WO2022228431A1 - 抗pd-l1单域抗体及其用途 - Google Patents

抗pd-l1单域抗体及其用途 Download PDF

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WO2022228431A1
WO2022228431A1 PCT/CN2022/089289 CN2022089289W WO2022228431A1 WO 2022228431 A1 WO2022228431 A1 WO 2022228431A1 CN 2022089289 W CN2022089289 W CN 2022089289W WO 2022228431 A1 WO2022228431 A1 WO 2022228431A1
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antibody
seq
antigen
amino acid
binding fragment
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PCT/CN2022/089289
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French (fr)
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周岳华
刘洪川
李理
张静
刘丹丹
冯辉
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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Definitions

  • the present invention provides single-domain antibodies or antigen-binding fragments thereof that specifically bind to PD-L1 and compositions comprising the same. Also provided are nucleic acid molecules encoding the antibodies of the invention or antigen-binding fragments thereof, vectors and host cells for expressing the antibodies of the invention or antigen-binding fragments thereof, and therapeutic and diagnostic methods and uses of the antibodies of the invention or antigen-binding fragments thereof.
  • PD-L1 Programmed death-ligand 1
  • CD274 cluster of differentiation 274
  • B7 homologous protein 1 B7homolog1, B7-H1
  • the tumor necrosis factor superfamily is a type I transmembrane glycoprotein composed of 290 amino acid residues, including an IgV-like domain, an IgC-like domain, a transmembrane hydrophobic domain, and an intracellular tail of 30 amino acids.
  • the complete molecular weight is 40kDa1.
  • PD-L1 mRNA is expressed in almost all tissues, but PD-L1 protein is only continuously expressed in a small number of tissues, including liver, lung, tonsil, and immune amnesty tissues such as eye and placenta.
  • PD-L1 is also expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, etc. 2 .
  • the receptor of PD-L1 is PD-1, which is mainly expressed on the surface of immune cells such as CD4+ T cells, CD8+ T cells, NKT cells, B cells and activated monocytes.
  • the combination of PD-L1 and PD-1 can initiate the phosphorylation of tyrosine residues in ITIM (immunoreceptor tyrosine inhibition module) in the cytoplasmic region of PD-1, which promotes the binding of tyrosine phospholipase to SHP2 and activates SHP2.
  • ITIM immunomunoreceptor tyrosine inhibition module
  • This combination can further inhibit the metabolism of T cells, inhibit the secretion of anti-apoptotic protein Bcl-X2, reduce the secretion of effector cytokines IL-2, IFN-r, induce T cell exhaustion and apoptosis, thereby reducing immune T cells Involved in the immune response, exercise negative regulatory function 4.
  • T cells recognize antigen and secrete IFN-r after activation.
  • T-cell-derived IFN-r will expand and maintain T-cell functions, such as up-regulating MHC molecules, enhancing antigen processing and presentation of target cells, and promoting T-cell differentiation.
  • IFN-r also induces PD-L1 expression in immune-inflamed tissue, preventing excessive immune damage to the tissue.
  • IFN-r can induce the expression of PD-L1 on the surface of conventional epithelial cells, vascular endothelial cells, myeloid cells, naive T cells and other cells5,6.
  • IFN-r-induced interferon regulatory factor 1 can also bind to the interferon regulatory factor binding sites at the first 200bp and 320bp of the PD-L1 transcription start site to regulate PD-L17 from the transcriptional level.
  • PD-L1 can bind to PD-1 on the surface of T cells to perform a negative regulatory function, thereby protecting the inflammatory site.
  • single domain antibody is cloned from the heavy chain variable region (VHH) of camelid natural heavy chain antibody.
  • VHH heavy chain variable region
  • Single-domain antibodies have excellent biological properties.
  • the molecular weight is 12-15KD, which is one-tenth of that of complete antibodies. While overcoming the disadvantages of large molecular weight of natural antibodies, they also have complete antigen-binding sites and have good tissue penetration. Permeability, high specificity, good water solubility. Because of its special structural properties, it combines the advantages of traditional antibodies and small molecule drugs, and almost perfectly overcomes the shortcomings of traditional antibodies, such as long development cycle, low stability, and harsh storage conditions. Therefore, there is an urgent need in the art to develop antibodies that can bind to PD-L1 and block the binding of PD-L1/PD-1, especially anti-PD-L1 single-domain antibodies.
  • the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, which has the advantages of high affinity and high specificity against human PD-L1.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof provided by the present invention can be used as stand-alone therapy or in combination with other therapies/or other anti-cancer agents, such as in the treatment of cancer.
  • the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region VH, wherein the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 2 and SEQ ID NO: 3 or consisting of; HCDR1, HCDR2 and HCDR3 with 2 or 3 amino acid differences.
  • the HCDR2 described herein with 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO:2 is SEQ ID NO:26.
  • the amino acid sequence of FR1 of the heavy chain variable region comprises or consists of FR1 of SEQ ID NO: 11 or 12, or comprises the same sequence as SEQ ID NO: 11 or 12.
  • the FR1 region of NO: 11 or 12 has or consists of FR1 with 1, 2 or 3 amino acid differences;
  • the amino acid sequence of FR2 of the heavy chain variable region comprises SEQ ID NO: 5, 10, 11, 14, 20 or FR2 of 24 or consisting of or comprising or consisting of a FR2 having 1, 2 or 3 amino acid differences from FR2 of SEQ ID NO: 5, 10, 11, 14, 20 or 24;
  • the heavy chain may
  • the amino acid sequence of the FR3 of the variable region comprises or consists of the FR3 of SEQ ID NO: 6, 7, 8, 11, 13 or 17, or comprises 1, 2 or 3 amino acid differences from the FR3 of SEQ ID NO: 11 FR3 or consists of;
  • the amino acid sequence of FR4 of the heavy chain variable region comprises or consists of FR4 of SEQ ID NO:11, or comprises 1, 2 or 3 amino acid differences from FR4 of SEQ ID NO:11 FR4 or consist of it.
  • the amino acid sequence of FR1 of the heavy chain variable region comprises or consists of FR1 of SEQ ID NO: 11 or 12, or comprises the same sequence as SEQ ID NO: 11 or 12.
  • the FR1 region of NO: 11 or 12 has or consists of FR1 with 1, 2 or 3 amino acid differences;
  • the amino acid sequence of FR2 of the heavy chain variable region comprises the FR2 of SEQ ID NO: 11, 14, 20 or 24 or consist of, or comprise or consist of, a FR2 having 1, 2 or 3 amino acid differences from FR2 of SEQ ID NO: 11, 14, 20 or 24;
  • the amino acid sequence of FR3 of the heavy chain variable region comprises FR3 of SEQ ID NO: 11 or consisting of, or comprising or consisting of, FR3 having 1, 2 or 3 amino acid differences from FR3 of SEQ ID NO: 11;
  • the amino acid sequence of FR4 of the heavy chain variable region Comprising or consisting of FR4 of SEQ ID NO:11, or comprising or consisting of FR4 having 1, 2 or 3 amino acid differences from FR4 of SEQ ID NO:11.
  • the amino acid sequence of FR1 of the heavy chain variable region comprises or consists of FR1 of SEQ ID NO: 11 or 12, or comprises the same sequence as SEQ ID NO: 11 or 12.
  • the FR1 region of NO: 11 or 12 has or consists of FR1 with 1, 2 or 3 amino acid differences;
  • the amino acid sequence of FR2 of the heavy chain variable region comprises the FR2 of SEQ ID NO: 11, 20 or 24 or consists of It consists of, or comprises or consists of a FR2 having 1, 2 or 3 amino acid differences with FR2 of SEQ ID NO: 11, 14, 20 or 24;
  • the amino acid sequence of FR3 of the heavy chain variable region comprises SEQ ID FR3 of NO: 11 or consisting of, or comprising or consisting of, FR3 having 1, 2 or 3 amino acid differences from FR3 of SEQ ID NO: 11;
  • the amino acid sequence of FR4 of the heavy chain variable region comprising SEQ ID NO: 11 FR4 of ID NO: 11 consists of, or comprises or consists of, FR4 having 1, 2 or 3 amino acid differences from FR4 of SEQ ID NO: 11.
  • the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively HCDR1, HCDR2 and HCDR3.
  • the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 26 and SEQ ID NO: 3, respectively HCDR1, HCDR2 and HCDR3.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH
  • the heavy chain variable region comprises a variable region selected from the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, respectively , 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25.
  • the amino acid sequence of is an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH, the heavy chain variable region comprises as SEQ ID NO: 4, 5, 6, 7, 8, The amino acid sequence shown in or consisting of any one of 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25.
  • the antibody or antigen-binding fragment thereof of the present invention comprises an Fc region which is an Fc region of human IgGl, IgG2, IgG3 or IgG4; preferably the Fc region of human IgGl.
  • the antibody or antigen-binding fragment thereof of the invention wherein the antibody comprises a heavy chain comprising, for example, SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, and 34
  • the antibody or antigen-binding fragment thereof of the invention wherein the antibody comprises a heavy chain comprising the group consisting of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33 and or consisting of the amino acid sequence shown in any one of 34.
  • the antibody or antigen-binding fragment thereof of the present invention wherein the antibody is a single domain antibody or a heavy chain antibody.
  • the antibody or antigen-binding fragment thereof of the present invention has one or more of the following properties:
  • the antibody or antigen-binding fragment thereof of the present invention wherein the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the antibody or antigen-binding fragment thereof of the present invention wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, Fv, scFv or sdAb.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention is of any IgG subtype, such as IgG1, IgG2, IgG3, or IgG4.
  • the invention also provides a multispecific antibody comprising the heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
  • the present invention also provides a single chain antibody comprising the heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
  • the present invention provides isolated nucleic acid molecules encoding the anti-PD-L1 antibodies or antigen-binding fragments thereof described herein.
  • the present invention provides a vector comprising the nucleic acid molecule described herein; preferably, the vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising a nucleic acid molecule described herein or a vector described herein, or expressing or containing an antibody or antigen-binding fragment thereof described herein; preferably, the host cell are eukaryotic cells, more preferably mammalian cells.
  • the present invention provides a method of making an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein, the method comprising culturing the antibody or antigen-binding fragment thereof as described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof described host cell and recover the expressed antibody or antigen-binding fragment thereof from said host cell.
  • the present invention also provides immunoconjugates comprising the antibodies or antigen-binding fragments thereof described herein and other agents, eg, therapeutic or diagnostic agents.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein or an immunoconjugate as described herein, and a pharmaceutically acceptable carrier or excipient agent.
  • the present invention provides a combination product comprising an antibody or antigen-binding fragment thereof as described herein, an immunoconjugate as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agent.
  • the present invention provides an antibody or antigen-binding fragment thereof, an immunoconjugate as described herein, a pharmaceutical composition as described herein, or a combination as described herein in the manufacture of a therapeutic and Use in a medicament for the prevention of a disease or condition mediated by PD-L1, preferably the disease or condition is cancer, more preferably the cancer is selected from those with elevated expression of PD-1, PD-L1 or PD-L2 cancer.
  • the present invention provides an antibody or antigen-binding fragment thereof, an immunoconjugate as described herein, a pharmaceutical composition as described herein, or a combination product as described herein for use in therapy and /or preventing a PD-L1 mediated disease or disorder, preferably the disease or disorder is cancer, more preferably the cancer is selected from cancers with elevated PD-1, PD-L1 or PD-L2 expression.
  • the present invention provides a method of treating and/or preventing a PD-L1 mediated disease or disorder, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody as described herein, or The antigen-binding fragment, the immunoconjugate described herein, the pharmaceutical composition described herein, or the combination product described herein, preferably the disease or disorder is cancer, more preferably the cancer is selected from the group consisting of PD-1, PD - Cancers with elevated L1 or PD-L2 expression.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, an immunoconjugate as described herein, a pharmaceutical composition as described herein, or a combination product as described herein.
  • the present invention provides a method for detecting the presence of PD-L1 in a sample using the antibody or antigen-binding fragment thereof of any of the embodiments herein, the method comprising causing any one of claims 1-9 to The antibody or its antigen-binding fragment or the immunoconjugate containing the antibody or its antigen-binding fragment and a diagnostic agent is contacted with the sample to be tested, and the presence of the antibody or its antigen-binding fragment and the PD-L1 binding is detected. and optionally the step of determining the amount of the conjugate.
  • Figure 1 FACS assay to detect the binding of chimeric antibody chC1 to human PD-L1 on the surface of MAB-BA-231 cells.
  • Figure 2 Specificity analysis of the chimeric antibody chC1.
  • A ELISA method to detect the binding specificity of chC1 to various recombinant proteins;
  • B FACS method to detect the binding specificity of chC1 to various cell lines.
  • Figure 3 Detection of humanized PD-L1 single domain antibody binding to PD-L1 by ELISA.
  • Figure 4 Detection of humanized PD-L1 single-domain antibody blocking the binding of PD-L1 to PD-1 by ELISA.
  • Figure 5 Inhibitory effect of humanized single domain antibody on MC38B7H1 tumor growth.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” should be construed as inclusive, that is, including at least one of the number or list of elements, but also including more than one, and optionally , additional unlisted items. Only under terms expressly stated to the contrary, such as “only one” or “indeed one” or when “consisting of” is used in a claim, will refer to only one of the listed numbers or one element of the list.
  • the terms "programmed cell death 1 ligand 1", “PD-L1", “B7 homolog 1 (B7-H1)”, “PD-L1 antigen”, “PDCD1 ligand 1” and “CD274" are interchangeable Used interchangeably, includes variants, isoforms, species homologues of human PD-L1, analogs with at least one common epitope with PD-L1 (see e.g. Butte (2008) Mol Immunol. 45:3567-3572 ).
  • anti-PD-L1 constructs of the invention may interact with PD-L1 from species other than human or other proteins structurally related to human PD-L1 (eg, human PD-L1 homologues) cross-react.
  • the anti-PD-L1 construct may be fully specific for human PD-L1 and exhibit no species cross-reactivity or other types of cross-reactivity.
  • human PD-L1 refers to the human sequence PD-L1, such as the complete amino acid sequence of human PD-L1 with GenBank accession number Q9NZQ7.
  • the human PD-L1 sequence may differ from the human PD-L1 with GenBank accession number Q9NZQ7, for example, by having conservative mutations or mutations in non-conserved regions, and the PD-L1 is identical to the human PD-L1 with GenBank accession number Q9NZQ7 -L1 has essentially the same biological function.
  • the biological function of human PD-L1 is to have epitopes in the extracellular domain of PD-L1 that are specifically bound by the anti-PD-L1 constructs of the present disclosure, or the biological function of human PD-L1 is the regulation of T cell activity.
  • PD-1 programmed cell death 1
  • CD80 human B7-1
  • antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric and camelized single domain antibodies.
  • HCAb heavy chain antibody
  • HCAb heavy chain only antibody
  • HCAb refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in quadrabodies. Camelids such as camels, llamas or alpacas are known to produce HCAbs.
  • single domain antibody refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the SdAb alone was able to bind to the antigen without pairing with the corresponding CDR-containing polypeptide.
  • the single domain antibody is engineered from a camelid HCAb, and its heavy chain variable domain is referred to herein as a " VHH " (variable domain of the heavy chain of a heavy chain antibody). ).
  • VHHs variable domain of the heavy chain of a heavy chain antibody.
  • Some VHHs are also known as Nanobodies.
  • Camelid sdAbs are among the smallest known antigen-binding antibody fragments.
  • a basic VHH has the following structure from N-terminal to C-terminal: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementary Decision areas 1 to 3.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media .
  • isolated does not mean the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • variable region refers to the amino-terminal domain of the heavy or light chain of an antibody.
  • variable domains of heavy and light chains may be referred to as " VH " and " VL ", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen binding site.
  • Heavy chain-only antibodies from camelid species have a single heavy chain variable region called " VHH ". Therefore VHH is a special type of VH .
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule comprising at least four peptide chains in nature: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • antibody-binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody that retains the parental At least some binding specificity of an antibody.
  • antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • a binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • CDR regions are loops in the variable domains of antibodies that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ( "antigen contact point”).
  • the CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • the CDRs located within the antibody heavy chain variable region domains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domains are referred to as LCDR1, LCDR2 and LCDR3.
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia based on the three-dimensional structure of the antibody and topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S.
  • the CDRs of the antibodies of the invention can be bounded by one skilled in the art according to any scheme in the art (eg different assignment systems or combinations).
  • the CDR boundaries of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. different assignment systems or combinations), resulting in their claimed CDR boundaries being different from the specific CDR boundaries defined by the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • constant domain refers to a portion of an immunoglobulin molecule that has an amino acid sequence that is more conserved relative to the rest of the immunoglobulin containing the antigen binding site (ie, the variable domain).
  • the constant domains contain the CH1, CH2 and CH3 domains of the heavy chain (collectively CH) and the CHL (or CL) domain of the light chain.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from the amino acid residue at Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, eg, during production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody.
  • composition of an intact antibody may comprise a population of antibodies with all K447 residues removed, a population of antibodies with none of the K447 residues removed, and a population of antibodies with a mixture of antibodies with and without K447 residues.
  • Suitable native sequence Fc regions for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides typically also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • diabody refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Variable domain
  • murine antibody or "hybridoma antibody” in the present disclosure is a monoclonal antibody against human PD-L1 prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with PD-L1 antigen, and hybridomas expressing antibodies with desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from rodent or the like antibodies (“parental antibodies”), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibody induces induction in human subjects as compared to the parental rodent antibody The likelihood of an adverse immune response is low.
  • humanized antibody refers to a form of antibody that contains sequences from human and non-human (eg, mouse, rat) antibodies.
  • humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • FR hypervariable loops Framework
  • a humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
  • murine/camelid CDR regions can be inserted into human germline framework regions using methods known in the art.
  • Fully human antibody refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4). Isotypes also include modified forms of one of these species, wherein modifications have been made to alter Fc function, eg, to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4
  • epitope refers to a protein determinant capable of specific binding by an antibody.
  • Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is the loss of binding to the former but not to the latter in the presence of a denaturing solvent.
  • cross-reactivity refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Thus, “cross-reactivity” should be understood as an inter-species reaction with the same molecule X expressed in different species.
  • the cross-reactive specificity of monoclonal antibodies recognizing human PD-L1, monkey, and/or murine PD-L1 (mouse or rat) can be determined by FACS analysis.
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant ( kdis and kon , respectively ) .
  • KD equilibrium dissociation constant
  • Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • does not bind to a protein or cell means, does not bind to the protein or cell, or does not bind to it with high affinity, ie binds the protein or cell with a K D of 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 -5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, more preferably 1.0 ⁇ 10 -2 M or higher.
  • high affinity for IgG antibodies refers to an antigen with a KD of 1.0 x 10 -6 M or less, preferably 5.0 x 10 -8 M or less, more preferably 1.0 x 10 -8 M or lower, 5.0 ⁇ 10 -9 M or lower, more preferably 1.0 ⁇ 10 -9 M or lower.
  • "high affinity” binding may vary.
  • "high affinity” binding of an IgM subtype means a KD of 10-6 M or lower, preferably 10-7 M or lower, more preferably 10-8 M or lower.
  • antibody-dependent cytotoxicity refers to cell-mediated immune defense in which immune system effector cells actively associate cell membrane surface antigens with antibodies, such as Claudin18. 2 Antibodies that bind to target cells such as cancer cells are lysed.
  • CDC complement-dependent cytotoxicity
  • IgG and IgM antibodies that, when bound to surface antigens, initiate the canonical complement pathway, including formation of membrane attack complexes and lysis of target cells.
  • the antibodies of the present invention when bound to Claudin 18.2, trigger CDC against cancer cells.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Human US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo).
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements thereof, as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from Any source, capable of integrating with the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked).
  • the recombinant construct will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be directed using both heterologous and non-heterologous (ie, endogenous) promoters.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.
  • Activation can have the same meaning, eg, activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
  • a “response/response”, eg, the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (eg, concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), wherein changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
  • percent (%) amino acid sequence identity or simply “identity” is defined as the maximum percent sequence identity obtained when amino acid sequences are aligned (and where necessary gaps are introduced), and no conservative substitutions are considered to be Following the portion of sequence identity, the percentage of amino acid residues in the candidate amino acid sequence that are identical to those in the reference amino acid sequence.
  • Sequence alignments to determine percent amino acid sequence identity can be performed using various methods in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • immune response refers to the action by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines and complement) by these cells or by the liver, which results in selective Damage, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of signals from one part of a cell to another of the cell. part.
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions leading to signal transduction.
  • the penultimate process usually involves nuclear events leading to changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, neutralization or antagonism of PD in vivo or in vitro - Potency for L1 activity, IC50 , in vivo stability of the antibody and immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (ie, generally with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of The ability of proteins to be expressed at high levels in mammalian cells.
  • the terms “treating” or “treating” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treating” or “treating” refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernible by a patient. In another embodiment, “treating” or “treating” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to a cynomolgus monkey.
  • Administration "in combination with” one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” mean that the PD-L1 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents, is effective in preventing Or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition.
  • a therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • the therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of the therapeutic agent will result in an improvement in the diagnostic criterion or parameter by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.
  • Cancer and “cancerous” refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Benign and malignant cancers as well as dormant tumors or micrometastases are included in this definition. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, cancer of the stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Cancer of salivary gland, kidney or kidney, liver, prostate, vulva, thyroid, liver, and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's Lymphoma (NHL), Small Lymphocytic (SL) NHL, Intermediate/Follicular NHL, Intermediate Diffuse NHL, High-Grade Immunoblastic NHL, High-Grade Lymphoblastic NHL, High-Grade Small Anu
  • lung cancer
  • the present invention provides anti-PD-L1 antibodies or antigen-binding fragments thereof.
  • anti-PD-L1 antibody refers to a PD-L1 protein or fragment thereof with sufficient affinity such that the Antibodies can be used as diagnostic and/or therapeutic agents targeting PD-L1.
  • the antibody or antigen-binding fragment thereof of the present invention has one or more of the following properties:
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH, wherein the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:3; or comprise 1, 2 or 3 amino acid differences from the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively HCDR1, HCDR2 and HCDR3.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH, wherein the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:3.
  • the heavy chain variable region comprises the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 26 and SEQ ID NO: 3, respectively HCDR1, HCDR2 and HCDR3.
  • the antibody or antigen-binding fragment thereof of the present invention comprises an Fc region, preferably, the Fc region is linked to the C-terminus of the heavy chain variable region.
  • the antibody of the invention further comprises a constant region CH1 between the variable region of the heavy chain and the Fc.
  • the Fc region is the Fc region of human IgGl, IgG2, IgG3 or IgG4; preferably the Fc region of human IgGl.
  • the antibodies of the invention are single domain antibodies or heavy chain antibodies.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the single domain antibody or heavy chain antibody of the present invention are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or respectively As shown in SEQ ID NO: 1, SEQ ID NO: 26 and SEQ ID NO: 3; the amino acid sequence of FR1 is shown in FR1 of SEQ ID NO: 11 or 12; the amino acid sequence of FR2 is shown in SEQ ID NO: 5 , FR2 of 10, 11, 14, 20 or 24, preferably FR2 of SEQ ID NO: 11, 14, 20 or 24, more preferably FR2 of SEQ ID NO: 11, 20 or 24; its FR3
  • the amino acid sequence is shown in FR3 of SEQ ID NO:6, 7, 8, 11, 13 or 17, preferably shown in FR3 of SEQ ID NO:11; the amino acid sequence of FR4 is shown in FR3 of SEQ ID NO:11 Show.
  • amino acid sequence of the single domain antibody is set forth in any of SEQ ID NOs: 5-25. In some embodiments, the amino acid sequence of the heavy chain antibody is set forth in any of SEQ ID NOs: 27-30.
  • amino acid changes include amino acid deletions, insertions or substitutions.
  • the anti-PD-L1 antibodies or antigen-binding fragments thereof of the invention include those that have been mutated by amino acid deletions, insertions, or substitutions, but are still related to the antibodies described above (particularly in the CDR regions depicted in the sequences above) Those antibodies that have amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions that have been mutated by amino acid deletions, insertions, or substitutions when compared to the CDR regions depicted in a particular sequence.
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants.
  • An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl - 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • the anti-PD-L1 antibody is a monoclonal antibody.
  • the anti-PD-L1 antibody is humanized.
  • Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13:1619-1633).
  • the anti-PD-L1 antibody is a fully human antibody.
  • Fully human antibodies can be prepared using a variety of techniques known in the art. Fully human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol 20:450-459 (2008).
  • the anti-PD-L1 antibody is a chimeric antibody.
  • At least a portion of the framework sequences of the anti-PD-L1 antibody are human consensus framework sequences.
  • the anti-PD-L1 antigen-binding fragment of the present invention is Fab, Fab', F(ab')2, Fab'-SH, Fv, scFv, dAb or sdAb.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention is of any IgG subtype, such as IgG1, IgG2, IgG3, or IgG4.
  • the anti-PD-L1 antibody molecule is in the form of a bispecific or multispecific antibody molecule.
  • a multispecific antibody molecule can be, for example, a trispecific antibody molecule comprising a first binding specificity for PD-L1 and second and third binding specificities for one or more molecules.
  • the present invention provides a polynucleotide encoding the anti-PD-L1 antibody or antigen-binding fragment thereof described herein.
  • the polynucleotide may comprise a polynucleotide encoding the amino acid sequence of the heavy chain variable region of an antibody, or a polynucleotide comprising the amino acid sequence encoding the heavy chain of an antibody.
  • the polynucleotides encoding the antibodies of the invention include those that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60, 70, or 60, 70 or more of the coding region corresponding to the CDRs depicted in the sequences described above. , 80, 90, 95 or 100% identical polynucleotides.
  • the present invention provides a vector comprising a polynucleotide as described herein, preferably, the vector is a eukaryotic expression vector.
  • the polynucleotides as described herein are contained in one or more expression vectors.
  • the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, and/or expressing or comprising an antibody or antigen-binding fragment thereof as described herein;
  • the host cells are eukaryotic cells, more preferably mammalian cells.
  • the present invention provides a method for preparing an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein, the method comprising under conditions suitable for expression of the antibody or antigen-binding fragment thereof
  • the host cells described herein are cultured to express the antibody or antigen-binding fragment thereof, and the expressed antibody or antigen-binding fragment thereof is recovered from the host cell.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, Monkey Kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • ATCC American Type Culture Collection
  • the present invention provides a method of making an anti-PD-L1 antibody, wherein the method comprises, when introducing an expression vector into a mammalian host cell, by culturing the host cell for a period of time sufficient to allow the antibody to develop in the host.
  • the antibody is produced by expression in the cell, or more preferably by secretion of the antibody into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • afucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic , because their carbohydrate structure is a normal component of native human serum IgG.
  • the present invention also encompasses anti-PD-L1 antibodies conjugated to other substances (immunoconjugates).
  • other agents such as therapeutic or diagnostic agents, such as cytotoxic or immunosuppressive or chemotherapeutic agents. Cytotoxic agents include any agent that is detrimental to cells.
  • the immunoconjugate contains a therapeutic agent that can be used to prevent or treat cancer.
  • the therapeutic agent is preferably a small molecule anticancer drug known in the art, such as chemotherapeutic agents and cytotoxic agents, and the like.
  • the immunoconjugate can be, for example, an antibody drug conjugate (ADC).
  • the immunoconjugate contains a diagnostic agent useful in the diagnosis of a disease such as cancer.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof or a conjugate thereof as described herein, and a pharmaceutically acceptable carrier or excipient Form.
  • a pharmaceutically acceptable carrier or excipient Form may incorporate suitable carriers, excipients and other agents in formulations for combined administration, thereby providing improved transfer, delivery, tolerance, and the like.
  • the conjugate in the pharmaceutical composition is a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a therapeutic agent.
  • composition refers to a formulation that allows the active ingredients contained therein to be present in a biologically effective form and does not contain additional ingredients that would have unacceptable toxicity to the subject to whom the formulation is administered.
  • compositions of the anti-PD-L1 antibodies described herein are preferably in the form of aqueous solutions or lyophilized formulations.
  • compositions or formulations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other .
  • the other active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a growth inhibitory agent, an antibiotic, or various anti-tumor or anti-cancer agents known, in a suitable amount effective for the intended use exist in combination.
  • the pharmaceutical compositions of the present invention further comprise compositions of polynucleotides encoding anti-PD-L1 antibodies or antigen-binding fragments thereof.
  • the present invention provides a combination product comprising an antibody or antigen-binding fragment thereof described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein, and one or more Additional therapeutic agents.
  • the additional therapeutic agent includes, but is not limited to, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulatory agents.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, an immunoconjugate described herein, a pharmaceutical composition described herein, or a combination product described herein prepared for use in therapy and/or Or use in a medicament for the prevention of a disease or condition mediated by PD-L1, preferably the disease or condition is cancer, more preferably the cancer is selected from cancers with elevated PD-1, PD-L1 or PD-L2 expression .
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, an immunoconjugate described herein, a pharmaceutical composition described herein, or a combination product described herein for use in therapy and/or Or prevent a PD-L1 mediated disease or disorder, preferably the disease or disorder is cancer, more preferably the cancer is selected from cancers with elevated PD-1, PD-L1 or PD-L2 expression.
  • the present invention provides a method of treating and/or preventing a PD-L1 mediated disease or disorder comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody described herein or The antigen-binding fragment thereof, the immunoconjugate described herein, the pharmaceutical composition described herein, or the combination product described herein, preferably the disease or disorder is cancer, more preferably the cancer is selected from the group consisting of PD-1, Cancers with elevated PD-L1 or PD-L2 expression.
  • the cancer or tumor described herein can be selected from melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer Cancer and rectal cancer; preferably these cancers or tumors have elevated PD-1, PD-L1 or PD-L2 expression. Whether the expression of PD-1, PD-L1 or PD-L2 in the cancer tissue of the patient is increased can be determined by comparing with the normal expression level of PD-1, PD-L1 or PD-L2 in the corresponding normal tissue of the normal population.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration Wait.
  • the present invention also provides for co-administration of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents) to a subject.
  • the therapy includes surgery and/or radiation therapy.
  • the methods or uses provided herein further comprise administering to an individual one or more therapies (eg, therapeutic modalities and/or other therapeutic agents).
  • therapies eg, therapeutic modalities and/or other therapeutic agents.
  • Antibodies of the invention can be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • PD-1 antibody, CTLA-4 antibody and LAG-3 antibody for example, PD-1 antibody, CTLA-4 antibody and LAG-3 antibody.
  • the application also provides the above-mentioned anti-PD-L1 antibodies or antigen-binding fragments thereof, immunoconjugates, pharmaceutical compositions or combination products of the antibodies or fragments thereof in preparation for the prevention and/or treatment of PD-L1-related diseases or disorders Use in medicine, eg, in medicine for tumors.
  • the invention provides a method of detecting the presence of PD-L1 in a sample using an antibody or antigen-binding fragment thereof as described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the detection includes the step of contacting the antibody or antigen-binding fragment or immunoconjugate described herein with a sample to be tested and detecting whether a complex comprising the antibody or antigen-binding fragment or immunoconjugate described herein is formed , and optionally the step of quantifying the complexes formed.
  • the compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other methods, and equivalent substitutions known to those skilled in the art
  • preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the present invention adopts the following abbreviations:
  • his-tag stands for histidine tag
  • Fc tag stands for crystallizable fragment tag
  • ECD stands for extracellular domain
  • PEI stands for polyethyleneimine
  • BSA stands for bovine serum albumin
  • PEG stands for polyethylene glycol
  • PBS stands for Phosphate Buffered Saline.
  • the hPD-L1 gene fragment (PD-L1 sequence: NP_054862.1, 19aa-238aa) was obtained by amplifying, and at the same time, a suitable restriction site was introduced, and then the gene fragment was inserted into the c-terminal and connected to the camel Fc fragment (aFc) in a eukaryotic transient expression vector encoding the gene.
  • aFc camel Fc fragment
  • Example 2 Screening and identification of antigens Preparation of human PD-L1-mouse Fc fusion protein (hPD-L1-mFc) and human PD-L1-histidine tag fusion protein (hPD-L1-his)
  • the hPD-L1 gene fragment (PD-L1 sequence: NP_054862.1, 19aa-238aa) was obtained by amplifying, and at the same time, a suitable restriction site was introduced, and then the gene fragment was inserted into the c-terminal and connected to the mouse Fc fragment (mFc) encoding gene or 6 ⁇ His Tag encoding gene in eukaryotic transient expression vector.
  • mFc mouse Fc fragment
  • 6 ⁇ His Tag 6 ⁇ His Tag
  • the camels were immunized with the recombinant protein hPD-L1-aFc prepared in Example 1, and the camels were immunized once every two weeks, 4 times in total.
  • 1 mL of blood was collected for immune titer detection. After the titer reached the requirements for building a library, 100 mL of blood was collected to separate PBMCs for building a library.
  • PBMC peripheral blood mononuclear cells
  • cDNA RNA sequence
  • primers for building a single-domain antibody library were designed and synthesized, and the antibody sequences were amplified by PCR.
  • the vector and amplified antibody fragments were digested with NcoI and NotI endonucleases.
  • the ligation product was constructed by the connection method of T4 ligase, and the ligation product was transferred into the TG1 strain by electrotransformation, cultured and stored.
  • the test results showed that after the camel immunization, the effective titer of VHH reached 1.6k, and the effective titer of total IgG reached 32k-64k.
  • the immunization was successful and met the requirements of subsequent library construction. After electroporation, the strains were serially diluted, and after overnight incubation on the plate, the colonies on the plate were counted and the library capacity was calculated. The calculation results show that the capacity of the constructed immune library is 1.3 ⁇ 10 8 .
  • the constructed camel immune library was screened by solid-phase screening to obtain specific phage-displayed single-domain antibodies.
  • Screening Screening of specific antibodies by solid-phase method. Coat the specific antigen on the surface of the immune tube, block the immune tube and the antibody library with a blocking agent, add the antibody library to the immune tube and incubate, then wash repeatedly, and finally use pH2.2 acid for elution, wash After de-neutralization to neutrality, it was incubated with XL-Blue strains in logarithmic growth phase for infection, and further phage display was performed to recover specific phage particles. Monoclonal identification was performed after 2-3 rounds of screening.
  • Variable region amino acid sequence of single domain antibody C1 (SEQ ID NO: 4):
  • HCDR1:APCMA SEQ ID NO: 1
  • HCDR3 ARATFYRCEGDASSYTY (SEQ ID NO: 3)
  • VHH antibody gene was obtained by PCR, and then the gene was cloned upstream of the gene encoding the Fc segment of the constant region of the human IgG1 heavy chain to construct a recombinant eukaryotic expression vector to obtain a camel chimeric single domain Antibody expression plasmids.
  • VHH-hFc single domain chimeric antibody
  • control antibody KN035 was synthesized and expressed according to the Envafolimab sequence published by WHO (KN035 sequence source: WHO Drug Information, Vol.33, No.3, 2019, Page 634-635, Envafolimab).
  • the binding activity of chCl and the control antibody KN035 was compared in parallel.
  • the Biacore assay results showed that the affinity (KD) of chC1 binding to free PD-L1 in the liquid phase was 0.317nM, while the affinity (KD) of the control antibody KN035 (Corning Jerry) was 0.405nM, as shown in Table 1.
  • Table 1 Affinity determination of single domain chimeric antibody and human PD-L1 recombinant protein
  • Assay method AHC capture method; antigen concentration: 12.5, 6.25, 3.125, 1.5625, 0.78125nM.
  • ELISA and FACS were used to analyze chC1 and PD-1, B7H3, B7H4, CTLA4, CD28, ICOS and other recombinant proteins, as well as chC1 and MBA-MA-231, HEL, THP -1. Binding of Raji, CHO, HEK-293 and other cell lines.
  • chC1 The inhibitory effect of chC1 on the binding of PD-L1 to PD-1 was analyzed by ELISA. The results showed that chC1 could effectively block the binding of recombinant human PD-L1 to its receptor PD-1, and its half effective inhibitory concentration (IC 50 ) was 0.59 ⁇ g/ml, while the IC 50 of the control antibody KN035 was 0.65 ⁇ g/ml .
  • the humanization transformation process involves the following steps: A. Align the gene sequence of the camel single domain antibody with the gene sequence of the human embryonic antibody to find out the sequence with high homology; B. Analyze and investigate HLA-DR Affinity, select the human embryonic framework sequence with low affinity; C. Using computer simulation technology, apply molecular docking to analyze the framework amino acid sequence of variable region and its surrounding, and investigate its spatial three-dimensional binding mode.
  • the key amino acid individuals in the camel single domain antibody gene sequence that can interact with PD-L1 and maintain the spatial framework are analyzed, and then grafted back to the selected human embryonic gene framework, On this basis, the amino acid sites in the framework region that must be retained are identified; D.
  • the embedded residues, the residues that have direct interaction with the CDR region, and the VL and Residues that have an important effect on the conformation of VH were backmutated, and amino acid residues that resulted in chemical instability in the CDR regions of the antibody were optimized. The optimization results are as follows:
  • HCDR2 ASVRGGLSPYYADSVKG (SEQ ID NO:2) optimized to ASVRGGLSPYYADVKG (SEQ ID NO:26)
  • variable regions of humanized anti-PD-L1 single domain antibodies were obtained:
  • the binding activities of the humanized and chimeric antibodies were compared in parallel. Using a Thermo Scientific microplate reader, the plate was coated with a fixed concentration of His-tagged human PD-L1 antigen (2.0 ⁇ g/mL), and after blocking with 2% BSA, the above-mentioned antibodies (C1-hu1 to C1-hu6) were added in a series of dilutions.
  • the inhibition of PD-L1 binding to PD-1 by humanized and chimeric antibodies was compared in parallel.
  • the plate was firstly coated with streptavidin (0.4 ⁇ g/mL), and then a fixed concentration of biotinylated PD-L1 antigen (0.2 ⁇ g/mL) was bound to it.
  • streptavidin 0.4 ⁇ g/mL
  • biotinylated PD-L1 antigen 0.2 ⁇ g/mL
  • the above antibodies were serially diluted with a fixed concentration of PD-1mFc (2.0 ⁇ g/mL) (wherein C1-hu1 to C1-hu6 No.
  • the full-length amino acid sequences of the heavy chains of the eight humanized anti-PD-L1 single-domain antibodies are as follows:
  • Example 7 The PD-1/PD-L1 pathway blocking test by the reporter gene method to detect the antibody PD-L1 nanobody
  • CHO/PD-L1 cells (Promega) were taken, digested and resuspended in F-12 Nutrient Mixture complete medium, and the cell density was adjusted to 5 ⁇ 10 5 cells/ml using complete medium according to the cell count results, and the cell suspension was transferred To the sample addition tank, add 100 ⁇ l/well to a 96-well plate using a pipette, and place it at 37°C in a 5% CO 2 incubator for 16-20 h; prepare Jurkat/PD-1 (Promega) cells the next day Suspension, resuspend the cells in assay medium according to the cell count results, and adjust the cell density to 2.5 ⁇ 10 6 cells/ml; remove the cell culture plate with CHO/PD-L1 cells from the incubator and use a multi-channel transfer Take out 100 ⁇ l of culture medium from each well of the liquid dispenser, add the antibody of the present invention in a gradient dilution at 40 ⁇ l/well, then transfer the Jurkat/PD-1 cell suspension to the sample addition tank, add 40
  • the anti-PD-L1 nanobodies of the present invention can effectively block the interaction of PD-1/PD-L1, promote the enhancement of NFAT signal, and have a dose-dependent effect of drug concentration.
  • the EC50 of C1-hu17, C1-hu18, C1-hu19, C1-hu20 and C1-hu21 activities were 0.5914nM, 0.5870nM, 0.6696nM, 0.5650nM, and 0.5843nM, respectively.
  • MC38B7H1 cells were inoculated subcutaneously on the right side of 6-8 week-old female B-hPD-L1 humanized mice at a concentration of 1 ⁇ 10 6 cells/0.1 mL, and when the tumor grew to about 104 mm 3 , an appropriate animal was selected to press the tumor Randomly divided by volume, 8 in each group, a total of 4 groups, namely: G1 saline control group, G2C1-hu7 (10mg/kg) group, G3C1-hu8 (10mg/kg) group and C1-hu10 (10mg/kg) group Group.
  • the route of administration in all groups was intraperitoneal injection, the administration dose was 10 mg/kg, and the administration concentration was 1 mg/ml.
  • the average tumor volume in the saline group was 1011 ⁇ 91 mm 3
  • the average tumor volumes in the C1-hu7 group, C1-hu8 group and C1-hu10 group were respectively were 663 ⁇ 104 mm 3 , 630 ⁇ 132 mm 3 and 675 ⁇ 84 mm 3 .
  • the relative tumor inhibition rates were 38.4%, 42.0% and 37.0%, respectively, and there was a statistical difference (p ⁇ 0.05). It is shown that the humanized antibody can significantly inhibit the growth of subcutaneously transplanted tumor of B-hPD-L1 humanized mouse MC38B7H1 cells.

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Abstract

提供了抗PD-L1单域抗体及其用途。具体而言，提供了抗PD-L1抗体或其抗原结合片段，其包含重链可变区VH，其中所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3或由其组成；或包含与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3。还提供了编码该抗体或其抗原结合片段的核酸分子，用于表达该抗体或其抗原结合片段的载体和宿主细胞，以及该抗体或其抗原结合片段的诊断方法和制药用途。

Description

抗PD-L1单域抗体及其用途 技术领域
本发明提供了与PD-L1特异性结合的单域抗体或其抗原结合片段和包含其的组合物。还提供了编码本发明抗体或其抗原结合片段的核酸分子，用于表达本发明抗体或其抗原结合片段的载体和宿主细胞，以及本发明抗体或其抗原结合片段的治疗和诊断方法和用途。
背景技术
程序性细胞死亡蛋白配体1(Programmed death-ligand 1,PD-L1)，又可称为分化簇274(cluster of differentiation 274，CD274)或者B7同源蛋白1(B7homolog1，B7-H1)，属于肿瘤坏死因子超家族，是由290个氨基酸残基组成的I型跨膜糖蛋白，包含一个IgV样区、一个IgC样区、一个跨膜疏水区和一个30个氨基酸的胞内尾部,完整分子量为40kDa1。PD-L1mRNA在几乎所有组织中都有表达，但PD-L1蛋白只在少部分组织中持续表达，包括肝脏、肺脏、扁桃体以及免疫特赦组织如眼、胎盘等。PD-L1也表达于活化的T细胞，B细胞，单核细胞，树突状细胞，巨噬细胞等2。
PD-L1的受体为PD-1，主要表达于CD4+T细胞、CD8+T细胞、NKT细胞、B细胞和活化的单核细胞等免疫细胞表面。PD-L1与PD-1结合可以启动PD-1胞浆区ITIM(免疫受体酪氨酸抑制作用模块)酪氨酸残基的磷酸化，促使酪氨酸磷脂酶与SHP2结合，活化SHP2,使下游Syk和PI3K发生去磷酸化从而传递终止信号，限制抗原呈递细胞或者树突状细胞与T细胞的相互作用3。这种结合还可以进一步抑制T细胞的代谢，抑制抗凋亡蛋白Bcl-X2的分泌，减少效应细胞因子IL-2,IFN-r的分泌，诱导T细胞耗竭和凋亡，从而降低免疫T细胞参与的免疫应答，行使负性调节功能4。
T细胞识别抗原并活化后会分泌IFN-r。T细胞来源的IFN-r会扩增和维持T细胞功能，比如上调MHC分子，增强目标细胞的抗原处理和呈递，促进T细胞分化。IFN-r同时也会诱导免疫炎症部位组织的PD-L1表达，防止过度免疫对组织造成伤害。IFN-r可以诱导常规上皮细胞，血管内皮细胞，髓样细胞，幼稚T细胞等细胞表面PD-L1的表达5,6。IFN-r诱导产生的干扰素调节因子1(IRF-1)也可以与PD-L1转录起始位点前200bp和320bp处的干扰素调节因子结合位点结合，从转录水平调节PD-L17。PD-L1可以与T细胞表面的PD-1结合行使负调节功能，从而保护炎性部位。
单域抗体作为一种新型的小分子抗体片段，由驼类天然的重链抗体重链可变区(VHH) 克隆获得。单域抗体具有优良的生物学特性，分子量12-15KD，是完整抗体的十分之一，在克服了天然抗体分子量大的弊端的同时又拥有完整的抗原结合位点，具有很好的组织穿透性，特异性高，水溶性好。因其特殊的结构性质，兼具了传统抗体与小分子药物的优势，几乎完美克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷。因此，本领域迫切需要开发能与PD-L1结合，且可以阻断PD-L1/PD-1结合的抗体，尤其是抗PD-L1单域抗体。
发明内容
本发明提供了抗PD-L1抗体或其抗原结合片段，其具有针对人PD-L1的高亲和力和高特异性等优势。本发明提供的抗PD-L1抗体或其抗原结合片段可作为独立的疗法或与其它疗法/或其他抗癌药剂联合，用于诸如癌症的治疗。
在一个方面，本发明提供一种抗PD-L1抗体或其抗原结合片段，其包含重链可变区VH，其中所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3或由其组成；或包含与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3。在一些实施方式中，本发明所述与SEQ ID NO:2所示氨基酸序列具有1、2或3个氨基酸差异的HCDR2是SEQ ID NO:26。
在一些实施方式中，本发明所述抗体或其抗原结合片段中，所述重链可变区的FR1的氨基酸序列包含SEQ ID NO:11或12的FR1或由其组成，或包含与SEQ ID NO:11或12的FR1区具有1、2或3个氨基酸差异的FR1或由其组成；所述重链可变区的FR2的氨基酸序列包含SEQ ID NO:5、10、11、14、20或24的FR2或由其组成，或包含与SEQ ID NO:5、10、11、14、20或24的FR2具有1、2或3个氨基酸差异的FR2或由其组成；所述重链可变区的FR3的氨基酸序列包含SEQ ID NO:6、7、8、11、13或17的FR3或由其组成，或包含与SEQ ID NO:11的FR3具有1、2或3个氨基酸差异的FR3或由其组成；所述重链可变区的FR4的氨基酸序列包含SEQ ID NO:11的FR4或由其组成，或包含与SEQ ID NO:11的FR4具有1、2或3个氨基酸差异的FR4或由其组成。
在一些实施方式中，本发明所述抗体或其抗原结合片段中，所述重链可变区的FR1的氨基酸序列包含SEQ ID NO:11或12的FR1或由其组成，或包含与SEQ ID NO:11或12的FR1区具有1、2或3个氨基酸差异的FR1或由其组成；所述重链可变区的FR2的氨基酸序列包含SEQ ID NO:11、14、20或24的FR2或由其组成，或包含与SEQ ID NO:11、14、20或24的FR2具有1、2或3个氨基酸差异的FR2或由其组成；所述重链可变区的FR3的氨基酸序列包含SEQ ID NO:11的FR3或由其组成，或包含与SEQ ID NO:11的FR3 具有1、2或3个氨基酸差异的FR3或由其组成；所述重链可变区的FR4的氨基酸序列包含SEQ ID NO:11的FR4或由其组成，或包含与SEQ ID NO:11的FR4具有1、2或3个氨基酸差异的FR4或由其组成。
在一些实施方式中，本发明所述抗体或其抗原结合片段中，所述重链可变区的FR1的氨基酸序列包含SEQ ID NO:11或12的FR1或由其组成，或包含与SEQ ID NO:11或12的FR1区具有1、2或3个氨基酸差异的FR1或由其组成；所述重链可变区的FR2的氨基酸序列包含SEQ ID NO:11、20或24的FR2或由其组成，或包含与SEQ ID NO:11、14、20或24的FR2具有1、2或3个氨基酸差异的FR2或由其组成；所述重链可变区的FR3的氨基酸序列包含SEQ ID NO:11的FR3或由其组成，或包含与SEQ ID NO:11的FR3具有1、2或3个氨基酸差异的FR3或由其组成；所述重链可变区的FR4的氨基酸序列包含SEQ ID NO:11的FR4或由其组成，或包含与SEQ ID NO:11的FR4具有1、2或3个氨基酸差异的FR4或由其组成。
在一些实施方式中，本发明所述抗体或其抗原结合片段中，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
在一些实施方式中，本发明所述抗体或其抗原结合片段中，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:26和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
在一些实施方式中，本发明所述抗体或其抗原结合片段，其包含重链可变区VH，所述重链可变区分别包含选自SEQ ID NO:4、5、6、7、8、9、10、11、12、13、14、15、17、18、19、20、21、22、23、24或25中任一项所示的氨基酸序列或由其组成，或包含与SEQ ID NO:4、5、6、7、8、9、10、11、12、13、14、15、17、18、19、20、21、22、23、24或25中任一项所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段，其包含重链可变区VH，所述重链可变区分别包含如SEQ ID NO:4、5、6、7、8、9、10、11、12、13、14、15、17、18、19、20、21、22、23、24或25中任一项所示的氨基酸序列或由其组成。
在一些实施方式中，本发明所述抗体或其抗原结合片段，其包含Fc区，所述Fc区是人IgG1、IgG2、IgG3或IgG4的Fc区；优选为人IgG1的Fc区。
在一些实施方式中，本发明所述抗体或其抗原结合片段，其中所述抗体包含重链，所述重链包含如SEQ ID NO:27、28、29、30、31、32、33和34中任一项所示的氨基酸序列或由其组成，或包含与SEQ ID NO:27、28、29或30中任一项所示的氨基酸序列具有 至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段，其中所述抗体包含重链，所述重链包含选自SEQ ID NO:27、28、29、30、31、32、33和34中任一项所示的氨基酸序列或由其组成。
在一些实施方式中，本发明所述的抗体或其抗原结合片段，其中所述抗体是单域抗体或重链抗体。
在一些实施方式中，本发明所述的抗体或其抗原结合片段，其具有以下特性中的一种或多种：
(1)以高亲和力结合PD-L1；
(2)阻断PD-L1的相关活性；
(3)具有良好的热稳定性。
在一些实施方式中，本发明所述的抗体或其抗原结合片段，其中所述抗体为嵌合抗体、人源化抗体或全人抗体。
在一些实施方式中，本发明所述的抗体或其抗原结合片段，其中所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
在一些实施方式中，本发明所述抗PD-L1抗体或其抗原结合片段是任何IgG亚型，如IgG1、IgG2、IgG3或IgG4。
本发明还提供了一种多特异性抗体，包含本文所述抗体或其抗原结合片段的重链可变区。
本发明还提供了一种单链抗体，包含本文所述抗体或其抗原结合片段的重链可变区。
在又一个方面，本发明提供了分离的核酸分子，其编码本文所述的抗PD-L1抗体或其抗原结合片段。
在又一个方面，本发明提供了载体，其包含本文所述的核酸分子；优选地，所述载体为真核表达载体。
在又一个方面，本发明提供了宿主细胞，其包含本文所述的核酸分子或本文所述的载体，，或表达或含有本文所述的抗体或其抗原结合片段；优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了制备如本文所述的抗PD-L1抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
在又一个方面，本发明还提供了免疫缀合物，其包含本文所述抗体或其抗原结合片段和其它物质，例如治疗剂或诊断剂。
在又一个方面，本发明提供了药物组合物，其包含如本文所述的抗PD-L1抗体或其抗原结合片段或本文所述的免疫缀合物，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了组合产品，其包含如本文所述的抗体或其抗原结合片段、本文所述的免疫缀合物或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、本文所述的免疫缀合物、本文所述的药物组合物或本文所述的组合产品在制备用于治疗和/或预防PD-L1介导的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选所述癌症选自具有PD-1、PD-L1或PD-L2表达升高的癌症。
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、本文所述的免疫缀合物、本文所述的药物组合物或本文所述的组合产品，其用于治疗和/或预防PD-L1介导的疾病或病症，优选所述疾病或病症为癌症，更优选所述癌症选自具有PD-1、PD-L1或PD-L2表达升高的癌症。
在又一个方面，本发明提供了治疗和/或预防PD-L1介导的疾病或病症的方法，其包括向有需要的受试者施用治疗或预防有效量的如本文所述的抗体或其抗原结合片段、本文所述的免疫缀合物、本文所述的药物组合物或本文所述的组合产品，优选所述疾病或病症为癌症，更优选所述癌症选自具有PD-1、PD-L1或PD-L2表达升高的癌症。
在又一个方面，本发明提供了试剂盒，其包括如本文所述的抗体或其抗原结合片段、本文所述的免疫缀合物、本文所述的药物组合物或本文所述的组合产品。
在又一个方面，本发明提供了使用本文任一实施方案所述的抗体或其抗原结合片段检测PD-L1在样品中的存在的方法，所述方法包括使权利要求1-9中任一项所述的抗体或其抗原结合片段或含所述抗体或其抗原结合片段与诊断剂的免疫缀合物与待检样品接触并检测是否存在该抗体或其抗原结合片段与PD-L1形成的结合物以及任选地确定该结合物含量的步骤。
附图说明
图1：FACS法检测嵌合抗体chC1与MAB-BA-231细胞表面人PD-L1的结合。
图2：嵌合抗体chC1的特异性分析。A：ELISA法检测chC1与多种重组蛋白的结合特异性；B：FACS法检测chC1与多种细胞株的结合特异性。
图3：ELISA法检测人源化PD-L1单域抗体与PD-L1的结合。
图4：ELISA法检测人源化PD-L1单域抗体阻断PD-L1与PD-1的结合。
图5：人源化单域抗体对MC38B7H1肿瘤生长的抑制作用。
具体实施方式
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值，包括但不限于±5％、±2％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
如本文所用，术语“或”应被理解为与如上定义的“和/或”具有相同的含义。例如，当分离列表中的项目时，“或”或“和/或”应被解释为包容性的，即，包括数量或元素列表中的至少一个，但也包括多于一个，以及任选地，额外的未列出的项目。只有明确指出相反的术语下，例如“只有一个”或“的确一个”或者在权利要求中使用“由...组成”时，将指的是仅列出的一个数字或列表的一个元素。
除非明确指出相反的情况，否则如本文所用，词“一”和“一个”应理解为“至少一个”。
术语“程序性细胞死亡1配体1”、“PD-L1”、“B7同源物1(B7-H1)”、“PD-L1抗原”、“PDCD1配体1”和“CD274”可互换使用，包括人PD-L1的变体、同种型、物种同源物，具有至少一个与PD-L1的共同表位的类似物(参见例如Butte(2008)Mol Immunol.45:3567-3572)。因此，在某些情况下，本发明的抗-PD-L1构建体可以与来自除了人之外的其它物种的PD-L1或结构上与人PD-L1相关的其它蛋白(例如人PD-L1同源物)交叉反应。在其它情况下，所述抗-PD-L1构建体可以对人PD-L1完全特异，并且不表现出物种交叉反应性或其它类型的交叉反应性。
术语“人PD-L1”是指人序列PD-L1，诸如具有GenBank登录号Q9NZQ7的人PD-L1的完全氨基酸序列。所述人PD-L1序列可以不同于具有GenBank登录号Q9NZQ7的人PD-L1，在于例如具有保守突变或在非保守区域中的突变，并且所述PD-L1与具有GenBank登录号Q9NZQ7的人PD-L1具有基本相同的生物学功能。例如，人PD-L1的生 物学功能是在PD-L1的胞外结构域中具有被本公开内容的抗-PD-L1构建体特异性结合的表位，或者人PD-L1的生物学功能是调节T细胞活性。
术语“程序性细胞死亡1(PD-1)”意图指属于免疫球蛋白超家族并且在T细胞和B细胞上表达的细胞表面受体。人B7-1(CD80)的氨基酸序列以Genbank登录号NP_005009公开。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单域抗体。
术语“重链抗体”、“仅重链抗体”或“HCAb”是指一种功能性抗体，其包含重链但是缺少通常在四链抗体中存在的轻链。已知骆驼科动物(诸如骆驼、美洲驼或羊驼)产生HCAb。
术语“单结构域抗体”、“单域抗体”或“sdAb”是指具有三个互补决定区(CDR)的单个抗原结合多肽。仅SdAb就能够在不与相应的含有CDR的多肽配对的情况下与抗原结合。在一些情况下，单结构域抗体从骆驼科HCAb工程化改造而得到，并且其重链可变结构域在本文中被称为“V HH”(重链抗体的重链的可变结构域)。一些V HH也被称为纳米抗体。骆驼科sdAb是最小的已知抗原结合抗体片段之一。一个基本的V HH具有以下从N末端至C末端的结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，其中FR1至FR4分别指框架区1至4，并且其中CDR1至CDR3指互补决定区1至3。
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基末端结构域。重链和轻链的可变结构域可以分别被称为“V H”和“V L”。这些结构域通常是抗体的最可变部分(相对于同一类的其它抗体而言)并且含有抗原结合位点。来自骆驼科物种的仅重链抗体具有被称为“V HH”的单个重链可变区。因此V HH是V H的特殊类型。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“全长抗体”，是指在天然存在时包含至少四条肽链的免疫球蛋白分子：两条重(H)链和两条轻(L)链通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3 组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab') 2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
“互补决定区”或＂CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变区结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。
本发明抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883；Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))、基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services,National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差 异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia、AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
术语“恒定结构域”是指具有相对于含有抗原结合位点的免疫球蛋白的其它部分(即可变结构域)更加保守的氨基酸序列的免疫球蛋白分子的一部分。恒定结构域含有重链的CH1、CH2和CH3结构域(统称CH)和轻链的CHL(或CL)结构域。
术语“Fc区”用于定义免疫球蛋白重链的C末端区域，包括天然序列Fc区和变体Fc区。虽然免疫球蛋白重链的Fc区的边界可以变化，但是人IgG重链Fc区通常被定义为从位置Cys226或从Pro230处的氨基酸残基延伸至其羧基末端。Fc区的C末端赖氨酸(依据EU编号系统的残基447)可以例如在该抗体的产生或纯化期间或通过重组工程化改造编码该抗体的重链的核酸被去除。因此，完整抗体的组成可以包含所有K447残基都被去除的抗体群体、K447残基都未被去除的抗体群体、以及具有和不具有K447残基的抗体的混合物的抗体群体。用于本文所述的抗体中的合适的天然序列Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。
术语“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
术语“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
术语“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链 (VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
术语“鼠源抗体”或“杂交瘤抗体”在本公开中为根据本领域知识和技能制备的抗人PD-L1的单克隆抗体。制备时用PD-L1抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。本发明的人源化抗体，可以使用本领域已知的方法将鼠源/骆驼源的CDR区插入人种系框架区。
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“表位”指能够与抗体特异性结合的蛋白质决定簇。表位通常由各种化学活性表面分子诸如氨基酸或糖侧链组成，并且通常具有特定三维结构特征以及特定电荷特征。构象性表位和非构象性表位的区别在于在变性溶剂存在下，与前者而非与后者的结合丧失。
本文中所描述的术语“交叉反应”指的是对人类、猴、和/或鼠源(小鼠或大鼠)相同靶分子的抗原片段的结合。因此，“交叉反应”应被理解为与在不同物种中表达的相同分子X的种属间反应。识别人PD-L1、猴、和/或鼠PD-L1(小鼠或大鼠)的单克隆抗体的交叉反应特异性可通过FACS分析确定。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分 子X对其配偶物Y的亲和力可以通常由平衡解离常数(K D)代表，平衡解离常数是解离速率常数和结合速率常数(分别是k dis和k on)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“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K D为1.0×10 -6M或更高，更优选1.0×10 -5M或更高，更优选1.0×10 -4M或更高、1.0×10 -3M或更高，更优选1.0×10 -2M或更高。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的K D为1.0×10 -6M或更低，优选5.0×10 -8M或更低，更优选1.0×10 -8M或更低、5.0×10 -9M或更低，更优选1.0×10 -9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指K D为10 -6M或更低，优选10 -7M或更低，更优选10 -8M或更低。
术语“抗体依赖的细胞毒性”、“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫防御，其中免疫系统效应细胞主动地将细胞膜表面抗原与抗体，例如Claudin18.2抗体，结合的靶细胞例如癌细胞裂解。
术语“补体依赖的细胞毒性”或“CDC”是指IgG和IgM抗体的效应功能，当与表面抗原结合时引发典型的补体途径，包括形成膜攻击复合体以及靶细胞裂解。本发明的抗体，与Claudin 18.2结合时，引发对癌细胞的CDC。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
术语“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
术语“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导 致基因表达的变化。
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗PD-L1活性的能力、IC 50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方式中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方式中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方式中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“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，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的PD-L1抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤，和白血病。此类癌症的更具体例子包括鳞状细胞癌，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃的癌或胃癌(包 括胃肠癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，肝瘤(hepatoma)，乳腺癌，结肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌或肾的癌，肝癌，前列腺癌，外阴癌，甲状腺癌，肝的癌，及各种类型的头颈癌，以及B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHL，中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。
抗PD-L1抗体
在一个方面，本发明提供了抗PD-L1抗体或其抗原结合片段。术语“抗PD-L1抗体”、“抗PD-L1”、“PD-L1抗体”或“结合PD-L1的抗体”是指能够以足够的亲合力结合PD-L1蛋白或其片段以致所述抗体可以用作靶向PD-L1中的诊断剂和/或治疗剂。
在一些实施方式中，本发明所述的抗体或其抗原结合片段，其具有以下特性中的一种或多种：
(1)以高亲和力结合人PD-L1；
(2)阻断PD-L1与其受体PD-1的结合；
(3)具有良好的热稳定性。
在一些实施方式中，本发明的抗PD-L1抗体或其抗原结合片段包含重链可变区VH,其中所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或包含与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3。
在一些实施方式中，本发明的抗PD-L1抗体或其抗原结合片段包含重链可变区VH,其中所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
在一些实施方式中，本发明所述抗体或其抗原结合片段中，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:26和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含Fc区，优选地，所述Fc区连接在重链可变区的C末端。在一些实施方式中，本发明的抗体的重链可变区和Fc之 间还包含恒定区CH1。在一些实施方式中，所述Fc区是人IgG1、IgG2、IgG3或IgG4的Fc区；优选为人IgG1的Fc区。
在一些实施方式中，本发明所述的抗体是单域抗体或重链抗体。
在一些实施方式中，本发明所述单域抗体或重链抗体的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示，或分别如SEQ ID NO:1、SEQ ID NO:26和SEQ ID NO:3所示；其FR1的氨基酸序列如SEQ ID NO:11或12的FR1所示；其FR2的氨基酸序列如SEQ ID NO:5、10、11、14、20或24的FR2所示，优选如SEQ ID NO:11、14、20或24的FR2所示，更优选如SEQ ID NO:11、20或24的FR2；其FR3的氨基酸序列如SEQ ID NO:6、7、8、11、13或17的FR3所示，优选如SEQ ID NO:11的FR3所示；其FR4的氨基酸序列如SEQ ID NO:11的FR3所示。
在一些实施方式中，所述单域抗体的氨基酸序列如SEQ ID NO:5-25中任一所示。在一些实施方式中，所述重链抗体的氨基酸序列如SEQ ID NO:27-30中任一所示。
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗PD-L1抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
在一些实施方式中，抗PD-L1抗体是单克隆抗体。
在一些实施方式中，抗PD-L1抗体是人源化的。用于使抗体人源化的不同方法是技 术人员已知的，如由Almagro&Fransson综述的，其内容通过提述完整并入本文(Almagro JC和Fransson J(2008)Frontiers inBioscience 13:1619-1633)。
在一些实施方式中，抗PD-L1抗体是全人抗体。可使用本领域中已知的各种技术来制各全人抗体。全人抗体一般描述于van Dijk和van de Winkel，Curr.Opin.Pharmacol 5:368-74(2001)以及Lonberg,Curr.Opin.Immunol 20:450-459(2008)。
在一些实施方式中，抗PD-L1抗体是嵌合抗体。
在一些实施方式中，至少部分的抗PD-L1抗体的框架序列是人共有框架序列。
在一些实施方式中，本发明所述抗PD-L1的抗原结合片段为Fab、Fab'、F(ab')2、Fab'-SH、Fv、scFv、dAb或sdAb。
在一些实施方式中，本发明所述抗PD-L1抗体或其抗原结合片段是任何IgG亚型，如IgG1、IgG2、IgG3或IgG4。
在一些实施方式中，抗PD-L1抗体分子是双特异性或多特异性抗体分子形式。多特异性抗体分子例如可以是三特异性抗体分子，其包含针对PD-L1的第一结合特异性和针对一种或多种的分子的第二及第三结合特异性。
抗体表达
在又一个方面，本发明提供了一种多核苷酸，其编码本文所述的抗PD-L1抗体或其抗原结合片段。所述多核苷酸可以包含编码抗体的重链可变区的氨基酸序列的多核苷酸，或包含编码抗体的重链的氨基酸序列的多核苷酸。
在一些实施方式中，编码本发明抗体的多核苷酸包括已通过核苷酸缺失、插入或置换突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少约60、70、80、90、95或100％同一性的多核苷酸。
在又一个方面，本发明提供了一种载体，其包含如本文所述的多核苷酸，优选地，所述载体为真核表达载体。在一些实施方式中，如本文所述的多核苷酸包含在一个或多个表达载体中。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸或如本文所述的表达载体，和/或表达或包含本文所述的抗体或其抗原结合片段；优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种用于制备如本文所述的抗PD-L1抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞中，使其表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。
在一个实施方式中，本发明提供制备抗PD-L1抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中，来产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
免疫辍合物
在一些实施方式中，本发明还涵盖与其他物质缀合的抗PD-L1抗体(免疫缀合物)。在一些实施方式中，其它物质例如治疗剂或诊断剂，如细胞毒素剂或免疫抑制剂或化疗剂。细胞毒素剂包含任何对细胞有害的药剂。
在一些实施方式中，所述免疫缀合物含有治疗剂，可用于预防或治疗癌症。所述治疗剂优选是本领域已知的小分子抗癌药，如化疗剂和细胞毒性剂等。所述免疫辍合物可以是，例如抗体药物偶联物(ADC)。
在一些实施方式中，所述免疫辍合物含有诊断剂，可用于疾病如癌症的诊断。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗PD-L1抗体或其抗原结合片段或本文所述的辍合物，和药学上可接受的载体或赋形剂。应理解，本发明提供的抗PD-L1抗体或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。优选地，药物组合物中的所述辍合物为含有本发明所述抗体或其抗原结合片段与治疗剂的辍合物。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的 形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗PD-L1抗体与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的抗PD-L1抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码抗PD-L1抗体或其抗原结合片段的多核苷酸的组合物。
在又一个方面，本发明提供了一种组合产品，其包含本文所述的抗体或其抗原结合片段、本文所述的免疫辍合物或本文所述的药物组合物，以及一种或多种另外的治疗剂。在一些实施方式中，所述另外的治疗剂包括但不限于化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂。
医药用途
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的免疫辍合物、本文所述的药物组合物或本文所述的组合产品在制备用于治疗和/或预防PD-L1介导的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选所述癌症选自具有PD-1、PD-L1或PD-L2表达升高的癌症。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的免疫辍合物、本文所述的药物组合物或本文所述的组合产品，其用于治疗和/或预防PD-L1介导的疾病或病症，优选所述疾病或病症为癌症，更优选所述癌症选自具有PD-1、PD-L1或PD-L2表达升高的癌症。
在又一个方面，本发明提供了一种治疗和/或预防PD-L1介导的疾病或病症的方法，其包括向有需要的受试者施用治疗或预防有效量的本文所述的抗体或其抗原结合片段、本文所述的免疫辍合物、本文所述的药物组合物或本文所述的组合产品，优选所述疾病或病症为癌症，更优选所述癌症选自具有PD-1、PD-L1或PD-L2表达升高的癌症。
在一些实施方式中，本文所述的癌症或肿瘤可以选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌；优选地这些癌症或肿瘤具有升高的PD-1、PD-L1或PD-L2表达。可通过与正常人群相应的正常组织中PD-1、PD-L1或PD-L2的正常表达水平相比较来判断患者癌组织中PD-1、PD-L1 或PD-L2的表达是否升高。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
本发明还提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体。例如，可以与至少一种另外的治疗剂共施用。例如，PD-1抗体、CTLA-4抗体和LAG-3抗体。
本申请还提供了上述抗PD-L1抗体或其抗原结合片段、抗体或其片段的免疫缀合物、药物组合物或组合产品在制备用于预防和/或治疗PD-L1相关疾病或病症的药物中的用途，例如肿瘤的药物中的用途。
用于诊断和检测的方法
在又一个方面，本发明提供了一种使用本文所述的抗体或其抗原结合片段检测PD-L1在样品中的存在的方法。术语“检测”用于本文中时，包括定量或定性检测。在一些实施方式中，所述样品是生物样品。在某些实施方式中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方式中，生物样品包含细胞或组织。所述检测包括使本文所述的抗体或其抗原结合片段或免疫辍合物与待检样品接触并检测是否形成了包含本文所述抗体或其抗原结合片段或免疫辍合物的复合物的步骤，以及任选得对所形成的复合物进行定量的步骤。
本发明包括所述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明采用下述缩略词：
his-tag代表组氨酸标签；
Fc tag代表可结晶片段标签；
ECD代表胞外结构域；
PEI代表聚乙烯亚胺；
BSA代表牛血清白蛋白；
PEG代表聚乙二醇；
PBS代表磷酸缓冲盐溶液。
实施例
通过以下实施例对本发明进行说明，但并不旨在对本发明作出任何限制。本文已经详细描述了本发明，其中也公开了其具体实施方式。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1、重组蛋白人PD-L1-骆驼Fc融合蛋白(hPD-L1-aFc)的制备
通过PCR的方法，扩增获得hPD-L1基因片段(PD-L1序列：NP_054862.1，19aa-238aa)，同时引入合适的酶切位点，随后将该基因片段插入c端连入骆驼Fc片段(aFc)编码基因的真核瞬时表达载体中。测序正确后，大提质粒获得大量质粒，转染HEK293细胞瞬时表达，通过Protein G柱亲和层析纯化获得hPD-L1-aFc融合蛋白。
实施例2、筛选及鉴定抗原人PD-L1-鼠Fc融合蛋白(hPD-L1-mFc)、人PD-L1-组氨酸标签融合蛋白(hPD-L1-his)的制备
通过PCR的方法，扩增获得hPD-L1基因片段(PD-L1序列：NP_054862.1，19aa-238aa)，同时引入合适的酶切位点，随后将该基因片段插入c端连入鼠Fc片段(mFc)编码基因或6×His Tag编码基因的真核瞬时表达载体中。测序正确后，大提质粒获得大量质粒，转染HEK293细胞瞬时表达。通过Protein A柱亲和层析纯化获得hPD-L1-hFc融合蛋白，镍柱亲和层析纯化获得hPD-L1-mFC或hPD-L1-his。
实施例3、单域抗体的筛选
3.1、免疫羊驼及骆驼免疫库的构建
用实施例1制备得到的重组蛋白hPD-L1-aFc进行骆驼免疫，骆驼免疫两周一次，共4次。每次注射0.8mg上述重组蛋白，佐以弗氏不完全佐剂，采取皮下多点注射的方式。免疫结束后2周采集1mL血用于免疫效价检测。效价达到建库要求后，采集100mL血 分离PBMC用于建库。免疫后采集外周血单核细胞(PBMC)，提取PBMC的总RNA反转获得cDNA，作为后续扩增VHH片段的模板。根据相关文献和数据库中检索到单域抗体的基因，设计并合成单域抗体库构建引物，PCR扩增出抗体序列。利用NcoI、NotI两种核酸内切酶对载体和扩增的抗体片段进行酶切。采用T4连接酶的连接方式构建连接产物，利用电转化法将连接产物转入到TG1菌种之中并进行培养、保存。检测结果显示，骆驼免疫后VHH有效效价达到1.6k，总IgG有效效价达到32k-64k，免疫成功，满足后续建库要求。电转之后对菌种进行梯度稀释，涂布培养板过夜培养之后，对数板上的菌落进行计数并计算库容量。计算结果显示所构建的免疫库库容量为1.3×10 8。
3.2、骆驼免疫库筛选
通过固相筛选的方法对所构建的骆驼免疫库进行筛选，获得特异性噬菌体展示单域抗体。
原始库呈现：骆驼免疫库转接至含有氨苄青霉素和四环素的2YT培养基，培养至对数生长期，加入M13辅助噬菌体，然后加入卡那霉素，在较低温度条件下呈现过夜。第二天收取培养上清，PEG沉淀对噬菌体进行浓缩，获得高滴度的抗体库呈现产物用于后续筛选。
筛选：采用固相法进行特异性抗体的筛选。将特异性抗原包被在免疫管表面，用封闭剂分别封闭免疫管和抗体库后，将抗体库加至免疫管中孵育，然后进行反复洗涤，最后用pH2.2的酸进行洗脱，洗脱物中和至中性后与对数生长期的XL-Blue菌种混合孵育进行感染，进一步进行噬菌体展示，回收特异性噬菌体颗粒。筛选2-3轮后进行单克隆鉴定。
鉴定：感染回收特异性噬菌体颗粒的XL-Blue涂布平板，长出克隆后进行单克隆鉴定。将单克隆挑取后进行培养，培养至对数增长期，加入M13辅助噬菌体感染，然后入卡那霉素，在30℃培养过夜。第二天取上清，用包被了PD-L1的酶联板进行ELISA反应。反应呈阳性的克隆提取噬粒(含有抗体基因的噬菌体展示载体)进行测序，确定VHH基因序列。
经过两轮固相筛选和单克隆鉴定及测序，获得高度富集的特异性噬菌体抗体C1。
C1可变区及CDR区氨基酸序列如下所示(Kabat方案)：
单域抗体C1的可变区氨基酸序列(SEQ ID NO:4)：
Figure PCTCN2022089289-appb-000001
HCDR1:APCMA(SEQ ID NO:1)
HCDR2:ASVRGGLSPYYADSVKG(SEQ ID NO:2)
HCDR3:ARATFYRCEGDASSYTY(SEQ ID NO:3)
实施例4、抗PD-L1单域嵌合抗体
4.1单域嵌合抗体制备
根据单克隆鉴定的测序结果，设计特异性引物，通过PCR获得VHH抗体基因，随后将基因克隆至人IgG1重链恒定区Fc段编码基因上游，构建重组真核表达载体，获得骆驼嵌合单域抗体表达质粒。通过293F细胞瞬时表达和蛋白亲和纯化，获得以C1为亲本的单域嵌合抗体(VHH-hFc)chC1。同时，根据WHO公布的Envafolimab序列合成并表达了对照抗体KN035(KN035序列来源：WHO Drug Information,Vol.33,No.3,2019,Page634-635,Envafolimab)。
4.2、单域嵌合抗体结合活性分析
对chC1与对照抗体KN035的结合活性进行了平行比较。Biacore测定结果显示，chC1与液相中游离PD-L1结合的亲和力(KD)为0.317nM，而对照抗体KN035(康宁杰瑞)的亲和力(KD)为0.405nM，具体见表1。
表1：单域嵌合抗体与人PD-L1重组蛋白亲和力测定
  ka(1/Ms) kd(1/s) KA(1/M) KD(M)
chC1 1.46E+06 4.62E-04 3.16E+09 3.17E-10
KN035 2.08E+06 8.43E-04 2.47E+09 4.05E-10
测定方法：AHC捕获法；抗原浓度：12.5、6.25、3.125、1.5625、0.78125nM。
FACS结果显示(图1)，chC1与MAB-BA-231细胞表面的人PD-L1的结合活性EC 50为8.1ng/ml，而对照抗体KN035的EC 50为8.8ng/ml。其中，NC-huIgG1为阴性对照抗体。
4.3、单域嵌合抗体特异性分析
为了考察chC1的特异性，采用ELISA和FACS两种方法，分析了chC1与PD-1、B7H3、B7H4、CTLA4、CD28、ICOS等多种重组蛋白，以及chC1与MBA-MA-231、HEL、THP-1、Raji、CHO、HEK-293等细胞株的结合情况。
结果显示，chC1及对照抗体KN035均特异性结合PD-L1重组蛋白及人PD-L1高表达细胞株(MBA-MA-231)，而不与其它几种重组蛋白以及非PD-L1高表达细胞株结合(图2，A和B)。
4.4、单域嵌合抗体阻断活性分析
通过ELISA法分析了chC1对PD-L1与PD-1结合的抑制作用。结果显示，chC1可以有效阻断重组人PD-L1与其受体PD-1的结合作用，其半数有效抑制浓度(IC 50)为0.59 μg/ml，而对照抗体KN035的IC 50为0.65μg/ml。
实施例5、单域抗体可变区的人源化改造
对于抗体可变区的人源化改造，首先在NCBI(http://www.ncbi.nlm.nih.gov/igblast/)网站中的人类免疫球蛋白基因数据库搜寻与骆驼单域抗体的cDNA序列同源的人类种系IgG基因，再藉由Kabat编号系统或IMGT编号系统定义可变区CDR的氨基酸序列及其精确边界。原则上将与骆驼单域抗体具有高同源性的人类IGHV选为人源化模板，藉由CDR嫁接实施抗体可变区的人源化。
根据上述获得的骆驼单域抗体的序列，进行人源化改造。简言之，人源化改造过程涉及以下步骤：A、把骆驼单域抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；B、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架序列；C、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析骆驼单域抗体基因序列中可与PD-L1作用以及维护空间构架的关键氨基酸个体，将其嫁接回已经选择的人胚胎系基因框架，并在此基础上标配出必须保留的框架区氨基酸位点；D、以单域抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并优化导致抗体CDR区化学不稳定的氨基酸残基。优化结果如下：
HCDR2:ASVRGGLSPYYADSVKG(SEQ ID NO:2)优化为ASVRGGLSPYYADVKG(SEQ ID NO:26)
在此基础上得到了以下多种人源化抗PD-L1单域抗体可变区：
C1-hu1:SEQ ID NO:5
Figure PCTCN2022089289-appb-000002
C1-hu2:SEQ ID NO:6
Figure PCTCN2022089289-appb-000003
C1-hu3:SEQ ID NO:7
Figure PCTCN2022089289-appb-000004
Figure PCTCN2022089289-appb-000005
C1-hu4:SEQ ID NO:8
Figure PCTCN2022089289-appb-000006
C1-hu5:SEQ ID NO:9
Figure PCTCN2022089289-appb-000007
C1-hu6:SEQ ID NO:10
Figure PCTCN2022089289-appb-000008
C1-hu7:SEQ ID NO:11
Figure PCTCN2022089289-appb-000009
C1-hu8:SEQ ID NO:12
Figure PCTCN2022089289-appb-000010
C1-hu9:SEQ ID NO:13
Figure PCTCN2022089289-appb-000011
C1-hu10:SEQ ID NO:14
Figure PCTCN2022089289-appb-000012
C1-hu11:SEQ ID NO:15
Figure PCTCN2022089289-appb-000013
Figure PCTCN2022089289-appb-000014
C1-hu12:SEQ ID NO:16
Figure PCTCN2022089289-appb-000015
C1-hu13:SEQ ID NO:17
Figure PCTCN2022089289-appb-000016
C1-hu14:SEQ ID NO:18
Figure PCTCN2022089289-appb-000017
C1-hu15:SEQ ID NO:19
Figure PCTCN2022089289-appb-000018
C1-hu16:SEQ ID NO:20
Figure PCTCN2022089289-appb-000019
C1-hu17：SEQ ID NO:21
Figure PCTCN2022089289-appb-000020
C1-hu18：SEQ ID NO:22
Figure PCTCN2022089289-appb-000021
C1-hu19：SEQ ID NO:23
Figure PCTCN2022089289-appb-000022
Figure PCTCN2022089289-appb-000023
C1-hu20：SEQ ID NO:24
Figure PCTCN2022089289-appb-000024
C1-hu21：SEQ ID NO:25
Figure PCTCN2022089289-appb-000025
实施例6、人源化PD-L1单域抗体的筛选
6.1、ELISA法检测人源化单域抗体与PD-L1的结合：
将人源化抗体与嵌合抗体的结合活性进行了平行比较。使用Thermo Scientific的酶标仪，用固定浓度的His标签人PD-L1抗原(2.0μg/mL)包板，2％BSA封闭后，加入梯度稀释的上述抗体(其中C1-hu1至C1-hu6号分子1μg/ml起始，2.5倍梯度稀释，共12个浓度；C1-hu7至C1-hu16号分子，0.1μg/ml起始，2.5倍梯度稀释，共8个浓度)，将HRP偶联的鼠抗人IgG4Fc(Southern Biotech，9200-05)稀释5000倍作为检测抗体进行检测，然后用0.1mg/ml TMB显色，最后用2M盐酸溶液终止反应，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合EC 50。相对结合活性％＝嵌合抗体C1的EC 50/人源化抗体的EC50*100％。
结果见表2和图3(A-G)。
6.2、ELISA法检测人源化单域抗体抑制PD-L1与PD-1的结合：
将人源化抗体与嵌合抗体对PD-L1与PD-1结合的抑制作用进行了平行比较。使用Thermo Scientific的酶标仪，首先用链霉亲和素(Streptavidin)0.4μg/mL包板，再固定浓度的生物素化的PD-L1抗原(0.2μg/mL)与其结合。2％BSA封闭后，用固定浓度的PD-1mFc(2.0μg/mL)，对上述抗体进行梯度稀释(其中C1-hu1至C1-hu6号分子10μg/ml起始，2.5倍梯度稀释，共12个浓度；C1-hu7至C1-hu16号分子1.5μg/ml起始，2.5倍梯度稀释，共8个浓度)并加样，使两者竞争结合PD-L1抗原。将HRP偶联的羊抗鼠IgG(Fc特异性)(Sigma，A2554)稀释5000倍作为检测抗体进行检测，然后用0.1mg/ml TMB显色，最后用2M盐酸溶液终止反应，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合IC 50。相对竞争抑制活性％＝嵌合抗体huC1的IC 50/人源化抗体的IC 50*100％。结果见表2和图4(A-G)。
表2：人源化PD-L1单域抗体相对结合活性及相对竞争抑制活性
样品名称 相对结合活性，％ 相对竞争抑制活性，％
C1-hu1 45.5 53.4
C1-hu2 56.5 73.3
C1-hu5 62.5 61.7
C1-hu7 89.8 84.8
C1-hu8 91.2 93.0
C1-hu10 88.2 98.1
C1-hu11 81.3 89.1
C1-hu15 / 68.1
C1-hu16 76.6 107.1
C1-hu17 75.2 86.8
C1-hu18 83.6 93.4
C1-hu19 81.4 99.1
C1-hu20 79.4 99.7
C1-hu21 76.0 101.0
注：“/”表示未检测。
结果表明，大部分人源化分子均具有一定的结合活性和竞争抑制活性，其中，C1-hu17、C1-hu18、C1-hu20和C1-hu21号分子具有更好的结合活性和竞争抑制活性。
8种人源化抗PD-L1单域抗体的重链全长氨基酸序列如下：
C1-hu17：SEQ ID NO:27
Figure PCTCN2022089289-appb-000026
C1-hu18：SEQ ID NO:28
Figure PCTCN2022089289-appb-000027
Figure PCTCN2022089289-appb-000028
C1-hu20：SEQ ID NO:29
Figure PCTCN2022089289-appb-000029
C1-hu21：SEQ ID NO:30
Figure PCTCN2022089289-appb-000030
C1-hu7：SEQ ID NO:31
Figure PCTCN2022089289-appb-000031
C1-hu8：SEQ ID NO:32
Figure PCTCN2022089289-appb-000032
Figure PCTCN2022089289-appb-000033
C1-hu10：SEQ ID NO:33
Figure PCTCN2022089289-appb-000034
C1-hu19：SEQ ID NO:34
Figure PCTCN2022089289-appb-000035
实施例7、报告基因法检测抗体PD-L1纳米抗体对PD-1/PD-L1通路阻断试验
取CHO/PD-L1细胞(Promega)，消化并用F-12Nutrient Mixture完全培养基重悬细胞，根据细胞计数结果使用完全培养基调整细胞密度至5×10 5个细胞/ml，将细胞悬液转移至加样槽中，使用移液器以100μl/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养16～20h；第二天制备Jurkat/PD-1(Promega)细胞悬液，根据细胞计数结果使用分析培养基重悬细胞，并调整细胞密度至2.5×10 6cells/ml；将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出，使用多道移液器每孔取出100μl培养液，按照40μl/孔加入梯度稀释的本发明抗体，然后将Jurkat/PD-1细胞悬液转移至加样槽中，以40μl/孔加入到细胞培养板中，置于37℃，5％CO 2培养箱培养5～6h。取出细胞培养板，置于室温放置5～10min，然后 每孔加入40μl ONE-Glo TM荧光素酶检测系统(promega，E6130)，置于混匀器上混匀5～10min，然后使用M1000pro多功能酶标仪(Tecan)检测化学发光信号值。
本发明抗PD-L1纳米抗体均能够有效地阻断PD-1/PD-L1的相互作用，促进NFAT信号的增强，并且有药物浓度剂量依赖效应。其中，C1-hu17、C1-hu18、C1-hu19、C1-hu20和C1-hu21活性的EC 50分别为0.5914nM，0.5870nM，0.6696nM，0.5650nM，0.5843nM。
实施例8、人源化单域抗体对小鼠肿瘤生长的抑制作用
将MC38B7H1细胞以1×10 6个/0.1mL浓度接种到6-8周龄雌性B-hPD-L1人源化小鼠的右侧皮下，待肿瘤生长到约104mm 3时选取合适的动物按肿瘤体积随机分组，每组8只，共4组，分别为：G1生理盐水对照组、G2C1-hu7(10mg/kg)组、G3C1-hu8(10mg/kg)组和C1-hu10(10mg/kg)组。所有组给药途径均为腹腔注射，给药剂量为10mg/kg，给药浓度为1mg/ml。每周给药2次，连续给药6次，末次给药4天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，计算相对肿瘤抑制率TGI％(TGI％＝(1-(Ti-T0)/(Vi-V0))×100％，Ti：治疗组在给药第i天的肿瘤体积均值；T0：治疗组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值)。
如图5所示，在实验结束时，即接种肿瘤细胞后第27天，生理盐水组平均肿瘤体积为1011±91mm 3，C1-hu7组、C1-hu8组和C1-hu10组平均肿瘤体积分别为663±104mm 3，630±132mm 3和675±84mm 3，与生理盐水组相比，相对肿瘤抑制率分别为38.4％、42.0％和37.0％，且具有统计学差异(p<0.05)。表明人源化抗体可以显著抑制B-hPD-L1人源化小鼠MC38B7H1细胞皮下移植瘤的生长。

Claims (19)

  1. 一种抗PD-L1抗体或其抗原结合片段，其包含重链可变区VH，其中所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3或由其组成；或包含与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；优选地，所述与SEQ ID NO:2所示氨基酸序列具有1、2或3个氨基酸差异的HCDR2是SEQ ID NO:26。
  2. 如权利要求1所述的抗体或其抗原结合片段，其中：
    所述重链可变区的FR1的氨基酸序列包含SEQ ID NO:11或12的FR1或由其组成，或包含与SEQ ID NO:11或12的FR1区具有1、2或3个氨基酸差异的FR1或由其组成；
    所述重链可变区的FR2的氨基酸序列包含SEQ ID NO:5、10、11、14、20或24的FR2或由其组成，或包含与SEQ ID NO:5、10、11、14、20或24的FR2具有1、2或3个氨基酸差异的FR2或由其组成；
    所述重链可变区的FR3的氨基酸序列包含SEQ ID NO:6、7、8、11、13或17的FR3或由其组成，或包含与SEQ ID NO:6、7、8、11、13或17的FR3具有1、2或3个氨基酸差异的FR3或由其组成；
    所述重链可变区的FR4的氨基酸序列包含SEQ ID NO:11的FR4或由其组成，或包含与SEQ ID NO:11的FR4具有1、2或3个氨基酸差异的FR4或由其组成；
    优选地，所述重链可变区的FR1的氨基酸序列包含SEQ ID NO:11或12的FR1或由其组成，或包含与SEQ ID NO:11或12的FR1区具有1、2或3个氨基酸差异的FR1或由其组成；所述重链可变区的FR2的氨基酸序列包含SEQ ID NO:11、20或24的FR2或由其组成，或包含与SEQ ID NO:11、14、20或24的FR2具有1、2或3个氨基酸差异的FR2或由其组成；所述重链可变区的FR3的氨基酸序列包含SEQ ID NO:11的FR3或由其组成，或包含与SEQ ID NO:11的FR3具有1、2或3个氨基酸差异的FR3或由其组成；所述重链可变区的FR4的氨基酸序列包含SEQ ID NO:11的FR4或由其组成，或包含与SEQ ID NO:11的FR4具有1、2或3个氨基酸差异的FR4或由其组成。
  3. 如权利要求1所述的抗体或其抗原结合片段，其包含重链可变区VH，所述重链可变区分别包含选自SEQ ID NO:4、5、6、7、8、9、10、11、12、13、14、15、17、18、 19、20、21、22、23、24或25中任一项所示的氨基酸序列或由其组成，或包含与SEQ ID NO:4、5、6、7、8、9、10、11、12、13、14、15、17、18、19、20、21、22、23、24或25中任一项所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  4. 如权利要求1-3中任一项所述的抗体或其抗原结合片段，其包含Fc区，所述Fc区是人IgG1、IgG2、IgG3或IgG4的Fc区；优选为人IgG1的Fc区。
  5. 如权利要求1-3中任一项所述的抗体或其抗原结合片段，其中所述抗体包含重链，所述重链包含选自SEQ ID NO:27、28、29、30、31、32、33和34中任一项所示的氨基酸序列或由其组成，或包含与SEQ ID NO:27、28、29、30、31、32、33和34中任一项所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  6. 如权利要求1-5中任一项所述的抗体或其抗原结合片段，其中所述抗体是单域抗体或重链抗体。
  7. 如权利要求1-5中任一项所述的抗体或其抗原结合片段，其具有以下特性中的一种或多种：
    (1)以高亲和力结合PD-L1；
    (2)阻断PD-L1与其受体PD-1的结合作用；
  8. 如权利要求1-7中任一项所述的抗体或其抗原结合片段，其中所述抗体为嵌合抗体、人源化抗体或全人抗体。
  9. 如权利要求1-7中任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
  10. 分离的核酸分子，其编码如权利要求1-9中任一项所述的抗PD-L1抗体或其抗原结合片段。
  11. 载体，其包含如权利要求10所述的核酸分子，优选地，所述载体为真核表达载体。
  12. 宿主细胞，其包含如权利要求10所述的核酸分子，或包含如权利要求11所述的载体，或表达如权利要求1-9中任一项所述的抗体或其抗原结合片段；优选地，所述宿主细胞是真核细胞，更优选为哺乳动物细胞。
  13. 制备如权利要求1-12中任一项所述的抗PD-L1抗体或其抗原结合片段的方法， 所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养权利要求12所述的宿主细胞，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
  14. 免疫缀合物，其包含如权利要求1-9中任一项所述抗体或其抗原结合片段和其它物质，例如治疗剂或诊断剂。
  15. 药物组合物，其包含如权利要求1-9中任一项所述的抗PD-L1抗体或其抗原结合片段或权利要求14所述的免疫缀合物，和药学上可接受的载体或赋形剂。
  16. 组合产品，其包含如权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求14所述的免疫缀合物或权利要求15所述的药物组合物，以及一种或多种另外的治疗剂。
  17. 如权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求14所述的免疫缀合物、权利要求15所述的药物组合物或权利要求16所述的组合产品在制备用于治疗和/或预防PD-L1介导的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选所述癌症选自具有PD-1、PD-L1或PD-L2表达升高的癌症。
  18. 试剂盒，其包括如权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求14所述的免疫缀合物、权利要求15所述的药物组合物或权利要求16所述的组合产品。
  19. 检测PD-L1在样品中的存在的方法，所述方法包括使权利要求1-9中任一项所述的抗体或其抗原结合片段或含所述抗体或其抗原结合片段与诊断剂的免疫缀合物与待检样品接触并检测是否存在该抗体或其抗原结合片段与PD-L1形成的结合物以及任选地确定该结合物含量的步骤。
PCT/CN2022/089289 2021-04-27 2022-04-26 抗pd-l1单域抗体及其用途 WO2022228431A1 (zh)

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