隆抗体的VH和VL序列或CDR的那些。一旦获得了编码VH和VL的DNA片段，这些DNA片段可以进一步通过标准的重组DNA技术进行操作，例如将可变区基因转变为全长抗体链基因、Fab片段基因或scFv基因。在这些操作中，编码VH或VL的DNA片段与编码另一蛋 白的另一DNA片段，例如抗体恒定区或柔性接头，可操作地连接。术语“可操作地连接”是指两个DNA片段连接在一起，从而两个DNA片段编码的氨基酸序列都在阅读框内。
编码VH区的分离DNA可以通过可操作地连接VH编码DNA与编码重链恒定区(CHl、CH2和CH3)的另一DNA分子而转变成全长重链基因。人重链恒定区基因的序列在领域内已知，且包括这些区域的DNA片段可以通过标准PCR扩增而获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是优选为IgG1恒定区。对于Fab片段重链基因，编码VH区的DNA可以可操作地与仅编码重链CH1恒定区的另一DNA分子连接。
编码VL区的分离DNA可以通过可操作地连接VL编码DNA与编码轻链恒定区CL的另一DNA分子而转变成全长轻链基因。人轻链恒定区基因的序列在领域内已知，且包括这些区域的DNA片段可以通过标准PCR扩增而获得。在优选实施方式中，轻链恒定区可以是κ和λ恒定区。
为创建scFv基因，编码VH和VL的DNA片段可以可操作地与编码柔性接头的另一片段连接，从而VH和VL序列可以作为连续的单链蛋白进行表达，其中VH和VL区域通过该柔性接头连接。
本申请的抗体或其抗原结合部分可以与治疗剂偶联，形成免疫偶联物(immunoconjugate)，例如抗体-药物偶联物(ADC)。合适的治疗剂包括细胞毒性分子、烷化剂、DNA小沟结合分子、DNA嵌入剂、DNA交联剂、组蛋白去乙酰化酶抑制剂、核输出抑制剂、蛋白酶体抑制剂、拓扑异构酶I或II的抑制剂、热激蛋白抑制剂、酪氨酸激酶抑制剂、抗生素和抗有丝分裂剂。在ADC中，抗体和治疗剂可以通过接头交联，该接头可切割，例如肽类接头、二硫类接头或腙类接头。更优选地，接头是肽类接头，例如Val-Cit、Ala-Val、Val-Ala-Val、Lys-Lys、Ala-Asn-Val、Val-Leu-Lys、Ala-Ala-Asn、Cit-Cit、Val-Lys、Lys、Cit、Ser或Glu。ADC可以如美国专利7,087,600、6,989,452、和7,129,261，PCT公开WO 02/096910、WO 07/038,658、WO 07/051,081、WO 07/059,404、WO 08/083,312、和WO 08/103,693，美国专利公开20060024317、20060004081、和20060247295中描述般进行制备。在抗体内化的情况下，ADC才有应用基础。内化型抗体可以与细胞毒性分子偶联，使得细胞毒性分子特定地对内化抗体的细胞造成损伤。特别地，细胞毒性分子可以经抗体内化而进入靶细胞中。细胞毒性分子可以是任何对靶细胞产生损害的小分子化合物或蛋白分子，例如微管蛋白聚合抑制剂、DNA损伤剂等。
另一方面，本申请涉及包含与至少一个其他功能分子如另一种肽或蛋白(例如，另一抗体或受体配体)相连接的本申请抗体或其抗原结合部分的双特异性分子，以生成与至少两个不同结合位点或靶向分子结合的双特异性分子。术语“双特异性分子”包括具有三种或更多种特异性的分子。
双特异性分子可以以多种形式和尺寸出现。在尺寸谱的一端，双特异性分子保 持传统抗体形式，除其具有两个结合臂且各臂具有不同特异性外，替代具有两个特异性相同的结合臂的情况。在另一极端的是双特异性分子，由两个经肽链连接的单链抗体片段(scFv)构成，称为Bs(scFv)2构建体。中间尺寸的双特异性分子包括由肽类接头连接的两个不同的F(ab)片段。这些和其他形式的双特异性分子可以通过基因改造、体细胞杂交或化学法进行制备。
本申请还提供包含PD-L1单链抗体scFv的嵌合抗原受体，该scFv包含本申请中所述的重链和轻链CDR、或重链和轻链可变区。
PD-L1嵌合抗原受体可以包含(a)含有PD-L1 scFv的胞外抗原结合域；(b)跨膜结构域；和(c)胞内信号转导结构域。
本申请还提供一种免疫细胞，如T细胞或NK细胞，其包含有本申请的嵌合抗原受体。
溶瘤病毒偏好地侵染并杀灭癌细胞。本申请的抗体或其抗原结合部分可以与溶瘤病毒一起使用。此外，编码本申请抗体或其抗原结合部分的溶瘤病毒可以引入人体中。
在另一方面，本申请提供一种组合物，其包含本申请的抗体或其抗原结合部分、核酸分子、表达载体、宿主细胞、免疫偶联物、嵌合抗原受体、免疫细胞、双特异抗体、和/或溶瘤病毒。在一些实施方式中，组合物为药物组合物，还包含药学上可接受的载体。组合物可以任选地包含一种或多种其他药学上的有效成分，例如另一抗肿瘤抗体、或免疫增强抗体，或者非抗体类抗肿瘤剂、或免疫增强剂。本申请的组合物可以与例如另一抗癌剂、或另一免疫增强剂联合使用。
药学组合物可以包含任何数量的赋形剂。可以使用的赋形剂包括载体、表面活性剂、增稠或乳化剂、固体粘合剂、分散或混悬剂、增溶剂、染色剂、矫味剂、涂层、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂及其组合。合适赋形剂的选择和使用在Gennaro，ed.，Remington：The Science and Practice of Pharmacy，20th Ed.(Lippincott Williams & Wilkins 2003)中有教导。
优选地，药物组合物适合于静脉内、肌内、皮下、肠道外、脊柱或表皮施用(例如通过注射或推注)。基于施用途径的不同，有效成分可以包在材料中，以保护其不受酸和可能使其失活的其他自然条件的影响。“肠道外施用”是指不同于肠道和局部外用的方式，通常通过注射进行，包括但不限于静脉内、肌内、动脉内、膜内、囊内、眶内、心脏内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬脑膜上和胸骨内注射和推注。或者，本申请的抗体可以通过非肠道外路径施用，例如外用、表皮施用或粘膜施用，例如鼻内、经口、阴道、直肠、舌下、或局部外用。
药物组合物可以为无菌水溶液或分散液的形式。它们也可以配制在微乳剂、脂质体或其他适于高浓度药物的有序结构中。
与载体材料一起制备成单剂型的有效成分的量将随着治疗主体和特定施用模 式而变，且基本上而言是产生疗效的组合物的量。以百分比计，该量为约0.01-约99％的与药学上可接受载体结合的有效成分。
给药方案经调整提供最佳的所需反应(例如，治疗反应)。例如，可以施用快速灌注剂，可以随时间推移施用多个分剂量，或者剂量可以随治疗情况的危急程度成比例降低或提高。特别有利的是，以方便施用和剂量均匀的剂量单位型配置肠道外组合物。剂量单位型是指物理上分开的单位，适于治疗主体的单次给药；各单位包含计算出来与药学载体一起产生所需疗效的预定量的有效成分。或者，抗体可以以缓释剂施用，这种情况下所需的施用频率降低。
对于抗体的施用，剂量可以为约0.001-100mg/kg宿主体重。示例性的治疗方案涉及每周施用一次。
“治疗有效量”的本申请药物组合物引起疾病症状严重程度的降低、无症状期频率和持续时间的增加。例如，对于带瘤受试者的治疗，“治疗有效量”，与未治疗受试者相比，将肿瘤生长抑制至少约20％、抑制至少约40％，甚至抑制至少约60％，且更特别地抑制至少约80％。治疗有效量的治疗抗体可以减小肿瘤尺寸，或者减轻受试者的症状，受试者可以是人或另一哺乳动物。
药物组合物可以是缓释试剂，包括植入体、和微胶囊递送系统。可以使用生物可降解、生物相容的聚合物，例如乙烯-醋酸乙烯、聚酸酐、聚乙醇酸、胶原蛋白、聚原酸酯、和聚乳酸。参见，例如，Sustained and Controlled Release Drug Delivery Systems，J.R.Robinson，ed.，Marcel Dekker,Inc.，New York，1978。
药学组合物可以经医学设备来给药，例如(1)无针皮下注射设备(例如，美国专利5,399,163；5,383,851；5,312,335；5,064,413；4,941,880；4,790,824；和4,596,556)；(2)微量输液泵(美国专利4,487,603)；(3)经皮给药设备(美国专利4,486,194)；(4)推注设备(美国专利4,447,233和4,447,224)；和(5)渗透设备(美国专利4,439,196和4,475,196)。
在某些实施方式中，本申请的组合物中的组分可以经配制，以确保合适的体内分布。例如，为确保本申请的治疗抗体或其抗原结合部分穿越血脑屏障，抗体可以配制在脂质体中，其还可以额外地包含靶向功能基团，以增强对特定细胞或器官的选择性输送。
本申请还涉及体内基因疗法，其中将编码本申请抗体或其抗原结合部分、免疫偶联物、或双特异性分子等的核酸分子直接引入受试者中。例如，将编码本申请抗体或抗原结合部分的核酸序列经由带有或不带有合适递送载体例如腺相关病毒载体的核酸构建体经局部注射而引入目标细胞。其他可供选择的病毒载体包括但不限于逆转录病毒、腺病毒、单纯疱疹病毒、和乳头状瘤病毒载体。病毒载体的体内物理转移可以通过所需核酸构建体或包含所需核酸序列的其他合适递送载体的局部注射、脂质体介导的转移、直接注射(裸露的DNA)、或微粒轰击(基因枪)而实现。
本申请的药物组合物具有多种体外和外内应用，涉及例如癌症的治疗，或者更笼统地讲，用于癌症等疾病患者的免疫增强。药物组合物可以施用至人受试者，以例如体内抑制肿瘤生长。
考虑到本申请药物组合物的抑制肿瘤细胞增殖和存活的能力，本申请提供抑制受试者中肿瘤细胞生长的方法，包括向该受试者施用本申请的药物组合物，从而在该受试者中肿瘤生长被抑制。可以由本申请抗体治疗的肿瘤的非限制性例子包括但不限于实体瘤。实体瘤包括，但不限于，黑色素瘤、非小细胞肺癌、肾细胞癌、膀胱癌、或头颈部鳞状细胞癌，不管是原发还是转移的。此外，难治的或复发的恶性肿瘤也可能可以用本申请的药物组合物进行治疗。
本申请提供本申请药物组合物与一种或多种其他抗体或非抗体类治疗剂一起施用的联合疗法，其能有效抑制受试者中的肿瘤生长。在一个实施方式中，本申请提供在受试者中抑制肿瘤生长的方法，包括向受试者施用本申请药物组合物以及一种或多种其他抗体，例如PD-1抗体。在某些实施方式中，受试者是人。在另一个方面，本申请提供癌症治疗方法，其中本申请的药物组合物与化疗剂一起施用，化疗剂可以是细胞毒性剂。其他可以与本申请药物组合物联合的疗法包括但不限于免疫原性剂施用、白介素2(IL-2)施用、放疗、手术或激素去除。
本申请的组合物还可以用于在受试者中解除或缓解免疫抑制。特别地，本申请还提供一种在受试者中解除或缓解免疫抑制的方法，包括向受试者施用有效量的本申请药物组合物。在一些实施方式中，该方法用于解除或缓解肿瘤微环境中的免疫抑制，包括向肿瘤位点施用有效量的本申请药物组合物。
本申请的组合物还可以用于增强免疫应答。具体地，本申请提供一种在受试者中增强免疫应答的方法，包括向受试者施用有效量的本申请药物组合物。
本文讨论的治疗剂的组合可以作为在药学可接受载体中的单一组合物同时施用，或者作为分开的组合物同时施用，其中各药剂处于药学可接受载体中。在另一个实施方式中，治疗剂的组合可以按序施用。
此外，如果进行多次联合疗法施用，且药剂按序施用，则在各时间点的按序施用的次序可以反转或保持相同，按序施用可以与同时施用或其任何组合相结合。
本申请的各方面和实施方式将参照附图和以下实施例进行讨论。其他方面和实施方式对于本领域技术人员是清楚的。在本文中描述的所有文献通过引用的方式全部并入本文。尽管本申请已经结合示例性实施方式进行了描述，很多等同修改和变化在给出本申请时对于本领域技术人员是清楚的。因而，本申请的示例性实施方式是示例性的，非限制性的。可以对所述实施方式做出多种变化，而不脱离本申请的宗旨和范围。
实施例1.表达抗人源PD-L1抗体的阳性噬菌体克隆的获得
将天然全人噬菌体展示文库，针对带有生物素标记的人源PD-L1蛋白(百普赛斯，PD1-H82E5)、稳定表达人PD-L1的细胞系(南京蓬勃，RD00868)、和 CHO-K1细胞系(南京蓬勃)进行三轮淘选，获得噬菌体文库淘选洗脱液。将中和后的噬菌体淘选洗脱液加入准备好的TG1菌液中，混匀后37℃静置侵染TG1宿主菌45分钟。充分侵染后，将菌液梯度稀释，涂布在带有相关抗性的琼脂平板上，倒置于37℃过夜培养。次日，准备分装有0.5mL2YT培养基(带有0.2％w/v的葡萄糖，及0.1mg/mL氨苄抗生素)的96孔无菌深孔板，用无菌枪头挑取平板中培养的单克隆菌落于对应孔板中，37℃ 220rpm振荡过夜培养(16-18小时)。次日取0.05-0.1mL过夜培养的单克隆菌液转接于新准备的分装有0.40-0.45mL含有终浓度0.1mg/mL氨苄抗生素的2YT培养基的无菌深孔板中，培养至OD600值约为0.6-0.8，加入一定比例的辅助噬菌体，振荡混匀后于37℃静置侵染45分钟，补加0.25mL 2YT培养基(含有0.1mg/mL氨苄抗生素，及加入后终浓度为0.05mg/mL的卡那霉素)，并于30℃ 220rpm振荡过夜培养(16-18小时)。将过夜表达的菌液于4000rpm离心10分钟，获得噬菌体展示表达上清，用于针对人源PD-L1蛋白(金斯瑞，Z03425)的间接ELISA结合检测及针对表达PD-L1的细胞的FACS结合检测，以获得表达全人源抗体的阳性噬菌体克隆。
具体地，在间接ELISA中，向ELISA酶标板加入100μl/孔的于碳酸盐缓冲液(CBS)包被试剂中的1μg/mL重组人源PD-L1-His蛋白(金斯瑞，Z03425)，4℃下包被过夜。用PBST(含0.05％吐温)洗涤板，并将板用300μl/孔的含3％脱脂牛奶的PBS于37℃封闭1小时。随后弃去封闭液，向板加入50μl噬菌体表达上清液及50μl 0.05％PBST，室温下孵育2小时。将板用0.05％PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗M13噬菌体抗体(义翘)室温孵育45分钟。将板用0.05％PBST洗涤六次，然后加入TMB显色液(金斯瑞)于室温避光孵育10分钟。通过加入50μl的1M HCl终止液(西格玛)终止反应，使用酶标仪在450nm下读板。
在FACS中，收集上述稳定表达人源PD-L1的PD-L1+细胞(蓬勃，RD00868)和PD-L1-CHO-K1细胞，用PBS清洗3次。在96孔板中依次加入1.0×105个PD-L1+细胞或CHO-K1细胞、50μl待测上清液、及50μl 3.5μg/mL带有生物素标记的抗噬菌体抗体，4℃孵育1小时。随后用PBS清洗细胞3次，添加100μl iFluor标记的链霉亲和素蛋白(杰克森，016-600-084)，于4℃孵育45分钟。最后用PBS清洗细胞3次，用FACS Calibur(BD)读取信号。
实施例2.单克隆PD-L1抗体的可变区测序及抗体的重组生产
根据实施例1中ELISA和FACS检测的结果，选择阳性克隆对应菌液进行培养、噬菌粒抽提、PCR及单克隆Sanger测序，最终获得3个阳性克隆。3个克隆对应的抗体ID号和抗体序列/序列号如表1所示。
表1.阳性克隆及其对应抗体的序列号

合成密码子优化后的可变区DNA片段，将其插入pcDNA3.4-Fc(HuIgG1)表达载体中，形成表达质粒。
将上述质粒转染ExpiCHO-S细胞，并于37℃摇瓶中培养7天后，收取上清用于抗体纯化。纯化之前，将管道和蛋白A柱用0.2M NaOH去热原，并将柱用含有0.05M Tris和1.5M NaCl(pH＝8.0)的缓冲液重新平衡。随后将收获的细胞培养物上清液，使用2×上述缓冲液1：1稀释并过滤除菌。将过滤的上清液和蛋白A柱室温孵育2小时，用并1×上述缓冲液洗涤柱后，使用无菌0.1M柠檬酸钠(pH3.5)洗脱抗体，将收集的洗脱液用九分之一体积的无菌1M Tris-HCl(pH＝9.0)中和。在无菌条件下，将产品缓冲液通过透析更换为PBS(pH7.4)，以除去任何的洗脱缓冲液，并对得到的样品进行浓缩。浓缩之后，使用1.43的消光系数Ec(0.1％)通过OD280nm对抗体进行定量。
纯化的抗体通过BioRad电泳系统用10％预制胶(金斯瑞)通过SDS-PAGE来分析。凝胶用Estain2.0(金斯瑞)染色并通过比较染色带估计分子大小和纯度。
表达出来的抗体的重链包含重链可变区和重链恒定区(Uniprot编号P01857-1)，轻链包含轻链可变区和轻链恒定区(Uniprot编号P01834)。
实施例3.单克隆抗体对人源PD-L1重组蛋白的结合力
使用间接ELISA来评估本申请的纯化抗体对于人源PD-L1-His重组蛋白的结合能力。
具体地，将ELISA板用100μl/孔的于碳酸盐缓冲液(CBS蓬勃自制)中的1μg/mL重组人源PD-L1重组蛋白(金斯瑞，Z03425)4℃包被过夜。用PBST(含0.05％吐温)洗涤ELISA板，并用含3％脱脂牛奶的PBS在37℃封闭1小时。随后弃去封闭液，向板加入100μl 3倍梯度稀释的本申请纯化抗体、阿维鲁单抗(南京蓬勃合成)、或IgG1同型对照抗体，起始浓度为15μg/mL(约100nM)，共计11个测试浓度梯度。板在37℃下孵育1小时，用0.05％PBST洗涤3次，并用100μl/孔的缀合辣根过氧化物酶的小鼠抗人IgG Fc片段(金斯瑞，A01854)37℃孵育0.5小时。将板用0.05％PBST洗涤六次，然后加入TMB显色液(金斯瑞)，于室温避光孵育15分钟，之后加入50μl的1M HCl终止液(西格玛)，以终止反应。使用酶标仪在450nm下读板。
本申请3种抗体与PD-L1蛋白结合的ELISA实验结果展示在图1中，各抗体的EC50值如表2所示。可以看出，与阿维鲁单抗相比，本申请的PD-L1抗体与重 组人源PD-L1抗原蛋白的结合能力相当。
表2.PD-L1抗体与PD-L1蛋白的结合力检测
实施例4.单克隆抗体与表达人源、猴源和小鼠源PD-L1的细胞的结合活性
分别收集表达人源、猴源和小鼠源PD-L1蛋白的稳定细胞系(蓬勃，RD00868；蓬勃，M00573；蓬勃，M00567)、以及PD-L1-CHO-K1细胞，用PBS清洗3次。在96孔板中加入1×105个检测细胞和三倍梯度稀释的纯化抗体或对照，抗体起始浓度为45μg/mL(约300nM)，孔内总体积为100μl，4℃孵育1小时。随后用PBS清洗细胞3次，添加100μl iFluor标记的山羊抗人IgG Fcγ特异片段抗体(杰克森，115-545-071)，4℃孵育45分钟。最后用PBS清洗细胞3次，用FACS分析仪(BD Calibur)读取信号。
图2-图4分别示出抗体与表达人源、猴源和小鼠源PD-L1蛋白的稳定细胞系的结合测试结果，EC50值示于表3。可以看出，本申请的所有抗体均与表达人源、猴源和小鼠源PD-L1的细胞结合，而不与PD-L1阴性细胞结合，这表明本申请的这3种抗体与人源、猴源和鼠源PD-L1细胞的结合是特异性的。
表3.全人源抗体与PD-L1相关细胞的FACS EC50检测
实施例5.单克隆抗体与人源PD-L1蛋白的SPR结合检测
使用表面等离子共振(SPR)生物传感器Biacore 8K(思拓凡)，测定本申请抗体与重组人源PD-L1蛋白的结合亲和力。
具体地，通过Fc捕获法将抗体固定在传感器芯片上，人源PD-L1蛋白(金斯瑞，Z03425)用作分析物。在流动池中以30μl/min的流速连续注射120秒七种不同浓度的人PD-L1(浓度范围为0.78125～200nM)来测量结合，后续注射缓冲液流解离360秒，测量抗体和抗原蛋白的解离情况。使用Biacore 8K评估软件获得解离(kd)和缔合(ka)速率常数的数据，平衡解离常数(KD)由kd与ka的比率计算得到。
SPR亲和力测定结果见表4，相关传感图见图5(A-D)。可以看出，本申请的抗体以及阿维鲁单抗与人源PD-L1抗原蛋白有着相当的结合亲和力水平，特别是AHP30005，平衡解离常数(KD)在10-10范围内。
表4.全人源抗体与人源PD-L1蛋白的亲和力测定
实施例6.单克隆抗体的人源PD-L1蛋白结合表位
使用间接ELISA来检测本申请抗体在人源PD-L1-His蛋白(金斯瑞，Z03425)上的结合表位与阿维鲁单抗以及阿替利珠单抗(南京蓬勃合成)的结合表位是否相同。
具体地，将ELISA板用100μl/孔的于CBS(蓬勃自制)中的1μg/mL重组人源PD-L1(His)蛋白(金斯瑞，Z03425)4℃包被过夜。用PBST(含0.05％吐温)洗涤板，并将板用300μl/孔含3％脱脂牛奶的PBS在37℃封闭1小时。随后弃去封闭液，向板加入50μl 3倍梯度稀释的本申请抗体或对照，起始浓度为15μg/mL(约100nM)，共计11个测试浓度梯度，在37℃孵育1小时。然后向板加入50μl 0.02μg/mL生物素化的阿维鲁单抗或阿替利珠单抗，在37℃孵育1小时。将板用PBST洗涤3次，并用100μl/孔的缀合辣根过氧化物酶的亲合素抗体(金斯瑞，M00091)37℃孵育0.5小时。将板用PBST洗涤六次，然后加入TMB显色液(金斯瑞)，于室温避光孵育10分钟。通过加入50μl的1M HCl终止液(西格玛)终止反应。使用酶标仪在450nm下读板。
图6示出加入生物素化阿维鲁单抗后的检测结果，图7示出加入生物素化阿替利珠单抗后的检测结果。阿维鲁单抗和阿替利珠单抗对各抗体与人源PD-L1的结合的抑制IC50如表5所示。
表5.全人源抗体与人源PD-L1蛋白的结合表位检测

由结果可以看出，阿维鲁单抗能够高效地抑制AHP30005以及AHP31319与人源PD-L1蛋白的结合，而对AHP30019与人源PD-L1蛋白的结合的影响很小。这表明，AHP30005以及AHP31319在PD-L1上的结合表位很可能与阿维鲁单抗有重叠，而AHP30019的结合表位很可能与阿维鲁单抗不重叠或几乎不重叠。
此外，阿替利珠单抗能够高效地抑制AHP30005与人源PD-L1蛋白的结合，对AHP31319与人源PD-L1蛋白的结合也有影响，而对AHP30019与人源PD-L1蛋白的结合几乎没有影响。这表明，AHP30005在PD-L1上的结合表位很可能与阿替利珠单抗重叠，AHP31319在PD-L1上的结合表位也很可能与阿替利珠单抗有重叠，而AHP30019的结合表位应当与阿替利珠单抗不重叠。
实施例7.单克隆抗体对PD-1与PD-L1的体外阻断活性
使用表达人PD-1蛋白和NFAT-RE诱导的荧光素酶报告基因的Jurkat细胞(GS-J2B/PD-1细胞系，蓬勃，RD00871)、以及表达人PD-L1和能够以非抗原依赖性方式激活TCR的细胞表面蛋白的CHO-K1细胞(GS-C3/PD-L1细胞系，蓬勃，RD00703)，通过PD-1与PD-L1阻断生物测定，经观察细胞依赖性生物发光，来检测本申请抗体对PD-1与PD-L1的阻断作用。其中，当上述Jurkat细胞与GS-C3/PD-L1细胞共培养时，GS-C3/PD-L1细胞上的PD-L1与Jurkat细胞上的PD-1相互作用，抑制Jurkat细胞的TCR信号转导以及NFAT-RE介导的荧光素酶活性；而加入阻断PD-1与PD-L1相互作用的PD-L1抗体后，会解除抑制性信号，从而引起TCR信号通路的激活和NFAT-RE介导的荧光素酶的发光。
具体地，GS-C3/PD-L1细胞去除培养基，用2mL DPBS洗涤。加入2mL胰酶，37℃消化5分钟。加入8mL细胞培养基终止消化，收集细胞悬液，800rpm离心5分钟。用1mL F12K细胞培养基(赛默飞，21127022)轻轻重悬细胞，制成单细胞悬液，细胞悬液稀释至～1.25×105/mL。将40μl细胞悬液转移到384孔白色平底检测板中，37℃、5％CO2培养箱中孵育过夜(16-20小时)。Jurkat细胞去除培养基，用2mL DPBS洗涤。加入2mL胰酶，37℃消化5分钟，加入8mL细胞培养基终止消化，收集细胞悬液，800rpm离心5分钟。用1000μl完全培养基轻轻重悬细胞，制成单细胞悬液，细胞悬液稀释至～7.5×105/mL。从培养箱中取出含有GS-C3/PD-L1细胞的384孔检测板，将测定板倒置在水槽上以去除培养基，然后将倒置的板子放在纸巾上5-10秒以吸干剩余的培养基。向含有GS-C3/PD-L1细胞的384孔检测板加入20μl三倍梯度稀释的本申请抗体或对照，抗体起始浓度 为170nM，以及20μl Jurkat细胞悬液，37℃、5％CO2中孵育6小时。加入40μl Bio-Lite荧光素酶检定体系试剂，用Pherastar酶标仪读数。
结果显示在图8中。可以看出，本申请的抗体全都能够以浓度依赖性的方式抑制PD-1与PD-L1的相互作用，其中AHP30005抗体的抑制效果最佳，优于阿替利珠单抗和阿维鲁单抗，AHP31319的抑制效果优于阿维鲁单抗。
本文中提及的氨基酸和核苷酸序列信息如下。
①AHP30005全人源抗体
重链可变区VH CDR1
重链可变区VH CDR2
重链可变区VH CDR3
重链可变区
轻链可变区VL CDR1
轻链可变区VL CDR2
轻链可变区VL CDR3
轻链可变区
②AHP31319全人源抗体
重链可变区VH CDR1
重链可变区VH CDR2
重链可变区VH CDR3
重链可变区

轻链可变区VL CDR1
轻链可变区VL CDR2
轻链可变区VL CDR3
轻链可变区
③AHP30019全人源抗体
重链可变区VH CDR1
重链可变区VH CDR2
重链可变区VH CDR3
重链可变区
轻链可变区VL CDR1
轻链可变区VL CDR2
轻链可变区VL CDR3
轻链可变区
尽管本申请已经结合一个或多个实施方式进行了描述，应当理解的是，本申请并不受限于这些实施方式。本申请中的描述意在涵盖所有变体形式以及等同物，均包含在所附权利要求的主旨和范围内。所有在本文中引用的文献通过引用的方式全部并入本文。

Claims (25)

  1. 一种分离的单克隆抗体或其抗原结合部分，其能够与PD-L1特异性结合，包含：
    i)重链可变区，其包含VH CDR1、VH CDR2和VH CDR3，其中VH CDR1、VH CDR2和VH CDR3分别包含(1)GRTALTYA(SEQ ID NO：1)、INWSGSMT(SEQ ID NO：2)、和AATRTAITMPPQRDGVDY(SEQ ID NO：3)；或(2)GSSFSLAV(SEQ ID NO：8)、ISSAAGT(SEQ ID NO：9)、和WRGAVPGDPYGR(SEQ ID NO：10)的氨基酸序列，或包含与上述氨基酸序列相比在各CDR具有1-3个氨基酸替换的氨基酸序列；和/或
    ii)轻链可变区，其包含VL CDR1、VL CDR2和VL CDR3，其中VL CDR1、VL CDR2和VL CDR3分别包含(1)QDISNY(SEQ ID NO：4)、DAS、和QQYDNLPRT(SEQ ID NO：5)；(2)QSVSNNY(SEQ ID NO：11)、GAS、和QQYGNSPGT(SEQ ID NO：12)；或(3)QSLVHSDGNTY(SEQ ID NO：15)、EVS和MQGTHWPWT(SEQ ID NO：16)的氨基酸序列，或包含与上述氨基酸序列相比在各CDR具有1-3个氨基酸替换的氨基酸序列。
  2. 根据权利要求1所述的分离的单克隆抗体或其抗原结合部分，包含重链可变区和轻链可变区，其中VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3分别包含(1)GRTALTYA(SEQ ID NO：1)、INWSGSMT(SEQ ID NO：2)、AATRTAITMPPQRDGVDY(SEQ ID NO：3)、QDISNY(SEQ ID NO：4)、DAS、和QQYDNLPRT(SEQ ID NO：5)；(2)GSSFSLAV(SEQ ID NO：8)、ISSAAGT(SEQ ID NO：9)、WRGAVPGDPYGR(SEQ ID NO：10)、QSVSNNY(SEQ ID NO：11)、GAS、和QQYGNSPGT(SEQ ID NO：12)；或(3)GSSFSLAV(SEQ ID NO：8)、ISSAAGT(SEQ ID NO：9)、WRGAVPGDPYGR(SEQ ID NO：10)、QSLVHSDGNTY(SEQ ID NO：15)、EVS和MQGTHWPWT(SEQ ID NO：16)的氨基酸序列。
  3. 根据权利要求1或2所述的分离的单克隆抗体或其抗原结合部分，其中重链可变区包含与SEQ ID NOs：6、13、或17具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的分离的单克隆抗体或其抗原结合部分，其中轻链可变区包含与SEQ ID NOs：7、14、或18具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
  5. 根据权利要求2-4中任一项所述的分离的单克隆抗体或其抗原结合部分，其中重链可变区和轻链可变区分别包含与(1)SEQ ID NOs：6和7；(2)SEQ ID NOs：13和14；或(3)SEQ ID NOs：17和18具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的分离的单克隆抗体或其抗原结合部分，还包含重链恒定区和/或轻链恒定区。
  7. 根据权利要求6所述的分离的单克隆抗体或其抗原结合部分，其中重链恒定区为IgG1重链恒定区。
  8. 根据权利要求7所述的分离的单克隆抗体或其抗原结合部分，其中重链可变区为包含如Uniprot编号P01857-1所示氨基酸序列的人IgG1重链恒定区。
  9. 根据权利要求6所述的分离的单克隆抗体或其抗原结合部分，其中轻链恒定区为κ轻链恒定区。
  10. 根据权利要求9所述的分离的单克隆抗体或其抗原结合部分，其中轻链恒定区为包含Uniprot编号P01834所示氨基酸序列的人κ轻链恒定区。
  11. 根据权利要求1-10中任一项所述的分离的单克隆抗体或其抗原结合部分，其为人源、或嵌合的。
  12. 根据权利要求1-11中任一项所述的分离的单克隆抗体或其抗原结合部分，其i)能够与人PD-L1结合，ii)能够与猴PD-L1结合，iii)能够与小鼠PD-L1结合，iv)能够阻断PD-1与PD-L1结合，和/或v)具有体内抗肿瘤效果。
  13. 一种免疫偶联物，其包含i)权利要求1-12中任一项所述的抗体或其抗原结合部分，和ii)细胞毒性分子，其中i)与ii)经接头连接或直接连接。
  14. 一种双特异性分子，其包含i)权利要求1-12中任一项所述的抗体或其抗原结合部分，和ii)第二功能基团，其中i)和ii)与不同抗原或同一抗原的不同表位结合，且i)和ii)相连接。
  15. 一种嵌合抗原受体，其包含i)胞外抗原结合域，ii)跨膜结构域，和iii) 胞内信号转导结构域，其中胞外抗原结合域包含权利要求1-12中任一项所述的抗体或其抗原结合部分。
  16. 一种免疫细胞，包含权利要求15所述的嵌合抗原受体。
  17. 一种溶瘤病毒，其表达权利要求1-12中任一项所述的抗体或其抗原结合部分。
  18. 一种核酸分子，其编码权利要求1-12中任一项所述的抗体或其抗原结合部分、权利要求13所述的免疫偶联物、权利要求14所述的双特异性分子或权利要求15所述的嵌合抗原受体。
  19. 一种表达载体，其包含权利要求18所述的核酸分子。
  20. 一种宿主细胞，其包含权利要求19所述的表达载体，或在其基因组内整合有权利要求18所述的核酸分子。
  21. 一种组合物，其包含权利要求1-12中任一项所述的抗体或其抗原结合部分、权利要求13所述的免疫偶联物、权利要求14所述的双特异性分子、权利要求15所述的嵌合抗原受体、权利要求16所述的免疫细胞、权利要求17所述的溶瘤病毒、权利要求18所述的核酸分子、权利要求19所述的表达载体或权利要求20所述的宿主细胞。
  22. 根据权利要求21所述的组合物，其为药物组合物，还包含药学上可接受的载体。
  23. 权利要求21或22所述的组合物在制备一种用于治疗或缓解PD-L1相关癌症的药物中的用途。
  24. 根据权利要求23所述的用途，其中PD-L1相关癌症为实体瘤。
  25. 根据权利要求24所述的用途，其中实体瘤为黑色素瘤、非小细胞肺癌、肾细胞癌、膀胱癌、或头颈部鳞状细胞癌。
PCT/CN2024/130101 2023-11-06 2024-11-06 靶向人源pd-l1的全人源抗体 Pending WO2025098364A1 (zh)

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