WO2018077189A1 - 高亲和力的可溶性pd-1分子 - Google Patents

高亲和力的可溶性pd-1分子 Download PDF

Info

Publication number
WO2018077189A1
WO2018077189A1 PCT/CN2017/107659 CN2017107659W WO2018077189A1 WO 2018077189 A1 WO2018077189 A1 WO 2018077189A1 CN 2017107659 W CN2017107659 W CN 2017107659W WO 2018077189 A1 WO2018077189 A1 WO 2018077189A1
Authority
WO
WIPO (PCT)
Prior art keywords
molecule
amino acid
seq
affinity
pdl
Prior art date
Application number
PCT/CN2017/107659
Other languages
English (en)
French (fr)
Inventor
李懿
李艳艳
Original Assignee
广东香雪精准医疗技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东香雪精准医疗技术有限公司 filed Critical 广东香雪精准医疗技术有限公司
Priority to CN201780074723.8A priority Critical patent/CN110023333B/zh
Publication of WO2018077189A1 publication Critical patent/WO2018077189A1/zh

Links

Images

Classifications

    • 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
    • C07K14/70521—CD28, CD152
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to high-affinity soluble programmed death receptor (PD-1) molecules capable of recognizing the programmed death receptor ligand PDL-1 with high affinity (Programmed Death) Ligand-1, PDL-1) molecule.
  • PD-1 programmed death receptor
  • the present invention relates to methods and uses for the preparation of such molecules.
  • PD-1 is an immunosuppressive receptor expressed on activated T cells and B cells, and its ligand is PDL-1 or PDL-2.
  • PD-1 belongs to the B7 family and is an Ig superfamily type I transmembrane glycoprotein with a size of 50-55 kD. It consists of three parts: extracellular IgV region, transmembrane region and intracellular region. It is found by structural and biochemical analysis. The proximal cysteine residue of the membrane, PD-1 is present as a monomer (Xuewu Zhang and Almo, Immunity, 2004, 20, 337-347). PD-1 interacts with the ligand PDL-1 and plays an important role in the negative regulation of the immune response.
  • a PD-1 molecule comprising a mutation in the amino acid sequence set forth in SEQ ID NO.
  • the amino acid sequence of the PD-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. Mutation or insertion of an amino acid residue to obtain the PD-1 molecule.
  • the amino acid sequence of the PD-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. Sex.
  • the affinity of the PD-1 molecule to the PDL-1 molecule is wild-type PD-1 molecule and PDL-1 At least 2 times the affinity of the molecule; preferably at least 10 times; more preferably at least 100 times; most preferably at least 200 times.
  • the affinity of the PD-1 molecule to the PDL-1 molecule is at least 500 times the affinity of the wild type PD-1 molecule to the PDL-1 molecule; preferably, at least 1000 fold; more preferably, At least 2000 times.
  • the mutated amino acid residue site in the PD-1 molecule is one or more of 30 to 60, and/or 85 to 105 amino acid residues, wherein the amino acid residue number is SEQ. The number shown in ID NO.1.
  • the mutated amino acid residue site in the PD-1 molecule is one or more of amino acid residues 31-37, 40-48, 56, and/or 89-103, wherein The amino acid residue numbering is given by the number shown in SEQ ID NO.
  • the number of mutated amino acid residue sites is n, wherein 1 ⁇ n ⁇ 15; preferably, 2 ⁇ n ⁇ 11; more preferably, 2 ⁇ n ⁇ 6, such as n can be 1 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the mutated amino acid residue sites in the PD-1 molecule include 91G, 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 93I, One or more of 95L, 97P, 98K, 99A, 100Q, 101I, 103E, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the mutated amino acid residue site in the PD-1 molecule comprises 91G, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the mutated amino acid residue site in the PD-1 molecule comprises 99A, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the mutated amino acid residue site in the PD-1 molecule further comprises 41N, 42Q, 43T, 48A, 95L, 97P, 98K and/or 100Q, wherein the amino acid residue numbering uses SEQ ID NO The number shown in .1.
  • the mutated PD-1 molecule comprises one or more amino acid residues selected from the group consisting of 91A, 91S, 91V or 91T; 31T; 33L; 35N or 35M; 37V, 37L or 37E; 40A or 40T; 41G, or 41L; 42N; 43V or 43G; 48G or 48S; 56L; 89M; 92V or 92Y; 93L; 95W or 95F; 97G; 98R, 98Y or 98P; 99P, 99V, 99I or 99F; Or 100W; 101V; and 103D; wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the mutated PD-1 molecule comprises 91V or 91S.
  • the mutated PD-1 molecule further comprises 99I or 99P.
  • the PD-1 molecule comprises: 91V and 99I; or
  • amino acid residue numbering is given by the number shown in SEQ ID NO.
  • amino acid sequence of the PD-1 molecule is selected from the group consisting of SEQ ID NO. 39, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41 or 43.
  • amino acid sequence of the PD-1 molecule is selected from the group consisting of SEQ ID NO. 39, 5, 11, 13, 15, or 43;
  • amino acid sequence of the PD-1 molecule is SEQ ID NO.
  • the PD-1 molecule is soluble.
  • the C- or N-terminus of the PD-1 molecule binds to a conjugate.
  • the conjugate that binds to the PD-1 molecule is a T cell receptor, preferably the T cell receptor is a high affinity T cell receptor.
  • the PD-1 molecule increases anti-CD3 mAb and anti-CD28 mAb-mediated PBMC proliferation by more than 15%, preferably 18% to 20%; and/or
  • the ratio of the PD-1 molecule to promote IFN- ⁇ release is about 20%; preferably, the ratio of promoting IFN- ⁇ release is increased to 40-50%.
  • a fusion protein comprising the PD-1 molecule of the first aspect of the invention is provided.
  • the fusion protein further comprises IgG4.
  • a multivalent PD-1 complex comprising at least two PD-1 molecules, and wherein at least one PD-1 molecule is the invention
  • the PD-1 molecule of one aspect; or the multivalent PD-1 complex comprises at least one fusion protein of the second aspect of the invention.
  • nucleic acid molecule comprising the PD-1 molecule of the first aspect of the invention, the fusion protein of the second aspect of the invention, or the third aspect of the invention is provided.
  • 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 integrated with an exogenous source;
  • the host cell contains or expresses the PD-1 molecule of the first aspect of the invention, the fusion protein of the second aspect of the invention, or the multivalent PD-1 complex of the third aspect of the invention.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the PD-1 molecule of the first aspect of the invention, or the fusion of the second aspect of the invention A protein, or a PD-1 complex of the third aspect of the invention.
  • a method of treating a disease comprising administering to a subject in need of treatment an appropriate amount of the PD-1 molecule of the first aspect of the invention, the fusion protein of the second aspect of the invention, or The PD-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 use of the PD-1 molecule of the first aspect of the invention, the fusion protein of the second aspect of the invention, or the PD-1 complex of the third aspect of the invention is used to prepare drugs for treating tumors.
  • a method of preparing the PD-1 of the first aspect of the invention comprising the steps of:
  • a PD-1 molecule comprising a mutation in the amino acid sequence set forth in SEQ ID NO.
  • Figure 1 shows the SDS-PAGE gel after purification of wild-type PD-1 protein.
  • M protein molecular weight Mark.
  • Figure 2 is a BIAcore map of the binding of a wild-type PDL-1 molecule to a PD-1 molecule.
  • Figure 3 shows that PD-1 and L5B7 recognize the flow-through detection of PD-L1 on the surface of H1299 cells, indicating that L5B7 has higher ability to recognize PDL-1 on the surface of H1299 cells than PD-1.
  • A anti-PDL-1 antibody (2.5 ul / sample) recognizes PDL-1 on the surface of H1299 cells;
  • B different concentrations of PD-1, L5B7 (concentration of 0.02mg/ml, 0.04mg/ml, 0.08mg) /ml) Flow cytometry to identify PDL-1 on the surface of H1299 cells, wherein the amount of SA-PE is 0.5 ul/sample;
  • C when the concentration is 0.08 mg/ml, the control group, PD-1, L5B7 recognize PDL Flow histogram of -1.
  • Figure 4a is a schematic diagram of eukaryotic expression of PD-1 mutant and its mutant (PD-1-IgG4 fusion protein schematic, note: the theoretical molecular weight of the dimer is about 80 kD, and the actual molecular weight after glycosylation is about 125 kD).
  • Figure 4b is a 8% SDS-PAGE gel electrophoresis pattern of the supernatant after 48 h.
  • M protein molecular weight Mark. 1, 2, PD-1-IgG4, L5B7-IgG4 Non-Reducing electrophoresis results; 3, 4, PD-1-IgG4, L5B7-IgG4 Reducing electrophoresis results.
  • Figure 4c is a functional validation map that promotes ImmTAC-IG4 mediated killing.
  • PD-1-IgG4, L5B7-IgG4 were shown to promote ImmTAC-IG4-mediated killing of LDH and CD25 and CD107a flow cytometry.
  • A LDH release, has been converted to the kill ratio.
  • B When the kill ratio is 1:1, the expression of CD25 and CD107a in CD8T cells in the killing reaction system is flow-through, and the amount of circulating antibody for detecting CD25 and CD107a is 2.5 ul/sample.
  • Figure 5 shows the proliferation of PBMCs stimulated by high-affinity PD-1 mutants; wherein AF in Figure 5a sequentially shows the flow of PBMCs that promote stimulation of high-affinity mutants L1B2, L2B12, L2F8, L2F10, L5B7, and L45.
  • Figure 5b is a statistical plot of the proliferation rate of PBMCs that each high affinity PD-1 mutant promotes stimulation.
  • Figure 6 shows that high affinity PD-1 mutants promote the release of IFN- ⁇ from stimulated PBMC;
  • Figure 6a shows the test The high affinity PD-1 mutation promotes the Elispot assay results of IFN- ⁇ release by stimulated PBMC;
  • 6b is a statistical plot of the ratio of IFN- ⁇ released by each high affinity PD-1 mutant promoting stimulation of PBMC.
  • the present inventors have unexpectedly discovered that soluble PD-1 molecules having high affinity for PDL-1 molecules can effectively enhance the killing ability of lymphocytes.
  • the present invention provides a soluble high affinity PD-1 molecule having an affinity for PDL-1 that is at least twice the affinity of the wild-type PD-1 molecule for PDL-1.
  • the PD-1 molecule of the present invention contains a mutation in the amino acid sequence shown in SEQ ID NO. More specifically, the amino acid sequence of the PD-1 molecule has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO.
  • Wild-type PD-1 molecule refers to an extracellular region of a wild-type PD-1 molecule, the amino acid sequence and the nucleotide sequence thereof are SEQ ID NO. 1 and SEQ ID, respectively. Shown in NO.2:
  • amino acid sequence and nucleotide sequence of PDL-1 are shown in SEQ ID NO. 3 and SEQ ID NO. 4, 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 with its ligand is higher than that of the wild type T cell receptor and its ligand.
  • a single-stranded autoreactive mouse 2C TCR with improved stability was screened by a yeast screening system, and its affinity for ligands was increased by about 100-fold compared to wild-type (9 nM) (Holler, PD et al. Natl. Acad. Sci. USA. 2000.97, 5387-5392).
  • 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.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • 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 a protein; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, arginine or lysine; a monosaccharide, a disaccharide 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; and/or nonionic surfactants such as
  • PD-1 (Programmed Death-1) is an immunosuppressive receptor expressed on activated T cells and B cells, and PDL-1 is a ligand thereof.
  • PD-1 belongs to the B7 family and is an Ig superfamily type I transmembrane glycoprotein with a size of 50-55 kD. It consists of three parts: extracellular IgV region, transmembrane region and intracellular region. It is found by structural and biochemical analysis. The proximal cysteine residue of the membrane, PD-1 is present as a monomer.
  • PD-1 interacts with its ligand PDL-1 (Programmed Death Ligand-1) and plays an important role in the negative regulation of immune response.
  • the present inventors have unexpectedly discovered that soluble PD-1 molecules having high affinity for PDL-1 molecules can effectively enhance the killing ability of lymphocytes.
  • the present invention provides a soluble high affinity for PDL-1 which is at least twice the affinity of wild-type PD-1 molecule for PDL-1. PD-1 molecule.
  • the binding affinity of the PD-1 molecule to PDL-1 in inverse proportion to the dissociation equilibrium constant K D
  • T 1/2 the binding half-life
  • K D dissociation equilibrium constant
  • T 1/2 the binding half-life
  • K off the binding half-life
  • 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 PD-1 molecule to the PDL-1 molecule in the present invention is 2.815E-06M, and the BIAcore binding map thereof is shown in FIG. 2 . 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 PD-1 molecule to the PDL-1 molecule is detected to be 1.408E-06M, the high affinity PDL-1 molecule is indicated to be PD.
  • the affinity of the -1 molecule is twice the affinity of the wild-type PD-1 molecule for PDL-1.
  • 1M 1000 ⁇ M
  • 1 ⁇ M 1000nM
  • 1nM 1000pM.
  • the affinity of the PD-1 molecule of the present invention to the PDL-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 PD-1 molecule to the PDL-1 molecule.
  • Multiplier preferably at least 10 times; more preferably, at least 50 times; most preferably at least 100 times.
  • the affinity of the PD-1 molecule to the PDL-1 molecule is at least 500 times the affinity of the wild type PD-1 molecule to the PDL-1 molecule; preferably, at least 1000 fold; more preferably, At least 2000 times.
  • the present invention is a high affinity for PD-1 and PDL-1 molecule affinity K D ⁇ 1.408E-06M; Preferably, 1.0E-06M ⁇ K D ⁇ 5.0E-06M ; more preferably, 1.0E-08M ⁇ K D ⁇ 5.0E-07M; most preferably, 1.0E-08M ⁇ K D ⁇ 1.0E-11M .
  • the high affinity PD-1 molecule of the invention contains one or more mutations in the amino acid sequence set forth in SEQ ID NO. Specifically, the amino acid sequence of the PD-1 molecule has at least 90% (preferably, at least 92%; more preferably, at least 94%) sequence identity to the amino acid sequence set forth in SEQ ID NO.
  • the amino acid residue sites mutated in the high affinity PD-1 molecule of the present invention include 91G, 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 93I, 95L.
  • amino acid residue sites can also be simply written as 91, 31, 33, 35, 37, 40, 41, 42, 43, 48, 56, 89, 92, 93, 95, 97, 98, The 99, 100, 101, and 103 positions, wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • the mutated PD-1 molecule comprises one or more amino acid residues selected from the group consisting of 91A, 91S, 91V or 91T; 31T; 33L; 35N, 35M; 37V, 37L or 37E; 40A, 40T; 41G, 41L; 42N; 43V, 43G; 48G, 48S; 56L; 89M; 92V, 92Y; 93L; 95W, 95F; 97G; 98R, 98Y or 98P; 99P, 99V, 99I or 99F; 100S, 100W; 101V; 103D wherein the amino acid residue numbering is the number shown in SEQ ID NO.
  • amino acid sequence of the PD-1 molecule is selected from the group consisting of SEQ ID NO. 39, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41 or 43;
  • SEQ ID NO. 40 6-8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42 and 44:
  • the wild type PD-1 molecule used in the present invention does not contain a transmembrane region.
  • the PD-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, Curr Opin Biotechnol, 1995, 6(1): 30-6).
  • PCR polymerase chain reaction
  • LIC linkage-independent cloning
  • the method of producing the high affinity PD-1 molecule of the present invention may be, but is not limited to, screening for a PD-1 having high affinity for PD-1 from a diverse library of phage particles displaying such PD-1 molecules, such as The literature (Li, et al., Nature Biotech, 2005, 23(3): 349-354).
  • the gene expressing the wild type PD-1 of the present invention or the gene expressing the wild type PD-1 of the present invention which is slightly modified can be used to prepare a template strand.
  • the changes required to produce the high affinity PD-1 of the invention are then introduced into the DNA encoding the template strand.
  • the PD-1 molecule of the invention may also be provided in the form of a multivalent complex.
  • the multivalent PD-1 of the present invention comprises a polymer formed by combining two, three, four or more PD-1 molecules of the present invention, such as a dimer can be prepared using an IgG FC segment, or p53 A tetrameric domain to produce a tetramer, or a complex of multiple PD-1 of the invention combined with another molecule.
  • the high affinity PD-1 molecules of the present 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 PD-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, Novel bi-specific agents for Targeted cancer thrapy. OncoImmunology, 2013, 2: 2, e22891).
  • 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 PD-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.
  • the meaning of "degenerate variant”, as used herein, "degenerate variant” in the present invention refers to a protein sequence having SEQ ID NO. 1 but having 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 PD-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 present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and PD-1 of the present invention, or a PD-1 complex of the present invention.
  • the present invention also provides a method for treating a disease comprising administering an appropriate amount of the PD-1 of the present invention, or the PD-1 complex of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment; in particular, the present invention
  • the PD-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), Ile(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 amino acids of similar or similar properties, the protein is usually not altered. The function. 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 PD-1 molecules which have been slightly modified with PD-1 of the present invention.
  • Modifications include: chemically derivatized forms of PD-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 PD-1 of the invention or in further processing steps. Such modification can be accomplished by exposing PD-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.
  • the PD-1 or PD-1 complex of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the PD-1, multivalent PD-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 PD-1 of the present invention may be used alone or in combination or coupled 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, dextrose, 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.
  • 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 PD-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 the 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 PD-1 or PD-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 PD-1 molecule having high affinity for PDL-1.
  • the high-affinity PD-1 molecule of the present invention can effectively increase the killing ability of lymphocytes.
  • the extracellular amino acid sequence and nucleotide sequence of wild-type PD-1 are SEQ ID NO. 1 and 2, respectively, and the target gene carrying the extracellular sequence of wild-type PD-1 is digested with NcoI and NotI, and subjected to NcoI.
  • the pET28a vector (Novagen) which was double digested with NotI, was ligated with a biotin tag.
  • the ligation product was transformed into E. coli DH5 ⁇ (Vazyme), coated with kanamycin-containing LB plate, inverted culture at 37 ° C overnight, and positive clones were picked for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and then extracted. Transformation of recombinant plasmid into E. coli Rosetta strain (TIANGEN), used for expression.
  • the above-mentioned Rosetta colony containing the recombinant plasmid pET28a-PD-1 was inoculated into LB medium containing kanamycin, cultured at 37 ° C until the OD600 was 0.6-0.8, IPTG was added to a final concentration of 0.7 mM, and culture was continued at 37 ° C for 4 h.
  • Cell pellet was harvested by centrifugation at 6000 g for 15 min, cell pellet was lysed with Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000 g for 15 min, and washed with Bugbuster (Merck) to remove cell debris and membrane fractions, centrifuged at 6000 g 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 8.1), 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 8.1
  • a buffer 50 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 6 M guanidine HCl, pH 8.1
  • DTT was further added to a final concentration of 20 mM, and treated at 37 ° C for 1 h.
  • the above treated PD-1 mixture 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), 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 K D , 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 8.5), and dialysis was continued for 24 h at 4 ° C. The dialysate was then replaced with the same fresh buffer for dialysis for 24 hours.
  • the sample was passed through 0.45 ⁇ m. The filter was filtered, vacuum degassed and injected onto an anion exchange column (HiTrap Q HP, GE Healthcare). The protein was purified by linear gradient elution with 0-1 M NaCl in 10 mM Tris-HCl pH 8.5 and the collected fractions were subjected to SDS-PAGE analysis.
  • the target PD-1 fraction was collected and further purified by a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target component was also subjected to SDS-PAGE analysis, and the results are shown in Fig. 1.
  • the binding activity of the wild-type PD-1 molecule to PDL-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.
  • PD-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 PDL-1 used in the present example are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and the expression, renaturation and purification processes thereof are the wild type PDL- in Example 1.
  • the expression, renaturation and purification processes of 1 are the same.
  • the process of biotinylation is as follows:
  • the purified PDL-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 0.05 MBicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA enzyme. (GST-BirA), the mixture was incubated overnight at room temperature, and biotinylation was detected by SDS-PAGE.
  • Biotinylated labeled PDL-1 molecules were concentrated to 500 ⁇ l using a Millipore ultrafiltration tube. Biotinylated PDL-1 was purified by gel filtration chromatography, and Superdex 75 10/300 gel was pre-equilibrated with filtered PBS. Filter column (GE General Electric Company), further load 500 ⁇ l of concentrated biotinylated PDL-1 molecule, then elute with PBS at a flow rate of 1 ml/min, and collect the collected components for SDS-PAGE analysis. The components of the target protein were concentrated using a Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the biotinylated PDL-1 molecule was dispensed at -80 °C.
  • the extracellular sequence of wild-type PD-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 PD-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 PD-1 molecule of the present invention was expressed, renatured and purified as described in Example 1, and its affinity for the PDL-1 molecule was determined as described in Example 2.
  • the affinity of the high-affinity PD-1 molecule obtained in the present invention to the PDL-1 molecule is at least 2-fold the affinity of the wild-type PD-1 molecule to the PDL-1 molecule, and the amino acid sequence thereof and its affinity with the PDL-1 molecule are as follows. Table 1 shows.
  • Example 4 L5B7 recognizes the ability of PD-1L on the surface of H1299 cells to be higher than PD-1
  • Biacore results showed that PD-1 mutants with improved affinity were obtained after screening, but whether this affinity change affects the binding of PDL-1 on the cell surface under physiological conditions still needs to be confirmed experimentally. Therefore, we selected H1299 cells with positive expression of PDL-1, added different concentrations of biotinylated PD-1 and L5B7 proteins, and analyzed the ability of PD-1 and L5B7 to recognize PDL-1 on the cell surface by flow cytometry.
  • eukaryotic expression is in the form of fusion expression with IgG4.
  • the eukaryotic expression sequence of PD-1 protein was optimized by Suzhou Heart Biotechnology Co., Ltd., and after splicing with IgG4 overlapPCR, it was ligated to the pGZFUSE plasmid vector through EcoRI and NheI sites, and transferred to Top 10 strain (through the same time) Mutation to obtain mutant clones of other affinities).
  • the cells were inoculated in 200 ml of LB medium at 1:1000, cultured at 37 ° C overnight, and collected on the next day, and a large amount of plasmid was extracted.
  • the concentration of the plasmid was measured by OD260/0D280, adjusted to 1 mg/ml, and stored at -20 degrees after dispensing. The large plasmid is ready for use.
  • a schematic representation of the fusion expressed protein is shown in Figure 4a.
  • the eukaryotic expression vector was constructed according to a, and after sequencing, the fusion protein expression was carried out using the 293T adherent cell expression system.
  • ImmTACs The ability of ImmTACs to redirect T cell-specific killing of tumor cells has been reported in several studies (Jakobsen, 2013; Oates et al., 2015).
  • the basic principle is that ImmTACs can mimic the key signal of T cell activation to exert effector function. On the one hand, it recognizes the MHC-peptide complex on the surface of tumor cells through its high affinity specific TCR, and on the other hand, it activates through its anti-CD3 antibody. The downstream signaling pathway of T cell activation, thereby targeting T cells specifically killing tumor cells. Therefore, in this study, the function of the PD-1/L5B7-IgG4 fusion protein was evaluated by whether the protein could be promoted. Figure 4c of the ImmTAC-IG4 molecule-mediated killing process of PBMC against Mel624 tumor cells.
  • CD25 and CD107a in CD8T cells of 1-IgG4 and L5B7-IgG4 fusion protein groups was up-regulated compared with the unproteinized group, and the up-regulation rate of L5B7-IgG4 group was higher than that of PD-1-IgG4 group, and CD25 was up-regulated from 10.9%. 13.2%, CD107a was increased from 16.9% to 19.8%, further validating the results of increased LDH release.
  • the fluorescent dye CFSE also known as CFDA SE (5,6-carboxyfluorescein diacetate, succinimidyl ester), is a fluorofluorescein diacetate succinimide ester, which is a fluorescent dye that penetrates cell membranes and has cells The specifically bound succinimide lipid group and the hydroxyfluorescein diacetate group with non-enzymatic hydrolysis make CFSE a good cell marker.
  • the fluorescent cytoplasmic proteins are evenly distributed to the second generation cells, so that the fluorescence intensity is reduced to half compared with the first generation cells; and so on, the third is obtained by splitting.
  • the fluorescence intensity of the cells will be weakened again than the second generation cells. This phenomenon can be detected by flow cytometry under the excitation light of 488 nm, and the cell fluorescence is continuously reduced by the detection of cell fluorescence intensity, and the cell division and proliferation are further analyzed.
  • PBMC proliferation was stimulated by plating at 1.5*10 5 cells/well in a 96-well flat bottom plate and adding 15 ⁇ g/ml anti-CD3 mAb and 7.5 ⁇ g/ml anti-CD28 mAb.
  • Example 7 High-affinity PD-1 molecule promotes IFN- ⁇ release from PBMC
  • anti-CD3 mAb and anti-CD28 mAb not only promote PBMC proliferation, but also promote PBMC release of IFN- ⁇ .
  • PBMC was tested by enzyme-linked immunospot (Elispot) and found that PD-1 monomeric protein could not promote the release of IFN- stimulated PBMC.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Toxicology (AREA)
  • Wood Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明提供了高亲和力的可溶性PD-1分子，具体地本发明提供了一种程序性死亡受体PD-1分子，其在野生型PD-1分子的基础上发生了突变，并且所述PD-1分子与其配体PDL-1分子的亲和力是野生型PD-1分子与野生型PDL-1分子的亲和力的至少2倍。同时，本发明的PD-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PD-1分子的核酸，以及本发明PD-1分子的复合物。本发明的PD-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PD-1分子 技术领域
本发明涉及生物技术领域，更具体地涉及高亲和力的可溶性程序性死亡受体(Programmed Death-1,PD-1)分子能够高亲和地识别程序性死亡受体配体PDL-1(Programmed Death Ligand-1,PDL-1)分子。本发明涉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，其配体为PDL-1或PDL-2。PD-1属于B7家族，是大小为50-55kD的Ig超家族Ⅰ型跨膜糖蛋白；由胞外IgV区、跨膜区、胞内区三部分组成，通过结构和生化分析发现，由于缺少膜近端半胱氨酸残基，PD-1以单体存在(Xuewu Zhang and Almo,Immunity,2004,20,337–347)。PD-1与配体PDL-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，研究人员已进行了大量研究。在侵袭性胰腺癌的鼠模型中，T.Nomi等(Clin.Cancer Res.2007,13：2151-2157)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PDL-1分子具有较高亲和力的PD-1分子。
本发明的再一目的是提供一种上述高亲和力的PD-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PD-1分子，所述PD-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PD-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PD-1分子。
在另一优选例中，所述PD-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PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1 分子的亲和力的至少2倍；优选地，至少10倍；更优选地，至少100倍；最优选地，至少200倍。
在另一优选例中，所述PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力的至少500倍；优选地，至少1000倍；更优选地，至少2000倍。
在另一优选例中，所述PD-1分子中突变的氨基酸残基位点为30～60、和/或85～105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PD-1分子中突变的氨基酸残基位点为31～37、40～48、56、和/或89～103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，2≤n≤11；更优选地，2≤n≤6，如n可以为1、2、3、4、5、6、7、8、9、10。
在另一优选例中，所述PD-1分子中突变的氨基酸残基位点包括91G、31V、33N、35Y、37M、40S、41N、42Q、43T、48A、56P、89L、92A、93I、95L、97P、98K、99A、100Q、101I、103E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PD-1分子中突变的氨基酸残基位点包括91G，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PD-1分子中突变的氨基酸残基位点包括99A，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PD-1分子中突变的氨基酸残基位点还包括41N、42Q、43T、48A、95L、97P、98K和/或100Q，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PD-1分子包括选自下组的一个或多个氨基酸残基：91A、91S、91V或91T；31T；33L；35N或35M；37V、37L或37E；40A或40T；41G、或41L；42N；43V或43G；48G或48S；56L；89M；92V或92Y；93L；95W或95F；97G；98R、98Y或98P；99P、99V、99I或99F；100S或100W；101V；和103D；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PD-1分子包括91V或91S。
在另一优选例中，突变后的PD-1分子还包括99I或99P。
在另一优选例中，所述PD-1分子包括：91V和99I；或
91S、98Y和99I；或
41L、42N、43G、48S、91V和99P；或
41G、43V、48G、91V和99P，
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PD-1分子的氨基酸序列选自SEQ ID NO.39、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41或43。
在另一优选例中，所述PD-1分子的氨基酸序列选自SEQ ID NO.39、5、11、13、15或43；
在另一优选例中，所述PD-1分子的氨基酸序列为SEQ ID NO.39。
在另一优选例中，所述PD-1分子是可溶的。
在另一优选例中，所述PD-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PD-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。
在一优选例中，所述PD-1分子使得anti-CD3mAb和anti-CD28mAb介导的PBMC增殖提高15％以上，优选18％～20％；和/或
所述PD-1分子促进IFN-γ释放的比率约20％左右；优选地，促进IFN-γ释放的比率增加到40～50％。
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PD-1分子。
在另一优选例中，所述融合蛋白还包括IgG4。
本发明的第三方面，提供了一种多价PD-1复合物，所述多价PD-1复合物包含至少两个PD-1分子，并且其中的至少一个PD-1分子为本发明第一方面所述的PD-1分子；或者所述多价PD-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所述的PD-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PD-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；或者
所述的宿主细胞含有或表达本发明第一方面所述的PD-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PD-1复合物。
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PD-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PD-1复合物。
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PD-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PD-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PD-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PD-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PD-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PD-1分子；
(ii)分离或纯化出所述的PD-1分子。
在本发明的第一方面，提供了一种PD-1分子，所述PD-1分子在SEQ ID NO.1所示的氨基酸序列中含有突变。
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1为野生型PD-1蛋白纯化后的SDS-PAGE胶图.M，蛋白分子量Mark.
图2为野生型PDL-1分子与PD-1分子结合的BIAcore图谱。
图3为PD-1、L5B7识别H1299细胞表面PD-L1的流式检测，显示L5B7识别H1299细胞表面PDL-1的能力高于PD-1。注：A，anti-PDL-1抗体(2.5ul/样品)识别H1299细胞表面的PDL-1；B，不同浓度的PD-1、L5B7(浓度为0.02mg/ml、0.04mg/ml、0.08mg/ml)识别H1299细胞表面的PDL-1的流式检测图，其中SA-PE的用量为0.5ul/样品；C，当浓度为0.08mg/ml时，对照组、PD-1、L5B7识别PDL-1的流式柱状图。
图4a为PD-1突变体及其突变体真核表达的示意图(PD-1-IgG4融合蛋白示意图，注：二聚体理论分子量为80kD左右，糖基化后实际分子量为125kD左右)。
图4b为48h小时后表达上清8％SDS-PAGE凝胶电泳图。注：M，蛋白分子量Mark。1、2，PD-1-IgG4、L5B7-IgG4的Non-Reducing电泳结果图；3、4，PD-1-IgG4、L5B7-IgG4的Reducing电泳结果图。
图4c为促进ImmTAC-IG4介导的杀伤的功能验证图。显示了PD-1-IgG4、L5B7-IgG4促进ImmTAC-IG4介导杀伤的LDH释放及CD25、CD107a流式检测。注：A，LDH释放，已经公式换算为杀伤比。B，当杀伤比为1:1时，杀伤反应体系中CD8T细胞的CD25、CD107a表达情况流式检测图，检测CD25、CD107a的流式抗体用量为2.5ul/样品。
图5显示了高亲和力PD-1突变体促进刺激的PBMC的增殖情况；其中图5a中的A-F依次显示了高亲和力突变体L1B2、L2B12、L2F8、L2F10、L5B7、L45促进刺激的PBMC增殖的流式图；5b是各高亲和力PD-1突变体促进刺激的PBMC增殖比率的统计图。
图6显示了高亲和力PD-1突变体促进刺激的PBMC释放IFN-γ；其中图6a显示了检 测高亲和力PD-1突变促进刺激的PBMC释放IFN-γ的Elispot实验结果；6b是各高亲和力PD-1突变体促进刺激的PBMC释放IFN-γ比率的统计图。
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PDL-1分子具有高亲和力的可溶性PD-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PDL-1的亲和力是野生型PD-1分子对PDL-1的亲和力至少两倍的可溶性高亲和力PD-1分子。
具体地，本发明中所述PD-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PD-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PD-1分子：本发明中所述的野生型PD-1分子是指野生型PD-1分子的胞外区，其氨基酸序列和核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示：
Figure PCTCN2017107659-appb-000001
PDL-1的氨基酸序列和核苷酸序列分别如SEQ ID NO.3和SEQ ID NO.4所示：
Figure PCTCN2017107659-appb-000002
Figure PCTCN2017107659-appb-000003
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较对应野生型T细胞受体与其配体亲和力提高的T细胞受体。例如，经过酵母筛选系统筛选得到稳定性提高的单链自身反应性鼠源2C TCR，其对配体的亲和力较野生型(9nM)提高了100倍左右(Holler,P.D.et al.Natl.Acad.Sci.USA.2000.97,5387-5392)。
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(Programmed Death-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为其配体。PD-1属于B7家族，是大小为50-55kD的Ig超家族Ⅰ型跨膜糖蛋白；由胞外IgV区、跨膜区、胞内区三部分组成，通过结构和生化分析发现，由于缺少膜近端半胱氨酸残基，PD-1以单体存在。PD-1与其配体PDL-1(Programmed Death Ligand-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PDL-1分子具有高亲和力的可溶性PD-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PDL-1的亲和力是野生型PD-1分子对PDL-1的亲和力至少两倍的可溶性高亲和力 PD-1分子。
可通过任何合适的方法测定上述PD-1分子与PDL-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PD-1分子对PDL-1分子的解离平衡常数KD为2.815E-06M，其BIAcore结合图谱如图2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PD-1分子对PDL-1分子的解离平衡常数KD为1.408E-06M，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PD-1分子对PDL-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PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力的至少2倍；优选地，至少10倍；更优选地，至少50倍；最优选地，至少100倍。
在另一优选例中，所述PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力的至少500倍；优选地，至少1000倍；更优选地，至少2000倍。
具体地，本发明高亲和力PD-1分子与PDL-1的亲和力KD≤1.408E-06M；优选地，1.0E-06M≤KD≤5.0E-06M；更优选地，1.0E-08M≤KD≤5.0E-07M；最优选地，1.0E-08M≤KD≤1.0E-11M。
本发明的高亲和力PD-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PD-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。
更具体地，本发明高亲和力PD-1分子中突变的氨基酸残基位点包括91G、31V、33N、35Y、37M、40S、41N、42Q、43T、48A、56P、89L、92A、93I、95L、97P、98K、99A、100Q、101I、103E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。基于本发明的技术内容，特别是公开的序列，本领域技术人员应该理解以上所述的氨基酸残基位点中的单字母是代表突变前该位点的氨基酸残基。因此，上述的“氨基酸残基位点”也可以简单写作第91、31、33、35、37、40、41、42、43、48、56、89、92、93、95、97、98、99、100、101、103位，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PD-1分子包括选自下组的一个或多个氨基酸残基：91A、91S、91V或91T；31T；33L；35N、35M；37V、37L或37E；40A、40T；41G、41L；42N；43V、43G；48G、48S；56L；89M；92V、92Y；93L；95W、95F；97G；98R、98Y或98P；99P、99V、99I或99F；100S、100W；101V；103D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PD-1分子的氨基酸序列选自SEQ ID NO.39、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41或43；
其编码核苷酸序列分别对应于：SEQ ID NO.40、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、42和44：
Figure PCTCN2017107659-appb-000004
Figure PCTCN2017107659-appb-000005
Figure PCTCN2017107659-appb-000006
Figure PCTCN2017107659-appb-000007
Figure PCTCN2017107659-appb-000008
为获得可溶性的高亲和力PD-1分子，本发明所用的野生型PD-1分子不含跨膜区。因此，在本发明的一个优选例中，所述PD-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,CurrOpinBiotechnol,1995,6(1):30-6)。
产生本发明的高亲和力PD-1分子的方法可以是但不限于从展示此类PD-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PD-1，如文献(Li,et al.,Nature Biotech,2005,23(3):349-354)中所述。
应理解，表达本发明野生型PD-1的基因或者表达略作修饰的本发明野生型PD-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PD-1所需的改变。
本发明的PD-1分子也可以多价复合体的形式提供。本发明的多价PD-1包含两个、三个、四个或更多个本发明PD-1分子相结合而形成的多聚物，如可以用IgG FC段制备二聚体，或p53的四聚结构域来产生四聚体，或多个本发明PD-1与另一分子结合而形成的复合物。
本发明的高亲和力PD-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PD-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,Novel bi-specific agents for targeted cancer thrapy.OncoImmunology,2013,2:2,e22891)。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PD-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PD-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PD-1、或本发明PD-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PD-1、或本发明PD-1复合物、或本发明的药物组合物；尤其是，本发明的PD-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)、Ile(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PD-1略作修饰后的PD-1分子。修饰(通常不改变一级结构)形式包括：本发明PD-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PD-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PD-1分子。这种修饰可以通过将PD-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PD-1。
本发明的PD-1或PD-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PD-1、多价PD-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PD-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PD-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PD-1或PD-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PDL-1具有高亲和力的PD-1分子。
(2)本发明的高亲和力PD-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。
实施例1野生型PD-1的表达、复性和纯化
野生型PD-1的胞外氨基酸序列及核苷酸序列分别为SEQ ID NO.1和2，将携带野生型PD-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(Novagen)该载体经过优化后带有biotin tag标签)连接。连接产物转化至E.coli DH5α(Vazyme)，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 Rosetta菌株 (TIANGEN)中,用于表达。
将上述含有重组质粒pET28a-PD-1的Rosetta菌落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0.7mM，37℃继续培养4h。6000g离心15min收获细胞沉淀物，用BugbusterMaster Mix(Merck)裂解细胞沉淀物，6000g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，6000g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 8.1)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PD-1包涵体蛋白中，加入2mL缓冲液(50mM Tris-HCl，200mM NaCl，2mM EDTA，6M guanidine HCl，pH 8.1)，再加入DTT至终浓度为20mM，37℃处理1h。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PD-1混合液，4℃搅拌30min，然后将复性液装入截留量为3.5KD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 8.5)，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，真空脱气后进样至阴离子交换柱(HiTrap Q HP，GE Healthcare)。用10mMTris-HCl pH8.5配制的0-1MNaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析。根据分析结果，收集目标PD-1组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300，GE Healthcare)纯化，目标组分也进行SDS-PAGE分析，结果如图1所示。
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PD-1分子与PDL-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PD-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PDL-1的氨基酸序列和核苷酸序列分别如SEQ ID NO.3、SEQ ID NO.4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Millipore超滤管将纯化的PDL-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0.05MBicine 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Millipore超滤管将生物素化标记后的PDL-1分子浓缩至500μl，采用凝胶过滤层析纯化生物素化的PDL-1，先用过滤过的PBS预平衡Superdex 75 10/300凝胶过滤柱(GE通用电气公司)，再加载500μl浓缩过的生物素化PDL-1分子，然后用PBS以1ml/min流速洗脱,将收集到的组分进行SDS-PAGE分析，根据结果合并含有目标蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，将生物素化的PDL-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PD-1分子与PDL-1分子的结合亲和力的KD值为2.815E-06M，其BIAcore结合图谱如图2所示。
实施例3高亲和力PD-1分子的产生
将实施例1中所述的野生型PD-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PD-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PD-1分子，并按实施例2中所述方法测定其与PDL-1分子的亲和力。本发明中获得的高亲和力PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力至少2倍，其氨基酸序列及其与PDL-1分子的亲和力数值如下表1所示。
表1 高亲和力克隆对PDL-1分子的BIAcore结果
Figure PCTCN2017107659-appb-000009
Figure PCTCN2017107659-appb-000010
实施例4L5B7识别H1299细胞表面PD-L1的能力高于PD-1
Biacore结果显示，经过筛选后确实得到了亲和力提高的PD-1突变体，但这种亲和力的改变是否会影响其与生理条件下细胞表面PDL-1的结合仍需实验确认。因此，我们选用PDL-1表达阳性的H1299细胞，加入不同浓度的生物素化PD-1、L5B7蛋白，流式细胞术分析PD-1、L5B7识别细胞表面PDL-1的能力。
图3结果显示，随着加入的PD-1、L5B7蛋白浓度的升高，其对PDL-1的识别能力逐渐增强；在同一浓度条件下，L5B7蛋白识别PDL-1的能力高于PD-1，这种识别能力的改变可能是由于L5B7的亲和力较高所引起的，与生化结果一致。
实施例5高亲和性PD-1分子双价融合蛋白产生
a表达载体构建
为了增加PD-1及其高亲和突变体在体内的稳定性及效价，真核表达采用与IgG4融合表达的形式。PD-1蛋白真核表达序列由苏州省心生物技术有限公司优化合成，与IgG4 overlapPCR拼接完成后，通过EcoRⅠ、NheⅠ位点连接到pGZFUSE质粒载体上，并化转到Top 10菌株中(同时通过突变获得其它亲和性的突变体克隆)。按1:1000接种于200ml LB培养基中，37度过夜培养后，于第二天收菌，进行质粒大量提取。OD260/0D280测质粒浓度，调整为1mg/ml，分装后与-20度保存。大提质粒备用。融合表达蛋白示意图如图4a。
b蛋白表达
根据a构建真核表达载体，经测序正确后，融合蛋白表达使用293T贴壁细胞表达系统。转染前一天，铺对数生长期293T细胞于10cm培养皿中，第二天以质粒：lipo 2000＝1:2(体积比)的条件转染细胞，4h后更换新鲜的FreestyleTM293培养基，48h后取少量上清液跑SDS-PAGE鉴定表达情况如图4b。72小时后收上清，使用0.22μm的滤膜过滤后，稀释至20倍体积提前预冷的10mM Tris-Hcl中(pH＝8.5)，进行阴离子与分子筛纯化。SDS-PAGE凝胶电泳结果显示：293T贴壁细胞表达的融合蛋白纯度很高。我们进一步纯化的目的有两个，一是置换蛋白所处溶液，防止在表达过程中或培养基本身存在的某些物质影响后续实验；二是浓缩蛋白，并进一步增加纯度，避免后续实验由于加入大量蛋白，或存在一些杂蛋白受到影响。
c融合蛋白功能鉴定
ImmTACs分子能够重定向T细胞特异性杀伤肿瘤细胞已在多篇研究中报道(Jakobsen,2013；Oates et al.,2015)。其基本原理是ImmTACs可以模拟T细胞活化发挥效应功能的关键信号，一方面通过其高亲和的特异性TCR识别肿瘤细胞表面的MHC-肽复合物，另一方面通过其anti-CD3抗体端激活T细胞活化的下游信号通路，从而定向T细胞特异性杀伤肿瘤细胞。因此，在本研究中评价PD-1/L5B7-IgG4融合蛋白的功能是通过该蛋白能否促进 ImmTAC-IG4分子介导的PBMC对Mel624肿瘤细胞的杀伤过程来评价的图4c。
结果发现，当ImmTAC的浓度为10-9M，E:T＝5：1和1:1(PBMC为效应细胞，Mel62为靶细胞)时，PD-1-IgG4、L5B7-IgG4融合蛋白均可以促进ImmTAC分子定向PBMC杀伤肿瘤细胞的LDH释放，且L5B7-IgG4融合蛋白促进LDH释放高于PD-1-IgG4组；收集杀伤比为1:1时反应体系中的细胞进行流式检测发现PD-1-IgG4、L5B7-IgG4融合蛋白组CD8T细胞的CD25、CD107a的表达较未加蛋白组均上调，且L5B7-IgG4组上调的比例高于加PD-1-IgG4组，CD25从10.9％上调至13.2％，CD107a从16.9％上调至19.8％，进一步验证了LDH释放增加的结果。说明PD-1-IgG4、L5B7-IgG4融合蛋白能够促进ImmTAC分子介导的PBMC对Mel624肿瘤细胞的杀伤，与其他文献中可溶性PD-1能够促进肿瘤特异性T细胞杀伤的研究结果一致，增加其亲和性后该促进作用进一步加强。
实施例6高亲和力PD-1分子促进活化的PBMC的增殖实验
进行本实验以验证本发明的高亲和力PD-1分子能够促进活化的PBMC增值。
荧光染料CFSE，也可称为CFDA SE(5,6-carboxyfluorescein diacetate,succinimidyl ester)，即羟基荧光素二醋酸盐琥珀酰亚胺脂，是一种可穿透细胞膜的荧光染料，具有与细胞特异性结合的琥珀酰亚胺脂基团和具有非酶促水解作用的羟基荧光素二醋酸盐基团，这使得CFSE成为一种良好的细胞标记物。当细胞进行分裂增殖时，具有荧光的胞质蛋白被平均分配到第二代细胞中，这样与第一代细胞相比，其荧光强度便会减弱至一半；以此类推，分裂得到的第三代细胞的荧光强度便会比第二代细胞再次减弱。这种现象可以在488nm的激发光下，采用流式细胞仪检测分析，通过检测到细胞荧光强度不断的降低，进一步分析得出细胞分裂增殖的情况。
在本实施例中，将新鲜分离的外周血单个核细胞(PBMC)经终浓度为1μM CFDA-SE染色后，用含10％FBS的RIPM-1640洗两遍终止CFDA-SE染色。以1.5*105个/孔铺于96-孔平底板，并加入15μg/ml anti-CD3mAb和7.5μg/ml anti-CD28mAb刺激PBMC增殖。结果显示，高亲和力PD-1分子不仅明显减少了未增殖细胞的数量；而且从荧光偏移量上看，加入高亲和力PD-1突变的组荧光左边更加向偏移，说明增殖强度更大(图5a)。统计PD-1蛋白及高亲和力突变体促进增殖的比率
Figure PCTCN2017107659-appb-000011
发现各高亲和力突变体能促进18％～20％的anti-CD3mAb和anti-CD28mAb介导的PBMC增殖，但是野生型PD-1并不能明显促进PBMC的增殖(图5b)。
实施例7高亲和力PD-1分子促进PBMC的IFN-γ释放实验
anti-CD3mAb和anti-CD28mAb不仅能促进PBMC增殖，而且可以促进PBMC释放IFN-γ。在本实施例中，PBMC经60μg/ml anti-CD3mAb和30μg/ml anti-CD28mAb刺激后，通过酶联免疫斑点(Elispot)实验，发现PD-1单体蛋白并不能促进刺激的PBMC释放IFN-γ，但是分别加入5μg/ml L1B2、5μg/ml L2B12、5μg/ml L2F8、5μg/ml L2F10、5μg/ml L5B7、5μg/ml L45高亲和力突变体蛋白后，检测IFN-γ释放的斑点明显增加(图 6a)，且斑点的增加和亲和力有明显的关系。统计不同高亲和力突变体促进IFN-γ释放的比率
Figure PCTCN2017107659-appb-000012
低亲和力的L1B2和L2B12促进IFN-γ释放的比率为20％左右，进一步提高亲和力，促进IFN-γ释放的比率增加到40～50％(图6b)。
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 (29)

  1. 一种PD-1分子，其特征在于，所述PD-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从而获得所述PD-1分子；所述PD-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PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力的至少2倍。
  2. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子的氨基酸序列与SEQ ID NO.1所示的氨基酸序列有92％；优选地，至少94％的序列相同性。
  3. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力的至少10倍；优选地，至少100倍；更优选地，至少200倍。
  4. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力的至少500倍；优选地，至少1000倍；更优选地，至少2000倍。
  5. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子中突变的氨基酸残基位点为第30～60和/或85～105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。
  6. 如权利要求5所述的PD-1分子，其特征在于，所述PD-1分子中突变的氨基酸残基位点为31～37、40～48、56和/或89～103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。
  7. 如权利要求1所述的PD-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，2≤n≤11；更优选地，2≤n≤6，如n可以为1、2、3、4、5、6、7、8、9、10。
  8. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子中突变的氨基酸残基位点包括91G、31V、33N、35Y、37M、40S、41N、42Q、43T、48A、56P、89L、92A、93I、95L、97P、98K、99A、100Q、101I和103E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。
  9. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子中突变的氨基酸残基位点包括91G，其中氨基酸残基编号采用SEQ ID NO.1所示的编号；和/或
    所述PD-1分子中突变的氨基酸残基位点包括99A，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PD-1分子中突变的氨基酸残基位点还包括41N、42Q、43T、48A、95L、97P、98K和/或100Q，其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
  10. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子包括选自下组的一个或多个氨基酸残基：91A、91S、91V或91T；31T；33L；35N或35M；37V、37L或37E； 40A或40T；41G或41L；42N；43V或43G；48G或48S；56L；89M；92V或92Y；93L；95W或95F；97G；98R、98Y或98P；99P、99V、99I或99F；100S或100W；101V；和103D；其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
  11. 如权利要求10所述的PD-1分子，其特征在于，所述PD-1分子包括91V或91S。
  12. 如权利要求10所述的PD-1分子，其特征在于，所述PD-1分子还包括99I或99P。
  13. 如权利要求10所述的PD-1分子，其特征在于，所述PD-1分子包括：91V和99I；或
    91S、98Y和99I；或
    41L、42N、43G、48S、91V和99P；或
    41G、43V、48G、91V、和99P；
    其中氨基酸残基编号采用SEQ ID NO.1所示的编号。
  14. 如权利要求10所述的PD-1分子，其特征在于，所述PD-1分子的氨基酸序列选自SEQ ID NO.39、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、41或43；
    优选地，所述PD-1分子的氨基酸序列选自SEQ ID NO.39、5、11、13、15或43；
    最优选地，所述PD-1分子的氨基酸序列为SEQ ID NO.39。
  15. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子是可溶的。
  16. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子的C或N末端结合有偶联物。
  17. 如权利要求16所述的PD-1分子，其特征在于，与所述PD-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。
  18. 如权利要求1所述的PD-1分子，其特征在于，所述PD-1分子使得anti-CD3mAb和anti-CD28mAb介导的PBMC增殖提高15％以上，优选18％～20％；和/或
    所述PD-1分子促进IFN-γ释放的比率约20％左右；优选地，促进IFN-γ释放的比率增加到40～50％。
  19. 一种融合蛋白，其特征在于，所述融合蛋白包括权利要求1-18中任一项所述的PD-1分子。
  20. 如权利要求19所述的融合蛋白，其特征在于，所述融合蛋白还包括IgG4。
  21. 一种多价PD-1复合物，其特征在于，所述多价PD-1复合物包含至少两个PD-1分子，并且其中的至少一个PD-1分子为权利要求1-18中任一项所述的PD-1分子；或者所述多价PD-1复合物包含至少一个权利要求19或20所述的融合蛋白。
  22. 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1-18中任一项所述的PD-1分子、权利要求19或20所述的融合蛋白、或权利要求21所述的多价PD-1复合物的核酸序列或其互补序列。
  23. 一种载体，其特征在于，所述的载体含有权利要求22所述的核酸分子。
  24. 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23所述的载体或染 色体中整合有外源的权利要求22所述的核酸分子；或者
    所述的宿主细胞含有或表达权利要求1-18中任一项所述的PD-1分子、权利要求19或20所述的融合蛋白、或权利要求21所述的多价PD-1复合物。
  25. 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1-18中任一项所述的PD-1分子、或权利要求19或20所述的融合蛋白、或权利要求21所述的PD-1复合物。
  26. 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1-18中任一项所述的PD-1分子、权利要求19或20所述的融合蛋白、或权利要求21所述的PD-1复合物、或权利要求25所述的药物组合物。
  27. 如权利要求26所述的方法，其特征在于，所述疾病为肿瘤。
  28. 权利要求1-18中任一项所述的PD-1分子、权利要求19或20所述的融合蛋白、或权利要求21所述的PD-1复合物的用途，用于制备治疗肿瘤的药物。
  29. 一种制备权利要求1-18中任一项所述的PD-1的方法，包括步骤：
    (i)培养权利要求24所述的宿主细胞，从而表达权利要求1-18中任一项所述的PD-1分子；
    (ii)分离或纯化出所述的PD-1分子。
PCT/CN2017/107659 2016-10-27 2017-10-25 高亲和力的可溶性pd-1分子 WO2018077189A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201780074723.8A CN110023333B (zh) 2016-10-27 2017-10-25 高亲和力的可溶性pd-1分子

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610959248.XA CN107987153A (zh) 2016-10-27 2016-10-27 高亲和力的可溶性pd-1分子
CN201610959248.X 2016-10-27

Publications (1)

Publication Number Publication Date
WO2018077189A1 true WO2018077189A1 (zh) 2018-05-03

Family

ID=62024327

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/107659 WO2018077189A1 (zh) 2016-10-27 2017-10-25 高亲和力的可溶性pd-1分子

Country Status (2)

Country Link
CN (2) CN107987153A (zh)
WO (1) WO2018077189A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021051661A1 (zh) * 2019-09-19 2021-03-25 北京伟杰信生物科技有限公司 重组犬pd-1融合蛋白及其制备方法与应用
CN114031682A (zh) * 2018-06-07 2022-02-11 江苏东抗生物医药科技有限公司 一种高亲和力的pd-1膜外区突变体的融合蛋白及其药物组合物和用途

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108794619B (zh) * 2018-05-31 2021-09-17 郑州大学 一种高亲和pd-1蛋白突变体
CN111714618B (zh) * 2019-03-22 2024-07-12 香雪生命科学技术（广东）有限公司 T细胞和高亲和力pd-1融合蛋白的组合
CN110478472B (zh) * 2019-09-29 2020-08-28 北京鼎成肽源生物技术有限公司 Pd-1封闭剂及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016023001A1 (en) * 2014-08-08 2016-02-11 The Board Of Trustees Of The Leland Stanford Junior University Multispecific high affinity pd-1 agents and methods of use

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR031699A1 (es) * 2000-05-26 2003-10-01 Bristol Myers Squibb Co Molecula mutante de ctla4 soluble que se enlaza a cd80 y/o cd86, molecula de acido nucleico que la codifica, vector y sistema vector que comprende a esta ultima, celula huesped que lo posee, metodo para producir una proteina mutante y dicha proteina, uso de la molecula mutante para preparar un medic
CN104356236B (zh) * 2005-07-01 2020-07-03 E.R.施贵宝&圣斯有限责任公司 抗程序性死亡配体1(pd-l1)的人单克隆抗体
CN105985427A (zh) * 2015-02-06 2016-10-05 广州市香雪制药股份有限公司 高亲和力ny-eso t细胞受体

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016023001A1 (en) * 2014-08-08 2016-02-11 The Board Of Trustees Of The Leland Stanford Junior University Multispecific high affinity pd-1 agents and methods of use

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114031682A (zh) * 2018-06-07 2022-02-11 江苏东抗生物医药科技有限公司 一种高亲和力的pd-1膜外区突变体的融合蛋白及其药物组合物和用途
CN114181296A (zh) * 2018-06-07 2022-03-15 江苏东抗生物医药科技有限公司 一种高亲和力的pd-1膜外区突变体的融合蛋白及其药物组合物和用途
CN114031682B (zh) * 2018-06-07 2023-06-30 江苏东抗生物医药科技有限公司 一种高亲和力的pd-1膜外区突变体的融合蛋白及其药物组合物和用途
CN114181296B (zh) * 2018-06-07 2023-06-30 江苏东抗生物医药科技有限公司 一种高亲和力的pd-1膜外区突变体的融合蛋白及其药物组合物和用途
WO2021051661A1 (zh) * 2019-09-19 2021-03-25 北京伟杰信生物科技有限公司 重组犬pd-1融合蛋白及其制备方法与应用

Also Published As

Publication number Publication date
CN110023333A (zh) 2019-07-16
CN107987153A (zh) 2018-05-04
CN110023333B (zh) 2023-08-25

Similar Documents

Publication Publication Date Title
WO2018077189A1 (zh) 高亲和力的可溶性pd-1分子
US11124557B2 (en) High-affinity and soluble PDL-1 molecule
US11292841B2 (en) Anti-PD-1 nano-antibody and application thereof
JP7048494B2 (ja) 修飾ｔ細胞に対する条件的活性型キメラ抗原受容体
KR20070097423A (ko) 흉선-특이성 단백질
TW202140540A (zh) 一種辨識hpv的t細胞受體
KR20190094382A (ko) Ny-eso에 대한 고친화력 tcr
CN113881680A (zh) T细胞抗原受体、其多聚体复合物及其制备方法和应用
JP2021536256A (ja) 修飾ｔ細胞に対する条件的活性型キメラ抗原受容体
TW202144403A (zh) 一種辨識ｈｐｖ１６的高親和力ｔｃｒ
TW202144402A (zh) 辨識ａｆｐ抗原的高親和力ｔｃｒ
WO2023241522A1 (zh) 靶向kras g12v突变多肽的t细胞受体及其用途
EP3936520A1 (en) High-affinity tcr for recognizing afp antigen
WO2022111475A1 (zh) 一种识别hpv抗原的tcr
CN110016074B (zh) Mage-a3人源化t细胞受体
CN114057864B (zh) 一种识别afp的高亲和力tcr
WO2021254458A1 (zh) 一种识别hpv抗原的高亲和力t细胞受体
WO2021228255A1 (zh) 一种识别afp抗原的高亲和力t细胞受体
KR20240057442A (ko) 이중특이성 nk 세포 작용제, 제조 방법 및 응용
WO2022206860A1 (zh) 针对afp的t细胞受体
WO2022166905A1 (zh) 一种针对hpv的高亲和力tcr
US20240043496A1 (en) Delta t-cell or gamma receptor chains or parts thereof for treating ovarian cancer
WO2023221959A1 (zh) 一种识别mage的高亲和力t细胞受体及其应用
CN117304301A (zh) 一种结合ssx2抗原的tcr分子及其应用
KR20220144327A (ko) Hla-dr 특이적 키메라 항원 수용체 및 이의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17865384

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 21/10/2019)

122 Ep: pct application non-entry in european phase

Ref document number: 17865384

Country of ref document: EP

Kind code of ref document: A1