WO2017059819A1 - 高亲和力的可溶性pdl-1分子 - Google Patents

高亲和力的可溶性pdl-1分子 Download PDF

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WO2017059819A1
WO2017059819A1 PCT/CN2016/101597 CN2016101597W WO2017059819A1 WO 2017059819 A1 WO2017059819 A1 WO 2017059819A1 CN 2016101597 W CN2016101597 W CN 2016101597W WO 2017059819 A1 WO2017059819 A1 WO 2017059819A1
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pdl
molecule
amino acid
complex
fusion protein
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French (fr)
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李懿
梁兆端
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Guangzhou Institute of Biomedicine and Health of CAS
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Guangzhou Institute of Biomedicine and Health of CAS
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Priority to CN201680058851.9A priority Critical patent/CN108137671B/zh
Priority to EP16853123.4A priority patent/EP3360893B1/en
Priority to US15/767,334 priority patent/US11124557B2/en
Priority to JP2018518441A priority patent/JP6650515B2/ja
Publication of WO2017059819A1 publication Critical patent/WO2017059819A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a soluble programmed death receptor ligand (Programmed Death Ligand-1, PDL-1) having high affinity for Programmed Death-1 (PD-1) molecules. a molecule, and a method of preparation and use of the molecule.
  • PDL-1 soluble programmed death receptor ligand
  • PD-1 is an immunosuppressive receptor expressed on activated T cells and B cells.
  • PDL-1 is a ligand of PD-1, belonging to the B7 family, with IgV and IgC-like regions, transmembrane regions and tails of the cytoplasmic region.
  • PDL-1 interacts with receptor PD-1 on lymphocytes in an immune response. The negative regulation plays an important role.
  • Many tumor cell lines and tumor cells highly express PDL-1 molecules (Konishi J et al., Clin. Cancer Res., 2004, 10(15): 5094-5100), which binds to PD-1 molecules on the surface of lymphocytes.
  • a further object of the present invention is to provide a method for preparing the above high affinity PDL-1 molecule and use thereof way.
  • a PDL-1 molecule comprising a mutation in the amino acid sequence set forth in SEQ ID NO: 1.
  • the amino acid sequence of the PDL-1 molecule is based on the amino acid sequence shown in SEQ ID NO.: 1 and one or more amino acid residues are subjected to the amino acid sequence shown in SEQ ID NO: 1. Mutation or insertion of an amino acid residue to obtain the PDL-1 molecule.
  • the amino acid sequence of the PDL-1 molecule is at least 90% (preferably, at least 92%; more preferably, at least 94%) identical to the amino acid sequence set forth in SEQ ID NO: 1. Sex.
  • the affinity of the PDL-1 molecule to the PD-1 molecule is at least 2 times the affinity of the wild-type PDL-1 molecule to the PD-1 molecule; preferably, at least 5 fold; more preferably, At least 10 times; most preferably at least 50 times.
  • the affinity of the PDL-1 molecule to the PD-1 molecule is at least 100 times the affinity of the wild type PDL-1 molecule to the PD-1 molecule; preferably, at least 200 fold; more preferably, At least 500 times.
  • the mutated amino acid residue in the PDL-1 molecule is one or more of amino acid residues 1 to 3, 35 to 50, and/or 95 to 105, wherein the amino acid residue
  • the base number is the number shown in SEQ ID NO: 1.
  • the mutated amino acid residue in the PDL-1 molecule is one or more of 1, 36 to 40, 47 to 48, and/or 95 to 101 amino acid residues, wherein the amino acid
  • the residue numbering is given by the number shown in SEQ ID NO: 1.
  • the number of mutated amino acid residue sites is n, wherein 1 ⁇ n ⁇ 15; preferably, 3 ⁇ n ⁇ 11; more preferably, 4 ⁇ n ⁇ 10, such as n can be 5 6, 6, 8, 9, 10.
  • the mutated amino acid residue site in the PDL-1 molecule comprises one or more of 1F, 36I, 38Y, 40E, 47I, 48Q, 95R, 97M, 99S, 101G or after 102G There is one or more amino acid insertions in which the amino acid residue numbering is the number shown in SEQ ID NO: 1.
  • the mutated amino acid residue sites in the PDL-1 molecule include 36I, 38Y, and 40E, wherein the amino acid residue numbering is the number shown in SEQ ID NO: 1.
  • the mutated amino acid residue site in the PDL-1 molecule further comprises 40E, and 47I, wherein the amino acid residue numbering is the number shown in SEQ ID NO: 1.
  • the mutated amino acid residue sites in the PDL-1 molecule further comprise 95R, and 97M, wherein the amino acid residue numbering is the number shown in SEQ ID NO: 1.
  • the mutated PDL-1 molecule comprises one or more amino acid residues selected from the group consisting of: 1W; 36Q, 36E, 36T or 36N; 38F, 38N, 38H or 38A; 40M, 40F, 40L, 40W or 40Y; 47L, 47V, 47F, 47S, 47Y, 47P or 47A; 48S; 95T, 95V or 95L; 97L; 99G or 99A; 101D, 101K, 101E or 101H; after insertion of amino acid G at 102G; The residue numbering is given by the number shown in SEQ ID NO: 1.
  • the PDL-1 molecule comprises: 36Q, 38H, and 40F;
  • the PDL-1 molecule further comprises: 48S, wherein the amino acid residue numbering is the number shown in SEQ ID NO: 1.
  • the PDL-1 molecule further comprises: 47V, and 48S; or
  • the PDL-1 molecule further comprises: 97L, wherein the amino acid residue numbering is the number shown in SEQ ID NO: 1.
  • the PDL-1 molecule further comprises: 95T, 97L, 99G, and 101K; or
  • amino acid sequence of the PDL-1 molecule is selected from one of SEQ ID NOs: 5-34.
  • the PDL-1 molecule is soluble.
  • the C or N terminus of the PDL-1 molecule binds to a conjugate.
  • the conjugate that binds to the PDL-1 molecule is a T cell receptor, preferably the T cell receptor is a high affinity T cell receptor.
  • a fusion protein comprising the PDL-1 molecule of the first aspect of the invention.
  • the fusion protein further comprises hIgG4Fc.
  • a multivalent PDL-1 complex comprising at least two PDL-1 molecules, and wherein at least one PDL-1 molecule is the invention a PDL-1 molecule as described in one aspect; or the multivalent PDL-1 complex comprises at least one second party of the invention The fusion protein described above.
  • a fourth aspect of the invention provides a nucleic acid molecule comprising a PDL-1 molecule encoding the first aspect of the invention, the fusion protein of the second aspect of the invention, or the third aspect of the invention The nucleic acid sequence of the multivalent PDL-1 complex or its complement.
  • a vector comprising the nucleic acid molecule of the fourth aspect of the invention is provided.
  • a host cell comprising the vector of the fifth aspect of the invention or the nucleic acid molecule of the fourth aspect of the invention in which the exogenous source is integrated with the chromosome is contained in the host cell.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the PDL-1 molecule of the first aspect of the invention, or the fusion of the second aspect of the invention, is provided A protein, or a PDL-1 complex of the third aspect of the invention.
  • the pharmaceutical composition further comprises ImmTAC and/or HATac.
  • a method of treating a disease comprising administering to a subject in need of treatment an appropriate amount of the PDL-1 molecule of the first aspect of the invention, the fusion protein of the second aspect of the invention, or The PDL-1 complex according to the third aspect of the invention, or the pharmaceutical composition according to the seventh aspect of the invention.
  • the disease is a tumor.
  • the ninth aspect of the invention provides the use of the PDL-1 molecule of the first aspect of the invention, the fusion protein of the second aspect of the invention, or the PDL-1 complex of the third aspect of the invention, It is used to prepare drugs for treating tumors.
  • a method of preparing the PDL-1 of the first aspect of the invention comprising the steps of:
  • Figure 1a and Figure 1b are the extracellular amino acid sequence and nucleotide sequence of the wild-type PDL-1 molecule, respectively.
  • Figure 2 is a SDS-PAGE gel of purified wild-type PDL-1 protein.
  • 3a and 3b are the amino acid sequence and nucleotide sequence of the renatured and purified PD-1 in Example 2 of the present invention, respectively.
  • Figure 4 is a BIAcore map of the binding of the wild-type PDL-1 molecule to the PD-1 molecule.
  • Figure 5 is the amino acid sequence of the high affinity PDL-1 molecule of the present invention, and the mutated or inserted amino acid residues are underlined.
  • Figures 6a and 6b are the alpha and beta chain amino acid sequences of ImmTAC (1G4), respectively.
  • Figure 7 shows that the high affinity PDL-1 molecule of the invention significantly enhances ImmTAC (1G4)-mediated PBMC killing of tumor cells.
  • Figure 8a is the amino acid sequence of the hIgG4 Fc molecule.
  • Figure 8b is the nucleotide sequence of the hIgG4 Fc molecule.
  • Figure 9 is a SDS-PAGE gel of purified wild-type PDL1-hIgG4Fc fusion protein.
  • Figure 10a shows that the high affinity PDL1-hIgG4Fc fusion protein of the present invention significantly enhances ImmTAC (1G4)-mediated PBMC killing of tumor cell Mel624.
  • Figure 10b shows that the high affinity PDL1-hIgG4Fc fusion protein of the present invention significantly enhances ImmTAC (1G4)-mediated PBMC killing of tumor cell NCI-H1299.
  • Figure 10c shows that the high affinity PDL1-hIgG4Fc fusion protein of the present invention significantly enhances ImmTAC (1G4)-mediated PBMC killing of tumor cells IM9.
  • Figure 10d shows that the high affinity PDL1-hIgG4Fc fusion protein of the present invention significantly enhances ImmTAC (1G4)-mediated PBMC killing of tumor cell MDA-MB-231.
  • the present inventors have unexpectedly discovered that soluble PDL-1 molecules having high affinity for PD-1 molecules can effectively enhance the killing ability of lymphocytes.
  • the present invention provides a soluble high affinity PDL-1 molecule having an affinity for PD-1 that is at least twice the affinity of the wild-type PDL-1 molecule for PD-1.
  • the PDL-1 molecule of the present invention contains a mutation in the amino acid sequence shown in SEQ ID NO: 1. More specifically, the amino acid sequence of the PDL-1 molecule has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
  • Wild-type PDL-1 molecule refers to the extracellular region of the wild-type PDL-1 molecule, and its amino acid sequence and nucleotide sequence are shown in Figures 1a and 1b, respectively.
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • T cells about 75% positive for CD4 and CD8
  • B cells about NK cells (about 25% combined).
  • High affinity T cell receptor refers to a T cell receptor whose affinity to its ligand is at least twice the affinity of the corresponding wild type T cell receptor to its ligand.
  • Tumor refers to all types of cancer cell growth or carcinogenesis, metastatic tissue or malignant transformed cells, tissues or organs, regardless of pathological type or stage of infection.
  • tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignant tumors of different organ systems, such as sarcoma, lung squamous cell carcinoma, and cancer.
  • sarcoma a malignant tumors of different organ systems
  • lung squamous cell carcinoma e.g., infected prostate, lung, breast, lymph, gastrointestinal (eg colon), and genitourinary tract (eg kidney, epithelial cells), pharynx.
  • Squamous cell carcinoma of the lung includes malignant tumors, for example, most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell cancer of the lung, small intestine cancer and esophageal cancer. Metastatic lesions of the above cancers can likewise be treated and prevented using the methods and compositions of the invention.
  • physiologically acceptable carriers are non-toxic to the cells or individuals to which they are exposed, in dosages and concentrations.
  • physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins Protein; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, Arginine or lysine; monosaccharides, disaccharides and other sugars, including glucose, mannose or dextrin; complexing agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; / or nonionic surfactants such as TWEENTM, polyethylene glycol
  • PD-1 is an immunosuppressive receptor expressed on activated T cells and B cells.
  • PDL-1 is a ligand of PD-1, belonging to the B7 family, with IgV and IgC-like regions, transmembrane regions and tails of the cytoplasmic region.
  • PDL-1 interacts with receptor PD-1 on lymphocytes in an immune response.
  • the negative regulation plays an important role.
  • the present inventors have unexpectedly discovered that soluble PDL-1 molecules having high affinity for PD-1 molecules can effectively enhance the killing ability of lymphocytes.
  • the present invention provides a soluble high affinity PDL-1 molecule having an affinity for PD-1 that is at least twice the affinity of the wild-type PDL-1 molecule for PD-1.
  • the binding affinity of the above PDL-1 molecule to PD-1 (in inverse proportion to the dissociation equilibrium constant K D ) and the binding half life (expressed as T 1/2 ) can be determined by any suitable method. It should be understood that doubling the affinity will result in a halving of K D . T 1/2 is calculated as In2 divided by the dissociation rate (K off ). Therefore, doubling K off will result in a halving of T 1/2 .
  • the same test protocol is used to detect binding affinity or binding half-life several times, for example 3 or more times, and the average of the results is taken. In a preferred embodiment, these measurements are performed using the surface plasmon resonance (BIAcore) method of the present invention.
  • the method detects that the dissociation equilibrium constant K D of the wild-type PDL-1 molecule to the PD-1 molecule in the present invention is 2.462E-05M, and the BIAcore binding map thereof is shown in FIG. 4 . Since the doubling of the affinity will result in a halving of K D , if the dissociation equilibrium constant K D of the high affinity PDL-1 molecule to the PD-1 molecule is detected to be 1.231E-05M, the high affinity PDL-1 molecule is indicated to PD.
  • the affinity of the -1 molecule is twice the affinity of the wild-type PDL-1 molecule for PD-1.
  • 1M 1000 ⁇ M
  • 1 ⁇ M 1000nM
  • 1nM 1000pM.
  • the affinity of the PDL-1 molecule of the present invention to the PD-1 molecule is determined by the preferred method for determining the affinity of the present invention to be at least 2 of the affinity of the wild-type PDL-1 molecule to the PD-1 molecule. Preferably; at least 5 times; more preferably at least 10 times; most preferably at least 50 times.
  • the affinity of the PDL-1 molecule to the PD-1 molecule is at least 100 times the affinity of the wild type PDL-1 molecule to the PD-1 molecule; preferably, at least 200 fold; more preferably, At least 500 times.
  • the present invention is a high affinity PDL-1 and PD-1 molecule affinity K D ⁇ 1.231E-05M; Preferably, 1.0E-06M ⁇ K D ⁇ 1.0E-05M ; more preferably, 1.0E-07M ⁇ K D ⁇ 1.0E-06M; most preferably, 1.0E-08M ⁇ K D ⁇ 1.0E-07M .
  • the high affinity PDL-1 molecule of the present invention contains one or more mutations in the amino acid sequence set forth in SEQ ID NO: 1.
  • the amino acid sequence of the PDL-1 molecule and the amino acid sequence of SEQ ID NO: 1 are at least 90% (preferably, at least 92%; more preferably, at least 94%, such as 95%, 96%, Sequence identity of 97%, 98%, 99%).
  • amino acid residue sites mutated in the high affinity PDL-1 molecule of the present invention include one or more of 1F, 36I, 38Y, 40E, 47I, 48Q, 95R, 97M, 99S, 101G or after 102G Insertion of one or more amino acids, wherein the amino acid residue numbering is the number shown in SEQ ID NO: 1.
  • the mutated PDL-1 molecule comprises one or more amino acid residues selected from the group consisting of: 1W; 36Q, 36E, 36T or 36N; 38F, 38N, 38H or 38A; 40M, 40F, 40L, 40W or 40Y; 47L, 47V, 47F, 47S, 47Y, 47P or 47A; 48S; 95T, 95V or 95L; 97L; 99G or 99A; 101D, 101K, 101E or 101H; after insertion of amino acid G at 102G; The residue numbering is given by the number shown in SEQ ID NO: 1.
  • amino acid sequence of the PDL-1 molecule is selected from one of SEQ ID NOs: 5-34.
  • the wild-type PDL-1 molecule used in the present invention does not contain a transmembrane region.
  • the PDL-1 molecule is soluble.
  • Mutations can be carried out by any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme-based cloning or linkage-independent cloning (LIC) methods. Many standard molecular biology textbooks detail these methods. For more details on polymerase chain reaction (PCR) mutagenesis and cloning based on restriction enzymes, see Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (Third Edition) CSHL Publishing house. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6).
  • PCR polymerase chain reaction
  • LIC linkage-independent cloning
  • Methods for producing high affinity PDL-1 molecules of the invention may be, but are not limited to, screening for a PDL-1 having high affinity for PD-1 from a diverse library of phage particles displaying such PDL-1 molecules, such as The literature (Li, et al (2005) Nature Biotech 23(3): 349-354).
  • the gene expressing the wild type PDL-1 of the present invention or the gene expressing the wild type PDL-1 of the present invention which is slightly modified can be used to prepare a template strand. Then introduced into the DNA encoding the template strand The desired changes in the high affinity PDL-1 of the invention.
  • the PDL-1 molecules of the invention may also be provided in the form of multivalent complexes.
  • the multivalent PDL-1 of the present invention comprises a polymer formed by combining two, three, four or more PDL-1 molecules of the present invention, and a dimer can be prepared using an IgG FC segment, as embodied in the present invention. As described in Example 5, or the tetrameric domain of p53 to produce a tetramer, or a complex of multiple PDL-1 of the invention in combination with another molecule.
  • the high affinity PDL-1 molecules of the invention may be used alone or in combination with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate is preferably a T cell receptor, and more preferably, the T cell receptor is a high affinity T cell receptor.
  • the high affinity PDL-1 molecules of the invention can also be used in combination with other molecules to produce an effective synergistic effect.
  • the other molecule is ImmTAC or HATac. Both molecules are capable of redirecting T cells to kill target cells.
  • the ImmTAC molecule is a fusion molecule of a soluble double-stranded TCR molecule containing an artificial interchain disulfide bond between the ⁇ constant region and an anti-CD3 antibody, and is specifically described in the literature (Joanne Oates, Bent K. Jakobsen. (ImmTACs) Novel bi- Specific agents for targeted cancer thrapy. OncoImmunology 2: 2, e22891, February 2013).
  • the HATac molecule is a High Affinity T-cell activation core, wherein one form of a soluble single-chain TCR molecule and a resistance can be obtained by linking the hydrophobic core-mutated ⁇ and ⁇ -chain variable domains.
  • the soluble single-chain TCR molecule can be found in particular in the patent document WO2014/206304.
  • the invention also relates to nucleic acid molecules encoding PDL-1 of the invention.
  • the nucleic acid molecule of the invention may be in the form of DNA or in the form of RNA.
  • the DNA can be a coding strand or a non-coding strand.
  • a nucleic acid sequence encoding a TCR of the invention may be the same or a degenerate variant of the nucleic acid sequence set forth in the Figures of the invention.
  • degenerate variant refers in the present invention to a protein sequence having SEQ ID NO: 1, but to the sequence of SEQ ID NO: Differential nucleic acid sequences.
  • the full length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification, recombinant methods or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding PDL-1 (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as host cells genetically engineered using the vectors or coding sequences of the invention.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier And the PDL-1 of the present invention or the PDL-1 complex of the present invention.
  • the present invention also provides a method of treating a disease comprising administering an appropriate amount of a PDL-1 of the present invention, or a PDL-1 complex of the present invention, or a pharmaceutical composition of the present invention to a subject in need of treatment; in particular, the present invention
  • the PDL-1 molecule is used in combination with other molecules, preferably the other molecule is ImmTAC or HATac.
  • amino acid names in this article are identified by the international common single letter, and the corresponding amino acid names are abbreviated as: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), I le (I), Leu (L), Lys (K), Met (M), Phe (F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V); in the art, when substituted with similar or similar amino acids, it usually does not change.
  • the function of the protein The addition of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the structure and function of the protein.
  • the present invention also encompasses a PDL-1 molecule which has been slightly modified with the PDL-1 of the present invention.
  • Modifications include: chemically derived forms of PDL-1 of the invention, such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of PDL-1 of the invention or in further processing steps. Such modification can be accomplished by exposing PDL-1 to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme.
  • Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine.
  • PDL-1 which has been modified to enhance its antiproteolytic properties or to optimize solubility properties.
  • the PDL-1 or PDL-1 complex of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the PDL-1, multivalent PDL-1 complexes of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in any suitable form (depending on the method desired for administration to a patient). It can be provided in unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It can include a plurality of said unit dosage forms.
  • the PDL-1 of the present invention may be used singly or in combination or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).
  • Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, Ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the therapeutic compositions can be formulated as injectables, such as liquid solutions or suspensions; solid forms such as liquid carriers, which may be formulated in solution or suspension prior to injection.
  • injectables such as liquid solutions or suspensions
  • solid forms such as liquid carriers, which may be formulated in solution or suspension prior to injection.
  • Once formulated into a composition of the invention it can be administered by conventional routes including, but not limited to, intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal. External includes subcutaneous, intramuscular or intravenous.
  • the subject to be prevented or treated may be an animal; especially a human.
  • a pharmaceutical composition of various dosage forms may be employed depending on the use.
  • an injection, an oral preparation, or the like can be exemplified.
  • These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, and the formulation process can be carried out in a customary manner depending on the dosage form.
  • compositions of the invention may also be administered in the form of sustained release agents.
  • the PDL-1 of the present invention can be incorporated into a pill or microcapsule in which a sustained release polymer is used as a carrier, and then the pill or microcapsule is surgically implanted into a tissue to be treated.
  • the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, and lactic acid polymer.
  • a lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.
  • the PDL-1 or PDL-1 complex of the present invention as an active ingredient can be reasonably determined according to the body weight, age, sex, and degree of symptoms of each patient to be treated. To determine, the physician will ultimately determine the reasonable amount.
  • the present invention obtains a PDL-1 molecule having high affinity for PD-1.
  • the high affinity PDL-1 molecule of the present invention can effectively increase the killing ability of lymphocytes.
  • the invention is further illustrated by the following specific examples. It is to be understood that the examples are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually published under conventional conditions, for example, (Sambrook and Russell et al., Molecular Cloning-A Laboratory Manual (Third Edition) (2001) CSHL Publishing Society) The conditions described in the conditions, or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.
  • the experimental materials and reagents used in the following examples are available from commercially available sources unless otherwise specified.
  • the extracellular amino acid sequence and nucleotide sequence of wild-type PDL-1 are SEQ ID NOS: 1 and 2, respectively, as shown in Fig. 1a and Fig. 1b, and the target gene carrying the extracellular sequence of wild-type PDL-1 is subjected to NcoI. It was digested with NotI and ligated with the pET28a vector digested with NcoI and NotI. The ligation product was transformed into E.col i DH5 ⁇ , coated with kanamycin-containing LB plate, inverted culture at 37 ° C overnight, and the positive clones were picked for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and the recombinant plasmid was extracted. Transformed to E. coli BL21 (DE3) for expression.
  • Cell pellet was harvested by centrifugation at 5000 rpm for 15 min, cell pellet was lysed with Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min, and washed with Bugbuster (Merck) to remove cell debris and membrane fractions, centrifuged at 6000 rpm for 15 min, and inclusion bodies were collected. .
  • the inclusion bodies were dissolved in a buffer (50 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 6 M guanidine HCl, pH 8.0), and the insolubles were removed by high-speed centrifugation. The supernatant was quantified by BCA and then dispensed at -80 ° C. Save spare.
  • a buffer 50 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 6 M guanidine HCl, pH 8.0
  • DTT was further added to a final concentration of 10 mM, and treated at 37 ° C for 30 min.
  • the above treated PDL-1 was added dropwise to 100 mL of refolding buffer (50 mM HEPES, pH 7.5, 500 mM L-arginine, 9 mM glutathione, 1 mM glutathione disulfide, 24 mM NaCl, 1 mM KCl), and stirred at 4 ° C for 30 min, then The reconstituted solution was placed in a cellulose membrane dialysis bag with a cut-off amount of 3.5 kDa, and the dialysis bag was placed in 2 L of pre-cooled water and slowly stirred at 4 ° C overnight.
  • refolding buffer 50 mM HEPES, pH 7.5, 500 mM L-arginine, 9 mM glutathione, 1 mM glutathione disulfide, 24 mM NaCl, 1 mM KCl
  • the dialysate was changed to 2 L of pre-cooled buffer (10 mM Tris-HCl pH 8.0), dialysis was continued for 24 h at 4 ° C, then the dialysate was replaced with the same fresh buffer for further dialysis for 24 hours, and the sample was filtered through 0.45 ⁇ m.
  • the PDL-1 component is concentrated and further filtered with a gel filtration column (Superdex 75 10/300, GE Purification, the target components were also subjected to SDS-PAGE analysis.
  • the eluted fraction for BIAcore analysis was further tested for purity using gel filtration.
  • the conditions were as follows: column Agilent Bio SEC-3 (300A, ⁇ 7.8 ⁇ 300 mm), mobile phase 150 mM phosphate buffer, flow rate 0.5 mL/min, column temperature 25 ° C, UV detection wavelength 214 nm.
  • the binding activity of wild-type PDL-1 molecule to PD-1 was detected using a BIAcore T200 real-time analysis system.
  • the anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the surface of the chip. Finally, the unreacted activated surface was blocked with a solution of ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000 RU.
  • a low concentration of streptavidin is passed over the surface of the coated antibody chip, then biotinylated PD-1 is passed through the detection channel, and the other channel is used as a reference channel, and 0.05 mM biotin is then 10 ⁇ L.
  • the flow rate of /min flows through the chip for 2 min, blocking the remaining binding sites of streptavidin.
  • the affinity was determined by single-cycle kinetic analysis.
  • PDL-1 was diluted to several different concentrations with HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) at 30 ⁇ L/min.
  • the flow rate which flows through the surface of the chip in turn, is 120 s for each injection and is dissociated for 600 s after the last injection.
  • the chip was regenerated with 10 mMGly-HCl, pH 1.75, after each round of assay. Kinetic parameters were calculated using BIAcore Evaluation software.
  • the amino acid sequence and nucleotide sequence of PD-1 used in this example are shown in Figures 3a and 3b, respectively, and the expression, renaturation and purification processes thereof are identical to the expression, renaturation and renaturation of wild-type PDL-1 in Example 1.
  • the purification process is the same.
  • the process of biotinylation is as follows:
  • the purified PD-1 molecule was concentrated using a Millipore ultrafiltration tube while the buffer was replaced with 10 mM Tris pH 8.0, followed by biotinylation reagent 0.05M Bicine pH 8.3, 10 mM ATP, 10 mM MgAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml.
  • the BirA enzyme (GST-BirA) was incubated overnight at room temperature and SDS-PAGE was used to determine if biotinylation was complete.
  • the biotinylated labeled PD-1 molecule was concentrated to 1 ml using a Mi llipore ultrafiltration tube, and biotinylated PD-1 was purified by gel filtration chromatography and filtered using an Akta Purifier (GE General Electric Company). pre-equilibrated in PBS HiPrep TM 16 / 60S200HR column (GE General Electric Co.), loaded 1ml the concentrated biotinylated PD-1 molecule, and then eluted at / min flow rate 1ml with PBS. The biotinylated PD-1 molecule appeared as a single peak elution at about 10 ml. The protein-containing fractions were pooled, concentrated using a Millipore ultrafiltration tube, protein concentration was determined by BCA method (Thermo), and biotinylated PD-1 molecules were dispensed at -80 °C.
  • the extracellular sequence of wild-type PDL-1 described in Example 1 was used as a template strand, and high affinity was performed according to the phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3): 349-354). Screening of PDL-1. After several rounds of screening, the phage library has a strong binding signal to PD-1, picking up the monoclonal and performing sequence analysis.
  • the high affinity PDL-1 molecule of the invention was expressed, renatured and purified as described in Example 1, and its affinity to the PD-1 molecule was determined as described in Example 2.
  • the affinity of the high-affinity PDL-1 molecule obtained in the present invention to the PD-1 molecule is at least 2-fold that of the wild-type PDL-1 molecule and the PD-1 molecule, and the amino acid sequence thereof is shown in Fig. 5, which is related to PD-
  • the affinity values of one molecule are shown in Table 1 below.
  • Example 4 Effect of high affinity PDL-1 molecule on the efficacy of PBMC (peripheral blood mononuclear cells) killing tumor cell line (A375)
  • This example uses a non-radioactive cytotoxicity assay to verify killing.
  • This test is a colorimetric substitution test for the 51Cr release cytotoxicity assay to quantify the lactate dehydrogenase (LDH) released after cell lysis.
  • LDH lactate dehydrogenase
  • the LDH released from the culture supernatant was tested using a 30-minute coupled enzyme assay which converted the tetrazolium salt (INT) to a red formazan product. The amount of color formed is proportional to the number of lysed cells.
  • Absorbance data at 490 nm was collected using a standard 96-well plate reader.
  • CytoTox96 non-radioactive cytotoxicity test (Promega) (G1780) Contains substrate mixture, assay buffer, lysis solution and stop solution; test medium: 10% FBS (heat inactivated, Gibbco, catalog number 10108-165), VIVO-15 (Lonza), catalog number: 04-418); Nunc microwell round bottom 96-well tissue culture plate (Nunc, catalog number 163320); Nunc-immunized plate Maxisorb (Nuk, Inc., catalog number 442404).
  • the target cells used in this example were A375 cells (available from ATCC, Cat. No. CRL-1619). Target cells were prepared in the test medium; the target cell concentration was adjusted to 2 x 10 ⁇ 5 cells/ml, thereby obtaining 2 x 10 ⁇ 4 cells/well, 100 ⁇ l.
  • the effector cells (PBMC cells) used in this example were obtained from fresh white membranes of healthy people, and the cells were resuspended in the test medium at 1 ⁇ 10 6 cells/ml to obtain 1 ⁇ 10 ⁇ 5 Cells/well, 100 ⁇ l.
  • the final concentration of ImmTAC in the experiment was 10 ⁇ -9M, and the storage concentration of ImmTAC was 13 ⁇ 10 ⁇ -6M, diluted 13,000 times, and directly added to the prepared effector cells.
  • the final concentration of different high affinity PDL1 was 30 ug/ml.
  • the alpha and beta chain amino acid sequences of ImmTAC used in this example are shown in Figures 6a and 6b, respectively.
  • the components of the assay were added to the plates in the following order: 100 ⁇ l of target cells (prepared as described above) were added to each well; 100 ⁇ l of effector cells (prepared as described above) were added to each well; different high affinity PDL1 was added to each well.
  • 50 ⁇ l of the supernatant from each well of the test plate was transferred to the corresponding well of a flat-bottom 96-well NuncVlnxisorb plate.
  • the substrate mixture was reconstituted using assay buffer (12 ml). 50 ⁇ l of the reconstituted substrate mixture was then added to each well of the plate.
  • the plate was covered with aluminum foil and incubated for 30 minutes at room temperature. 50 ⁇ l of the stop solution was added to each well of the plate to terminate the reaction.
  • the absorbance at 490 nm was recorded on an Elisa plate reader within 1 hour after the addition of the stop solution.
  • PBMC+A375+1G4+PDL1 is a wild-type PDL-1 molecule.
  • the increase or decrease in the cracking rate in the figure is relative to the "PBMC + A375 + 1 G4" histogram.
  • the specific calculation method is
  • a human immunoglobulin fragment crystallizable (hIgG4Fc) was used to construct a dimer of a high affinity PDL1 molecule. Specifically, the 3' end of the extracellular nucleotide sequence of the high affinity PDL-1 and the 5' end of the hIgG4 Fc nucleotide sequence are ligated by overlap PCR to obtain a fusion protein nucleic acid sequence and an amino acid sequence, and the hIgG4 Fc amino acid sequence and the nucleic acid sequence are respectively SEQ ID NOS: 37 and 38 are shown in Figures 8a and 8b.
  • the gene of interest carrying the fusion protein sequence was digested with EcoRI and NotI, and ligated with the pcDNA3.1(+) vector digested with EcoRI and NotI.
  • the ligated product was transformed into TOP10, and the LB plate containing ampicillin was applied and cultured at 37 ° C overnight.
  • the positive clones were picked for PCR screening, and the positive recombinants were sequenced. After confirming the sequence, the recombinant plasmid was transiently transferred to 293T cells. Used for expression.
  • the dimer of the wild-type PDL1 molecule is constructed in the same manner as above, and the dimer of the wild-type PDL1 molecule is expressed in the present invention as PDL1-hIgG4.
  • the dimer of the high affinity PDL1 molecule is expressed in the present invention as a high affinity PDL1 molecule number - hIgG4, such as L2D7-hIgG4.
  • 1 ⁇ 10 ⁇ 7 293T cells (purchased from ATCC, Cat. No. CRL-11268) were inoculated into a 10 cm cell culture dish, and washed 12 times with PBS and replaced with opti-MEM medium.
  • Add Lipo2000: recombinant plasmid (1ug/ul) 2:1 to opti-MEM, let stand for 5 min at room temperature, add opti-MEM containing lipo2000 dropwise to opti-MEM containing recombinant plasmid, mix and let stand at room temperature. After 20 min, 293T cells were replaced with opti-MEM medium, and after 4 hours, they were replaced with freestyle TM 293 medium and cultured in a 5% CO 2 37 ° C incubator for 3 days.
  • the culture supernatant was collected, filtered through a 0.45 ⁇ m filter, and the protein was purified by anion exchange column (HiTrap Q HP, GE Healthcare) using a linear gradient of 0-mM NaCl prepared in 20 mM Tris-HCl pH 7.0.
  • the components were subjected to SDS-PAGE analysis, and PDL1-hIgG4Fc was used as an example, and the SDS-PAGE gel map is shown in Fig. 9.
  • the fractions were concentrated and further purified using a gel filtration column (Superdex 20010/300, GE Healthcare), and the target components were also subjected to SDS-PAGE analysis.
  • the materials used were as follows: 10 cm cell culture dish (greiner, catalog number: 627160), opti-MEM medium (gibco, catalog number: 31985-070), Lipo2000, freestyle TM 293 medium (gibco, catalog number: 12338-018) , PureLink TM HiPure Plasmid Maxiprep Kit (invitrogen, catalog number: K2100-06)
  • Example 6 Effect of high affinity PDL1-hIgG4Fc fusion protein on the efficacy of PBMC (peripheral blood mononuclear cells) in killing tumor cell lines
  • This example uses a non-radioactive cytotoxicity assay to verify killing.
  • This test is a colorimetric substitution test for the 51Cr release cytotoxicity assay to quantify the lactate dehydrogenase (LDH) released after cell lysis.
  • LDH lactate dehydrogenase
  • the LDH released from the culture supernatant was tested using a 30-minute coupled enzyme assay which converted the tetrazolium salt (INT) to the red formazan product. The amount of color formed is proportional to the number of lysed cells.
  • Absorbance data at 490 nm was collected using a standard 96-well plate reader.
  • CytoTox 96 non-radioactive cytotoxicity test (Promega) (G1780) containing substrate mixture, assay buffer, lysis solution and stop solution; test medium: 10% FBS (heat inactivated, Gibb Company, catalog number 10108-165), VIVO-15 (Lonza), catalog number: 04-418); Nunc microwell round bottom 96-well tissue culture plate (Nunc, catalog number 163320); Nunc-immunization Flat Maxisorb (Newark, catalog number 442404).
  • the target cells used in this example were Mel624 cells (a gift from Cassian Yee Laboratories), NCI-H1299 cells (purchased from ATCC, Cat. No. CRL-5803), IM9 cells (purchased from ATCC, Cat. No. CRL-159), MDA-MB- 231 cells (purchased from ATCC, article number CRM-HTB-26).
  • Target cells were prepared in the test medium; the target cell concentration was adjusted to 2 x 10 ⁇ 5 cells/ml, thereby obtaining 2 x 10 ⁇ 4 cells/well, 100 ⁇ l.
  • the effector cells (PBMC cells) used in this example were obtained from fresh white membranes of healthy people, and the cells were resuspended in the test medium at 1 ⁇ 10 6 cells/ml to obtain 1 ⁇ 10 ⁇ 5 Cells/well, 100 ⁇ l.
  • the final concentration of ImmTAC in the experiment was 10 ⁇ -9M, and the storage concentration of ImmTAC was 5 ⁇ 10 ⁇ -6M, diluted 5000 times, and directly added to the prepared effector cells.
  • the final concentration of the different high affinity PDL1-hIgG4Fc fusion proteins was 20 ug/ml.
  • the alpha and beta chain amino acid sequences of ImmTAC (1G4) used in this example are shown in Figures 6a and 6b, respectively.
  • the components of the assay were added to the plates in the following order: 100 ⁇ l of target cells (prepared as described above) were added to each well; 100 ⁇ l of effector cells (prepared as described above) were added to each well; different high affinity PDL1-hIgG4Fc fusion proteins were added to each well.
  • 50 ⁇ l of the supernatant from each well of the test plate was transferred to the corresponding well of a flat-bottom 96-well NuncVlnxisorb plate.
  • the substrate mixture was reconstituted using assay buffer (12 ml). 50 ⁇ l of the reconstituted substrate mixture was then added to each well of the plate.
  • the plate was covered with aluminum foil and incubated for 30 minutes at room temperature. 50 ⁇ l of the stop solution was added to each well of the plate to terminate the reaction.
  • the absorbance at 490 nm was recorded on an Elisa plate reader within 1 hour after the addition of the stop solution.
  • PDL1-hlgG4 is a wild-type PDL-1 fusion molecule in the histogram of subscript "PBMC+Mel624+1G4+PDL1-hlgG4" in Figure 10a.
  • the increase or decrease in the cracking rate in the figure is relative to the "PBMC + Mel624 + 1 G4" histogram.
  • the specific calculation method is
  • This result shows that a part of the high affinity PDL-1 fusion molecule of the present invention significantly enhances the killing effect of PBMC on tumor cell Mel624.
  • PDL1-hlgG4 is a wild-type PDL-1 fusion molecule.
  • the increase or decrease in the cleavage rate in the figure is relative to the "PBMC+NCI-H1299+1G4" histogram.
  • the specific calculation method is
  • This result shows that part of the high affinity PDL-1 fusion molecule of the present invention significantly enhances the killing effect of PBMC on tumor cell NCI-H1299.
  • PDL1-hlgG4 is a wild-type PDL-1 fusion molecule.
  • the increase or decrease in the cleavage rate in the figure is relative to the "PBMC+IM9+1G4+PDL1-hlgG4" histogram.
  • the specific calculation method is
  • This result shows a part of the high affinity PDL-1 of the present invention.
  • the fusion molecule significantly increased the killing effect of PBMC on tumor cell IM9.
  • PDL1-hlgG4 is a wild-type PDL-1 fusion molecule.
  • the increase or decrease in the cleavage rate in the figure is relative to the "PBMC+MDA-MB-231+1G4+PDL1-hlgG4" histogram.

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Abstract

本发明提供了一种PDL-1分子，所述PDL-1分子与PD-1分子的亲和力是野生型PDL-1分子与PD-1分子的亲和力的至少2倍。同时，本发明的PDL-1分子能够有效地提高淋巴细胞的杀伤能力。另外，本发明还提供了编码本发明PDL-1分子的核酸，以及本发明PDL-1分子的复合物。本发明的PDL-1分子可以单独使用，也可与其他分子联用。

Description

高亲和力的可溶性PDL-1分子 技术领域
本发明涉及生物技术领域，更具体地涉及对程序性死亡受体(Programmed Death-1,PD-1)分子具有高亲和力的可溶性程序性死亡受体配体(Programmed Death Ligand-1,PDL-1)分子，以及该分子的制备方法和用途。
背景技术
PD-1是在激活的T细胞和B细胞上表达的免疫抑制性受体。PDL-1是PD-1的配体，属于B7家族，具有IgV和IgC样区、跨膜区及胞浆区尾部，PDL-1与淋巴细胞上的受体PD-1相互作用，在免疫应答的负调控方面发挥着重要作用。许多肿瘤细胞系和肿瘤细胞高表达PDL-1分子(Konishi J et al.，Clin.Cancer Res.，2004，10(15):5094－5100)，其与淋巴细胞表面的PD-1分子结合后，削弱了机体的抗肿瘤免疫应答(Radziewicz H et al.，J Virol，2007，81(6):2545－2553)，从而导致肿瘤免疫逃逸的发生。研究发现在宫颈癌和肝癌中，有近半数的肿瘤浸润CD8+T细胞表达PD-1分子，其与肿瘤细胞表达的PDL-1结合后，可能导致CTL细胞的耗尽及凋亡(Dong H et al.,J Mol Med(Berl)，2003，81(5):281－287；Karim R et al.，Clin Cancer Res,2009，15(20):6341－6347；Zhao Q et al.,Eur J Immunol,2011,41(8):2314－2322)。
针对上述肿瘤免疫逃逸问题，阻断淋巴细胞表面的PD-1与肿瘤细胞表面的PDL-1的相互作用可使淋巴细胞的免疫性提高，因此，有助于由免疫系统清除肿瘤细胞。针对这一问题，进行了大量研究。在侵袭性胰腺癌的鼠模型中，T.Nomi等(Clin.Cancer Res.13：2151-2157(2007))证明阻断PD-1与PDL-1相互作用的治疗功效。本领域技术人员致力于研究PDL-1与PD-1的相互作用，以期找到提高淋巴细胞杀伤能力的有效途径。
发明内容
本发明的目的在于提供一种对PD-1分子具有较高亲和力的PDL-1分子。
本发明的再一目的是提供一种上述高亲和力的PDL-1分子的制备方法及用 途。
在本发明的第一方面，提供了一种PDL-1分子，所述PDL-1分子在SEQ ID NO:1所示的氨基酸序列中含有突变。
在另一优选例中，所述PDL-1分子的氨基酸序列基于SEQ ID NO.:1所示的氨基酸序列，并且对SEQ ID NO:1所示的氨基酸序列进行一个或多个氨基酸残基的突变或氨基酸残基的插入从而获得所述PDL-1分子。
在另一优选例中，所述PDL-1分子的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少90％(优选地，至少92％；更优选地，至少94％)的序列相同性。
在另一优选例中，所述PDL-1分子与PD-1分子的亲和力是野生型PDL-1分子与PD-1分子的亲和力的至少2倍；优选地，至少5倍；更优选地，至少10倍；最优选地，至少50倍。
在另一优选例中，所述PDL-1分子与PD-1分子的亲和力是野生型PDL-1分子与PD-1分子的亲和力的至少100倍；优选地，至少200倍；更优选地，至少500倍。
在另一优选例中，所述PDL-1分子中突变的氨基酸残基位点为1～3、35～50、和/或95～105位氨基酸残基中的一个或多个，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子中突变的氨基酸残基位点为1、36～40、47～48和/或95～101位氨基酸残基中的一个或多个，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，突变的氨基酸残基位点的数量为n，其中1≤n≤15；优选地，3≤n≤11；更优选地，4≤n≤10，如n可以为5、6、7、8、9、10。在另一优选例中，所述PDL-1分子中突变的氨基酸残基位点包括1F、36I、38Y、40E、47I、48Q、95R、97M、99S、101G中的一个或多个或102G后有一个或多个氨基酸的插入，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子中突变的氨基酸残基位点包括36I、38Y、和40E，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子中突变的氨基酸残基位点还包括40E、和47I，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子中突变的氨基酸残基位点还包括95R、和97M，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，突变后的PDL-1分子包括选自下组的一个或多个氨基酸残基：1W；36Q、36E、36T或36N；38F、38N、38H或38A；40M、40F、40L、40W或40Y；47L、47V、47F、47S、47Y、47P或47A；48S；95T、95V或95L；97L；99G或99A；101D、101K、101E或101H；102G后插入氨基酸G；其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子包括：36Q、38H、和40F；或
36E、38H、和40F；或
36Q、38F、和40M；或
36Q、38F、和40M,其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子还包括：48S,其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子还包括：47V、和48S；或
47F、和48S,其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子还包括：97L，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子还包括：95T、97L、99G、和101K；或
95V、97L、99A、和101K，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子的氨基酸序列选自SEQ ID NO:5-34之一。
在另一优选例中，所述PDL-1分子是可溶的。
在另一优选例中，所述PDL-1分子的C或N末端结合有偶联物。
在另一优选例中，与所述PDL-1分子结合的偶联物为T细胞受体，优选地，所述T细胞受体为高亲和性T细胞受体。
本发明的第二方面，提供了一种融合蛋白，所述融合蛋白包括本发明第一方面所述的PDL-1分子。
在另一优选例中，所述融合蛋白还包括hIgG4Fc。
本发明的第三方面，提供了一种多价PDL-1复合物，所述多价PDL-1复合物包含至少两个PDL-1分子，并且其中的至少一个PDL-1分子为本发明第一方面所述的PDL-1分子；或者所述多价PDL-1复合物包含至少一个本发明第二方 面所述的融合蛋白。
本发明的第四方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面PDL-1分子、本发明第二方面所述的融合蛋白、或本发明第三方面所述的多价PDL-1复合物的核酸序列或其互补序列。
本发明第五方面，提供了一种载体，所述的载体含有本发明第四方面所述的核酸分子。
本发明的第六方面，提供了一种宿主细胞，所述的宿主细胞中含有本发明第五方面所述的载体或染色体中整合有外源的本发明第四方面所述的核酸分子。
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的PDL-1分子、或本发明第二方面所述的融合蛋白、或本发明第三方面所述的PDL-1复合物。
在另一优选例中，所述药物组合物中还包括ImmTAC和/或HATac。
本发明的第八方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的PDL-1分子、本发明第二方面所述的融合蛋白、或本发明第三方面所述的PDL-1复合物、或本发明的第七方面所述的药物组合物。
在另一优选例中，所述疾病为肿瘤。
本发明的第九方面，提供了本发明第一方面所述的PDL-1分子、本发明第二方面所述的融合蛋白、或本发明第三方面所述的PDL-1复合物的用途，用于制备治疗肿瘤的药物。
本发明的第十方面，提供了一种制备本发明第一方面所述的PDL-1的方法，包括步骤：
(i)培养本发明第六方面所述的宿主细胞，从而表达本发明第一方面所述的PDL-1分子；
(ii)分离或纯化出所述的PDL-1分子。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1a和图1b分别为野生型PDL-1分子的胞外氨基酸序列和核苷酸序列。
图2为野生型PDL-1蛋白纯化后的SDS-PAGE胶图。
图3a和图3b分别为本发明实施例2中所复性、纯化的PD-1的氨基酸序列和核苷酸序列。
图4为野生型PDL-1分子与PD-1分子结合的BIAcore图谱。
图5为本发明高亲和性PDL-1分子的氨基酸序列，突变或插入的氨基酸残基以加下划线表示。
图6a和图6b分别为ImmTAC(1G4)的α和β链氨基酸序列。
图7显示了本发明高亲和力PDL-1分子明显提高ImmTAC(1G4)介导PBMC杀伤肿瘤细胞。
图8a为hIgG4Fc分子的氨基酸序列。
图8b为hIgG4Fc分子的核苷酸序列。
图9为野生型PDL1-hIgG4Fc融合蛋白纯化后的SDS-PAGE胶图。
图10a显示了本发明高亲和力PDL1-hIgG4Fc融合蛋白显著提高ImmTAC(1G4)介导PBMC杀伤肿瘤细胞Mel624。
图10b显示了本发明高亲和力PDL1-hIgG4Fc融合蛋白显著提高ImmTAC(1G4)介导PBMC杀伤肿瘤细胞NCI-H1299。
图10c显示了本发明高亲和力PDL1-hIgG4Fc融合蛋白显著提高ImmTAC(1G4)介导PBMC杀伤肿瘤细胞IM9。
图10d显示了本发明高亲和性力PDL1-hIgG4Fc融合蛋白显著提高ImmTAC(1G4)介导PBMC杀伤肿瘤细胞MDA-MB-231。
具体实施方式
本发明通过广泛而深入的研究，意外地发现对PD-1分子具有高亲和力的可溶性PDL-1分子能够有效地提高淋巴细胞的杀伤能力。因此，本发明提供了一种对PD-1的亲和力是野生型PDL-1分子对PD-1的亲和力至少两倍的可溶性高亲和力PDL-1分子。
具体地，本发明中所述PDL-1分子在SEQ ID NO:1所示的氨基酸序列中含有突变。更具体地，所述PDL-1分子的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少90％的序列相同性。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且其意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
术语
野生型PDL-1分子：本发明中所述的野生型PDL-1分子是指野生型PDL-1分子的胞外区，其氨基酸序列和核苷酸序列分别如图1a和1b所示。
PBMC：外周血单个核细胞(PBMC)是具有圆形胞核的血细胞，如淋巴细胞或单核细胞。这些血细胞是免疫系统抵抗感染并适应于入侵者的关键组分。淋巴细胞群体由T细胞(CD4和CD8阳性约75％)、B细胞和NK细胞(合并约25％)组成。
高亲和性T细胞受体：是指与其配体的亲和力是相应野生型T细胞受体与其配体亲和力至少两倍的T细胞受体。
肿瘤：指包括所有类型的癌细胞生长或致癌过程，转移性组织或恶性转化细胞、组织或器官，不管病理类型或侵染的阶段。肿瘤的实施例非限制性地包括：实体瘤，软组织瘤，和转移性病灶。实体瘤的实施例包括：不同器官系统的恶性肿瘤，例如肉瘤，肺鳞癌和癌症。例如：感染的前列腺，肺，乳房，淋巴，肠胃(例如：结肠)，和生殖泌尿道(例如：肾脏，上皮细胞)，咽头。肺鳞癌包括恶性肿瘤，例如，多数的结肠癌，直肠癌，肾细胞癌，肝癌，肺部的非小细胞癌，小肠癌和食道癌。上述癌症的转移性病变可同样用本发明的方法和组合物来治疗和预防。
药用载体：还称作赋形剂或稳定剂，使用剂量和浓度对其暴露的细胞或个体无毒。经常地，生理可接受载体是pH缓冲的水溶液。生理可接受载体的例子包括缓冲剂如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸；低分子量(小于约10个残基)多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸、谷氨酰胺、天冬酰胺、 精氨酸或赖氨酸；单糖、二糖和其他糖类，包括葡萄糖、甘露糖或糊精；络合剂如EDTA；糖醇如甘露醇或山梨醇；成盐反离子如钠；和/或非离子表面活性剂如TWEENTM、聚乙二醇(PEG)和PLURONICSTM。
发明详述
PD-1是在激活的T细胞和B细胞上表达的免疫抑制性受体。PDL-1是PD-1的配体，属于B7家族，具有IgV和IgC样区、跨膜区及胞浆区尾部，PDL-1与淋巴细胞上的受体PD-1相互作用，在免疫应答的负调控方面发挥着重要作用。本发明通过广泛而深入的研究，意外地发现对PD-1分子具有高亲和力的可溶性PDL-1分子能够有效地提高淋巴细胞的杀伤能力。因此，本发明提供了一种对PD-1的亲和力是野生型PDL-1分子对PD-1的亲和力至少两倍的可溶性高亲和力PDL-1分子。
可通过任何合适的方法测定上述PDL-1分子与PD-1的结合亲和力(与解离平衡常数KD成反比)和结合半衰期(表示为T1/2)。应了解，亲和力翻倍将导致KD减半。T1/2计算为In2除以解离速率(Koff)。因此，Koff翻倍会导致T1/2减半。优选采用相同的试验方案检测结合亲和力或结合半衰期数次，例如3次或更多，取结果的平均值。在优选的实施方式中，采用本发明实施例中的表面等离振子共振(BIAcore)方法进行这些检测。
该方法检测到本发明中野生型PDL-1分子对PD-1分子的解离平衡常数KD为2.462E-05M，其BIAcore结合图谱如图4所示。由于亲和力翻倍将导致KD减半，所以若检测到高亲和力PDL-1分子对PD-1分子的解离平衡常数KD为1.231E-05M，则说明该高亲和力PDL-1分子对PD-1分子的亲和力是野生型PDL-1分子对PD-1的亲和力的2倍。本领域技术人员熟知KD值单位间的换算关系，即1M＝1000μM，1μM＝1000nM，1nM＝1000pM。
在本发明的一个优选例中，利用本发明优选的测定亲和力的方式测得本发明PDL-1分子与PD-1分子的亲和力是野生型PDL-1分子与PD-1分子的亲和力的至少2倍；优选地，至少5倍；更优选地，至少10倍；最优选地，至少50倍。
在另一优选例中，所述PDL-1分子与PD-1分子的亲和力是野生型PDL-1分子与PD-1分子的亲和力的至少100倍；优选地，至少200倍；更优选地，至少500倍。
具体地，本发明高亲和力PDL-1分子与PD-1的亲和力KD≤1.231E-05M；优选地，1.0E-06M≤KD≤1.0E-05M；更优选地，1.0E-07M≤KD≤1.0E-06M；最优选地，1.0E-08M≤KD≤1.0E-07M。
本发明的高亲和力PDL-1分子在SEQ ID NO:1所示的氨基酸序列中含有一个或多个突变。具体地，所述PDL-1分子的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少90％(优选地，至少92％；更优选地，至少94％，如95％、96％、97％、98％、99％)的序列相同性。
更具体地，本发明高亲和力PDL-1分子中突变的氨基酸残基位点包括1F、36I、38Y、40E、47I、48Q、95R、97M、99S、101G中的一个或多个或102G后有一个或多个氨基酸的插入，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，突变后的PDL-1分子包括选自下组的一个或多个氨基酸残基：1W；36Q、36E、36T或36N；38F、38N、38H或38A；40M、40F、40L、40W或40Y；47L、47V、47F、47S、47Y、47P或47A；48S；95T、95V或95L；97L；99G或99A；101D、101K、101E或101H；102G后插入氨基酸G；其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
在另一优选例中，所述PDL-1分子的氨基酸序列选自SEQ ID NO:5-34之一。
为获得可溶性的高亲和力PDL-1分子，本发明所用的野生型PDL-1分子不含跨膜区。因此，在本发明的一个优选例中，所述PDL-1分子是可溶的。
可采用任何合适的方法进行突变，包括但不限于依据聚合酶链式反应(PCR)的那些、依据限制性酶的克隆或不依赖连接的克隆(LIC)方法。许多标准分子生物学教材详述了这些方法。聚合酶链式反应(PCR)诱变和依据限制性酶的克隆的更多细节可参见Sambrook和Russell,(2001)分子克隆-实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)CSHL出版社。LIC方法的更多信息可见(Rashtchian,(1995)Curr Opin Biotechnol 6(1):30-6)。
产生本发明的高亲和力PDL-1分子的方法可以是但不限于从展示此类PDL-1分子的噬菌体颗粒的多样性文库中筛选出对PD-1具有高亲和性的PDL-1，如文献(Li,et al(2005)Nature Biotech 23(3):349-354)中所述。
应理解，表达本发明野生型PDL-1的基因或者表达略作修饰的本发明野生型PDL-1的基因都可用来制备模板链。然后在编码该模板链的DNA中引入产生 本发明的高亲和力PDL-1所需的改变。
本发明的PDL-1分子也可以多价复合体的形式提供。本发明的多价PDL-1包含两个、三个、四个或更多个本发明PDL-1分子相结合而形成的多聚物，可以用IgG FC段制备二聚体，如本发明实施例5中所述，或p53的四聚结构域来产生四聚体，或多个本发明PDL-1与另一分子结合而形成的复合物。
本发明的高亲和力PDL-1分子可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物优选为T细胞受体，更优选地，所述T细胞受体为高亲和性T细胞受体。
本发明的高亲和力PDL-1分子也可与其他分子联用，产生有效的协同作用。优选地，所述其他分子为ImmTAC或HATac。两种分子都能够重新定向T细胞，从而起到杀伤靶细胞的作用。所述ImmTAC分子是αβ恒定区之间含有人工链间二硫键的可溶性双链TCR分子与抗CD3抗体的融合分子，具体可参见文献(Joanne Oates，Bent K.Jakobsen.(ImmTACs)Novel bi-specific agents for targeted cancer thrapy.OncoImmunology2:2,e22891,February2013)。所述HATac分子是高亲和性T细胞活化芯(High Affinity T-cell activation core),其中一种形式可由疏水芯突变的α与β链可变域连接而成的可溶性单链TCR分子与抗CD3抗体的融合分子，所述可溶性单链TCR分子具体可参见专利文献WO2014/206304。
本发明还涉及编码本发明PDL-1的核酸分子。本发明的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码本发明TCR的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。举例说明“简并的变异体”的含义，如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:1的蛋白序列，但与SEQ ID NO:2的序列有差别的核酸序列。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明PDL-1(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的核酸分子的载体，以及用本发明的载体或编码序列经基因工程产生的宿主细胞。
本发明还提供一种药物组合物，所述药物组合物含有药学上可接受的载体 以及本发明PDL-1、或本发明PDL-1复合物。
本发明还提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明PDL-1、或本发明PDL-1复合物、或本发明的药物组合物；尤其是，本发明的PDL-1分子与其他分子联用，优选地，其他分子为ImmTAC或HATac。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、I le(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)；在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。
本发明还包括对本发明PDL-1略作修饰后的PDL-1分子。修饰(通常不改变一级结构)形式包括：本发明PDL-1的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在本发明PDL-1的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的PDL-1分子。这种修饰可以通过将PDL-1暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的PDL-1。
本发明的PDL-1或PDL-1复合物可与药学上可接受的载体一起在药物组合物中提供。本发明的PDL-1、多价PDL-1复合物通常作为无菌药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。该药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。此外，本发明的PDL-1可以单用，也可与其他治疗剂结合或偶联在一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、 乙醇、佐剂、及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼内、肌内、静脉内、皮下、皮内、或局部给药，优选为胃肠外包括皮下、肌肉内或静脉内。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有针剂、口服剂等。这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明PDL-1可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明PDL-1或PDL-1复合物，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定，最终由医师决定合理的用量。
本发明的主要优点在于：
(1)本发明获得了对PD-1具有高亲和力的PDL-1分子。
(2)本发明的高亲和力PDL-1分子能够有效地提高淋巴细胞的杀伤能力。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社) 中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1野生型PDL-1的表达、复性和纯化
野生型PDL-1的胞外氨基酸序列及核苷酸序列分别为SEQ ID NO:1和2，如图1a和图1b所示，将携带野生型PDL-1的胞外序列的目的基因经NcoⅠ和NotⅠ双酶切，与经过NcoⅠ和NotⅠ双酶切的pET28a载体连接。连接产物转化至E.col i DH5α，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3),用于表达。
将上述含有重组质粒pET28a-PDL-1的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中,37℃培养至OD600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀物，用BugbusterMaster Mix(Merck)裂解细胞沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min,收集包涵体。将包涵体溶解在缓冲液(50mM Tris-HCl,200mM NaCl,2mM EDTA,6M guanidine HCl,pH 8.0)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向7mg溶解的PDL-1包涵体蛋白中，加入2mL缓冲液(50mM Tris-HCl,200mM NaCl,2mM EDTA,6M guanidine HCl,pH 8.0)，再加入DTT至终浓度为10mM,37℃处理30min。用注射器向100mL复性缓冲液(50mM HEPES,pH 7.5,500mM L-arginine,9mM glutathione,1mM glutathione disulfide,24mM NaCl,1mM KCl)中滴加上述处理后的PDL-1，4℃搅拌30min，然后将复性液装入截留量为3.5kDa的纤维素膜透析袋,透析袋置于2L预冷的水中，4℃缓慢搅拌过夜。24小时后，将透析液换成2L预冷的缓冲液(10mMTris-HCl pH 8.0)，4℃继续透析24h，然后将透析液换成相同的新鲜缓冲液继续透析24小时，样品经0.45μm滤膜过滤，真空脱气后通过阴离子交换柱(HiTrap Q HP,GE Healthcare)，用10mMTris-HCl pH 8.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析，其SDS-PAGE胶图如图2所示。PDL-1组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300,GE  Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
用于BIAcore分析的洗脱组分进一步采用凝胶过滤法测试其纯度。条件为：色谱柱Agilent Bio SEC-3(300A，φ7.8×300mm)，流动相为150mM磷酸盐缓冲液，流速0.5mL/min，柱温25℃，紫外检测波长214nm。
实施例2结合表征
BIAcore分析
使用BIAcore T200实时分析系统检测野生型PDL-1分子与PD-1的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH 4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15，000RU。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将生物素化的PD-1流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将PDL-1用HEPES-EP缓冲液(10mM HEPES,150mMNaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH 1.75的10mMGly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
本实施例中所用的PD-1的氨基酸序列和核苷酸序列分别如图3a和3b所示，其表达、复性和纯化过程与实施例1中野生型PDL-1的表达、复性和纯化过程相同。其生物素化的过程如下：
a.生物素化
用Mill ipore超滤管将纯化的PD-1分子浓缩，同时将缓冲液置换为10mMTris pH 8.0，然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mMMgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
b.纯化生物素化后的复合物
用Mi llipore超滤管将生物素化标记后的PD-1分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的PD-1，利用Akta纯化仪(GE通用电气公司)，用 过滤过的PBS预平衡HiPrepTM 16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化PD-1分子，然后用PBS以1ml/min流速洗脱。生物素化的PD-1分子在约10ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Mill ipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，将生物素化的PD-1分子分装保存在-80℃。
通过本实施例上述过程检测到野生型PDL-1分子与PD-1分子的结合亲和力的KD值为2.462E-05M，其BIAcore结合图谱如图4所示。
实施例3高亲和力PDL-1分子的产生
将实施例1中所述的野生型PDL-1的胞外序列作为模板链，根据Li等((2005)Nature Biotech 23(3):349-354)描述的噬菌体展示和筛选方法，进行高亲和力PDL-1的筛选。经过几轮筛选后的噬菌体文库均和PD-1有较强的结合信号，从中挑取单克隆，并进行序列分析。
按照实施例1中所述方法表达、复性和纯化本发明高亲和性PDL-1分子，并按实施例2中所述方法测定其与PD-1分子的亲和力。本发明中获得的高亲和力PDL-1分子与PD-1分子的亲和力是野生型PDL-1分子与PD-1分子的亲和力的至少2倍，其氨基酸序列如图5所示，其与PD-1分子的亲和力数值如下表1所示。
表1
Figure PCTCN2016101597-appb-000001
Figure PCTCN2016101597-appb-000002
实施例4高亲和力PDL-1分子对PBMC(外周血单个核细胞)杀伤肿瘤细胞株(A375)效力的影响
本实施例采用非放射性细胞毒性试验来验证杀伤作用。该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的乳酸脱氢酶(LDH)。采用30-分钟偶联的酶试验检测培养上清液中释放的LDH，其将四唑盐(INT)转化成红色甲暨产物。形成的颜色量与裂解细胞的数量成比例。利用标准96孔平板读数计收集490nm的吸光度数据。
所用材料如下：CytoTox96非放射性细胞毒性试验(普罗迈格公司)(G1780) 含有底物混合物、试验缓冲液、裂解溶液和终止溶液；试验培养基:10％FBS(热灭活的，吉布可公司，目录号10108-165)、VIVO-15(Lonza)，目录号:04-418)；Nunc微孔圆底96孔组织培养板(纽克公司(Nunc)，目录号163320)；Nunc-免疫平板Maxisorb(纽克公司，目录号442404)。
本实施例所用的靶细胞是A375细胞(购自ATCC，货号CRL-1619)。在试验培养基中制备靶细胞；靶细胞浓度调节至2x10^5个细胞/毫升，从而得到2x10^4个细胞/孔，100μl。
本实施例中所用的效应细胞(PBMC细胞)是从健康人的新鲜白膜中分选获得，细胞以1×10^6个细胞/ml重悬在试验培养基中，从而得到1x10^5个细胞/孔，100μl。
试验中ImmTAC终浓度为10^-9M，ImmTAC的储存浓度为13X10^-6M，稀释13000倍，直接加入制备好的效应细胞中。不同高亲和力PDL1的终浓度为30ug/ml。本实施例中所用的ImmTAC的α和β链氨基酸序列分别如图6a和6b所示。
采用以下顺序将试验的诸组分加入平板:100μl靶细胞(如上所述制备)加入各孔；100μl效应细胞(如上所述制备)加入各孔；不同高亲和力PDL1加入各孔。
如下所述制备几个对照:效应细胞自发释放：仅有200μl效应细胞；靶细胞自发释放:仅有200μl靶细胞；靶细胞最大释放:仅有200μl靶细胞。所有孔一式三份制备，终体积为200μl。以250x g离心平板4分钟，然后在37℃温育24小时。将20μl裂解溶液加入靶细胞最大释放对照孔，45分钟后收集上清液。以250x g离心平板4分钟。将试验平板各孔的50μl清液转移至平底96孔NuncVlnxisorb板的相应孔。利用试验缓冲液(12m1)重建底物混合物。然后将50μl重建的底物混合物加入平板的各孔。平板盖上铝箔，室温下温育30分钟。将50μl终止溶液加入平板的各孔以终止反应。加入终止溶液后1小时内用Elisa平板读数计记录490nm的吸光度。
结果计算:从实验、靶细胞自发释放吸光度值中扣除背景的平均吸光度值,从靶细胞最大释放对照获得的吸光度值中扣除体积校正对照的平均吸光度值。前两步骤中获得的校正值用于下式以计算细胞毒性百分比:％细胞毒性＝100x(实验－靶细胞自发-效应细胞自发)/(靶细胞最大－靶细胞自发)。
实验结果如图7所示，图7中下标为“PBMC+A375+1G4+PDL1”的柱状图中 PDL1为野生型PDL-1分子。图中裂解率的提高或降低是相对于“PBMC+A375+1G4”柱状图而言。具体计算方式为|(“PBMC+A375+1G4+PDL-1分子”-“PBMC+A375+1G4”)|/“PBMC+A375+1G4”。该结果显示本发明的部分高亲和力PDL-1分子明显提高PBMC对肿瘤细胞的杀伤作用。
实施例5高亲和力PDL1二聚体蛋白的构建、表达和纯化
利用人的免疫球蛋白可结晶片段(human immunoglobulin fragment crystallizable,hIgG4Fc)来构建高亲和力PDL1分子的二聚体。具体地，将高亲和力PDL-1的胞外核苷酸序列3’端与hIgG4Fc核苷酸序列5’端通过overlap PCR连接，获得融合蛋白核酸序列和氨基酸序列，hIgG4Fc氨基酸序列和核酸序列分别为SEQ ID NO:37和38，如图8a和图8b所示。将携带融合蛋白序列的目的基因经EcoRⅠ和NotⅠ双酶切，与经过EcoRⅠ和NotⅠ双酶切的pcDNA3.1(+)载体连接。连接产物转化至TOP10，涂布含氨苄青霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒瞬转至293T细胞，用于表达。野生型PDL1分子的二聚体构建方式同上，野生型PDL1分子的二聚体在本发明中的表达方式为PDL1-hIgG4。高亲和力PDL1分子的二聚体在本发明中的表达方式为高亲和力PDL1分子编号-hIgG4，如L2D7-hIgG4。
1X10^7个293T细胞(购自ATCC，货号CRL-11268)接种于10cm细胞培养皿，12h后用PBS洗1次，并置换成opti-MEM培养基。以Lipo2000:重组质粒(1ug/ul)＝2:1的比例分别加入opti-MEM中，室温静置5min，含lipo2000的opti-MEM逐滴加入含重组质粒的opti-MEM，混合，室温静置20min，加入已置换opti-MEM培养基的293T细胞，4h后换成freestyleTM 293培养基，于5％CO2 37℃培养箱培养3d。
收集培养上清，经0.45μm滤膜过滤，通过阴离子交换柱(HiTrap Q HP,GE Healthcare)，用20mM Tris-HCl pH 7.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析，以PDL1-hIgG4Fc为例，其SDS-PAGE胶图如图9所示。组分浓缩后进一步用凝胶过滤柱(Superdex 20010/300,GE Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
所用材料如下：10cm细胞培养皿(greiner,目录号：627160)、opti-MEM培养基(gibco，目录号：31985-070)、Lipo2000、freestyleTM 293培养基(gibco， 目录号：12338-018)、PureLinkTM HiPure Plasmid Maxiprep Kit(invitrogen，目录号：K2100-06)
实施例6高亲和力PDL1-hIgG4Fc融合蛋白对PBMC(外周血单核细胞)杀伤肿瘤细胞株效力的影响
本实施例采用非放射性细胞毒性试验来验证杀伤作用。该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的乳酸脱氢酶(LDH)。采用30-分钟偶联的酶试验检测培养上清液中释放的LDH，其将四唑盐(INT)转化成红色甲瓒产物。形成的颜色量与裂解细胞的数量成比例。利用标准96孔平板读数计收集490nm的吸光度数据。
所用材料如下：CytoTox96非放射性细胞毒性试验(普罗迈格公司)(G1780)含有底物混合物、试验缓冲液、裂解溶液和终止溶液；试验培养基:10％FBS(热灭活的，吉布可公司，目录号10108-165)、VIVO-15(Lonza)，目录号:04-418)；Nunc微孔圆底96孔组织培养板(纽克公司(Nunc)，目录号163320)；Nunc-免疫平板Maxisorb(纽克公司，目录号442404)。
本实施例所用的靶细胞是Mel624细胞(Cassian Yee实验室惠赠)、NCI-H1299细胞(购自ATCC，货号CRL-5803)、IM9细胞(购自ATCC，货号CRL-159)、MDA-MB-231细胞(购自ATCC，货号CRM-HTB-26)。在试验培养基中制备靶细胞；靶细胞浓度调节至2x10^5个细胞/毫升，从而得到2x10^4个细胞/孔，100μl。
本实施例中所用的效应细胞(PBMC细胞)是从健康人的新鲜白膜中分选获得，细胞以1×10^6个细胞/ml重悬在试验培养基中，从而得到1x10^5个细胞/孔，100μl。
试验中ImmTAC终浓度为10^-9M，ImmTAC的储存浓度为5X10^-6M，稀释5000倍，直接加入制备好的效应细胞中。不同高亲和力PDL1-hIgG4Fc融合蛋白的终浓度为20ug/ml。本实施例中所用的ImmTAC(1G4)的α和β链氨基酸序列分别如图6a和6b所示。
采用以下顺序将试验的诸组分加入平板:100μl靶细胞(如上所述制备)加入各孔；100μl效应细胞(如上所述制备)加入各孔；不同高亲和力PDL1-hIgG4Fc融合蛋白加入各孔。
如下所述制备几个对照:效应细胞自发释放：仅有200μl效应细胞；靶细 胞自发释放:仅有200μl靶细胞；靶细胞最大释放:仅有200μl靶细胞。所有孔一式三份制备，终体积为200μl。以250x g离心平板4分钟，然后在37℃温育20小时。将20μl裂解溶液加入靶细胞最大释放对照孔，45分钟后收集上清液。以250x g离心平板4分钟。将试验平板各孔的50μl清液转移至平底96孔NuncVlnxisorb板的相应孔。利用试验缓冲液(12m1)重建底物混合物。然后将50μl重建的底物混合物加入平板的各孔。平板盖上铝箔，室温下温育30分钟。将50μl终止溶液加入平板的各孔以终止反应。加入终止溶液后1小时内用Elisa平板读数计记录490nm的吸光度。
结果计算:从实验、靶细胞自发释放吸光度值中扣除背景的平均吸光度值,从靶细胞最大释放对照获得的吸光度值中扣除体积校正对照的平均吸光度值。前两步骤中获得的校正值用于下式以计算细胞毒性百分比:％细胞毒性＝100x(实验－靶细胞自发-效应细胞自发/(靶细胞最大－靶细胞自发)。
实验结果如图10a、图10b、图10c和图10d所示。
图10a中下标为“PBMC+Mel624+1G4+PDL1-hlgG4”的柱状图中PDL1-hlgG4为野生型PDL-1融合分子。图中裂解率的提高或降低是相对于“PBMC+Mel624+1G4”柱状图而言。具体计算方式为|(“PBMC+Mel624+1G4+高亲和力PDL1-hlgG4分子”-“PBMC+Mel624+1G4+PDL1-hlgG4分子”)|/“PBMC+Mel624+1G4+PDL1-hlgG4分子”。该结果显示本发明的部分高亲和力PDL-1融合分子显著提高PBMC对肿瘤细胞Mel624的杀伤作用。
图10b中下标为“PBMC+NCI-H1299+1G4+PDL1-hlgG4”的柱状图中PDL1-hlgG4为野生型PDL-1融合分子。图中裂解率的提高或降低是相对于“PBMC+NCI-H1299+1G4”柱状图而言。具体计算方式为|(“PBMC+NCI-H1299+1G4+高亲和力PDL1-hlgG4分子”-“PBMC+NCI-H1299+1G4+PDL1-hlgG4分子”)|/“PBMC+NCI-H1299+1G4+PDL1-hlgG4分子”。该结果显示本发明的部分高亲和力PDL-1融合分子显著提高PBMC对肿瘤细胞NCI-H1299的杀伤作用。
图10c中下标为“PBMC+IM9+1G4+PDL1-hlgG4”的柱状图中PDL1-hlgG4为野生型PDL-1融合分子。图中裂解率的提高或降低是相对于“PBMC+IM9+1G4+PDL1-hlgG4”柱状图而言。具体计算方式为|(“PBMC+IM9+1G4+高亲和力PDL1-hlgG4分子”-“PBMC+IM9+1G4+PDL1-hlgG4分子”)|/“PBMC+IM9+1G4+PDL1-hlgG4分子”。该结果显示本发明的部分高亲和力PDL-1 融合分子显著提高PBMC对肿瘤细胞IM9的杀伤作用。
图10d中下标为“PBMC+MDA-MB-231+1G4+PDL1-hlgG4”的柱状图中PDL1-hlgG4为野生型PDL-1融合分子。图中裂解率的提高或降低是相对于“PBMC+MDA-MB-231+1G4+PDL1-hlgG4”柱状图而言。具体计算方式为|(“PBMC+MDA-MB-231+1G4+高亲和力PDL1-hlgG4分子”-“PBMC+MDA-MB-231+1G4+PDL1-hlgG4分子”)|/“PBMC+MDA-MB-231+1G4+PDL1-hlgG4分子”。该结果显示本发明的部分高亲和力PDL-1融合分子显著提高PBMC对肿瘤细胞MDA-MB-231的杀伤作用。
实验结果如图10a、10b、10c和10d所示，该结果显示本发明高亲和力PDL1-hIgG4Fc融合蛋白可显著提高ImmTAC(1G4)介导的PBMC杀伤肿瘤细胞。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种PDL-1分子，其特征在于，所述PDL-1分子的氨基酸序列基于SEQ ID NO.:1所示的氨基酸序列，并且对SEQ ID NO:1所示的氨基酸序列进行一个或多个氨基酸残基的突变或氨基酸残基的插入从而获得所述PDL-1分子；优选地，所述PDL-1分子的氨基酸序列与SEQ ID NO:1所示的氨基酸序列有至少90％的序列相同性；更优选地，所述PDL-1分子与PD-1分子的亲和力是野生型PDL-1分子与PD-1分子的亲和力的至少2倍。
  2. 如权利要求1所述的PDL-1分子，其特征在于，所述PDL-1分子中突变的氨基酸残基位点为1～3、35～50、和/或95～105位氨基酸残基中的一个或多个，其中氨基酸残基编号采用SEQ ID NO:1所示的编号。
  3. 如权利要求1所述的PDL-1分子，其特征在于，突变的氨基酸残基位点的数量为n，其中1≤n≤15；优选地，3≤n≤11；更优选地，4≤n≤10，如n可以为5、6、7、8、9、10。
  4. 如权利要求1所述的PDL-1分子，其特征在于，所述PDL-1分子中突变的氨基酸残基位点包括1F、36I、38Y、40E、47I、48Q、95R、97M、99S、和101G中的一个或多个或在102G后有一个或多个氨基酸残基的插入，其中氨基酸残基编号采用SEQ ID NO:1所示的编号；
    优选地，所述PDL-1分子包括选自下组的一个或多个氨基酸残基：1W；36Q、36E、36T或36N；38F、38N、38H或38A；40M、40F、40L、40W或40Y；47L、47V、47F、47S、47Y、47P或47A；48S；95T、95V或95L；97L；99G或99A；101D、101K、101E或101H；102G后插入氨基酸G；其中氨基酸残基编号采用SEQ ID NO:1所示的编号；
    更优选地，所述PDL-1分子的氨基酸序列选自SEQ ID NO:5-34之一。
  5. 一种融合蛋白，其特征在于，所述融合蛋白包括权利要求1-4任一所述的PDL-1分子。
  6. 如权利要求5所述的融合蛋白，其特征在于，所述融合蛋白还包括hIgG4Fc。
  7. 一种多价PDL-1复合物，其特征在于，所述多价PDL-1复合物包含至少两个PDL-1分子，并且其中的至少一个PDL-1分子为上述权利要求中任一项所述的PDL-1分子；或者所述多价PDL-1复合物包含至少一个权利要求5所述的 融合蛋白。
  8. 如权利要求7所述的多价PDL-1复合物，其特征在于，所述多价PDL-1复合物为二价PDL-1复合物、三价PDL-1复合物或四价PDL-1复合物。
  9. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1所述的PDL-1分子、权利要求5所述的融合蛋白、或权利要求7所述的多价PDL-1复合物的核酸序列或其互补序列。
  10. 一种载体，其特征在于，所述的载体含有权利要求9中所述的核酸分子。
  11. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求10中所述的载体或染色体中整合有外源的权利要求9中所述的核酸分子。
  12. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1-4中任一项所述的PDL-1分子、或权利要求5所述的融合蛋白、或权利要求7中所述的PDL-1复合物。
  13. 如权利要求12所述的药物组合物，其特征在于，所述组合物还包括ImmTAC和/或HATac。
  14. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1-4中任一所述的PDL-1分子、或权利要求5所述的融合蛋白、或权利要求7中所述的PDL-1复合物、或权利要求12中所述的药物组合物。
  15. 权利要求1-4任一项所述的PDL-1分子、或权利要求5所述的融合蛋白、权利要求7中所述的PDL-1复合物的用途，其特征在于，用于制备治疗肿瘤的药物。
  16. 一种制备权利要求1-4中任一所述的PDL-1的方法，其特征在于，包括步骤：
    (i)培养权利要求11所述的宿主细胞，从而表达权利要求1-4中任一所述的PDL-1分子；
    (ii)分离或纯化出所述的PDL-1分子。
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EP3360893B1 (en) 2021-08-18
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JP2018530337A (ja) 2018-10-18
JP6650515B2 (ja) 2020-02-19
US20180291081A1 (en) 2018-10-11
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EP3360893A1 (en) 2018-08-15

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