WO2017020802A1 - 针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白 - Google Patents

针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白 Download PDF

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WO2017020802A1
WO2017020802A1 PCT/CN2016/092680 CN2016092680W WO2017020802A1 WO 2017020802 A1 WO2017020802 A1 WO 2017020802A1 CN 2016092680 W CN2016092680 W CN 2016092680W WO 2017020802 A1 WO2017020802 A1 WO 2017020802A1
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antibody
binding molecule
cancer
tumor
cells
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PCT/CN2016/092680
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English (en)
French (fr)
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徐霆
董艳荣
王媲琳
陈亭
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苏州康宁杰瑞生物科技有限公司
思路迪（北京）医药科技有限公司
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Priority to JP2018524526A priority Critical patent/JP6946289B2/ja
Priority to CA2994339A priority patent/CA2994339C/en
Priority to BR112018002130-0A priority patent/BR112018002130A2/zh
Priority to MYPI2018700386A priority patent/MY195474A/en
Priority to EP16832290.7A priority patent/EP3348571A4/en
Priority to MX2018001387A priority patent/MX2018001387A/es
Priority to AU2016302951A priority patent/AU2016302951B2/en
Priority to CN201680031072.XA priority patent/CN107849130B/zh
Application filed by 苏州康宁杰瑞生物科技有限公司, 思路迪（北京）医药科技有限公司 filed Critical 苏州康宁杰瑞生物科技有限公司
Priority to NZ73949916A priority patent/NZ739499A/en
Priority to US15/748,438 priority patent/US20180291103A1/en
Priority to RU2018107427A priority patent/RU2715595C2/ru
Priority to KR1020187005843A priority patent/KR102138447B1/ko
Publication of WO2017020802A1 publication Critical patent/WO2017020802A1/zh
Priority to PH12018500233A priority patent/PH12018500233A1/en

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Definitions

  • the present invention relates to the field of medical biology, and discloses a single domain antibody directed against a programmed death ligand (PD-L1) and a derivative thereof.
  • the present invention discloses a Programmed Death Ligand 1 (PD-L1) binding molecule and its use, particularly in the treatment and/or prevention, or diagnosis of PD-L1-related diseases such as tumors.
  • PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA.
  • Two cell surface glycoprotein ligands, PD-L1 and PD-L2 have been identified that have been shown to down-regulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al.
  • Both PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs that bind to PD-1 but do not bind to other CD28 family members (Blank et al. 2004). It has also been shown that upregulation of PD-L1 expression on the cell surface is stimulated by IFN- ⁇ stimulation.
  • PD-L1 has been found in several murine and human cancers, including human lung cancer, ovarian cancer, colon cancer, melanoma, and various myeloma (Iwai et al. (2002), PNAS 99: 12293-7; Ohigashi et al. 2005), Clin Cancer Res 11: 2947-53).
  • the results have shown that PD-L1, which is highly expressed in tumor cells, plays an important role in the immune escape of tumors by increasing the apoptosis of T cells.
  • the researchers found that the P815 tumor cell line transfected with the PD-L1 gene can resist the cleavage of specific CTL in vitro, and it is more tumorigenic and invasive after being inoculated into mice.
  • anti-PD-L1 antibodies that are capable of binding with PD-L1 with high affinity and that block PD-1 binding to PD-L1, particularly anti-PD-L1 heavy chain single domain antibodies.
  • the inventors of the present invention obtained an anti-PD-L1 heavy chain single domain antibody (VHH) having high specificity, high affinity and high stability by screening using phage display technology.
  • VHH anti-PD-L1 heavy chain single domain antibody
  • the invention provides a PD-L1 binding molecule comprising an immunoglobulin single variable domain consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 and a human immunoglobulin Fc region.
  • the invention features a nucleic acid molecule encoding a PD-L1 binding molecule, and an expression vector and host cell comprising the nucleic acid molecule.
  • the invention also relates to a pharmaceutical composition comprising a PD-L1 binding molecule of the invention.
  • the invention also relates to methods of making the PD-L1 binding molecules described herein.
  • the invention further relates to the use of the PD-L1 binding molecules of the invention and to the use of pharmaceutical compositions, in particular for the prevention and/or treatment of diseases associated with PD-L1.
  • Figure 1 Shows the blocking effect of PD-L1 heavy chain single domain antibody on PD-1/PD-L1 interaction.
  • Figure 2 Shows the binding curve of PD-L1 heavy chain single domain antibody to PD-L1 antigen protein.
  • FIG. 1 Blocking curves showing the interaction of PD-L1 heavy chain single domain antibody with PD-1 and PD-L1.
  • Figure 5 Shows the binding curve of PD-L1 single domain antibody Fc fusion protein to PD-L1 (ELISA method).
  • FIG. 1 Blocking curve of PD-L1 single domain antibody Fc fusion protein to PD-L1/CD80 interaction (competitive ELISA method).
  • Figure 9 shows the interaction of PD-L1 single domain antibody Fc fusion protein with Jurket-PD1/PDL1 Blocking curve (FACS method)
  • FIG. 10 Flow cytometry detects the specificity of PD-L1 single domain antibody Fc fusion protein for PD-L1 protein binding.
  • Figure 11 Flow cytometry assay for binding of the PD-L1 single domain antibody Fc fusion protein to the monkey PD-L1 protein.
  • Figure 12 Shows that the PD-L1 single domain antibody Fc fusion protein recognizes a population of PD-L1 positive cells on a tissue section of a patient.
  • Figure 13 Shows the activation of PBMC by the PD-L1 single domain antibody Fc fusion protein.
  • Figure 14 Shows the activation of CD4+ T cells by the PD-L1 single domain antibody Fc fusion protein.
  • FIG. 15 Shows that PD-L1 single domain antibody Fc fusion protein stimulates secretion of IL-2.
  • Figure 16 Shows CDC and ADCC activity of PD-L1 single domain antibody Fc fusion protein with mutant Fc.
  • FIG. 1 Tumor growth curves after treatment with PD-L1 single domain antibody Fc fusion protein.
  • Figure 18 Inhibition of tumor growth by PD-L1 single domain antibody Fc fusion protein of different doses.
  • FIG. 19 Comparison of tumor growth inhibitory activity of hu56V2-Fc with 2.41.
  • Figure 20 Shows the effect of base and oxidative treatment on the activity of the PD-L1 single domain antibody Fc fusion protein.
  • antibody or “immunoglobulin” are used interchangeably unless otherwise indicated. Whether referred to herein as a heavy chain antibody or a conventional 4-chain antibody, are used as general terms to include full length antibodies, individual chains thereof, and all portions, domains or fragments thereof (including but not limited to antigen binding domains or fragments) , for example, a VHH domain or a VH/VL domain, respectively.
  • sequence as used herein (eg, in terms of "immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) is generally understood. To include both a related amino acid sequence and a nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited interpretation is required herein.
  • domain refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for the individual functional properties of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the rest of the protein and/or domain.
  • immunoglobulin domain refers to a spherical region of an antibody chain (eg, a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a spherical region.
  • An immunoglobulin domain is characterized in that it maintains an immunoglobulin folding characteristic of an antibody molecule consisting of two intercalations of about seven antiparallel beta-sheet strands, optionally arranged by a conserved disulfide bond, in two beta sheets. .
  • immunoglobulin variable domain refers to substantially referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3”, respectively.
  • An immunoglobulin domain consisting of "FR3”, and four “framework regions” of "framework region 4" or “FR4", wherein the framework regions are referred to in the art and hereinafter as “complementarity determining region 1", respectively.
  • the three “complementarity determining regions” or “CDRs” of "CDR1", “complementarity determining region 2" or “CDR2", and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the immunoglobulin variable domain confers specificity to the antigen for the antibody by having an antigen binding site.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding an antigenic epitope without pairing with other immunoglobulin variable domains.
  • An example of an immunoglobulin single variable domain within the meaning of the invention is a "domain antibody”, such as an immunoglobulin single variable domain VH and VL (VH domain and VL domain).
  • Another example of an immunoglobulin single variable domain is the "VHH domain” of Camelidae (or simply "VHH") as defined below.
  • VHH domain also known as heavy chain single domain antibody, VHH, VH H domain, VHH antibody fragment and VHH antibody, is an antigen called “heavy chain antibody” (ie “antibody lacking light chain”) Binding to the variable domains of immunoglobulins (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally transition antibodies devoid of light chains”; Nature 363,446 -448 (1993)).
  • the term “VHH domain” is used to bind the variable domain to a heavy chain variable domain (referred to herein as a "VH domain”) present in a conventional 4-chain antibody and to a conventional 4-chain antibody.
  • the light chain variable domain (which is referred to herein as the "VL domain") is distinguished.
  • the VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • -FR1 contains amino acid residues at positions 1-30,
  • - CDR1 contains amino acid residues at positions 31-35
  • -FR2 contains amino acids at positions 36-49,
  • - CDR2 contains amino acid residues at positions 50-65
  • -FR3 contains amino acid residues at positions 66-94
  • - CDR3 comprises amino acid residues at positions 95-102, and
  • - FR4 contains the amino acid residues at positions 103-113.
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (ie, according to One or more positions of the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more than the number of amino acid residues allowed by the Kabat number. base).
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • the total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domain structural and functional properties of the VHH domain and the polypeptides containing the same can be summarized as follows:
  • VHH domain which has been naturally "designed" to bind to antigen functionality in the absence of a light chain variable domain and without interaction with a light chain variable domain, can be used as a single and relatively small function
  • a sex antigen binds to a structural unit, domain or polypeptide. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies.
  • VH and VL domains are not normally suitable for practical application as single antigen-binding proteins or immunoglobulin single variable domains, but need to be
  • a functional antigen binding unit such as in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain).
  • VHH domain alone or as part of a larger polypeptide provides many advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (eg, Fab- or F(ab')2-fragments).
  • scFv single-chain antigen-binding protein
  • conventional antibody fragments eg, Fab- or F(ab')2-fragments.
  • VHH domain can be expressed from a single gene and does not require post-translational folding or modification
  • VHH domain can be easily engineered into a multivalent and multispecific format (formatted);
  • VHH domain is highly soluble and has no aggregation tendency
  • VHH domain is highly stable to heat, pH, proteases and other denaturing agents or conditions, and thus can be used without refrigeration equipment during preparation, storage or transportation, thereby achieving cost savings, time and environment;
  • VHH domain is easy to prepare and relatively inexpensive, even on the scale required for production;
  • the -VHH domain is relatively small compared to conventional 4-chain antibodies and their antigen-binding fragments (approximately 15 kDa or 1/10 of the size of conventional IgG) and thus displays higher compared to conventional 4-chain antibodies and their antigen-binding fragments. Tissue permeability and can be administered at higher doses;
  • the -VHH domain can display so-called cavity binding properties (especially due to its extended CDR3 loop compared to conventional VH domains), such that targets and epitopes that are unreachable to conventional 4-chain antibodies and antigen-binding fragments thereof can be reached.
  • the VHH domain derived from the camelid family can be substituted for one or more amino acid residues in the amino acid sequence of the original VHH sequence by one or more amino acid residues present at corresponding positions in the VH domain of the human conventional 4-chain antibody.
  • Humanization The humanized VHH domain may contain one or more fully human framework region sequences, and in a particular embodiment, may comprise a human framework region sequence of IGHV3.
  • the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen binding molecule or antigen binding protein (eg, an immunoglobulin single variable domain of the invention) can bind.
  • the specificity can be determined based on the affinity and/or affinity of the antigen binding molecule.
  • the affinity expressed by the dissociation equilibrium constant (KD) of the antigen and the antigen binding protein is a measure of the binding strength between the epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the epitope and the antigen binding molecule The stronger the bond strength (or the affinity can also be expressed as the association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner, depending on the particular antigen of interest.
  • Affinity is a measure of the strength of binding between an antigen binding molecule (eg, an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide comprising the same) and a related antigen. Affinity is related to both the affinity between the antigen binding site on its antigen binding molecule and the number of related binding sites present on the antigen binding molecule.
  • the specific binding molecule will be preferably 10 -7 to 10 -11 moles per liter (M), more preferably 10 -8 to 10 -11 moles per liter, even more preferably 10 as measured in the Biacore or KinExA assay.
  • KD dissociation constant
  • KA association constant
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays described herein (eg, Radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays.
  • SPR surface plasmon resonance
  • RIA Radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competitive assays sandwich competitive assays.
  • amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as are well known and agreed upon in the art.
  • amino acid difference refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence.
  • substitution will preferably be a conservative amino acid substitution, which means that the amino acid residue is replaced by another amino acid residue of similar chemical structure and its effect on the function, activity or other biological properties of the polypeptide. Smaller or substantially unaffected.
  • conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably such that one amino acid within groups (i)-(v) is substituted with another amino acid residue within the same group: (i) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) Polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) Polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is replaced by Ala Or Pro substituted; His is replaced by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Substituent; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is replaced by Trp or Phe; Val is substituted by Ile or Leu.
  • a reaction medium or medium that is derived from its natural biological source and/or obtains the polypeptide or nucleic acid molecule when it has been associated with at least one other component normally associated with it in the source or medium (medium) eg
  • a polypeptide or nucleic acid molecule is considered “substantially separated” when it is separated from another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component.
  • the polypeptide or nucleic acid molecule is considered to be “based" when it has been purified at least 2 fold, in particular at least 10 fold, more particularly at least 100 fold and up to 1000 fold or more.
  • the "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable for chromatographic techniques, such as polyacrylamide gel electrophoresis).
  • subject means a mammal, especially a primate, especially a human.
  • the present invention provides a PD-L1 binding molecule capable of specifically binding to PD-L1 and consisting of the amino acid sequence of the formula ALB, wherein A represents an immunoglobulin single variable domain and L represents an amino acid linker Or absent, and B represents the human immunoglobulin Fc region,
  • immunoglobulin single variable domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6,
  • the PD-L1 binding molecule is capable of forming a homodimer in the form of (ALB) 2 through the human immunoglobulin Fc region.
  • human immunoglobulin Fc region refers to a constant region derived from human IgG1, IgG2, IgG3 or IgG4 (the amino acid sequence of the constant region thereof is referred to the entry P01857, P01859 in the protein database of www.uniprot.org, respectively).
  • the Fc region of P01860, P01861) comprising a hinge region (Hinge) or a partial hinge region, a CH2 region, and a CH3 region of an immunoglobulin constant region.
  • the amino acid sequence of the "human immunoglobulin Fc region” can increase or eliminate Fc-mediated ADCC or CDC activity or enhance or attenuate the affinity of FcRn by mutating 1-5 amino acids on the CH2 region; Mutation of 1-4 amino acids in the Hinge region increases protein stability.
  • the "human immunoglobulin Fc region” does not include a mutation that promotes or prevents the formation of an Fc heterodimer, and does not include a modification that increases the sequence of other functional proteins at the nitrogen or carbon terminal of the Fc fragment.
  • the human immunoglobulin Fc region is mutated to remove ADCC and CDC activity.
  • the amino acid sequence of the immunoglobulin Fc region is selected from the group consisting of SEQ ID NOs: 7-9.
  • the PD-L1 binding molecules of the invention will generally be in the form of homodimers under the action of the human immunoglobulin Fc region.
  • amino acid linker refers to a non-functional amino acid sequence that is 1-20 amino acid residues in length and has no secondary structure or higher.
  • the amino acid linker is, for example, a flexible linker such as GGGGS, GS, GAP, and the like.
  • the PD-L1 binding molecule of the invention is selected from the group consisting of SEQ ID NO: amino acid sequence composition of 10-27.
  • the PD-L1 binding molecule of the invention has at least one of the following characteristics:
  • the PD-L1 binding molecule of the present invention may have a KD value of less than 1 ⁇ 10 -7 M, preferably less than 1 ⁇ 10 -8 M, more preferably less than 1 ⁇ 10 -9 M, more preferably less than 1 ⁇ . 10 -10 M, especially more preferably less than 1 x 10 -11 M.
  • a PD-L1 binding molecule of the invention is capable of specifically binding to human PD-L1 and blocking the interaction of PD-L1 and PD-1. In some embodiments, a PD-L1 binding molecule of the invention is capable of specifically binding to human PD-L1 and blocking the interaction of PD-L1 and CD80.
  • the PD-L1 binding molecules of the invention are capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60% More preferably, it is at least about 70%, more preferably at least about 80%.
  • the PD-L1 binding molecule of the present invention is resistant to alkali treatment and oxidation treatment.
  • the PD-L1 binding molecule activity of the present invention remains unchanged after treatment with a strong base (e.g., 500 mM ammonium bicarbonate) for about 8 hours, preferably about 16 hours, more preferably about 24 hours or more preferably about 32 hours.
  • a strong base e.g., 500 mM ammonium bicarbonate
  • an oxidizing agent 1% hydrogen peroxide
  • the PD-L1 binding molecule of the present invention has stability at a high concentration.
  • the PD-L1 binding molecules of the invention remain stable without aggregation.
  • the invention features a nucleic acid molecule encoding a PD-L1 binding molecule of the invention.
  • the nucleic acid of the invention may be RNA, DNA or cDNA.
  • the nucleic acid of the invention is a substantially isolated nucleic acid.
  • the nucleic acids of the invention may also be in the form of a vector, which may be present in the vector and/or may be part of a vector such as a plasmid, a co-end plasmid or YAC.
  • the vector may especially be an expression vector, ie A vector in which the PD-L1 binding molecule is expressed in vitro and/or in vivo (i.e., in a suitable host cell, host organism, and/or expression system) can be provided.
  • the expression vector typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.).
  • regulatory elements and other elements useful or necessary for expression of the PD-L1 binding molecule of the present invention such as a promoter, an enhancer, a terminator, an integration factor, a selection marker, a leader sequence, a reporter gene.
  • the nucleic acids of the invention may be prepared or obtained in a known manner (for example by automated DNA synthesis and/or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptides of the invention presented herein, and/or may be from a suitable natural Sources are separated.
  • the invention in another aspect, relates to a host cell expressing or capable of expressing one or more PD-L1 binding molecules of the invention and/or comprising a nucleic acid or vector of the invention.
  • Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include Gram-negative bacterial strains (eg, Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (eg, Bacillus) A cell of a genus Bacillus, a strain of Streptomyces, a strain of Staphylococcus, and a strain of Lactococcus.
  • Gram-negative bacterial strains eg, Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains eg, Bacillus
  • Bacillus A cell of a genus Bacillus, a strain of Streptomyces, a strain of Staphylococcus, and a strain of Lactococcus.
  • Suitable fungal cells include cells of the genus Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (eg, Saccharomyces cerevisiae), fission Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Han A cell of the species of the genus Hansenula.
  • Saccharomyces eg, Saccharomyces cerevisiae
  • fission Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cell in the art for expressing a heterologous protein can also be used in the present invention.
  • the invention also provides a method of producing a PD-L1 binding molecule of the invention, the method generally comprising the steps of:
  • the PD-L1 binding molecules of the invention are produced using mammalian cells.
  • the PD-L1 binding molecule of the present invention can achieve high expression in mammalian cells.
  • the expression level can be up to about 5 g/L, preferably about 6 g/L, preferably about 7 g/L, preferably about 8 g/L, more preferably about 9 g/L or more preferably about 10 g/L or more.
  • the PD-L1 binding molecule of the invention may be produced in an intracellular manner (eg, in the cytoplasm, in the periplasm, or in inclusion bodies) in a cell as described above, followed by isolation from the host cell and optionally further purification; or it may be a cell It is produced in an external manner (for example in a medium in which the host cells are cultured), followed by isolation from the medium and optionally further purification.
  • the PD-L1 binding molecules of the invention can also be obtained by other methods of protein production known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.
  • the invention provides a composition, such as a pharmaceutical composition, comprising a PD-L1 binding molecule of the invention in one or a combination formulated with a pharmaceutically acceptable carrier.
  • a composition such as a pharmaceutical composition, comprising a PD-L1 binding molecule of the invention in one or a combination formulated with a pharmaceutically acceptable carrier.
  • Such compositions may comprise one or a combination (e.g., two or more different) PD-L1 binding molecules of the invention.
  • the PD-L1 binding molecules of the invention will generally be in the form of homodimers under the action of the human immunoglobulin Fc region.
  • compositions of the invention may also be administered in combination therapy, i.e., in combination with other agents.
  • a combination therapy can include a PD-L1 binding molecule of the invention in combination with at least one other anti-tumor drug.
  • a PD-L1 binding molecule of the invention can be used in combination with an antibody that targets other tumor-specific antigens.
  • antibodies that target other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, antibodies against EGFR variants, anti-VEGFa antibodies, anti-HER2 antibodies, or anti-CMET antibodies.
  • the antibody is a monoclonal antibody.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., the antibody molecule, immunoconjugate
  • the active compound can be encapsulated in a material to protect the compound from acids and other natural conditions that can inactivate the compound.
  • compositions of the invention may comprise one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound without causing any undesirable toxicological effects (see, eg, Berge, SM et al. (1977) J. Pharm. Sci. 66:1). -19).
  • Examples of such salts include acid addition salts and base addition salts.
  • the acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and from, for example, aliphatic monocarboxylic acids and dicarboxylic acids, phenyl groups.
  • Non-toxic organic acid-derived salts such as substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids.
  • Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, and such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine
  • Non-toxic organic amine derived salts such as choline, diethanolamine, ethylenediamine, procaine.
  • compositions of the invention may also contain a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA) ), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • the prevention of the presence of microorganisms can be ensured by a sterilization procedure or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like.
  • various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like.
  • isotonic agents for example, sugars, polyols such as mannitol, sorbitol or sodium oxide.
  • Prolonged absorption of the injectable drug can be achieved by the addition of a delay absorbent such as monostearate and gelatin to the composition.
  • the pharmaceutically acceptable carrier includes a sterile aqueous solution or dispersion and a powder for the temporary preparation of a sterile injectable solution or dispersion.
  • a sterile aqueous solution or dispersion and a powder for the temporary preparation of a sterile injectable solution or dispersion.
  • Supplementary active compounds can also be incorporated into the compositions.
  • compositions must generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations.
  • the carrier can be a solvent or dispersing agent containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • Sterile injectable solutions can be prepared by incorporating the active compound in a suitable amount in a suitable solvent, and, if necessary, one or a combination of the ingredients listed above, followed by sterile microfiltration.
  • dispersing agents are prepared by incorporating the active compound into a sterile vehicle which may contain a base dispersion medium and the other ingredients listed above.
  • the preferred preparation methods are vacuum drying and lyophilization (lyophilization), a solution which is pre-sterilized in such a manner as a powder of the active ingredient plus any additional desired ingredients.
  • the amount of active ingredient that can be combined with the carrier materials in a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient that can be combined with the carrier materials in a single dosage form is generally the amount of the composition that produces the therapeutic effect. Typically, this amount will range from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, by weight of the active ingredient, and pharmaceutically acceptable The carrier is combined.
  • the dosage regimen can be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be reduced or increased proportionally as needed for an emergency condition of the treatment condition. It is especially advantageous to formulate the parenteral compositions in dosage unit form for ease of administration and uniformity.
  • Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound which is calculated to produce the active compound in association with the required pharmaceutical carrier. The desired therapeutic effect.
  • the specific description of the dosage unit form of the present invention is limited to and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) inherent in the art for formulating such sensitivity for treating an individual. Limitation of active compounds.
  • the dosage range is about 0.0001 to 100 mg/kg, which is more Often 0.01 to 20 mg/kg of recipient weight.
  • the dose may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or in the range of 1-20 mg/kg.
  • An exemplary treatment regimen requires weekly dosing, biweekly, biweekly, biweekly, monthly, once every 3 months, every 3-6 months, or initial dosing interval Slightly shorter (eg once a week to once every three weeks) the post-dosing interval is lengthened (eg once a month to once every 3-6 months).
  • the antibody molecule can also be administered as a sustained release formulation, in which case less frequent dosing is required.
  • the dose and frequency will vary depending on the half-life of the antibody molecule in the patient.
  • human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies.
  • the dosage and frequency of administration vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over long periods of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient exhibits partial or complete improvement in disease symptoms. Thereafter, the patient can be administered in a prophylactic regimen.
  • the actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to achieve an amount of active ingredient which is effective to achieve the desired therapeutic response to a particular patient, composition and mode of administration without toxicity to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, and the therapeutic Duration, other drugs, compounds and/or materials used in conjunction with the particular composition of the application, age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.
  • the "therapeutically effective amount" of the PD-L1 binding molecule of the present invention preferably results in a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic phase of the disease, or prevention of damage or disability caused by the pain of the disease.
  • a "therapeutically effective amount” preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, relative to a subject not receiving treatment. More preferably, it is at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.
  • the ability to inhibit tumor growth can be evaluated in animal model systems that predict the efficacy of human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art.
  • Therapeutically effective amount Therapeutic compounds can reduce tumor size or otherwise alleviate the symptoms of the subject. One skilled in the art can determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • compositions of the invention may be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will appreciate that the route and/or manner of administration will vary depending on the desired result.
  • Preferred routes of administration for the PD-L1 binding molecules of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually injections, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.
  • the PD-L1 binding molecules of the invention may also be administered by parenteral routes, such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • parenteral routes such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compound can be prepared with carriers which are not fast-released, such as a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microcapsule delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • Therapeutic compositions can be administered using medical devices well known in the art.
  • the therapeutic compositions of the present invention can be administered by a needleless hypodermic injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • a needleless hypodermic injection device such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • Examples of well-known implants and modules that can be used in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispersing a drug at a controlled rate; U.S. Patent No. 4,486,194 The patent discloses a therapeutic device for administration through the skin; U.S. Patent No.
  • the PD-L1 binding molecules of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) blocks many highly hydrophilic compounds.
  • the therapeutic compounds of the invention can be formulated, for example, in liposomes.
  • liposomes For a method of preparing a liposome, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548 and 5,399,331.
  • Liposomes comprise one or more targeting moieties that can be selectively transported into a particular cell or organ to enhance targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685 ).
  • targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannoside (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PGBloeman et al. 1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); Surfactant Protein A Receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); P120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.
  • the present invention provides the use and method of the PD-L1 binding molecule, nucleic acid molecule, host cell and pharmaceutical composition of the present invention for preventing and/or treating a PD-L1-related disease.
  • the PD-L1-related diseases which can be prevented and/or treated with the PD-L1 binding molecule of the present invention are described in detail below.
  • Blockade of PD-L1 by the PD-L1 binding molecule of the invention enhances the immune response to cancer cells in a patient.
  • PD-L1 is enriched in a variety of human cancers (Dong et al. (2002) Nat Med. 8: 787-9).
  • the interaction of PD-1 with PD-L1 leads to a decrease in lymphocytes infiltrating tumors, a decrease in T cell receptor-mediated proliferation, and an immune escape of cancer cells (Dong et al. (2003) J Mol Med 81:281-7; Blank et al. (2004) Cancer Immunol Immunother [epub]; Konishi et al (2004) Clin Cancer Res 10: 5094-5100).
  • the effect can reverse immunosuppression.
  • the interaction between PD-L2 and PD-1 is also blocked, the effect is synergistic (Iwai et al. (2002) PNAS 99: 12293-7; Brown et al. (2003) J Immunol 170: 1257- 66).
  • the PD-L1 binding molecule of the present invention can be used alone to inhibit the growth of cancerous tumors.
  • the PD-L1 binding molecules of the invention can be used in combination with other anti-tumor therapies, for example, in combination with other immunogenic agents, standard cancer therapies, or other antibody molecules.
  • the invention provides a method of preventing and/or treating cancer comprising administering to the subject a therapeutically effective amount of a PD-L1 binding molecule of the invention, inhibiting tumor cell growth in a subject.
  • Preferred cancers which can be prevented and/or treated using the PD-L1 binding molecules of the invention include cancers which are generally responsive to immunotherapy.
  • Non-limiting examples of preferred cancers that can be treated include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (eg, metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant blood.
  • Disease, head and neck cancer glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine tumor and osteosarcoma.
  • cancers examples include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrium Cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, Renal squamous cell carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis,
  • CNS central nervous system
  • the PD-L1 binding molecules of the invention can be transfected with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells transfected with genes encoding immunostimulatory cytokines Used in combination (He et al. (2004) J. Immunol 173:4919-28).
  • immunogenic agents include peptides of melanoma antigens, such as peptides of gp100, MAGE antigen, Trp-2, MART1 and/or tyrosinase, or expressed cells after transfection Tumor cells of the factor GM-CSF.
  • a PD-L1 blocker (such as an anti-PD-L1 antibody, such as a PD-L1 binding molecule of the invention) may be most effective when combined with a tumor vaccination regimen.
  • GM-CSF has been shown to be a strong activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proa Natl. Acad. Sci U.S.A. 90: 3539-43).
  • tumor-specific antigens are differentiation antigens expressed in tumors and tumor-producing cells, such as gp100, MAGE antigen, and Trp-2. More importantly, many of these antigens were shown to be targets for tumor-specific T cells found in the host.
  • the PD-L1 binding molecules of the invention can be used in combination with recombinantly produced tumor-specific proteins and/or peptides to generate an immune response against these proteins. These proteins are normally considered to be autoantigens by the immune system and are therefore tolerated.
  • Tumor antigens may also include protein telomerase, which is required for telomere synthesis of chromosomes and is expressed in more than 85% of human cancers, but only in a limited number of autologous tissues (Kim, N, et al. (1994). ) Science 266: 2011-2013).
  • the tumor antigen can also be a "new antigen" expressed by cancer cells, such as a fusion protein that changes a protein sequence due to somatic mutation or that produces two unrelated sequences (eg, bcr-abl in the Philadelphia chromosome).
  • tumor vaccines may include proteins from viruses associated with human cancer, such as human papillomavirus (HPV), hepatitis virus (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV).
  • HPV human papillomavirus
  • HBV and HCV hepatitis virus
  • KHSV Kaposi's herpes sarcoma virus
  • PD-L1 blocker such as an anti-PD-L1 antibody, such as the PD-L1 of the present invention
  • Another form of tumor-specific antigen that is used in combination with a molecule is a purified heat shock protein (HSP) isolated from tumor tissue itself.
  • HSP purified heat shock protein
  • heat shock proteins contain fragments of proteins from tumor cells that are very effective in delivering to antigen presenting cells to elicit tumor immunity (Suot, R and Srivastava, P (1995) Science 269: 1585-1588; Tamura, Y. Et al. (1997) Science 278: 117-120).
  • DCs Dendritic cells
  • DCs Dendritic cells
  • DCs are strong antigen presenting cells that can be used to elicit antigen-specific responses.
  • DCs can be produced in vitro and carry various protein and peptide antigens as well as tumor cell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332).
  • DCs can also be transduced by genetic means to express these tumor antigens as well. DCs have been directly fused to tumor cells for immunization (Kugler, A. et al. (2000) Nature Medicine 6: 332-336).
  • DC immunization can be effectively combined with a PD-L1 blocker (such as an anti-PD-L1 antibody, such as the PD-L1 binding molecule of the present invention) to activate a stronger anti-tumor response.
  • a PD-L1 blocker such as an anti-PD-L1 antibody, such as the PD-L1 binding molecule of the present invention
  • CAR-T the full name of Chimeric Antigen Receptor T-Cell Immunotherapy, is another effective cell therapy for malignant tumors.
  • a chimeric antigen receptor T cell (CAR-T cell) is an antigen binding portion of an antibody capable of recognizing a certain tumor antigen and an intracellular portion of a CD3- ⁇ chain or Fc ⁇ RI ⁇ is coupled in vitro as a chimeric protein.
  • the transduction method transfects a patient's T cells to express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • co-stimulatory molecular signal sequences can also be introduced to increase the cytotoxic activity, proliferation and survival time of T cells, and promote the release of cytokines.
  • PD-L1 blockers (such as anti-PD-L1 antibodies, such as the PD-L1 binding molecules of the invention) can be combined with CAR-T cell therapy to activate a stronger anti-tumor response.
  • the PD-L1 binding molecules of the invention can also be combined with standard cancer therapies.
  • the PD-L1 binding molecules of the invention can be effectively combined with a chemotherapy regimen. In these examples, it can reduce the dose of chemotherapeutic agent administered (Mokyr, M. et al. (1998) Cancer Research 58: 5301-5304).
  • An example of such a combination is the use of an anti-PD-L1 antibody in combination with ampicillin for the treatment of melanoma.
  • Another example of such a combination is the anti-PD-L1 antibody in combination with interleukin-2 (IL-2) for the treatment of melanoma.
  • IL-2 interleukin-2
  • the scientific principle of the combination of the PD-L1 binding molecule of the present invention and chemotherapy is cell death, which is the result of the cytotoxic effects of most chemotherapeutic compounds and should result in elevated levels of tumor antigens in the antigen presentation pathway. Can cooperate with cell death through PD-L1 blockade Other combination therapies for action have radiotherapy, surgery, and hormone deprivation. These protocols all produce a source of tumor antigen in the host.
  • An angiogenesis inhibitor can also be combined with the PD-L1 binding molecule of the invention. Inhibition of angiogenesis results in tumor cell death, which can provide tumor antigens to the host's antigen presentation pathway.
  • the PD-L1 binding molecules of the invention can also be used in combination with antibodies that target other tumor-specific antigens.
  • antibodies that target other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, antibodies against EGFR variants, anti-VEGFa antibodies, anti-HER2 antibodies, or anti-CMET antibodies.
  • the antibody is a monoclonal antibody.
  • the PD-L1 binding molecule of the present invention can also be used in combination with a bispecific antigen that targets Fc ⁇ or Fc ⁇ receptor expression effector cells to tumor cells (see, for example, US Patent Nos. 5,922.845 and 5,837,243).
  • Bispecific antibodies can also be utilized to target two different antigens.
  • macrophages have been targeted to tumor sites using anti-Fc receptor/anti-tumor antigen (eg, Her-2/neu) bispecific antibodies. This targeting can activate tumor-specific responses more efficiently.
  • the T cell aspect of these responses can be enhanced with PD-L1 blockers.
  • antigen can be delivered directly to DC using a bispecific antibody that binds to tumor antigens and dendritic cell-specific cell surface markers.
  • Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating tumor-expressing immunosuppressive proteins. In particular, it includes TGF- ⁇ (Kehr L J. et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. and O'Garra, A. (1992) Immunology Today 13: 198- 200) and Fas ligand (Hahne, M. et al. (1996) Science 274: 1363-1365). An antibody of each of these can be used in combination with the PD-L1 binding molecule of the present invention to counteract the action of an immunosuppressive agent and to facilitate a tumor immune response of the host.
  • T cell helper activity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in combination with the PD-L1 binding molecule of the present invention (Ito, N. et al. (2000). ) Immunobiology 201 (5) 527-40). It is also possible to combine T cell costimulatory molecules such as OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) in order to increase the level of T cell activation.
  • OX-40 Weinberg, A. et al. (2000) Immunol 164: 2160-2169
  • 4-1BB Melero, I. et al. (1997) in order to increase the level of T cell activation.
  • Bone marrow transplantation is currently used to treat a variety of tumors of hematopoietic origin. Graft versus host disease is a consequence of this treatment, and the response of the graft to the tumor can provide a therapeutic benefit.
  • PD-L1 blockers can be utilized to increase the effectiveness of tumor-specific T cells.
  • Ex vivo activation in the presence of the PD-L1 binding molecule of the invention is expected to increase the frequency and activity of adoptively transferred T cells. Accordingly, the present invention also provides a method of activating an immune cell (such as PBMC or T cell) ex vivo, comprising contacting the immune cell with a PD-L1 binding molecule of the present invention.
  • an immune cell such as PBMC or T cell
  • Another aspect of the present invention provides a method for preventing and/or treating an infectious disease in a subject, comprising administering to the subject a PD-L1 binding molecule of the present invention, such that the infectious disease of the subject is prevented and / or treatment.
  • PD-L1 blockers can be used alone or in combination with vaccines to stimulate immune responses to pathogens, toxins and autoantigens.
  • pathogens to which this treatment method can be particularly applied include pathogens that currently have no effective vaccine, or pathogens that are not fully effective in conventional vaccines. These include, but are not limited to, HIV, hepatitis virus (A, B, C), influenza virus, herpes virus, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.
  • PD-L1 blockers are particularly useful against infections that have been established by pathogens such as HIV, which present altered antigens during infection. Upon administration of an anti-human PD-L1 antibody, these new epitopes are recognized as foreign sources, resulting in a strong T cell response that is unaffected by the negative signal of PD-L1.
  • Virus adenovirus, influenza virus, arbovirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, small Virus, vaccinia virus, HTLV virus, Dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, JC virus and arbovirus encephalitis virus.
  • pathogenic bacteria that cause infectious diseases that can be treated by the methods of the invention include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, Neisseria gonorrhoeae, Krebs Bacillus, Proteus, R., Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botox, Bacillus, Bacillus, Leptospira, and Lyme Bacterial bacteria.
  • pathogenic fungi that cause infectious diseases treatable by the methods of the invention include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Muscular, Absidia, Rhizopus), S. sphaericus, dermatitis bud, Brazil coccidioides, Coccidioides and collagen Cytoplasmic bacteria.
  • the PD-L1 blocker can be combined with other forms of immunotherapy such as cytokine therapy (eg, interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, Specific antibody treatment provides enhanced presentation of tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).
  • cytokine therapy eg, interferon, GM-CSF, G-CSF, IL-2
  • bispecific antibody therapy provides enhanced presentation of tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).
  • Anti-PD-L1 antibodies can stimulate and amplify autoimmune responses. Therefore, it is conceivable to design an vaccination regimen using an anti-PD-L1 antibody in combination with various autoproteins to efficiently produce an immune response against these autologous proteins for disease treatment.
  • Alzheimer's disease involves the improper accumulation of A ⁇ peptides in amyloid deposits in the brain; an antibody response to amyloid can clear these amyloid deposits (Schenk et al. (1999) Nature 400: 173- 177).
  • Other self-proteins can also be used as targets, such as IgE, which is involved in the treatment of allergies and asthma, and in rheumatoid arthritis. TNF ⁇ .
  • anti-PD-L1 antibodies can be used to induce antibody responses to various hormones.
  • the response of neutralizing antibodies to reproductive hormones can be used for contraception.
  • the response of neutralizing antibodies to hormones and other soluble factors required for specific tumor growth can also be considered as a possible vaccination target.
  • a similar method using an anti-PD-L1 antibody can be used to induce a therapeutic autoimmune response to treat patients with inappropriate autoantigen accumulation, such as amyloid deposits including A ⁇ in Alzheimer's disease.
  • Cytokines such as TNF ⁇ and IgE.
  • Anti-PD-L1 antibodies can also be used to treat diseases such as chronic inflammatory diseases such as lichen planus, T cell-mediated chronic inflammatory skin mucosal disease (Youngnak-Piboonratanakit et al. (2004) Immunol Letters 94; 215-22). . Accordingly, in one aspect, the invention provides a method of eliminating chronic inflammatory disease with T cells comprising administering to a subject a PD-L1 binding molecule of the invention.
  • One aspect of the invention provides the use of a PD-L1 binding molecule of the invention as a vaccine adjuvant.
  • a PD-L1 binding molecule of the invention By co-administering an anti-PD-L1 antibody and a target antigen (eg, a vaccine), an anti-PD-L1 antibody can be utilized to increase a specific immune response against the antigen.
  • a target antigen eg, a vaccine
  • one aspect of the invention provides a method of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) an antigen; and (ii) a PD-L1 binding molecule of the invention such that The immune response of the antigen is enhanced.
  • the antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen.
  • Non-limiting examples of such antigens include those described in the above sections, such as the tumor antigens (or tumor vaccines) described above, or antigens from the above viruses, bacteria or other pathogens.
  • kits comprising a PD-L1 binding molecule, immunoconjugate or pharmaceutical composition of the invention, and instructions for use.
  • the kit may further comprise at least one additional reagent or one or more additional PD-L1 binding molecules of the invention (eg, a binding molecule that binds to a different epitope of PD-L1).
  • Kits typically include a label indicating the intended use of the contents of the kit. The term label is included on the kit or with the kit Any written or recorded material provided or otherwise provided with the kit.
  • the PDL1-Fc fusion protein for immunization (SEQ ID NO: 28) was expressed by CHO cells (pCDNA4, Invitrogen, Cat V86220) and purified by Protein A affinity chromatography.
  • a Xinjiang Bactrian camel (Camelus bactrianus) was selected for immunization.
  • 100 ml of peripheral blood lymphocytes of camel were extracted and total RNA was extracted using an RNA extraction kit supplied by QIAGEN, and the extracted RNA was reverse-transcribed into cDNA using the Super-Script III FIRST STRANDSUPERMIX kit according to the instructions.
  • a nucleic acid fragment encoding a variable region of a heavy chain antibody is amplified by nested PCR:
  • Upstream primer GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 29);
  • Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 30).
  • Upstream primer GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 31);
  • Downstream primer GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT (SEQ ID NO: 32).
  • the target heavy chain single domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pCDisplay-3 (Creative Biolabs, Cat: VPT4023) using restriction endonucleases (purchased from NEB) PstI and NotI.
  • the product was subsequently electrotransformed into E. coli electroporation competent cells TG1, and a heavy chain single domain antibody phage display library against PD-L1 was constructed and assayed.
  • the size of the storage capacity was calculated to be 1.33 ⁇ 10 8 by gradient dilution plating.
  • To detect the insertion rate of the library 24 clones were randomly selected for colony PCR. The results show that the insertion rate has reached 100%.
  • the plate was coated with PDL1-Fc fusion protein at 10 ⁇ g/well and placed at 4 ° C overnight. The next day, after blocking with 1% skim milk for 2 hours at room temperature, 100 ⁇ l of phage (8 ⁇ 10 11 tfu, camel heavy chain single domain antibody phage display library constructed from 1.1) was added and allowed to act at room temperature for 1 hour. Thereafter, it was washed 5 times with PBST (0.05% Tween 20 in PBS) to wash away unbound phage. Finally, the phage specifically binding to PD-L1 was dissociated with triethylamine (100 mM), and E.
  • PBST 0.05% Tween 20 in PBS
  • a secondary anti-mouse anti-mouse alkaline phosphatase-labeled antibody purchased from Amytech Co., Ltd.
  • an alkaline phosphatase coloring solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the sample well OD value is more than 3 times the OD value of the control well, it is judged as a positive clone hole.
  • the bacteria of the positive clone wells were transferred to an LB liquid containing 100 ⁇ g per ml of ampicillin to extract a plasmid and perform sequencing.
  • the protein sequences of the individual clones were analyzed according to the sequence alignment software Vector NTI. Clones having the same CDR1, CDR2, and CDR3 sequences were regarded as the same antibody strain, and clones having different CDR sequences were regarded as different antibody strains.
  • the coding sequence of the heavy chain single domain antibody obtained by sequencing analysis was subcloned into the expression vector PET32b (Novagen, product number: 69016-3), and the correct recombinant plasmid identified by sequencing was transformed into the expression host strain BL1 (DE3). (Tiangen Biotechnology, product number: CB105-02), which was coated on a plate containing 100 ⁇ g of LB solid medium per ml of ampicillin at 37 ° C overnight. Single colonies were selected for inoculation and culture overnight. The next day, the overnight strains were transferred and expanded, and shaken at 37 ° C until the OD value reached 0.6-1, induced by 0.5 mM IPTG, and shaken overnight at 28 °C.
  • the bacteria were collected by centrifugation, and the cells were disrupted to obtain a crude antibody extract.
  • the antibody protein is then purified by a nickel ion affinity chromatography column. Finally, an antibody protein with a purity of more than 90% is obtained.
  • the plate was coated with PDL1-Fc fusion protein at 4 ° C overnight, and 100 ng of the heavy chain single domain antibody obtained in Example 2.1 (control group was a single domain antibody not bound to PDL1-Fc protein) was added to each well, and reacted at room temperature for 1 hour.
  • a mouse anti-His tag antibody purchased from Beijing Kangwei Century Biotechnology Co., Ltd.
  • a secondary anti-goat goat anti-mouse horseradish peroxidase-labeled antibody (Shenzhou, Catalina, Catalina: SSA007200) was added and reacted at room temperature for 1 hour.
  • a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the plate was coated with Fc protein at 4 ° C overnight, and 100 ng of the heavy chain single domain antibody obtained in Example 2.1 (the control group was a single domain antibody against other unrelated targets) was added to each well, and reacted at room temperature for 1 hour. After washing, an anti-rabbit anti-human Fc antibody (purchased from Shanghai Puxin Biotechnology Co., Ltd.) was added and reacted at room temperature for 1 hour. After washing, a secondary anti-goat anti-rabbit horseradish peroxidase-labeled antibody (purchased from Shanghai Puxin Biotechnology Co., Ltd.) was added and reacted at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • Antibody strain OD for PD-L1 OD (for Fc) OD (PD-L1/Fc) OD (PD-L1/blank) SEQ ID NO PDL1-56dAb 2.931 0.068 43.10294118 54.27777778 1 blank 0.054 0.072 0.75 1
  • Mouse PDL1-Fc protein (SEQ ID NO: 33) was obtained by expression of HEK293 cells (pCDNA4, Invitrogen, Cat V86220).
  • the mouse PDL1-Fc fusion protein was coated at 0.5 ⁇ g/well with a plate at 4 ° C overnight, and 100 ng of the heavy chain single domain antibody obtained in Example 2.1 (the control group was a single domain antibody against other unrelated targets) was added to each well at room temperature. Reaction for 1 hour. After washing, a primary anti-His tag antibody was added and reacted at room temperature for 1 hour. After washing, add secondary anti-goat anti-mouse horseradish peroxidase-labeled antibody, room temperature Reaction for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm. The results are shown in Table 2.
  • Antibody strain OD for mouse PID-L1
  • PDL1-56dAb 0.075 blank 0.096
  • the heavy chain single domain antibody of human PD-L1 of the present invention does not bind to the mouse PDL1-Fc protein.
  • the PDL1-Fc protein and the PD1-Fc protein were expressed by HEK293 cells (pCDNA4, Invitrogen, Cat V86220).
  • the biotinylated protein PD1-Fc-Biotin was obtained using a Biotinlytion kit from Thermo.
  • PDL1-Fc fusion protein 0.5 ⁇ g/well 4°C overnight coated plate, then add 100 ng of the heavy chain single domain antibody obtained in Example 2.1 per well (control group is a single domain antibody against other unrelated targets, or just buffer) And 10 ⁇ g of PD1-Fc-Biotin (no antibody or protein was added to the blank group, only an equal volume of buffer was added), and the reaction was carried out for 1 hour at room temperature. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1 hour. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm. When the sample OD value is ⁇ 0.8 compared to the control OD value, the antibody is considered to have a blocking effect.
  • the antibody strain 56 exhibited a blocking effect on the PD-1/PD-L1 interaction.
  • HEK293 cells (293-PDL1 cells) transiently expressing human PD-L1 protein on the membrane were obtained by transient transfection of human HEK293 cells with a plasmid carrying the human PD-L1 full-length protein gene (pCDNA4, Invitrogen, Cat V86220).
  • 293-PDL1 cells were taken and resuspended in 0.5% PBS-BSA buffer in a 96-well plate, and the above-mentioned antibody to be detected was added.
  • a negative control was also set up, and the negative control was 2 ⁇ g of the single target Domain antibody. All samples were spiked with 0.3 ⁇ g of hPD-1-Fc-biotin and the secondary antibody was eBioscience's SA-PE. After staining, flow cytometry was performed. If the fluorescence value is increased after the addition of the antibody to the blank direction than the antibody-free group, the antibody is considered to block the interaction of PD-L1 and PD-1 on the cell surface. In this way, antibodies capable of blocking the binding of PD-L1 antigen and PD-1 on the cell surface were identified.
  • the obtained PD-L1 heavy chain single domain antibody was coated with 0.5 ⁇ g/well at 4 ° C overnight, followed by addition of a gradient dilution series of PDL1-Fc fusion protein, and reacted at room temperature for 1 hour. After washing, a goat anti-human IgG-Fc horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, horseradish peroxidase coloring solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis. The four-parameter fit was used to obtain the antibody-to-PD-L1 binding curve and EC50 value (about 5 ng/ml). The results are shown in Figure 2.
  • the plate was coated overnight with PDL1-Fc fusion protein 0.5 ⁇ g/well 4°C, followed by a gradient dilution series of 100 uL PD-L1 blocking single domain antibody Fc fusion protein per well (100 ⁇ g/mL PD1-Fc- in the dilution) Biotin), reacted at room temperature for 1 hour. Thereafter, SA-HRP (purchased from Sigma) was added, and the mixture was reacted at room temperature for 1 hour. Thereafter, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the humanization method is carried out by a method of resurfacing the protein surface amino acid and a VHH CDR grafting to a universal framework.
  • the humanization procedure was as follows: homology modeling was performed on antibody strain 56, and the modeling software was Modeller 9.
  • the reference homologous sequence is the NbBcII10 antibody (PDB number: 3DWT), and the relative solvent accessibility of the amino acid is calculated from the three-dimensional structure of the protein. If an amino acid of antibody strain 56 is exposed to a solvent, it is replaced with an amino acid at the same position as the reference human antibody DP-47 sequence, and finally all substitutions are completed.
  • VHH humanized universal framework transplantation method The specific steps of the VHH humanized universal framework transplantation method are as follows: Firstly, the universal humanized VHH framework h-NbBcII10FGLA (PDB number: 3EAK) designed by Cécile Vincke et al. based on sequence homology is obtained.
  • the framework is based on Nanobodies.
  • the NbBcII10 antibody (PDB number: 3DWT) was subjected to humanization of protein surface amino acid with reference to human antibody DP-47, and the partial amino acid FGLA of VHH sequence framework 2 (framework-2) was engineered.
  • the antibody strain 56 was humanized to obtain a humanized variant of five antibody strains 56.
  • Table 4 lists the sequence numbers of these humanized variants as well as the amino acid changes therein, wherein the amino acid residue numbers are numbered according to Kabat.
  • Figure 4 shows the alignment of the humanized sequences.
  • the amino acid sequence of human IgG1-Fc region was obtained from the constant region amino acid sequence of human immunoglobulin gamma1 (IgG1) on the protein database Uniprot (P01857).
  • a nucleic acid fragment encoding human IgG1-Fc was obtained from human PBMC total RNA by reverse transcription PCR, and an encoding nucleic acid fragment of the fusion protein of PD-L1 single domain antibody and Fc obtained in the above Example was obtained by overlapping PCR. This was then subcloned into the vector pCDNA4 (Invitrogen, Cat V86220).
  • An Fc region sequence that removes ADCC activity or CDC activity by site-directed mutagenesis can also be used, such as SEQ ID NO: 8 or 9.
  • Recombinantly constructed single domain antibody-Fc fusion protein particles were transfected into HEK293 cells for antibody expression.
  • the recombinant expression plasmid was diluted with Freestyle 293 medium and added to the desired PET (Polyethylenimine) solution.
  • Each group of plasmid/PET mixture was separately added to the HEK293 cell suspension, and placed at 37 ° C, 10% CO 2 , 90 rpm; Add 50 ⁇ g/LIGF-1. After 4 hours, add EX293 medium, 2 mM glutamine and 50 ⁇ g/LIGF-1, 135 rpm. support. Add 3.8 mM VPA after 24 hours. After 5 to 6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target PD-L1 single domain antibody-Fc fusion protein.
  • the sequence of the obtained PD-L1 single domain antibody-Fc fusion protein is shown in SEQ ID NOs: 10-27.
  • TM004 is an anti-PD-L1 antibody of Roche, and its antibody gene was cloned by the method of YW243.55.S70.hIgG in the patent US20130045201A1, and cloned into the vector pCDNA4.
  • the recombinantly constructed plasmid was transiently transfected with HEK293 cells by the same method in 4.1.
  • the obtained anti-PD-L1 antibody of MedImmune LLC was renamed to 2.41H90P; the anti-PD-L1 antibody of Roche was renamed to 243.55.
  • the expression level of the PD-L1 single domain antibody Fc fusion protein of the present invention is higher than 200 mg/L, while the expression level of antibody 2.41H90P is about 80 mg/L, and the expression level of antibody 243.55 It is about 40 mg/L. This result indicates that the PD-L1 single domain antibody Fc fusion protein of the present invention is more stable in structure than the other two known PD-L1 antibodies, and can obtain higher expression levels.
  • Example 5 Identification of the function of the PD-L1 single domain antibody Fc fusion protein
  • the PDL1-Chis protein (SEQ ID NO: 35) was obtained by transient expression of HEK293 and purification by nickel column affinity chromatography. The obtained PDL1-Chis protein was coated at 0.5 ⁇ g/well at 4 ° C overnight, and then a gradient dilution series of the PD-L1 single domain antibody Fc fusion protein obtained in the above Example was added, and reacted at room temperature for 1 hour. After washing, a goat anti-human IgG-Fc horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fit was used to obtain the antibody-to-PD-L1 binding curve and EC50 value (all test antibody EC50 values were about 150 ng/mL) to reflect the antibody pair.
  • Fig. 5 The results are shown in Fig. 5, in which the ordinate is OD405, the abscissa is the PD-L1 single domain antibody Fc fusion protein concentration (unit ng/mL); the inverted triangle, the positive triangle, and the square represent three different humanizations of the antibody strain 56, respectively.
  • Forms of Fc fusion proteins hu56v1-Fc, hu56v2-Fc, hu56v5-Fc.
  • the affinity of the three proteins for PD-L1 is comparable.
  • the binding kinetics of the PD-L1 single domain antibody Fc fusion protein obtained in the above examples against recombinant human PD-L1 was measured by surface plasmon resonance (SRP) method using a BIAcore X100 instrument.
  • Recombinant human PDL1-Fc was directly coated on a CM5 biosensor chip to obtain approximately 1000 response units (RU).
  • the antibody was serially diluted three-fold (1.37 nm to 1000 nm) with HBS-EP+1 ⁇ buffer (GE, cat#BR-1006-69), injected at 25 °C for 120 s, and the dissociation time was 30 min. 10 mM glycine-HCl (pH 2.0) was regenerated for 120 s.
  • Binding rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2).
  • the equilibrium dissociation constant (kD) is calculated as the ratio koff/kon.
  • the binding affinities of the measured anti-PD-L1 antibodies are shown in Table 5.
  • the results showed that the affinity of PDL1-56-Fc protein to PD-L1 target protein was significantly higher than that of two PD-L1 antibodies known in the art.
  • Binding to the PD-L1 antigen is difficult to dissociate, further indicating that PDL1-56-Fc acts as a blocking antibody and is superior in nature to two known PD-L1 antibodies.
  • PDL1-Fc fusion protein 0.5 ⁇ g/well 4°C overnight coated plate, followed by gradient dilution series of PD-L1 single domain antibody Fc fusion protein obtained in the above example, 100 uL per well (diluted solution) Containing 100 ug/mL PD1-Fc-Biotin), reacted at room temperature for 1 hour. After washing, SA-HRP (purchased from Sigma) was added and reacted at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the data processing and mapping analysis were performed by the application software SotfMax Pro v5.4.
  • the blocking curve and IC50 value of the antibody to PDL1-PD1 were obtained by four-parameter fitting.
  • the results are shown in Figure 6, in which the ordinate is OD405 and the abscissa is the PD-L1 single domain antibody Fc fusion protein concentration (in ng/mL); the inverted triangle, the positive triangle, and the square represent three different humanizations of the antibody strain 56, respectively.
  • Forms of Fc fusion proteins hu56v1-Fc, hu56v2-Fc, hu56v5-Fc. The ability of the three proteins to block the PDL1-PD1 interaction is comparable.
  • the CD80-Fc protein (SEQ ID NO: 36) was obtained by expression of HEK293 cells.
  • the biotinylated protein CD80-Fc-Biotin was obtained using a Biotinlytion kit from Thermo.
  • PDL1-Fc fusion protein 0.5 ⁇ g/well 4°C overnight coated plate, followed by a gradient dilution series of PD-L1 single domain antibody Fc fusion protein obtained in the above example, 100 uL per well (300ug/mL CD80 in the dilution) Fc-Biotin), reacted at room temperature for 1 hour. After washing, SA-HRP (purchased from Sigma) was added and reacted at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the four-parameter fitting was used to obtain the PDL1-CD80 blocking curve and IC50 value of the antibody.
  • the results are shown in Figure 7, in which the ordinate is OD405 and the abscissa is the concentration (in ng/mL) of the PD-L1 single domain antibody Fc fusion protein hu56V2-Fc.
  • the results showed that the PD-L1 blocking single domain antibody Fc fusion protein hu56V2-Fc can effectively block the interaction between PD-L1 and CD80.
  • Human HEK293 cells transiently expressed monkey PD-L1 protein on the membrane by transient transfection of a plasmid containing the full length gene of human PD-L1.
  • each group was added with PD1-muFc (SEQ ID NO: 39) at a working concentration of 2 ug/ml, and then different concentrations of KN035 were added, incubated on ice for 30 min, and washed 3 Afterwards, PE-labeled goat anti-mouse secondary antibody was added as a detection antibody, and after 30 min incubation on ice, the fluorescence intensity was measured by flow cytometry.
  • the Jurket cell line was transformed into a plasmid containing the full-length gene of human PD1, and a Jurket cell line stably expressing human PD1 protein was obtained, which was named Jurket-PD1.
  • Human HEK293 cells transiently expressed human PD-L1 protein on the membrane by transient transfection of a plasmid carrying the full length gene of human B7 family protein (pCDNA4, Invitrogen, Cat V86220).
  • the plasmid also fused the EGFP protein to the C-terminus of the target protein, so that the expression level of the B7 family protein on the membrane can be examined by the intensity of green fluorescence.
  • the constructed transient transfected cell lines include: 293-PDL1-EGFP, 293-PDL2-EGFP, 293-B7H3-EGFP, 293-B7H3-EGFP.
  • the constructed cells were resuspended in 0.5% PBS-BSA Buffer, hu56V2-Fc antibody was added, and a negative control was set to 2 ⁇ g of single domain antibody against other unrelated targets, and incubated on ice for 20 min. After washing, eBioscience secondary anti-hIg-PE was added and incubated on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ l of 0.5% PBS-BSA Buffer and detected by flow cytometry.
  • Human HEK293 cells transiently expressed monkey PD-L1 protein (SEQ ID NO: 37) on the membrane by transient transfection of a plasmid containing the full length cDNA of monkey PD-L1.
  • the plasmid also fused the EGFP protein to the C-terminus of the target protein, so that the expression level of the monkey PD-L1 protein on the membrane can be examined by the intensity of green fluorescence.
  • the constructed cells were resuspended in 0.5% PBS-BSA Buffer, hu56V2-Fc antibody was added, and incubated on ice for 20 min. After washing, eBioscience secondary anti-hIg-PE was added and incubated on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ l of 0.5% PBS-BSA Buffer and detected by flow cytometry.
  • 5.8PD-L1 single domain antibody Fc fusion protein can effectively identify PD-L1 positive cell population on patient tissue sections
  • Tumor tissue sections of PD-L1-positive lung cancer patients were stained with 5 ug/mL hu56V2-Fc antibody as primary antibody, and goat anti-human HRP-labeled antibody (Perkin-Elmer, Cat: NEF802001EA) was used as the secondary antibody for 1.5 hr at room temperature. The final color.
  • the results are shown in Figure 12.
  • the hu56V2-Fc antibody can effectively recognize the PD-L1 positive cell population on the tissue sections of lung cancer patients, and can simultaneously recognize PD-L1-positive tumor cells and PD-L1-positive immune cells.
  • PBMC peripheral blood mononuclear cells
  • a gradient dilution series of 2.5 ug/mL anti-CD3 antibody and PD-L1 single domain antibody Fc fusion protein hu56V2-Fc (also named KN035 in the experiment) was coated on a cell culture plate overnight at 4 °C. On the next day, 1 x 10 5 PBMC cells were added to each well. After 5 days of culture, the supernatant was taken, and the level of IFN- ⁇ in the supernatant was measured using an IFN- ⁇ ELISA test kit (ebioscience).
  • the PD-L1 single domain antibody Fc fusion protein can be combined with anti-CD3 antibody.
  • the PD-L1 single domain antibody Fc fusion protein enhances the activation of PBMC cells. At the same time, this activation effect is concentration dependent.
  • Peripheral blood mononuclear cells PBMC were isolated from healthy donors' peripheral blood-enriched leukocytes using human lymphocyte separation fluid (Tianjin Minyang) density gradient centrifugation. Then, it was cultured in serum-free RPMI1640 medium for 1-2 hours to remove unattached cells, and the cells were cultured in RPMI containing 10% FBS, 10 ng/ml GM-CSF, and 20 ng/mL IL-4. After 5-6 days of culture, 10 ng/ml of TNF- ⁇ was added and incubated for 24 hours to obtain mature dendritic cells (DC cells).
  • DC cells mature dendritic cells
  • the DC cells obtained by this method were resuspended in RPMI complete medium, 2 x 10 5 /ml. Then 50 ⁇ l was added to each well in a 96-well U-shaped bottom plate (Costar: 3799) and cultured in an incubator.
  • CD4+ T cells were isolated from another donor PBMC using a magnetic bead separation kit (Miltenyi Biotec: 130-096-533) according to the method described.
  • the 1 ⁇ 10 4 dendritic cells obtained by the above method were mixed with 1 ⁇ 10 5 CD4+ T cells, resuspended in RPMI complete medium and added to a 96-well culture plate, and 50 ⁇ l of the cell mixture was added to each well; 100 ⁇ l of each well was diluted in each well.
  • hu56V2-Fc in RPMI complete medium with a final antibody concentration of 0.1 ⁇ g/ml or 0 ⁇ g/ml. After 5-7 days of culture, the supernatant was taken, and the level of IFN-? in the supernatant was measured using an IFN- ⁇ ELISA assay kit (ebioscience).
  • the PD-L1 single domain antibody Fc fusion protein can enhance the ⁇ -interferon secretion of CD4+ T cells in the mixed lymphocyte reaction, that is, the PD-L1 blocking type single domain antibody Fc fusion protein enhances the activation of T cells.
  • GM-CSF + 25 ng/ml IL-4 was cultured in PBMC of test subject 1, and 6 days later, DC cells were harvested by TNF- ⁇ (50 ng/ml) for 24 hours; and then used in PBMC of tester 2 CD4+T cell was sorted by magnetic bead separation kit (Miltenyi Biotec: 130-096-533); DC cells were added to 96-well U-shaped bottom plate according to 10 4 /well, and 10 5 cells/well of CD4 were added after 2-4 hours.
  • the gray histogram indicates stimulation of the PD-L1 single domain antibody Fc fusion protein.
  • the black histogram indicates MedImmune LLC anti-PD-L1 antibody 2.41H90P.
  • PD-L1 single domain antibody Fc fusion egg can enhance the ability of CD4+ T cells to secrete IFN- ⁇ in mixed lymphocyte reaction with increasing concentration, and PD-L1 single domain antibody Fc fusion egg activated T cell at the same concentration The ability is slightly stronger than MedImmune LLC's anti-PD-L1 antibody 2.41H90P.
  • T cell activation system the Jurkat/Raji-PDL1 co-culture system, to detect the effect of PD-L1 single domain antibody Fc fusion protein on T cell activation and compared it with MedImmune LLC anti-PD-L1 antibody.
  • the system uses Jurkat cells (T cells) as effector cells, anti-human CD3 Antibody as the first signal of Jurkat cell activation; Raji-PDL1 cells stably expressing human PDL1 by genetic engineering, B7 family molecules on the surface CD80 provides a second costimulatory signal to activate Jurket cells, while PDL1, which is highly expressed on the cell surface, acts as a negative regulator to inhibit Jurket cell activation by binding to PD1.
  • hu56V1-Fcm1 and 2.41H90P antibody proteins were prepared using 10% FBS+1640+150ng/ml anti-CD3; Jurkat and Raji-PDL1 cells were adjusted to 3 ⁇ 10 6 cells/ml and 1.5 ⁇ 10 with 10% FBS, respectively. 6 cells/ml, 50 ul per well, and placed at 37 ° C for 24 hours, 100 ul of the culture supernatant was taken out, and the expression level of IL-2 was measured using a kit.
  • Figure 15 shows that the black histogram indicates that the PD-L1 single domain antibody Fc fusion protein stimulates secretion of IL-2, and the gray histogram indicates MedImmune LLC anti-PD-L1 antibody 2.41H90P.
  • the PD-L1 single domain antibody Fc fusion protein can enhance the ability of Jurkat cells to secrete IL-2 in the mixed lymphocyte reaction with increasing concentration, and its ability to activate Jurkat cells is slightly stronger than that of MedImmune LLC at the same concentration.
  • PD-L1 antibody 2.41H90P The PD-L1 single domain antibody Fc fusion protein can enhance the ability of Jurkat cells to secrete IL-2 in the mixed lymphocyte reaction with increasing concentration, and its ability to activate Jurkat cells is slightly stronger than that of MedImmune LLC at the same concentration.
  • Table 6 shows that the average KD of the PD-L1 single domain antibody Fc fusion protein and FcRn is about 5.1E-07M. There was no significant difference in affinity between the mutant Fc (Fcm1) and the wild-type Fc.
  • PBMCs were effector cells with 8 ⁇ 10 5 cells/well; Raji-PDL1 stably expressing human PDL1 protein was used as the target cell, and the number of cells was 2 ⁇ 10 5 cells/ Adding different concentrations of hu56V1-Fcm1 or Rituxan protein as a positive control, using CytoTox after 6 hours incubation at 37 °C
  • the non-radioactive cytotoxicity test kit detects ADCC activity (%) at a concentration.
  • Figure 16A shows that hu56V1-Fcm1 has no significant ADCC activity compared to the positive control Rituxan.
  • Raji-PDL1 cells were used as target cells, the number of cells was 2 ⁇ 10 4 cells/well, 5% cynomolgus monkey serum was supplied with complement, and different concentrations of hu56V1-Fcm1 and positive control Rituxan were added. After incubation at 37 °C for 2 h, CCK- was used. 8 Test the CDC activity of the sample.
  • Figure 16B shows that hu56V1-Fcm1 has no CDC activity in the concentration range of 0.02 ug/ml to 20 ug/ml compared to the positive control.
  • NOD/SCID mice transplanted subcutaneously expressing human PD-L1 melanoma cell line A375 (ATCC, CRL-1619 TM ) and peripheral blood mononuclear cells of human PBMC experiments to achieve this purpose.
  • A375 and PBMC were mixed in a ratio of 5:1 before injection, and a total of 100 ⁇ l (containing 5 million A375, 1 million PBMC) was injected subcutaneously.
  • the antibody was administered intraperitoneally for the first time 24 hours after tumor inoculation, followed by weekly administration at a dose of 0.3 mg/kg; PBS was used as a negative control. 4-6 mice per experimental group. Tumor formation was observed twice a week, and the long and short diameters of the tumor were measured with vernier calipers. The tumor volume was calculated and the tumor growth curve was plotted (see Figure 17A). It can be seen that the antibody hu56V2-Fc can be dosed at 0.3 mg/kg. Significant inhibition of tumor growth.
  • mice The same in vivo model was used to examine the PD-L1 single domain antibody Fc fusion protein, hu56V1-Fcm1, which also did not recognize mouse PD-L1.
  • A375 and human PBMCs were mixed 4:1 and inoculated subcutaneously in NOD-SCID mice. After 4 hours, different doses of hu56V1-Fcm1 (0.1, 0.3, 1, 3, 10 mg/kg) were administered intraperitoneally. Antitumor effect of NOD-SCID mouse A375/human PBMCs xenografts after weekly administration. PBS was used as a negative control. 4-6 mice per experimental group.
  • hu56V1-Fcm1 dose groups (0.1 ⁇ 10mg/kg) had significant anti-tumor effect on NOD-SCID mice A375/human PBMCs xenografts, but no dose-related effects.
  • the antibody hu56V1-Fcm1 significantly inhibited tumor growth at a dose of 0.1 mg/kg.
  • NOD-SCID mouse A375/human PBMCs xenograft model for investigation.
  • A375 was mixed with human PBMCs 4:1 and inoculated subcutaneously in NOD-SCID mice.
  • hu56V1-Fcm1 0.3 mg/kg was intraperitoneally injected, and then administered intraperitoneally every three days.
  • the final number of administrations was 1, 2, 3, and 4 times, respectively.
  • Tumor formation was observed every three days, and the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume was calculated until the 33rd day from the first administration.
  • the tumor growth curve was plotted (Fig. 18). The results showed that the number of administrations in the study period had significant antitumor effect on NOD-SCID mice A375/human PBMCs xenografts.
  • mice transplanted subcutaneously expressing human PD-L1 melanoma cell line A375 (ATCC, CRL-1619 TM ) and peripheral blood mononuclear cells of human PBMC experiments to achieve this purpose.
  • A375 and PBMC were mixed in a ratio of 5:1 before injection, and a total volume of 100 ⁇ l (containing 5 million A375, 1 million PBMC) was injected subcutaneously.
  • the antibody was administered intraperitoneally for the first time 24 hours after tumor inoculation, and then administered weekly. Once, the dose was 1 mg/kg.
  • the drug-administered group included hu56V2-Fc (expressed as hu56) and MedImmune LLC's anti-PD-L1 antibody group (expressed as 2.41), and PBS was used as a negative control.
  • 4-6 mice per experimental group Tumor formation was observed twice a week, and the long and short diameters of the tumor were measured with a vernier caliper, the tumor volume was calculated, and a tumor growth curve was drawn (see Fig. 19A).
  • the anti-PD-L1 antibody of MedImmune LLC was basically ineffective in this model, and the tumor volume had exceeded the negative control group on the 35th day, so the administration and tumor volume measurement were stopped. It can be seen that under this model, hu56V2-Fc inhibited the growth of A375 tumors at a dose of 1 mg/kg significantly better than MedImmune LLC's anti-PD-L1 antibody 2.41H90P.
  • A375 and PBMC were mixed in a ratio of 1:1 before injection, and a total volume of 100 ⁇ l (including 5 million A375, 5 million PBMC) was injected subcutaneously.
  • the antibody was administered intraperitoneally for the first time in 24 hours after tumor inoculation with anti-PD from MedImmune LLC.
  • the -L1 antibody (2.41H90P) was administered once a week at a dose of 1 mg/kg; PBS was used as a negative control.
  • mice per experimental group. Tumor formation was observed twice a week, and the long and short diameters of the tumor were measured with a vernier caliper, the tumor volume was calculated, and a tumor growth curve was drawn (see Fig. 19B). It can be seen that after increasing the ratio of PMBC, the anti-PD-L1 antibody of MedImmune LLC showed an antitumor effect in an in vivo model.
  • TGI (1 - administration group tumor volume / control tumor volume) x 100%
  • Example 6 Stability study of PD-L1 single domain antibody Fc fusion protein
  • the biological activity of the PD-L1 single domain antibody Fc fusion protein obtained in the above examples before and after the treatment was examined using a competition ELISA method. As shown in Figure 20, alkaline and oxidative disruption did not affect the activity of the candidate PD-L1 single domain antibody Fc fusion protein, and the competitive ELISA activity after 38 hours of alkaline treatment was 103% relative to 0 hours. The competitive ELISA activity for 8 hours of oxidation was 106% relative to 0 hours.
  • the PD-L1 single domain antibody Fc fusion protein was concentrated by UF/DF and exchanged into PBS buffer. The formation tendency of the polymer was examined by SE-HPLC.
  • the purity of the PD-L1 single domain antibody Fc fusion protein by SE-HPLC was 96.8%, and the aggregate increased by about 2.4% compared to the low concentration ( ⁇ 2 mg/mL).
  • the protein solution did not show turbidity or aggregation during the entire concentration process.

Abstract

本发明涉及医药生物领域，提供了针对程序性死亡配体（PD-L1）的单域抗体及其衍生蛋白。本发明还提供PD-L1结合分子在治疗和/或预防、诊断PD-L1相关疾病例如肿瘤中的用途。

Description

针对程序性死亡配体(PD-L1)的单域抗体及其衍生蛋白 技术领域
本发明涉及医药生物领域，公开了针对程序性死亡配体(PD-L1)的单域抗体及其衍生蛋白。具体而言，本发明公开了一种程序性死亡配体1(PD-L1)结合分子及其用途，特别是在治疗和/或预防、或诊断PD-L1相关疾病例如肿瘤中的用途。
背景技术
程序性死亡-1(PD-1)是CD28受体家族的成员，该家族包括CD28、CTLA-4、ICOS、PD-1和BTLA。该家族的最初成员CD28和ICOS通过添加单克隆抗体后增强T细胞增殖的功能而发现(Hutloff等(1999)，Nature397：263-266；Hansen等(1980)，Immunogenics 10：247-260)。已经鉴定了PD-1的两种细胞表面糖蛋白配体，PD-L1和PD-L2，已经表明它们在与PD-1结合后下调T细胞活化和细胞因子分泌(Freeman等(2000)，J Exp Med192：1027-34；Latchman等(2001)，Nat Immunol 2：261-8；Cater等(2002)，Eur J Immunol 32：634-43；Ohigashi等(2005)，Clin Cancer Res 11：2947-53)。PD-L1(B7-H1)和PD-L2(B7-DC)都是可与PD-1结合但是不与其他CD28家族成员结合的B7同源物(Blank等2004)。也已经显示通过IFN-γ刺激上调细胞表面上PD-L1的表达。
PD-L1的表达已经在几种鼠和人类癌症中发现，包括人肺癌、卵巢癌、结肠癌、黑色素瘤和各种骨髓瘤(Iwai等(2002)，PNAS 99：12293-7；Ohigashi等(2005)，Clin Cancer Res 11：2947-53)。已有的结果显示，肿瘤细胞高表达的PD-L1通过增加T细胞的凋亡从而在肿瘤的免疫逃逸中起着重要的作用。研究者发现，转染PD-L1基因的P815肿瘤细胞系在体外可抵制特异性CTL的裂解，将其接种小鼠体内后具有更强的致瘤性和侵袭性。这些生物学特性均可通过阻断PD-L1而逆转。敲除PD-1基因的小鼠，阻断PD-L1/PD-1通路，则接种肿瘤细胞不能形成肿瘤(Dong等(2002)，Nat Med 8：793-800)。也已经提示PD-L1可能与肠粘膜炎症有关，并且PD-L1的抑制防止了与结 肠炎有关的萎缩病(Kanai等(2003)，J Immunol 171：4156-63)
本领域仍需要能够与PD-L1高亲和力结合，并且能够阻断PD-1与PD-L1结合的抗PD-L1抗体，特别是抗PD-L1重链单域抗体。
发明概述
本发明的发明人利用噬菌体展示技术，通过筛选得到了具有高特异性、高亲和力和高稳定性的抗PD-L1重链单域抗体(VHH)。
在第一方面，本发明提供了一种PD-L1结合分子，其包括由选自SEQ ID NO：1-6的氨基酸序列组成的免疫球蛋白单一可变结构域和人免疫球蛋白Fc区。
在另一方面中，本发明涉及编码PD-L1结合分子的核酸分子以及含有所述核酸分子的表达载体和宿主细胞。
本发明还涉及包含本发明的PD-L1结合分子的药物组合物。
本发明还涉及制备本文所述PD-L1结合分子的方法。
本发明还涉及本发明所述PD-L1结合分子以及药物组合物的应用，尤其是预防和/或治疗与PD-L1相关的疾病的用途和方法。
附图说明
图1.示出PD-L1重链单域抗体对PD-1/PD-L1相互作用的阻断效应。
图2.示出PD-L1重链单域抗体对PD-L1抗原蛋白的结合曲线。
图3.示出PD-L1重链单域抗体对PD-1与PD-L1相互作用的阻断曲线。
图4.示出抗体株56的5种人源化变体的序列比对结果。
图5.示出PD-L1单域抗体Fc融合蛋白对PD-L1的结合曲线(ELISA法)。
图6.示出PD-L1单域抗体Fc融合蛋白对PD-L1/PD-1相互作用的阻断曲线(竞争ELISA法)。
图7.示出PD-L1单域抗体Fc融合蛋白对PD-L1/CD80相互作用的阻断曲线(竞争ELISA法)。
图8.示出PD-L1单域抗体Fc融合蛋白对293-PDL1/PD1相互作用的阻断曲线(FACS法)
图9.示出PD-L1单域抗体Fc融合蛋白对Jurket-PD1/PDL1相互作用 的阻断曲线(FACS法)
图10.流式细胞仪检测PD-L1单域抗体Fc融合蛋白对PD-L1蛋白结合的特异性。
图11.流式细胞仪检测PD-L1单域抗体Fc融合蛋白对猴PD-L1蛋白的结合。
图12.示出PD-L1单域抗体Fc融合蛋白识别患者组织切片上的PD-L1阳性细胞群。
图13.示出PD-L1单域抗体Fc融合蛋白对PBMC的激活作用。
图14.示出PD-L1单域抗体Fc融合蛋白对CD4+T细胞的激活作用。
图15.示出PD-L1单域抗体Fc融合蛋白刺激分泌IL-2。
图16.示出带有突变Fc的PD-L1单域抗体Fc融合蛋白的CDC以及ADCC活性。
图17.示出用PD-L1单域抗体Fc融合蛋白处理后的肿瘤生长曲线。
图18.示出不同给药次数的PD-L1单域抗体Fc融合蛋白对肿瘤生长的抑制活性。
图19.示出hu56V2-Fc与2.41的肿瘤生长抑制活性的比较。A：A375∶PBMC＝5∶1；B：A375∶PBMC＝1∶1。
图20.示出碱及氧化处理对PD-L1单域抗体Fc融合蛋白活性的影响。
发明详述
定义
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人，“Molecular Cloning：A Laboratory Manual”(第2版)，第1-3卷，Cold Spring Harbor Laboratory Press(1989)；Lewin，“Genes IV”，Oxford University Press，New York，(1990)；及Roitt等人，“Immunology”(第2版)，Gower Medical Publishing，London，New York(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在 本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用，术语(多肽或蛋白的)“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或结构域的功能。
如本文所用的术语“免疫球蛋白结构域”是指抗体链(例如常规4链抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征，其由排列在两个β折叠中任选由保守二硫键稳定的约7个反平行β折叠股的2层夹层组成。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
如本文所用的术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原表位的免疫球蛋白可变结构域。本发明含义中的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH及VL(VH结构域及VL结构域)。免疫球蛋白单一可变结构域的另一实例为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH结构域、VHH抗 体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.：“Naturally occurring antibodies devoid of light chains”；Nature363,446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本发明的上下文中，术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”以及
Figure PCTCN2016092680-appb-000001
及
Figure PCTCN2016092680-appb-000002
结构域”(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。
例如Riechmann及Muyldermans，J.Immunol.Methods 231,25-38(1999)的图2中所示，对于骆驼科的VHH结构域所应用的氨基酸残基，根据Kabat等人给出的VH结构域的一般编号法来编号(“Sequence of proteins of imnunological interest”，US Public Health Services，NIH Bethesda，MD，公开案第91号)。根据该编号法，
-FR1包含在位置1-30处的氨基酸残基，
-CDR1包含在位置31-35处的氨基酸残基，
-FR2包含在位置36-49处的氨基酸，
-CDR2包含在位置50-65处的氨基酸残基，
-FR3包含在位置66-94处的氨基酸残基，
-CDR3包含在位置95-102处的氨基酸残基，且
-FR4包含在位置103-113处的氨基酸残基。
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残 基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述替代方法还可以类似地应用于VHH结构域。然而，除非另有说明，否则在本说明书、权利要求书及附图中，将遵循如上所述的根据Kabat且适于VHH结构域的编号。
VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本文所述的目的。
VHH结构域及含有其的多肽的其他结构特性及功能性质可总结如下：
VHH结构域(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。此区分VHH结构域与常规4链抗体的VH及VL结构域，这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于这些独特性质，使用VHH结构域一单独或作为较大多肽的一部分一提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：
-仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；
-VHH结构域可自单一基因表达且不需要翻译后折叠或修饰；
-VHH结构域可容易地改造成多价及多特异性格式(格式化)；
-VHH结构域高度可溶且无聚集趋势；
-VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；
-VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；
-VHH结构域与常规4链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；
-VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.，Journal of Immunological Methods，240(2000)185-195；Li et al.，J Biol Chem.，287(2012)13713-13721；Deffar et al.，African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
源自骆驼科的VHH结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而经“人源化”。人源化VHH结构域可含有一个或多个完全人框架区序列，且在一具体实施方案中，可含IGHV3的人框架区序列。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)分子可结合的不同类型抗原或表位的数目。可基于抗原结合分子的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合分子之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合分子(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合分子上的抗原结合位点之间的亲和力，以及存在于抗原结合分子上的相关结合位点的数目。
通常，特异性的结合分子将以如于Biacore或KinExA测定中测量的优选10-7至10-11摩尔/升(M)、更优选10-8至10-11摩尔/升、甚至更优选 10-9至10-11、甚至更优选10-10至10-11或更低的解离常数(KD)，和/或以至少107M-1、优选至少108M-1、更优选至少109M-1，更优选至少1010M-1、例如至少1011M-1的缔合常数(KA)结合所要结合的抗原。任何大于10-4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“基本上分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“基 本上分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“基本上分离的”多肽或核酸分子优选基本上为均质的。
如本文所用的术语“对象”意指哺乳动物，尤其灵长类动物，尤其是人。
本发明的PD-L1结合分子
在第一方面，本发明提供了一种PD-L1结合分子，其能够特异性结合PD-L1且由式A-L-B的氨基酸序列组成，其中A表示免疫球蛋白单一可变结构域，L表示氨基酸接头或不存在，且B表示人免疫球蛋白Fc区，
其中所述免疫球蛋白单一可变结构域由选自SEQ ID NO：1-6的氨基酸序列组成，
所述PD-L1结合分子能够通过所述人免疫球蛋白Fc区形成(A-L-B)2形式的同二聚体。
如本发明所用，术语“人免疫球蛋白Fc区”指的是来自人IgG1、IgG2、IgG3或IgG4的恒定区(其恒定区氨基酸序列分别参考www.uniprot.org蛋白数据库中的条目P01857、P01859、P01860、P01861)的Fc区，其包含免疫球蛋白恒定区的铰链区(Hinge)或部分铰链区、CH2区、以及CH3区。本发明中，“人免疫球蛋白Fc区”的氨基酸序列可通过突变CH2区上的1-5个氨基酸以增加或去除Fc介导的ADCC或CDC活性或是增强或减弱FcRn的亲和力；或者通过突变Hinge区的1-4个氨基酸增加蛋白的稳定性。但是，本发明中，“人免疫球蛋白Fc区”不包括促进或阻止形成Fc异二聚体的突变，也不包括在Fc片段的氮端或碳端增加其他功能性蛋白序列的修饰。
在一个优选的实施方式中，所述人免疫球蛋白Fc区被突变以去除ADCC和CDC活性。在一个具体实施方式中，所述免疫球蛋白Fc区的氨基酸序列选自SEQ ID NO：7-9。
本领域技术人员将理解，在人免疫球蛋白Fc区的作用下，本发明的PD-L1结合分子通常将以同二聚体的形式存在。
如本发明所用，术语“氨基酸接头”指的是长1-20个氨基酸残基、无二级以上结构的非功能性氨基酸序列。所述氨基酸接头例如是柔性接头，例如GGGGS，GS，GAP等。
在一些实施方案中，本发明的PD-L1结合分子由选自SEQ ID  NO：10-27的氨基酸序列组成。
本发明的PD-L1结合分子具有下述特征中的至少一个：
(a)结合人PD-L1的KD值小于1×10-7M；
(b)阻断PD-L1和PD-1的相互作用；
(c)增强PBMC和T细胞的活化；
(d)抑制肿瘤生长。
本发明的所述PD-L1结合分子结合PD-L1的KD值可以小于1×10-7M，优选小于1×10-8M、更优选小于1×10-9M、更优选小于1×10-10M、尤其更优选小于1×10-11M。
在一些实施方案中，本发明的PD-L1结合分子能够特异性结合人PD-L1并阻断PD-L1和PD-1的相互作用。在一些实施方案中，本发明的PD-L1结合分子能够特异性结合人PD-L1并阻断PD-L1和CD80的相互作用。
本发明的PD-L1结合分子能够抑制肿瘤生长至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。
此外，本发明的PD-L1结合分子对碱处理和氧化处理具有抗性。例如，强碱(如500mM碳酸氢铵)处理约8小时、优选约16小时、更优选约24小时或更优选约32小时后，本发明的PD-L1结合分子活性保持不变。或者，氧化剂(1％双氧水)处理约2小时、优选约4小时或更优选约8小时后，本发明的PD-L1结合分子活性保持不变。
此外，本发明的PD-L1结合分子具有在高浓度下的稳定性。例如，在约100mg/ml、更优选约150mg/ml、更优选约200mg/ml或更优选约250mg/ml的浓度下，本发明的PD-L1结合分子保持稳定而不出现聚集。
核酸、载体、宿主细胞
在另一方面中，本发明涉及编码本发明的PD-L1结合分子的核酸分子。本发明的核酸可为RNA、DNA或cDNA。根据本发明的一个实施方案，本发明的核酸是基本上分离的核酸。
本发明的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即 可提供PD-L1结合分子体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本发明的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本发明的PD-L1结合分子的表达有用或必需的调控元件及其他元件的具体实例，例如启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本发明的核酸可基于关于本文给出的本发明的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
在另一方面中，本发明涉及表达或能够表达一种或多种本发明的PD-L1结合分子和/或含有本发明的核酸或载体的宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本发明还提供产生本发明的PD-L1结合分子的方法，所述方法通常包含以下步骤：
-在允许表达本发明的PD-L1结合分子的条件下培养本发明的宿主细胞；及
-从培养物回收由所述宿主细胞表达的PD-L1结合分子；及
-任选进一步纯化和/或修饰本发明的PD-L1结合分子。
在一个优选的实施方案中，本发明的PD-L1结合分子使用哺乳动物细胞产生。本发明的PD-L1结合分子可以在哺乳动物细胞中获得高表达。例如，表达水平可达大约5g/L、优选大约6g/L、优选大约7g/L、优选大约8g/L、更优选大约9g/L或更优选大约10g/L或者更高。
本发明的PD-L1结合分子可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本发明的PD-L1结合分子的方法中的蛋白分离及纯化技术为本领域技术人员所公知。
然而，本发明的PD-L1结合分子也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
药物组合物
另一方面，本发明提供一种组合物，例如药物组合物，其含有与药学上可接受的载体配制在一起的一种或组合的本发明的PD-L1结合分子。这样的组合物可以包含一种或组合的(例如两种或多种不同的)本发明的PD-L1结合分子。本领域技术人员将理解，在人免疫球蛋白Fc区的作用下，本发明的PD-L1结合分子通常将以同二聚体的形式存在。
本发明的药物组合物也可以在联合治疗中施用，即与其他药剂联用。例如，联合治疗可包括本发明的PD-L1结合分子联合至少一种其他的抗肿瘤药物。例如，本发明的PD-L1结合分子可以与靶向其它肿瘤特异性抗原的抗体联合使用。所述靶向其它肿瘤特异性抗原的抗体包括但不限于，抗EGFR抗体、抗EGFR变体的抗体、抗VEGFa抗体、抗HER2抗体、或抗CMET抗体。优选所述抗体是单克隆抗体。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即抗体分子、免疫缀合物包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用。
本发明的药物组合物可包含一种或多种药学上可接受的盐。“药学上可接受的盐”是指保持了亲代化合物的所需生物活性而不引起任何不期望的毒理学作用的盐(参见如Berge，S.M.等(1977)J.Pharm.Sci.66：1-19)。这样的盐的例子包括酸加成盐和碱加成盐。酸加成盐包括那些由诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等无毒性无机酸衍生的盐，以及由诸如脂族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳族酸、脂族和芳族磺酸等无毒性有机酸衍生的盐。碱加成盐包括那些由诸如钠、钾、镁、钙等碱土金属衍生的盐，以及由诸如N，N’-二苄基乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等无毒性有机胺衍生的盐。
本发明的药物组合物也可含有药学上可接受的抗氧化剂。药学上可接受的抗氧化剂的例子包括：(1)水溶性抗氧化剂，如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠，亚硫酸钠等；(2)油溶性抗氧化剂，如棕榈酸抗坏血酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚等；和(3)金属螯合剂，如柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等。
这些组合物还可含有佐剂，如防腐剂、润湿剂、乳化剂和分散剂。
可以通过灭菌程序或通过包含诸如对羟基苯甲酸酯、氯代丁醇、苯酚山梨酸等各种抗细菌剂和抗真菌剂确保防止存在微生物。在很多情况下，组合物中优选包含等渗剂，例如，糖、多元醇例如甘露糖醇、山梨糖醇或氧化钠。通过在组合物中加入延迟吸收剂，例如单硬脂酸盐和明胶，可实现注射型药物延长的吸收。
药学上可接受的载体包括无菌水溶液或分散液和用于临时制备无菌注射液或分散液的粉末剂。这些用于药学活性物质的介质和试剂的使用是本领域公知的。常规介质或试剂，除了任何与活性化合物不相容的范 围外，都可能在本发明的药物组合物中。还可以向组合物中掺入补充的活性化合物。
治疗性组合物一般必须是无菌的并且在制备和贮存条件下稳定的。可以将组合物配制成溶液、微乳状液、脂质体或其他适合高药物浓度的有序结构。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液态聚乙二醇等)及其合适的混合物的溶剂或分散剂。例如，通过使用包衣，例如卵磷脂，在分散液的情况下通过保持所需的颗粒大小，以及通过使用表面活性剂，可以保持适当的流动性。
通过将活性化合物以需要的量混入合适的溶剂中，并且根据需要加入以上列举的成分中的一种或其组合，接着无菌微过滤，可制备无菌注射液。通常，通过将活性化合物掺入到含有基本分散介质和上面所列其他所需成分的无菌载体中制备分散剂。对于用于制备无菌注射液的无菌粉末剂，优选的制备方法是真空干燥和冷冻干燥(冻干)，由其预先无菌过滤的溶液得到活性成分加任何额外所需成分的粉末。
可以与载体材料组合制备单一剂量形式的活性成分的量根据所治疗的对象和特定给药方式而不同。可以与载体材料组合制备单一剂量形式的活性成分的量一般是产生治疗效果的组合物的量。通常，以100％计，这个量的范围是大约0.01％至大约99％的活性成分，优选大约0.1％至大约70％，最优选大约1％至大约30％的活性成分，与药学上可接受的载体相组合。
可以调节剂量方案以提供最佳的期望的反应(例如，治疗反应)。例如，可以施用单一推注，可以随时间施用几次分开的剂量，或者根据治疗状况的紧急情况所需，可以按比例减小或增加剂量。特别有利的是将肠胃外组合物配制成容易给药并且剂量均匀的剂量单位形式。此处使用的剂量单位形式是指适合作为单位剂量用于所治疗的对象的物理不连续单位；每个单位含有预定量的活性化合物，经计算该预定量的活性化合物与需要的药物载体组合产生所需的治疗效果。对本发明剂量单位形式的具体说明限定于且直接依赖于(a)活性化合物的独特特性和要达到的特定治疗效果，和(b)本领域中固有的对于配制这种用于治疗个体敏感性的活性化合物的限制。
对于抗体分子的给药而言，剂量范围为约0.0001至100mg/kg，更通 常为0.01至20mg/kg受者体重。例如，剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重，10mg/kg体重或20mg/kg体重，或在1-20mg/kg范围内。示例性的治疗方案需要每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。
或者，抗体分子也可以作为持续释放制剂来给药，在此情况中需要频率较低的给药。剂量和频率根据抗体分子在患者中的半衰期而不同。通常，人抗体表现出最长的半衰期，之后是人源化抗体、嵌合抗体和非人类抗体。给药剂量和频率根据处理是预防性的还是治疗性的而不同。在预防性应用中，在长时间内以较不频繁的间隔给予相对较低的剂量。有些患者在余生中持续接受处理。在治疗性应用中，有时需要以较短的间隔给予较高的剂量，直到疾病的进展减轻或停止，优选直到患者表现为疾病症状部分或完全改善。之后，可以以预防性方案给患者给药。
本发明药物组合物中活性成分的实际剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物或其酯、盐或酰胺的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
本发明的PD-L1结合分子的“治疗有效量”优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于PD-L1相关肿瘤的治疗，相对于未接受治疗的对象，“治疗有效量”优选地将细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型系统中评价。或者，也可以通过检查抑制细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。治疗有效量的 治疗性化合物能够减小肿瘤大小，或者以其他方式缓解对象的症状。本领域技术人员可以根据如下因素确定这种量，如对象的大小、对象症状的严重性和选择的特定组合物或给药途径。
本发明的组合物可以利用本领域公知的一种或多种方法通过一种或多种给药途径给药。本领域技术人员应当理解，给药途径和/或方式根据期望的结果而不同。本发明PD-L1结合分子的优选给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊柱或其他肠胃外给药途径，例如注射或输注。本文使用的短语“肠胃外给药”是指除肠和局部给药以外的给药模式，通常是注射，包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。
或者，本发明的PD-L1结合分子也可以通过非肠胃外途径给药，如局部、表皮或粘膜途径给药，例如，鼻内、经口、阴道、直肠、舌下或局部。
活性化合物可以与保护化合物不被快速释放的载体一起制备，例如控释制剂，包括植入物、透皮贴剂和微胶囊递送系统。可以使用生物可降解的、生物相容的聚合物，例如乙烯乙酸乙烯酯、聚酐类、聚乙醇酸、胶原、聚原酸酯和聚乳酸。制备这样的制剂的很多方法受专利保护或者通常为本领域技术人员所公知。参见，例如，Sustainedand controlled Release Drug Delivery Systems，J.R.Robinson，ed.，Marcel Dekker，Inc.，New York，1978。
治疗性组合物可应用本领域公知的医疗装置给药。例如，在一个优选实施方案中，本发明的治疗组合物可用无针皮下注射装置给药，如在美国专利No.5,399,163；5,383,851；5,312,335；5,064,413；4,941，880；4,790,824；或4,596,556中公开的装置。可用于本发明的公知的植入物和模块的例子包括：美国专利No.4,487,603，该专利公开了用于以受控速率分散药物的可植入微量输注泵；美国专利No.4,486，194，该专利公开了用于通过皮肤给药的治疗装置；美国专利No.4,447,233，该专利公开了用于以精确的输注速率递送药物的医用输注泵；美国专利No.4,447,224，该专利公开了用于连续递送药物的变流可植入输注装置；美国专利No.4,439，196，该专利公开了具有多腔区室的渗透药物递送系 统：和美国专利No.4,475，196，该专利公开了一种渗透药物递送系统。这些专利引入本文作为参考。本领域技术人员公知许多其他这样的植入物、递送系统和模块。
在某些实施方案中，本发明的PD-L1结合分子可配制为确保在体内的正确分布。例如，血-脑屏障(BBB)阻止了许多高度亲水性的化合物。为了确保本发明的治疗性化合物能够跨过BBB(如果需要时)，可将它们配制在如脂质体中。至于制备脂质体的方法，参见，例如，美国专利4,522,811；5,374,548和5,399,331。脂质体包含可被选择性地转运入特定细胞或器官内的一个或多个靶向部分，从而增强靶向药物递送(参见，例如，V.V.Ranade(1989)J.Clin.Pharmacol.29：685)。靶向部分的例子包括叶酸或生物素(参见，例如，Low等的美国专利5,416,016)；甘露糖苷(Umezawa等(1988)Biochem.Biophys.Res.Commun.153：1038)；抗体(P.G.Bloeman等(1995)FEBS Lett.357：140；M.Owais等(1995)Antimicrob.Agents Chemother.39：180)；表面活性剂蛋白A受体(Briscoe等(1995)Am.J.Physiol.1233：134)；p120(Schreier等(1994)J.Biol.Chem.269：9090)；也参见K.Keinanen；M.L.Laukkanen(1994)FEBS Lett.346：123；J.J.Killion；I.J.Fidler(1994)Immunomethods 4：273。
疾病预防和治疗
在另一方面，本发明提供了本发明所述PD-L1结合分子、核酸分子、宿主细胞及药物组合物在预防和/或治疗与PD-L1相关的疾病中用途和方法。可用本发明的PD-L1结合分子预防和/或治疗的PD-L1相关的疾病如下详述。
癌症
本发明的PD-L1结合分子对PD-L1的阻断可以增强患者中对癌细胞的免疫应答。PD-L1富含于多种人类癌中(Dong等(2002)Nat Med.8：787-9)。PD-1与PD-L1的相互作用导致浸润肿瘤的淋巴细胞减少，T细胞受体介导的增殖减少，以及癌细胞的免疫逃逸(Dong等(2003)J Mol Med81：281-7；Blank等(2004)Cancer Immunol Immunother[epub]；Konishi等(2004)Clin Cancer Res 10：5094-5100)。抑制PD-L1与PD-1的局部相互 作用可以逆转免疫抑制，当PD-L2与PD-1的相互作用也被阻断时，效应是协同的(Iwai等(2002)PNAS 99：12293-7；Brown等(2003)J Immunol170：1257-66)。本发明的PD-L1结合分子可以单独使用，以抑制癌性肿瘤的生长。或者如以下所述，本发明的PD-L1结合分子可以与其它抗肿瘤治疗手段联合使用，例如与其他免疫原性剂、标准癌症疗法或其他抗体分子联合使用。
因此，在一个实施方案中，本发明提供一种预防和/或治疗癌症的方法，包括给该对象施用治疗有效量的本发明的PD-L1结合分子，抑制对象中的肿瘤细胞生长。
使用本发明的PD-L1结合分子可以预防和/或治疗的优选的癌症包括一般对免疫治疗有应答的癌症。可治疗的优选癌症的非限制性的例子包括肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤(例如转移的恶性黑色素瘤)、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本发明的方法治疗的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病，包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。本发明也可用于治疗转移性癌，特别是表达PD-L1的转移性癌(Iwai等(2005)Int Immunol 17：133-144)。
任选地，本发明的PD-L1结合分子可以与免疫原性剂如癌细胞、纯化的肿瘤抗原(包括重组蛋白、肽和碳水化合物分子)、用编码免疫刺激细胞因子的基因转染的细胞联用(He等(2004)J.Immunol 173：4919-28)。可以应用的免疫原性剂的非限制性实例包括黑素瘤抗原的肽，如gp100的肽、MAGE抗原、Trp-2、MART1和/或酪氨酸酶，或转染后表达细胞 因子GM-CSF的肿瘤细胞。
在人类中，已经表明一些肿瘤是具有免疫原性的，如黑素瘤。预期通过使用本发明的PD-L1结合分子阻断PD-L1来促进T细胞活化，可以激活宿主中的肿瘤应答。当与肿瘤接种方案联合时，PD-L1阻断剂(如抗PD-L1抗体，例如本发明的PD-L1结合分子)可能最有效。已经设计了针对肿瘤接种的许多实验策略(参见Rosenberg，S.，2000，Development of Cancer Vaccines，ASCO Educational Book Spring：60-62；Logothetis，C，2000，ASCO Educational Book Spring：300-302；Khayat，D.2000，ASCO Educational Book Spring：414-428；Foon，K.2000，ASCO Educational Book Spring：730-738；参见Restifo，N.和Sznol，M.，Cancer Vaccines，Ch.61，pp.3023-3043，DeVita，V.等(eds.)，1997，Cancer：Principles and Practice of Oncology.第五版)。在这些策略之一中，使用自体或异体肿瘤细胞制备疫苗。已经证明，当肿瘤细胞被转导而表达GM-CSF时，这些细胞疫苗最有效。已经表明GM-CSF是用于肿瘤接种的抗原呈递的强激活剂(Dranoff等(1993)Proa Natl.Acad.Sci U.S.A.90：3539-43)。
各种肿瘤中基因表达和大规模基因表达模式的研究鉴定出许多所谓的肿瘤特异性抗原(Rosenberg，SA(1999)Immunity 10：281-7)。在许多情况下，这些肿瘤特异性抗原是在肿瘤和产生肿瘤的细胞中表达的分化抗原，例如gp100、MAGE抗原和Trp-2。更重要的是，证明这些抗原中的许多是在宿主中发现的肿瘤特异性T细胞的靶标。本发明的PD-L1结合分子可以与重组产生的肿瘤特异性蛋白和/或肽组合使用，以产生针对这些蛋白质的免疫应答。这些蛋白质在正常情况下被免疫系统看作自身抗原，因此对其耐受。肿瘤抗原也可以包括蛋白质端粒酶，该酶是染色体的端粒合成所必需的，并且在85％以上的人类癌中表达，而仅在有限数量的自身组织中表达(Kim，N等(1994)Science 266：2011-2013)。肿瘤抗原也可以是癌细胞表达的“新抗原”，例如由于体细胞突变改变蛋白质序列或产生两种无关序列的融合蛋白(例如，Philadelphia染色体中的bcr-abl)。
其他肿瘤疫苗可以包括来自与人类癌症有关的病毒的蛋白质，如人类乳头瘤病毒(HPV)、肝炎病毒(HBV和HCV)和卡波西疱疹肉瘤病毒(KHSV)。可以与PD-L1阻断剂(如抗PD-L1抗体，例如本发明的PD-L1 结合分子)联合应用的另外一种形式的肿瘤特异性抗原是从肿瘤组织本身中分离的纯化的热休克蛋白(HSP)。这些热休克蛋白含有来自肿瘤细胞的蛋白质的片段，这些HSP在向抗原呈递细胞递送以引发肿瘤免疫方面非常有效(Suot，R和Srivastava，P(1995)Science 269：1585-1588；Tamura，Y.等(1997)Science 278：117-120)。
树突细胞(DC)是强抗原呈递细胞，可以用来引发抗原特异性应答。DC可以在体外产生，并且载有各种蛋白质和肽抗原以及肿瘤细胞提取物(Nestle，F.等(1998)Nature Medicine 4：328-332)。DC也可以通过遗传手段转导，从而也表达这些肿瘤抗原。已经为了免疫而直接将DC融合到肿瘤细胞上(Kugler，A.等(2000)Nature Medicine 6：332-336)。作为接种方法，DC免疫可以与PD-L1阻断剂(如抗PD-L1抗体，例如本发明的PD-L1结合分子)有效地组合，以激活更强的抗肿瘤应答。
CAR-T，全称是嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor T-Cell Immunotherapy)是另一种有效的恶性肿瘤的细胞治疗方法。嵌合抗原受体T细胞(CAR-T细胞)是将能识别某种肿瘤抗原的抗体的抗原结合部与CD3-ζ链或FcεRIγ的胞内部分在体外偶联为一个嵌合蛋白，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。同时，还可以引入共刺激分子信号序列以提高T细胞的细胞毒活性、增殖性与存活时间，促进细胞因子的释放。患者的T细胞被“重编码”后，可在体外扩增生成大量肿瘤特异性的CAR-T细胞并回输患者体内，实现肿瘤治疗的目的。PD-L1阻断剂(如抗PD-L1抗体，例如本发明的PD-L1结合分子)可以与CAR-T细胞疗法联合，激活更强的抗肿瘤应答。
本发明的PD-L1结合分子也可以与标准癌症治疗组合。本发明的PD-L1结合分子可以与化疗方案有效地组合。在这些例子中，它可以减少施用的化疗剂的剂量(Mokyr，M.等(1998)Cancer Research 58：5301-5304)。这种组合的一个例子是抗PD-L1抗体与氨烯咪胺联用治疗黑素瘤。这种组合的另外一个实例是抗PD-L1抗体与白介素-2(IL-2)联用治疗黑素瘤。本发明的PD-L1结合分子和化学疗法联用的科学原理是细胞死亡，这是大多数化疗化合物的细胞毒性作用的结果，应会导致抗原呈递途径中的肿瘤抗原水平升高。可以通过细胞死亡与PD-L1阻断协同 作用的其他联合治疗有放疗、手术和激素剥夺。这些方案都在宿主中产生肿瘤抗原的来源。血管发生抑制剂也可以与本发明的PD-L1结合分子组合。血管发生的抑制导致肿瘤细胞死亡，这可以将肿瘤抗原提供给宿主的抗原呈递途径。
本发明的PD-L1结合分子还可以与靶向其它肿瘤特异性抗原的抗体联合使用。所述靶向其它肿瘤特异性抗原的抗体包括但不限于，抗EGFR抗体、抗EGFR变体的抗体、抗VEGFa抗体、抗HER2抗体、或抗CMET抗体。优选所述抗体是单克隆抗体。
本发明的PD-L1结合分子也可以与将Fcα或Fcγ受体表达效应细胞靶向至肿瘤细胞的双特异性抗原联合应用(参见，例如US Patent Nos.5,922.845和5,837,243)。也可以利用双特异性抗体靶向两种不同的抗原。例如，已经利用抗-Fc受体/抗肿瘤抗原(例如Her-2/neu)双特异性抗体将巨噬细胞靶向肿瘤部位。这种靶向可以更有效地激活肿瘤特异性应答。利用PD-L1阻断剂可以加强这些应答的T细胞方面。或者，可以利用结合肿瘤抗原和树突细胞特异性细胞表面标记的双特异性抗体将抗原直接递送至DC。
肿瘤通过多种机制逃避宿主的免疫监视。其中许多机制可以通过灭活肿瘤表达的免疫抑制性蛋白质来克服。尤其包括TGF-β(KehrL J.等(1986)J.Exp.Med.163：1037-1050)、IL-10(Howard，M.和O′Garra，A.(1992)Immunology Today 13：198-200)和Fas配体(Hahne，M.等(1996)Science 274：1363-1365)。其中每种的抗体可以与本发明的PD-L1结合分子联用，来抵抗免疫抑制剂的作用，并且有利于宿主的肿瘤免疫应答。
可以用于激活宿主免疫应答的其他抗体可以与本发明的PD-L1结合分子联用。抗-CD40抗体能够有效地替代T细胞辅助活性(Ridge，J.等(1998)Nature 393：474-478)，并且可以与本发明的PD-L1结合分子联用(Ito，N.等(2000)Immunobiology 201(5)527-40)。也可以为了提高T细胞活化的水平而联合对T细胞共刺激分子如OX-40(Weinberg，A.等(2000)Immunol 164：2160-2169)、4-1BB(Melero，I.等(1997)Nature Medicine 3：682-685(1997)和ICOS(Hutloff，A.等(1999)Nature 397：262-266)的活化抗体以及阻断阴性共刺激分子如CTLA-4(例如，美国专利No.5,811,097)或BTLA(Watanabe，N.等(2003)Nat Immunol 4：670-9)、B7-H4(Sica， GL等(2003)Immunity 18：849-61)的活性的抗体。
骨髓移植当前用来治疗造血来源的多种肿瘤。移植物抗宿主疾病是这种治疗的一种后果，移植物对抗肿瘤的应答可以获得治疗性益处。可以利用PD-L1阻断剂提高肿瘤特异性T细胞的有效性。也有几种实验治疗方案涉及抗原特异性T细胞的离体激活和扩增以及这些细胞向受体内的过继转移，以用抗原特异性T细胞对抗肿瘤(Greenberg，R.和Riddell，S.(1999)Science 285：546-51)。这些方法也可以用来激活T细胞对传染原如CMV的应答。预期在本发明的PD-L1结合分子存在下离体激活可以提高过继转移的T细胞的频率和活性。因此，本发明还提供了一种离体激活免疫细胞(如PBMC或T细胞)的方法，包括使所述免疫细胞与本发明的PD-L1结合分子接触。
感染性疾病
本发明的其他方法用于治疗暴露于特定毒素或病原体的患者。因此，本发明的另一方面提供一种预防和/或治疗对象中的感染性疾病的方法，包括给该对象施用本发明的PD-L1结合分子，使得所述对象的感染性疾病得到预防和/或治疗。
类似于对于如上所述的肿瘤的应用，PD-L1阻断剂可以单独使用，或者作为佐剂与疫苗组合使用来刺激对病原体、毒素和自身抗原的免疫应答。特别可以应用该治疗方法的病原体的实例包括当前没有有效疫苗的病原体，或常规疫苗不完全有效的病原体。其中包括但不限于HIV、肝炎病毒(甲、乙、丙)、流感病毒、疱疹病毒、贾第虫、疟疾、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。PD-L1阻断剂特别可用于对抗诸如HIV等病原体已建立的感染，其在感染过程中呈现改变的抗原。在抗人PD-L1抗体给药时，这些新的表位被作为外源物识别，从而引起不受PD-L1的负信号影响的强T细胞应答。
引起可用本发明的方法治疗的感染性疾病的病原体病毒的一些实例包括HIV、肝炎(甲、乙、丙)、疱疹病毒(例如VZV、HSV-1、HAV-6，HSV-II和CMV、EB病毒)、腺病毒、流感病毒、虫媒病毒、埃可病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、流行性腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、 登革热病毒、乳头瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病毒、JC病毒和虫媒病毒脑炎病毒。
引起可用本发明的方法治疗的感染性疾病的病原体细菌的一些实例包括衣原体、立克次氏体菌、分枝杆菌、葡萄球菌、链球菌、肺炎球菌、脑膜炎球菌和淋球菌、克雷伯氏杆菌、变形菌、雷氏菌、假单胞菌、军团杆菌、白喉杆菌、沙门氏菌、芽孢杆菌、霍乱菌、破伤风菌、肉毒杆菌、炭疽杆菌、鼠疫杆菌、钩端螺旋体、和莱姆病细菌。
引起可用本发明的方法治疗的感染性疾病的病原体真菌的一些实例包括假丝酵母(白假丝酵母、克鲁斯假丝酵母、光滑假丝酵母、热带假丝酵母等)、新型隐球菌、曲霉属(烟曲霉、黑曲霉等)、毛霉属(毛霉、犁头霉、根霉)、申克孢子丝菌、皮炎芽生菌、巴西副球孢子菌、粗球孢子菌和夹膜组织胞浆菌。
引起可用本发明的方法治疗的感染性疾病的病原体寄生虫的一些实例包括溶组织内阿米巴、结肠小袋纤毛虫、福氏耐格里阿米巴、棘阿米巴属的种、兰伯贾第虫、隐孢子虫属的种、卡氏肺囊虫、间日疟原虫、果氏巴贝虫、布氏锥虫、克氏锥虫、杜氏利什曼原虫、鼠弓形体、巴西日圆线虫。
在所有上述的方法中，PD-L1阻断剂可以与其他形式的免疫疗法如细胞因子治疗(例如干扰素、GM-CSF、G-CSF、IL-2)或双特异性抗体治疗联合，双特异性抗体治疗提供增强的肿瘤抗原的呈递(参见，例如，Holliger(1993)Proc.Natl.Acad.Sci.USA 90：6444-6448；Poljak(1994)Structure 2：1121-1123)。
自身免疫反应
抗PD-L1抗体可以激起和扩大自身免疫应答。因此，可以考虑利用抗PD-L1抗体联合多种自身蛋白质来设计接种方案，以有效地产生对抗这些自身蛋白质的免疫应答，用于疾病治疗。
例如，阿尔茨海默病涉及Aβ肽在脑中淀粉样蛋白沉积物中的不当的积累；针对淀粉样蛋白的抗体应答能够清除这些淀粉样蛋白沉积物(Schenk等(1999)Nature 400：173-177)。也可以使用其他自身蛋白作为靶标，如涉及治疗变态反应和哮喘的IgE，和涉及类风湿性关节炎的 TNFα。最后，可以利用抗PD-L1抗体诱导抗体对各种激素的应答。中和抗体对生殖激素的应答可以用于避孕。中和抗体对激素和特定肿瘤生长所需的其他可溶性因子的应答也可以被认为是可能的接种靶标。
如上所述，应用抗PD-L1抗体的类似方法可以用来诱导治疗性自身免疫应答，以治疗具有不恰当的自身抗原积累的患者，如淀粉状蛋白沉积物包括阿尔茨海默病中的Aβ、细胞因子如TNFα和IgE。
慢性炎性疾病
抗PD-L1抗体也可以用来治疗如下疾病，如慢性炎性疾病，如扁平苔藓、T细胞介导的慢性炎性皮肤粘膜病(Youngnak-Piboonratanakit等(2004)Immunol Letters 94；215-22)。因此，在一个方面，本发明提供一种用T细胞消除慢性炎性疾病的方法，包括给对象施用本发明的PD-L1结合分子。
疫苗佐剂
本发明的一个方面提供了本发明的PD-L1结合分子作为疫苗佐剂的用途。通过共施用抗PD-L1抗体和目标抗原(例如疫苗)，可以利用抗PD-L1抗体提高针对抗原的特异性免疫应答。
因此，本发明的一个方面提供了增强对象中对抗原的免疫应答的方法，包括给该对象施用：(i)抗原；和(ii)本发明的PD-L1结合分子，使得所述对象中对抗原的免疫应答得到加强。所述抗原可以是，例如，肿瘤抗原、病毒抗原、细菌抗原或来自病原体的抗原。这些抗原的非限定性实例包括以上章节中所述的那些，例如以上所述的肿瘤抗原(或肿瘤疫苗)，或来自上述病毒、细菌或其他病原体的抗原。
试剂盒
本发明的范围内还包括试剂盒，该试剂盒包括本发明的PD-L1结合分子、免疫缀合物或药物组合物，以及使用说明。该试剂盒可以进一步包括至少一种另外的试剂或一种或多种另外的本发明的PD-L1结合分子(例如，结合PD-L1不同表位的的结合分子)。试剂盒一般包括表明试剂盒内容物的预期用途的标签。术语标签包括在试剂盒上或与试剂盒一起 提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1：针对PD-L1的重链单域抗体的筛选
1.1文库的构建：
免疫用的PDL1-Fc融合蛋白(SEQ ID NO：28)由CHO细胞表达(pCDNA4，Invitrogen，Cat V86220)，经Protein A亲和层析纯化得到。选取一只新疆双峰驼(Camelus bactrianus)进行免疫。4次免疫结束后，提取骆驼100ml外周血的淋巴细胞并使用QIAGEN公司提供的RNA提取试剂盒提取总RNA，使用Super-Script III FIRST STRANDSUPERMIX试剂盒按照说明书将提取的RNA反转录成cDNA。用巢式PCR扩增编码重链抗体的可变区的核酸片段：
第一轮PCR：
上游引物：GTCCTGGCTGCTCTTCTACAAGGC(SEQ ID NO：29)；
下游引物：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO：30)。
第二轮PCR：
以第一轮PCR产物作模板，
上游引物：GATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO：31)；
下游引物：GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO：32)。
回收目标重链单域抗体核酸片段，并使用限制性内切酶(购自NEB)PstI及NotI将其克隆进入噬菌体展示用载体pCDisplay-3(Creative Biolabs，Cat：VPT4023)中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对PD-L1的重链单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为1.33×108。为检测文库的插入率，随机选取24个克隆做菌落PCR。结果显示插入率已达到100％。
1.2针对PD-L1的重链单域抗体淘选：
用PDL1-Fc融合蛋白10μg/孔包被平板，4℃放置过夜。第二天用1％脱脂奶室温封闭2小时后，加入100μl噬菌体(8×1011tfu，来自1.1所构建的 骆驼重链单域抗体噬菌展示文库)，在室温下作用1小时。之后用PBST(PBS中含有0.05％吐温20)洗5遍，以洗掉不结合的噬菌体。最后用三乙基胺(100mM)将与PD-L1特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复3-4轮。由此，阳性的克隆被富集，达到了利用噬菌体展示技术筛取抗体库中PD-L1特异抗体的目的。
1.3用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆
3-4轮淘选后，获得的PD-L1结合阳性的噬菌体感染空白大肠杆菌并铺板。随后挑选96个单菌落分别培养，生产并纯化噬菌体。用PDL1-Fc融合蛋白包被平板4℃过夜，将获得的样品噬菌体(对照组为空白噬菌体)加入，室温下反应1小时。洗涤之后加入一抗小鼠抗-HA标签抗体(购自北京康为世纪生物科技有限公司)，室温反应1小时。洗涤之后加入二抗山羊抗-小鼠碱性磷酸酶标记抗体(购自艾美捷科技有限公司)，室温反应1小时。洗涤之后加入碱性磷酸酶显色液，405nm波长读取吸收值。当样品孔OD值大于对照孔OD值3倍以上时，判为阳性克隆孔。将阳性克隆孔的菌转移至含有100微克每毫升氨苄霉素的LB液体中培养以便提取质粒并进行测序。
根据序列比对软件Vector NTI分析各个克隆的蛋白序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株。
实施例2：针对PD-L1的重链单域抗体的初步评价鉴定
2.1重链单域抗体在宿主菌大肠杆菌中表达、纯化：
将测序分析所获得的重链单域抗体的编码序列亚克隆至表达载体PET32b(Novagen，产品号：69016-3)中，并将测序鉴定正确的重组质粒转化到表达型宿主菌BL1(DE3)(天根生化科技，产品号：CB105-02)中，其涂布在含有100微克每毫升氨苄青霉素的LB固体培养基的板上，37℃过夜。挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，37℃摇床培养至OD值达到0.6-1时，0.5mM IPTG诱导，28℃摇床培养过夜。第二天，离心收菌，并将菌体破碎以获得抗体粗提液。然后镍离子亲和层析柱纯化抗体蛋白。最终得到纯度达90％以上的抗体蛋白。
2.2检测候选PD-L1重链单域抗体对人PD-L1蛋白的特异性结合
用PDL1-Fc融合蛋白包被平板4℃过夜，每孔加入100ng实施例2.1所得的重链单域抗体(对照组为不结合PDL1-Fc蛋白的单域抗体)，室温下反应1小时。洗涤之后加入一抗小鼠抗-His标签抗体(购自北京康为世纪生物科技有限公司)，室温反应1小时。洗涤之后加入二抗山羊抗小鼠辣根过氧化物酶标记抗体(义翘神州，Cat：SSA007200)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。
用Fc蛋白包被平板4℃过夜，每孔加入100ng实施例2.1所得的重链单域抗体(对照组为针对其他不相关靶标的单域抗体)，室温下反应1小时。洗涤之后加入一抗兔抗人Fc抗体(购自上海普欣生物技术有限公司)，室温反应1小时。洗涤之后加入二抗山羊抗兔辣根过氧化物酶标记抗体(购自上海普欣生物技术有限公司)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。
其中当对PDL1-Fc蛋白的OD值除以空白对照OD值的比值＞＝4时，判断候选抗体能结合PDL1-Fc蛋白；同时上述能结合PDL1-Fc抗原蛋白的抗体，其结合PDL1-Fc的OD值除以其结合Fc蛋白的OD值，比值＞＝5时，则认为该候选抗体能特异性结合PD-L1部分而非Fc部分。
结果显示筛选出的一个抗体株，抗体株56，能特异性结合PD-L1，而不与Fc结合。具体结果见下表1：
表1
抗体株 OD(对PD-L1) OD(对Fc) OD(PD-L1/Fc) OD(PD-L1/空白) SEQ ID NO
PDL1-56dAb 2.931 0.068 43.10294118 54.27777778 1
空白 0.054 0.072 0.75 1  
2.3PD-L1重链单域抗体对小鼠PD-L1蛋白的结合情况
小鼠PDL1-Fc蛋白(SEQ ID NO：33)由HEK293细胞表达获得(pCDNA4，Invitrogen，Cat V86220)。
用小鼠PDL1-Fc融合蛋白0.5μg/孔包被平板4℃过夜，每孔加入100ng实施例2.1所得的重链单域抗体(对照组为针对其他不相关靶标的单域抗体)，室温下反应1小时。洗涤之后加入一抗小鼠抗-His标签抗体，室温反应1小时。洗涤之后加入二抗山羊抗小鼠辣根过氧化物酶标记抗体，室温 反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。结果见表2。
表2
抗体株 OD(对小鼠PID-L1)
PDL1-56dAb 0.075
空白 0.096
可见，本发明的人PD-L1的重链单域抗体不结合小鼠PDL1-Fc蛋白。2.4竞争ELISA考察PD-L1重链单域抗体对PD-1与PD-L1相互作用的阻断效果
PDL1-Fc蛋白与PD1-Fc蛋白(SEQ ID NO：34)由HEK293细胞表达获得(pCDNA4，Invitrogen，Cat V86220)。利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白PD1-Fc-Biotin。
PDL1-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入100ng实施例2.1所得的重链单域抗体(对照组为针对其他不相关靶标的单域抗体，或只是缓冲液)以及10μg PD1-Fc-Biotin(空白组不加入任何抗体或蛋白，只加入等体积缓冲液)，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)，室温反应1小时。之后加入显色液，405nm波长读取吸收值。当样品OD值比对照OD值＜0.8时，则认为抗体有阻断效果。
结果如表3所示，抗体株56表现出对PD-1/PD-L1相互作用的阻断效应。
表3
样品 OD
对照1 2.335
对照2 2.413
空白 0.079
PDL1-56 0.129
2.5通过FACS考察PD-L1重链单域抗体对细胞表面PD-L1与PD-1相互作用的阻断效果
通过向人HEK293细胞瞬时转染带有人PD-L1全长蛋白基因的质粒(pCDNA4，Invitrogen，Cat V86220)，获得在膜上瞬时表达人PD-L1蛋白的HEK293细胞(293-PDL1细胞)。
取293-PDL1细胞，在96孔板重悬于0.5％PBS-BSA缓冲液中，加入上述待检测抗体。同时设置阴性对照，阴性对照为2μg的针对其他靶标的单 域抗体。所有样本加入0.3μg hPD-1-Fc-biotin，二抗为eBioscience的SA-PE，染色完毕后流式细胞仪进行检测。如果加入抗体后荧光值比不加抗体组向空白方向迁移，则认为该抗体能阻断细胞表面PD-L1和PD-1的相互作用。以此方法鉴定能阻断细胞表面PD-L1抗原和PD-1结合的抗体。
结果如图1显示，抗体株56表现出对PD-1/PD-L1相互作用的阻断效应。
2.6PD-L1重链单域抗体对PD-L1抗原蛋白的结合曲线
用得到的PD-L1重链单域抗体0.5μg/孔4℃过夜包被平板，随后加入PDL1-Fc融合蛋白的梯度稀释系列，室温下反应1小时。洗涤之后加入山羊抗人IgG-Fc辣根过氧化物酶标记抗体，室温反应1小时。洗涤之后加入辣根过氧化物酶显色液，405nm波长读取吸收值。应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对PD-L1结合曲线及EC50值(约为5ng/ml)。结果见图2。
2.7PD-L1重链单域抗体对PD-1与PD-L1相互作用的阻断曲线
用PDL1-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入100uL PD-L1阻断型单域抗体Fc融合蛋白的梯度稀释系列(稀释液中含有100μg/mL PD1-Fc-Biotin)，室温下反应1小时。之后加入SA-HRP(购自Sigma公司)，室温反应1小时。之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体株56对PD-L1/PD-1阻断曲线及IC50值(143ng/mL)。结果见图3。实施例3：PD-L1单域抗体的人源化
人源化方法采用蛋白表面氨基酸人源化(resurfacing)的方法及VHH人源化通用框架移植法(CDR grafting to a universal framework)完成。
人源化步骤如下：对抗体株56进行同源建模，建模软件为Modeller9。参考同源序列为NbBcII10抗体(PDB编号为：3DWT)，并根据蛋白三维结构计算氨基酸的相对溶剂可及性(relative solvent accessibility)。如抗体株56的某个氨基酸暴露在溶剂内，则替换为参考人抗体DP-47序列相同位置的氨基酸，最终完成全部替换。
VHH人源化通用框架移植法具体步骤如下：首先获取Cécile Vincke等人根据序列同源性设计完成的通用性人源化VHH框架h-NbBcII10FGLA(PDB编号为：3EAK)，该框架设计基于纳米抗体NbBcII10抗体(PDB编号为：3DWT)，参考人源抗体DP-47进行蛋白表面氨基酸人源化，并改造VHH序列框架2(framework-2)的部分氨基酸FGLA完成。我们直接使用h-NbBcII10FGLA作为框架，将CDR替换为抗体株56的CDR区，完成抗体的人源化。
对抗体株56进行人源化，获得5种抗体株56的人源化变体。表4列出这些人源化变体的序列编号以及其中的氨基酸变化，其中氨基酸残基编号比照Kabat编号。图4显示人源化序列的比对结果。
表4
Figure PCTCN2016092680-appb-000003
实施例4：用哺乳动物细胞制备PD-L1阻断型抗体蛋白
4.1制备PD-L1单域抗体的Fc融合蛋白
根据蛋白数据库Uniprot上人免疫球蛋白gamma1(IgG1)的恒定区氨基酸序列(P01857)，得到人IgG1-Fc区氨基酸序列(SEQ ID NO：7)。通过逆转录PCR，从人PBMC总RNA中获得编码人IgG1-Fc的核酸片段，再通过overlapping PCR得到由上述实施例获得的PD-L1单域抗体与Fc的融合蛋白的编码核酸片段。之后再亚克隆至载体pCDNA4(Invitrogen，Cat V86220)。也可使用经定点突变去除ADCC活性或CDC活性的Fc区序列，如SEQ ID NO：8或9。
重组构建的单域抗体-Fc融合蛋白质粒转染HEK293细胞进行抗体表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PET(Polyethylenimine)溶液，将每组质粒/PET混合物分别加入HEK293细胞悬液中，放置在37℃，10％C02，90rpm中培养；同时补加50μg/LIGF-1。四小时后再补加EX293培养基，2mM谷氨酰胺和50μg/LIGF-1，135rpm培 养。24小时后加3.8mM VPA。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，纯化得到目标PD-L1单域抗体-Fc融合蛋白。
所获得的PD-L1单域抗体-Fc融合蛋白的序列示于SEQ ID NO：10-27。
4.2制备MedImmune LLC公司和Roche公司的PD-L1抗体
MedImmune LLC公司的抗PD-L1抗体，参照专利US20130034559中2.14H9的方法克隆抗体基因，并将其克隆进载体pCDNA4中。TM004为Roche公司的抗PD-L1抗体，参照专利US20130045201A1中YW243.55.S70.hIgG的方法克隆其抗体基因，并将其克隆进载体pCDNA4中。
重组构建的质粒通过4.1相同的方法进行HEK293细胞的瞬时转染表达，得到的MedImmune LLC公司的抗PD-L1抗体重新命名为2.41H90P；Roche公司的抗PD-L1抗体重新命名为243.55。
4.3PD-L1单域抗体Fc融合蛋白与两种已知PD-L1抗体表达结果比较
利用相同的表达体系和瞬时转染条件，本发明的PD-L1单域抗体Fc融合蛋白的表达水平均高于为200mg/L，而抗体2.41H90P表达水平约为80mg/L，抗体243.55表达水平约为40mg/L。该结果表明，本发明的PD-L1单域抗体Fc融合蛋白相比另两种已知PD-L1抗体，其结构更为稳定，能够获得更高表达水平。
实施例5：鉴定PD-L1单域抗体Fc融合蛋白的功能
5.1PD-L1单域抗体Fc融合蛋白对PD-L1结合能力鉴定(ELISA法)
PDL1-Chis蛋白(SEQ ID NO：35)由HEK293瞬时表达及镍柱亲和层析纯化获得。得到的PDL1-Chis蛋白0.5μg/孔4℃过夜包被平板，之后加入上述实施例获得的PD-L1单域抗体Fc融合蛋白的梯度稀释系列，室温下反应1小时。洗涤之后加入山羊抗人IgG-Fc辣根过氧化物酶标记抗体，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对PD-L1结合曲线及EC50值(所有供试抗体EC50值均为约150ng/mL)以反映抗体对PD-L1的亲和能力。
结果见图5，其中纵坐标为OD405，横坐标为PD-L1单域抗体Fc融合蛋白浓度(单位ng/mL)；倒三角、正三角、方形分别代表抗体株56的三种不同人源化形式的Fc融合蛋白：hu56v1-Fc、hu56v2-Fc、hu56v5-Fc。三种蛋白对PD-L1的亲和力相当。
5.2鉴定PD-L1单域抗体Fc融合蛋白对PD-L1结合能力(SPR法)并与已知抗体比较
上述实施例获得的PD-L1单域抗体Fc融合蛋白针对重组的人PD-L1的结合动力学通过表面等离振子共振(surface plasmon resonance，SRP)方法，使用BIAcore X100仪器测量。重组的人PDL1-Fc直接包被于CM5生物传感器芯片上以获得大约1000应答单位(response units，RU)。对于动力学测量，将抗体用HBS-EP+1×缓冲液(GE，cat#BR-1006-69)三倍连续稀释(1.37nm至1000nm)，在25℃进样120s，解离时间为30min，10mM甘氨酸-HCl(pH2.0)再生120s。使用简单一对一Languir结合模型(BIAcore评价软件3.2版(BIAcore Evaluation Software version 3.2))计算结合速率(kon)和解离速率(koff)。平衡解离常数(kD)以比率koff/kon计算。
测量的抗PD-L1抗体的结合亲和力见表5。结果表明PDL1-56-Fc蛋白对PD-L1靶标蛋白的亲和力显著高于两个本领域已知PD-L1抗体，其较高的Ka及较低的Kd值说明该抗体融合蛋白能更快速的结合PD-L1抗原并很难解离下来，这进一步说明PDL1-56-Fc作为一个阻断型抗体，其性质更优于两个已知PD-L1抗体。
表5
抗体 Ka Kd KD
PDL1-56-Fc 1.796E+6 1.432E-5 7.975E-12
PDL1-hu56V2-Fc 2.123E+6 1.820E-5 8.573E-12
PDL1-56 3.323E+6 8.213E-4 2.472E-10
2.41H90P 7.949E+5 6.160E-5 7.750E-11
243.55 4.481E+5 6.055E-5 1.351E-10
5.3鉴定PD-L1单域抗体Fc融合蛋白对PDL1-PD1相互作用的阻断效果(竞争ELISA法)
PDL1-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入上述实施例获得的PD-L1单域抗体Fc融合蛋白的梯度稀释系列，每孔100uL(稀释液中 含有100ug/mL PD1-Fc-Biotin)，室温下反应1小时。洗涤之后加入SA-HRP(购自Sigma公司)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对PDL1-PD1阻断曲线及IC50值。结果见图6，其中纵坐标为OD405，横坐标为PD-L1单域抗体Fc融合蛋白浓度(单位ng/mL)；倒三角、正三角、方形分别代表抗体株56的三种不同人源化形式的Fc融合蛋白：hu56v1-Fc、hu56v2-Fc、hu56v5-Fc。三种蛋白对PDL1-PD1相互作用的阻断能力相当。
5.4鉴定PD-L1单域抗体Fc融合蛋白对PD-L1/CD80相互作用的阻断能力(竞争ELISA法)
CD80-Fc蛋白(SEQ ID NO：36)由HEK293细胞表达获得。利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白CD80-Fc-Biotin。
PDL1-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入上述实施例获得的PD-L1单域抗体Fc融合蛋白的梯度稀释系列，每孔100uL(稀释液中含有300ug/mL CD80-Fc-Biotin)，室温下反应1小时。洗涤之后加入SA-HRP(购自Sigma公司)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。
应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对PDL1-CD80阻断曲线及IC50值。结果见图7，其中纵坐标为OD405，横坐标为PD-L1单域抗体Fc融合蛋白hu56V2-Fc的浓度(单位ng/mL)。结果显示，PD-L1阻断型单域抗体Fc融合蛋白hu56V2-Fc能有效阻断PD-L1与CD80之间的相互作用。
5.5鉴定PD-L1单域抗体Fc融合蛋白对PD-L1/PD1相互作用的阻断能力(FACS法)
人HEK293细胞通过瞬时转染包含人PD-L1全长基因的质粒，于膜上瞬时表达猴PD-L1蛋白。
按照5×105cells/tube分组，每组加入工作浓度2ug/ml的PD1-muFc(SEQ ID NO：39)，再加入不同浓度的KN035，冰上孵育30min，洗涤3 次后加入PE标记的羊抗鼠二抗作为检测抗体，冰上孵育30min后，流式细胞仪检测荧光强度。
应用软件GraphPad Prism进行数据处理和作图分析，通过四参数拟合，得到抗体对293细胞膜表达PDL1与可溶PD1直接相互作用的阻断曲线及IC50值。结果见图8，其中纵坐标为MFI，横坐标为PD-L1单域抗体Fc融合蛋白hu56V1-Fcm1的浓度(单位μg/mL)。结果显示，PD-L1阻断型单域抗体Fc融合蛋白能有效阻断293细胞膜表达PDL1与PD1之间的相互作用。
Jurket细胞株转入并整合包含人PD1全长基因的质粒，得到稳定表达人PD1蛋白的Jurket细胞株，命名为Jurket-PD1。
取Jurkat-PD1细胞与生物素标记的PDL1-muFc(SEQ ID NO：38)蛋白(30ug/ml)冰上孵育30min后，加入梯度稀释的PD-L1单域抗体Fc融合蛋白hu56V1-Fcm1，冰上孵育1h，PBS洗涤3次，加入1∶250稀释的Streptavidin PE，冰上孵育30min，PBS洗涤3次后，流式细胞仪检测荧光强度。
应用软件GraphPad Prism进行数据处理和作图分析，通过四参数拟合，得到抗体对Jurkat-PD1与可溶PDL1-muFc蛋白直接相互作用的阻断曲线及IC50值。结果见图9，其中纵坐标为MFI，横坐标为PD-L1单域抗体Fc融合蛋白hu56V1-Fcm1的浓度(单位μg/mL)。结果显示，PD-L1阻断型单域抗体Fc融合蛋白能有效阻断Jurkat-PD1与PD-L1之间的相互作用。
5.6分析PD-L1单域抗体Fc融合蛋白对PD-L1蛋白结合的特异性
人HEK293细胞通过瞬时转染带有人B7家族蛋白全长基因的质粒(pCDNA4，Invitrogen，Cat V86220)，于膜上瞬时表达人PD-L1蛋白。该质粒还使得目标蛋白C端融合EGFP蛋白，从而可以通过绿色荧光强度来考察膜上B7家族蛋白的表达水平。构建好的瞬时转染细胞株包括：293-PDL1-EGFP，293-PDL2-EGFP，293-B7H3-EGFP，293-B7H3-EGFP。
取构建好的细胞，重悬于0.5％PBS-BSA Buffer中，加入hu56V2-Fc抗体，同时设置阴性对照为2μg的针对其他不相关靶标的单域抗体，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μl的0.5％PBS-BSA Buffer中，流式细胞仪进行检测。
结果如图10所示。上排为对照组，下排为样品组。可以清楚的看到，hu56V2-Fc抗体只特异性结合人PD-L1蛋白，而不与其他B7家族蛋白结合。
5.7PD-L1单域抗体Fc融合蛋白对猴PD-L1蛋白的结合
人HEK293细胞通过瞬时转染包含猴PD-L1全长基因的质粒，于膜上瞬时表达猴PD-L1蛋白(SEQ ID NO：37)。该质粒上还使得目标蛋白C端融合EGFP蛋白，从而可以通过绿色荧光强度来考察膜上猴PD-L1蛋白的表达水平。
取构建好的细胞，重悬于0.5％PBS-BSA Buffer中，加入hu56V2-Fc抗体，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μl 0.5％PBS-BSA Buffer中，流式细胞仪进行检测。
结果如图11所示。可以清楚的看到，hu56V2-Fc抗体能有效与猴PD-L1蛋白结合。
5.8PD-L1单域抗体Fc融合蛋白可有效识别病人组织切片上的PD-L1阳性细胞群
PD-L1阳性肺癌病人的肿瘤组织切片，用5ug/mL的hu56V2-Fc抗体作为一抗过夜染色，用山羊抗人HRP标记抗体(Perkin-Elmer，Cat：NEF802001EA)为二抗室温反应1.5hr，最后显色。
结果如图12，hu56V2-Fc抗体能有效识别肺癌病人组织切片上的PD-L1阳性细胞群，且能同时识别PD-L1阳性的肿瘤细胞和PD-L1阳性的免疫细胞。
5.9PD-L1单域抗体Fc融合蛋白在对PBMC的激活作用
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞(PBMC)。
将2.5ug/mL抗-CD3抗体以及PD-L1单域抗体Fc融合蛋白hu56V2-Fc(实验中也命名为KN035)的梯度稀释系列在4℃过夜包被于细胞培养板上。第二天每孔中加入1x105个PBMC细胞。培养5天后取上清，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ的水平。
结果见图13。可见PD-L1单域抗体Fc融合蛋白配合anti-CD3抗体可 以增强PBMC中细胞分泌γ-干扰素，即PD-L1单域抗体Fc融合蛋白增强了PBMC细胞的活化。同时这种激活效果具有浓度依赖性。
5.10在树突细胞-T细胞混合淋巴反应中测定PD-L1单域抗体Fc融合蛋白对CD4+T细胞的激活作用，并与MedImmune LLC公司的抗PD-L1抗体进行比较
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞PBMC。然后将其用无血清的RPMI1640培养基培养1-2小时，除去未贴壁的细胞，并将细胞培养于含10％FBS，10ng/ml GM-CSF以及20ng/mL IL-4的RPMI中。培养5-6天后，加入10ng/ml的TNF-α并孵育24小时，获得成熟的树突细胞(DC细胞)。
将通过此方法获得的DC细胞重悬于RPMI完全培养基中，2x105/ml。然后在96孔U形底板(Costar：3799)中每孔加入50μl，在培养箱中培养。
利用磁珠分离试剂盒(Miltenyi Biotec：130-096-533)按照说明书方法从另一个供体PBMC中分离CD4+T细胞。
将通过上述方法获得的1x104个树突细胞和1x105个CD4+T细胞混合，于RPMI完全培养基中重悬并加入96孔培养板，每孔加入50μl细胞混液；每孔加入100μl稀释于RPMI完全培养基中的hu56V2-Fc，抗体终浓度为0.1μg/ml或0μg/ml。培养5-7天后取上清，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ的水平。
结果见图14A。可见PD-L1单域抗体Fc融合蛋白可以增强混合淋巴细胞反应中CD4+T细胞的γ-干扰素分泌，即PD-L1阻断型单域抗体Fc融合蛋白增强了T细胞的活化。
50ng/ml GM-CSF+25ng/ml IL-4培养供试者1的PBMC，6天后，用TNF-α(50ng/ml)成熟24h，收集DC细胞；再从供试者2的PBMC中用磁珠分离试剂盒(Miltenyi Biotec：130-096-533)分选出CD4+T cell；DC细胞按照104/well加入到96孔U形底板，2-4h后加入105cells/well的CD4+T；每孔加入不同浓度的PD-L1单域抗体Fc融合蛋白hu56V1-Fcm1或者MedImmune LLC公司的抗PD-L1抗体2.41H90P；培养5天后，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ的水平。
结果见图14B，其中灰色柱状图指示PD-L1单域抗体Fc融合蛋白刺激 分泌IFN-γ的情况，黑色柱状图指示MedImmune LLC公司的抗PD-L1抗体2.41H90P。PD-L1单域抗体Fc融合蛋可随浓度增加而增强混合淋巴细胞反应中CD4+T细胞分泌IFN-γ的能力，且相同浓度条件下，PD-L1单域抗体Fc融合蛋活化T细胞的能力略强于MedImmune LLC公司的抗PD-L1抗体2.41H90P。
5.11PD-L1单域抗体Fc融合蛋白在Jurkat/Raji-PDL1混合培养体系刺激T细胞分泌IL-2作用并与MedImmune LLC公司的抗PD-L1抗体进行比较
我们构建了一个T细胞活化体系即Jurkat/Raji-PDL1共培养体系来检测PD-L1单域抗体Fc融合蛋白对T细胞活化的作用并与MedImmune LLC公司的抗PD-L1抗体进行比较。
该体系采用Jurkat细胞(T细胞)作为效应细胞，以anti-human CD3Antibody作为Jurkat细胞活化的第一信号；通过基因工程改造、稳定表达人PDL1的Raji细胞Raji-PDL1细胞，其表面的B7家族分子中CD80提供第二共刺激信号活化Jurket细胞，同时其细胞表面高表达的PDL1作为负调节因子通过结合PD1抑制Jurket细胞活化。
使用10％FBS+1640+150ng/ml anti-CD3配制梯度稀释的hu56V1-Fcm1和2.41H90P抗体蛋白；Jurkat和Raji-PDL1细胞用10％FBS分别调整至3×106cells/ml和1.5×106cells/ml，每孔加入50ul，37℃放置24小时后，取出100ul培养上清，使用试剂盒检测IL-2的表达量。
图15显示，其中黑色柱状图指示PD-L1单域抗体Fc融合蛋白刺激分泌IL-2的情况，灰色柱状图指示MedImmune LLC公司的抗PD-L1抗体2.41H90P。PD-L1单域抗体Fc融合蛋白可随浓度增加而增强该混合淋巴细胞反应中Jurkat细胞分泌IL-2的能力，且相同浓度条件下，其活化Jurkat细胞的能力略强于MedImmune LLC公司的抗PD-L1抗体2.41H90P。
5.12PD-L1单域抗体Fc融合蛋白与FcRn的亲和力
生物素化的PD-L1单域抗体Fc融合蛋白hu56V1-Fc，hu56V2-Fc，hu56V1-Fcm1以及hu56V2-Fcm1稀释至10ug/mL固化到SA biosensor上，人FcRn蛋白(RnD Systems，货号8639-FC-050)稀释至200nM、100nM、50nM、25nM、12.5nM 5个梯度。使用Fortebio公司的Octet K2检测其相 互作用，其中固化100s，结合60s，解离30s。
表6显示，PD-L1单域抗体Fc融合蛋白与FcRn的平均KD约为5.1E-07M。突变Fc(Fcm1)与野生型Fc相比，亲和力没有明显差异。
表6
样品名称 KD(M) kon(1/Ms) kdis(1/s)
hu56V1-Fc 5.13E-07 1.95E+05 1.00E-01
hu56V2-Fc 5.05E-07 2.10E+05 1.06E-01
hu56V1-Fcm1 5.10E-07 1.89E+05 9.63E-02
hu56V2-Fcm 1 5.10E-07 2.34E+05 1.19E-01
5.13带有突变Fc的PD-L1单域抗体Fc融合蛋白的CDC以及ADCC活性考察
以300IU/ml IL-2活化24小时后PBMCs为效应细胞，细胞数为8×105cells/well；以稳定表达人PDL1蛋白的Raji-PDL1为靶细胞，细胞数为2×105cells/well；加入不同浓度的hu56V1-Fcm1或者作为阳性对照的Rituxan蛋白，37℃培养6小时后使用CytoTox 
Figure PCTCN2016092680-appb-000004
非放射性细胞毒性检测试剂盒检测浓度下的ADCC活性(％)。
图16A显示，与阳性对照Rituxan相比，hu56V1-Fcm1无明显ADCC活性。
以Raji-PDL1细胞为靶细胞，细胞数为2×104cells/well，5％食蟹猴血清提供补体，加入不同浓度的hu56V1-Fcm1和阳性对照Rituxan，37℃培养2h后，使用CCK-8检测样品的CDC活性。
图16B显示，与阳性对照相比，hu56V1-Fcm1在0.02ug/ml至20ug/ml浓度范围内，无CDC活性。
5.14PD-L1单域抗体Fc融合蛋白对肿瘤生长的抑制活性
对于不能识别小鼠PD-L1的PD-L1单域抗体Fc融合蛋白，hu56V2-Fc，采用免疫缺陷NOD/SCID(非肥胖型糖尿病/重症联合免疫缺陷)小鼠研究其体内活性。通过对NOD/SCID小鼠皮下移植表达人PD-L1的黑色素瘤细胞系A375(ATCC，CRL-1619TM)和人的外周血单个核细胞PBMC的实验来实现此研究目的。A375和PBMC在注射前按5∶1的比例混合，皮下注射总体 积100μl(含500万A375，100万PBMC)。抗体在肿瘤接种后24小时第一次腹腔注射给药，之后每周给药一次，给药剂量为0.3mg/kg；PBS作为阴性对照。每个实验组4-6只小鼠。每周两次观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，绘制肿瘤生长曲线图(参见图17A)，可以看出抗体hu56V2-Fc在0.3mg/kg剂量下就可以显著抑制肿瘤生长。
使用相同的体内模型考察同样不识别小鼠PD-L1的PD-L1单域抗体Fc融合蛋白，hu56V1-Fcm1。A375与人PBMCs按4∶1混合后接种于NOD-SCID小鼠皮下，4小时后腹腔注射给予不同剂量的hu56V1-Fcm1(0.1、0.3、1、3、10mg/kg)，研究一周一次连续4周给药后对NOD-SCID小鼠A375/human PBMCs异体移植瘤的抑瘤作用。PBS作为阴性对照。每个实验组4-6只小鼠。每周两次观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，绘制肿瘤生长曲线图(参见图17B)。结果显示，hu56V1-Fcm1各剂量组(0.1～10mg/kg)均对NOD-SCID小鼠A375/human PBMCs异体移植瘤具有显著抑瘤作用，但无明显剂量相关性。抗体hu56V1-Fcm1在0.1mg/kg剂量下就可以显著抑制肿瘤生长。
5.15不同给药次数的PD-L1单域抗体Fc融合蛋白对肿瘤生长的抑制活性
继续使用上述NOD-SCID小鼠A375/human PBMCs异体移植瘤模型进行考察。A375与人PBMCs按4∶1混合后接种于NOD-SCID小鼠皮下，4小时后腹腔注射给予hu56V1-Fcm1(0.3mg/kg)，之后每三天腹腔给药一次。最终给药次数分别为1，2，3，4次。每三天观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，直至第一次给药算起第33天。绘制肿瘤生长曲线图(图18)，结果显示考察周期内各给药次数均对NOD-SCID小鼠A375/human PBMCs异体移植瘤具有显著抑瘤作用。
5.16PD-L1单域抗体Fc融合蛋白对肿瘤生长的抑制活性并与MedImmune LLC公司的抗PD-L1抗体进行比较
通过对NOD/SCID小鼠皮下移植表达人PD-L1的黑色素瘤细胞系A375(ATCC，CRL-1619TM)和人的外周血单个核细胞PBMC的实验来实现此研究目的。A375和PBMC在注射前按5∶1的比例混合，皮下注射总体积100μl(含500万A375，100万PBMC)，抗体在肿瘤接种后24小时第一次 腹腔注射给药，之后每周给药一次，给药剂量为1mg/kg。给药组包括hu56V2-Fc(表示为hu56)以及MedImmune LLC公司的抗PD-L1抗体组(表示为2.41)，PBS作为阴性对照。每个实验组4-6只小鼠。每周两次观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，绘制肿瘤生长曲线图(参见图19A)。其中MedImmune LLC公司的抗PD-L1抗体在此模型下基本无效，且在第35天肿瘤体积已经超出阴性对照组，因此停止给药及肿瘤体积测量。可以看出在该模型下，hu56V2-Fc在1mg/kg剂量下抑制A375肿瘤生长的效果显著优于MedImmune LLC公司的抗PD-L1抗体2.41H90P。
由于上述体系下MedImmune LLC公司的抗PD-L1抗体没有显示出肿瘤抑制效果，有可能是由于体系内PBMC的激活不足以抑制肿瘤细胞的生长。因此增加混合细胞中PBMC的含量重复考察MedImmune LLC公司的抗PD-L1抗体的抑瘤效果。
A375和PBMC在注射前按1∶1的比例混合，皮下注射总体积100μl(含500万A375，500万PBMC)，抗体在肿瘤接种后24小时第一次腹腔注射给药MedImmune LLC公司的抗PD-L1抗体(2.41H90P)，之后每周给药一次，给药剂量为1mg/kg；PBS作为阴性对照。每个实验组4-6只小鼠。每周两次观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，绘制肿瘤生长曲线图(参见图19B)。可以看出，增加PMBC的比例后，MedImmune LLC公司的抗PD-L1抗体在体内模型中表现出了抑瘤效果。
计算抗体在第42天的平均肿瘤抑制率(TGI＝(1-给药组肿瘤体积/对照组肿瘤体积)x100％)，列入下表7：
表7
Figure PCTCN2016092680-appb-000005
上述体内抑瘤实验结果表明，本发明的PD-L1阻断型单域抗体Fc融合蛋白在对黑色素瘤A375的裸鼠体内模型中，表现出显著优于已知PD-L1阻断型抗体(MedImmune LLC公司的抗PD-L1抗体)的抑瘤效果。
实施例6：PD-L1单域抗体Fc融合蛋白稳定性研究
6.1PD-L1单域抗体Fc融合蛋白对碱性破坏及氧化破坏的抗性研究
使用500mM碳酸氢铵作为碱性破坏试剂，37℃处理38小时。选择1％双氧水作为氧化剂，室温处理8小时。
使用竞争ELISA法检测处理前后的上述实施例获得的PD-L1单域抗体Fc融合蛋白的生物学活性变化。如图20可知，碱性及氧化破坏不影响候选PD-L1单域抗体Fc融合蛋白的活性，38小时碱处理后的竞争ELISA活性相对0小时为103％。8小时氧化的竞争ELISA活性相对0小时为106％。
6.2PD-L1单域抗体Fc融合蛋白在高浓缩下的稳定性
将PD-L1单域抗体Fc融合蛋白通过UF/DF浓缩并换液到PBS缓冲液中。并通过SE-HPLC检测其聚体的形成趋势。
浓缩至200mg/mL时，PD-L1单域抗体Fc融合蛋白的SE-HPLC检测纯度为96.8％，与低浓度(～2mg/mL)时相比，聚体增加了约2.4％。整个浓缩过程中，蛋白溶液未出现浑浊或聚集现象。
Figure PCTCN2016092680-appb-000006
Figure PCTCN2016092680-appb-000007
Figure PCTCN2016092680-appb-000008
Figure PCTCN2016092680-appb-000009
Figure PCTCN2016092680-appb-000010

Claims (24)

  1. 程序性死亡配体1(PD-L1)结合分子，其能够特异性结合PD-L1且由式A-L-B的氨基酸序列组成，其中A表示免疫球蛋白单一可变结构域，L不存在或表示氨基酸接头，且B表示人免疫球蛋白Fc区，
    其中所述免疫球蛋白单一可变结构域由选自SEQ ID NO:1-6的氨基酸序列组成，
    所述PD-L1结合分子能够通过所述人免疫球蛋白Fc区形成同二聚体。
  2. 权利要求1的PD-L1结合分子，其中所述人免疫球蛋白Fc区是人IgG1、IgG2、IgG3或IgG4的Fc区。
  3. 权利要求1的PD-L1结合分子，其中所述人免疫球蛋白Fc区被突变以去除ADCC和CDC活性
  4. 权利要求1的PD-L1结合分子，其中所述免疫球蛋白Fc区的氨基酸序列选自SEQ ID NO:7-9。
  5. 权利要求1-4中任一项的PD-L1结合分子，其中所述氨基酸接头长1-20个氨基酸。
  6. 权利要求1-5中任一项的PD-L1结合分子，其由选自SEQ ID NO:10-27的氨基酸序列组成。
  7. 权利要求1-6中任一项的PD-L1结合分子，其具有下述特征中的至少一个：
    (a)与人PD-L1结合的KD值小于1×10-7M；
    (b)阻断PD-L1和PD-1的相互作用；
    (c)增强PBMC和/或T细胞的活化；
    (d)抑制肿瘤生长。
  8. 权利要求1-7中任一项的PD-L1结合分子，其结合PD-L1的KD值小于1×10-7M，优选小于1×10-8M、更优选小于1×10-9M、更优选小于1×10-10M、尤其更优选小于1×10-11M。
  9. 核酸分子，其编码权利要求1-8中任一项的PD-L1结合分子。
  10. 表达载体，其包含与表达调控元件可操作地连接的权利要求9的核酸分子。
  11. 宿主细胞，其包含权利要求9的核酸分子或以权利要求10的表达 载体转化，并能够表达所述PD-L1结合分子。
  12. 产生权利要求1-8中任一项的PD-L1结合分子的方法，包括：
    a)在允许所述PD-L1结合分子表达的条件下培养权利要求11的宿主细胞；
    b)从得自步骤a)的培养物回收由所述宿主细胞表达的PD-L1结合分子；及
    c)任选进一步纯化和/或修饰得自步骤b)的PD-L1结合分子。
  13. 药物组合物，其包含权利要求1-8任一项的PD-L1结合分子以及药学上可接受的载体。
  14. 一种在对象中预防和/或治疗癌症的方法，包括给所述对象施用有效量的权利要求1-8任一项的PD-L1结合分子或权利要求13的药物组合物。
  15. 权利要求14的方法，其还包括给所述对象施用其它抗肿瘤治疗手段。
  16. 权利要求15的方法，其中所述其它抗肿瘤治疗手段包括化疗、放疗或靶向其它肿瘤特异性抗原的抗体。
  17. 权利要求16的方法，其中所述靶向其它肿瘤特异性抗原的抗体包括抗EGFR抗体、抗EGFR变体的抗体、抗VEGFa抗体、抗HER2抗体、或抗CMET抗体。
  18. 权利要求17的方法，其中所述靶向其它肿瘤特异性抗原的抗体是单克隆抗体。
  19. 权利要求14-18中任一项的方法，其中所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体癌、骨肉瘤。
  20. 一种在对象中预防和/或治疗感染性疾病的方法，包括给所述对象施用有效量的权利要求1-8任一项的PD-L1结合分子或权利要求13的药物组合物。
  21. 权利要求20的方法，其中所述感染性疾病由选自以下的病原体引起：HIV、肝炎病毒、流感病毒、疱疹病毒、贾第虫、疟原虫、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。
  22. 一种在对象中预防和/或治疗慢性炎性疾病的方法，包括给所述对象施用有效量的权利要求1-8任一项的PD-L1结合分子或权利要求13的药物组合物。
  23. 权利要求22的方法，其中所述慢性炎性疾病为扁平苔藓或T细胞介导的慢性炎性皮肤粘膜病。
  24. 权利要求1-8任一项的PD-L1结合分子或权利要求13的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗癌症、感染性疾病或慢性炎性疾病。
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US20180291103A1 (en) 2018-10-11
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