CN110023333A - The soluble PD-1 molecule of high-affinity - Google Patents

The soluble PD-1 molecule of high-affinity Download PDF

Info

Publication number
CN110023333A
CN110023333A CN201780074723.8A CN201780074723A CN110023333A CN 110023333 A CN110023333 A CN 110023333A CN 201780074723 A CN201780074723 A CN 201780074723A CN 110023333 A CN110023333 A CN 110023333A
Authority
CN
China
Prior art keywords
molecule
amino acid
affinity
seq
acid residues
Prior art date
Legal status (The legal status 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 status listed.)
Granted
Application number
CN201780074723.8A
Other languages
Chinese (zh)
Other versions
CN110023333B (en
Inventor
李懿
李艳艳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiangxue Life Science Technology Guangdong Co ltd
Original Assignee
GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
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 GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd filed Critical GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Publication of CN110023333A publication Critical patent/CN110023333A/en
Application granted granted Critical
Publication of CN110023333B publication Critical patent/CN110023333B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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

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

The present invention provides the soluble PD-1 molecules of high-affinity, specifically the present invention provides a kind of programmed death receptor PD-1 molecules, it is mutated on the basis of wild type PD-1 molecule, and the affinity of the PD-1 molecule and its ligand PDL-1 molecule is at least 2 times of the affinity of wild type PD-1 molecule and wild type PDL-1 molecule.Meanwhile PD-1 molecule of the invention can effectively improve the killing ability of lymphocyte.In addition, the present invention also provides the nucleic acid for encoding PD-1 molecule of the present invention, and the compound of PD-1 molecule of the present invention.PD-1 molecule of the invention can be used alone, and can also be combined with other molecules.

Description

The soluble PD-1 molecule of high-affinity Technical field
The present invention relates to field of biotechnology, relate more specifically to soluble programmed death receptor (the Programmed Death-1 of high-affinity, PD-1) molecule being capable of height affine ground recognizer death receptor ligand PDL-1 (Programmed Death Ligand-1, PDL-1) molecule.The present invention relates to the preparation method of the molecule and purposes.
Background technique
PD-1 is the inhibitive ability of immunity receptor expressed in the T cell and B cell of activation, and ligand is PDL-1 or PDL-2.PD-1 belongs to B7 family, is the I type transmembrane glycoprotein of Ig superfamily that size is 50-55kD；It is made of the extracellular area IgV, transmembrane region, intracellular region three parts, is found by structure and biochemical analysis, due to lacking film proximal end cysteine residues, PD-1 has (Xuewu Zhang and Almo, Immunity, 2004,20,337-347) with monomer.PD-1 and ligand PDL-1 interacts, and plays an important role in terms of the negative regulation of immune response.Many tumor cell lines and tumour cell height express PDL-1 molecule (Konishi J et al., Clin.Cancer Res., 2004,10 (15): 5094-5100), in conjunction with the PD-1 molecule of lymphocytic cell surface after, weaken anti-tumor immune response (the Radziewicz H et al. of body, J Virol, 2007,81 (6): 2545-2553), so as to cause the generation of tumor immune escape.Research finds in cervical carcinoma and liver cancer there is the tumor-infiltrated CD8 of nearly half+T cell express PD-1 molecule, in conjunction with the PDL-1 of tumor cells expression after, may cause CTL cell exhaust and apoptosis (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).
For above-mentioned tumor immune escape problem, blocks the interaction of the PD-1 of the lymphocytic cell surface and PDL-1 of tumor cell surface that the immunity of lymphocyte can be made to improve, therefore, help to remove tumour cell by immune system.For this problem, researcher has been carried out numerous studies.In the mouse model of aggressive cancer of pancreas, T.Nomi etc. (Clin.Cancer Res.2007,13:2151-2157) proves to block the therapeutic efficiency of PD-1 and PDL-1 interaction.Those skilled in the art are dedicated to studying the interaction of PDL-1 and PD-1, to find the effective way for improving Lymphocvte Killer ability.
Summary of the invention
The purpose of the present invention is to provide the PD-1 molecules that a kind of pair of PDL-1 molecule has higher affinity.
Another object of the present invention is to provide a kind of Preparation method and use of the PD-1 molecule of above-mentioned high-affinity.
In the first aspect of the present invention, a kind of PD-1 molecule is provided, the PD-1 molecule contains mutation in the amino acid sequence shown in SEQ ID NO.1.
In another preferred example, the amino acid sequence of the PD-1 molecule is based on amino acid sequence shown in SEQ ID NO.:1, and carries out the mutation of one or more amino acid residues or the insertion of amino acid residue to amino acid sequence shown in SEQ ID NO.1 to obtain the PD-1 molecule.
In another preferred example, the amino acid sequence of the PD-1 molecule has at least 90% (preferably, at least 92% with amino acid sequence shown in SEQ ID NO.1；It is highly preferred that sequence identity at least 94%).
In another preferred example, the affinity of the PD-1 molecule and PDL-1 molecule is wild type PD-1 molecule and PDL-1 At least 2 times of the affinity of molecule；Preferably, at least 10 times；It is highly preferred that at least 100 times；Most preferably, at least 200 times.
In another preferred example, the affinity of the PD-1 molecule and PDL-1 molecule is at least 500 times of the affinity of wild type PD-1 molecule and PDL-1 molecule；Preferably, at least 1000 times；It is highly preferred that at least 2000 times.
In another preferred example, the acid residues sites being mutated in the PD-1 molecule are one or more of 30~60, and/or 85~105 amino acids residues, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the acid residues sites being mutated in the PD-1 molecule are one or more of 31~37,40~48,56, and/or 89~103 amino acids residues, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the quantity of the acid residues sites of mutation is n, wherein 1≤n≤15；Preferably, 2≤n≤11；It is highly preferred that 2≤n≤6, if n can be 1,2,3,4,5,6,7,8,9,10.
In another preferred example, the acid residues sites being mutated in the PD-1 molecule include one or more of 91G, 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 93I, 95L, 97P, 98K, 99A, 100Q, 101I, 103E, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the acid residues sites being mutated in the PD-1 molecule include 91G, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the acid residues sites being mutated in the PD-1 molecule include 99A, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the acid residues sites being mutated in the PD-1 molecule further include 41N, 42Q, 43T, 48A, 95L, 97P, 98K and/or 100Q, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the PD-1 molecule after mutation includes one or more amino acid residues selected from the group below: 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；100S or 100W；101V；And 103D；Wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the PD-1 molecule after mutation includes 91V or 91S.
In another preferred example, the PD-1 molecule after mutation further includes 99I or 99P.
In another preferred example, the PD-1 molecule includes: 91V and 99I；Or
91S, 98Y and 99I；Or
41L, 42N, 43G, 48S, 91V and 99P；Or
41G, 43V, 48G, 91V and 99P,
Wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the amino acid sequence of the PD-1 molecule is selected from SEQ ID NO.39,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,41 or 43.
In another preferred example, the amino acid sequence of the PD-1 molecule is selected from SEQ ID NO.39,5,11,13,15 or 43；
In another preferred example, the amino acid sequence of the PD-1 molecule is SEQ ID NO.39.
In another preferred example, the PD-1 molecule is soluble.
In another preferred example, the C of the PD-1 molecule or N-terminal are combined with conjugate.
In another preferred example, the conjugate in conjunction with the PD-1 molecule is T cell receptor, it is preferable that the T cell receptor is high-affinity T cell receptor.
In a preferred example, the PBMC proliferation that the PD-1 molecule mediates anti-CD3mAb and anti-CD28mAb improves 15% or more, preferably 18%~20%；And/or
The PD-1 molecule promotes the ratio about 20% or so of IFN-γ release；Preferably, promoting the ratio of IFN-γ release increases to 40~50%.
The second aspect of the present invention, provides a kind of fusion protein, and the fusion protein includes PD-1 molecule described in first aspect present invention.
In another preferred example, the fusion protein further includes IgG4.
The third aspect of the present invention provides a kind of multivalence PD-1 compound, and the multivalence PD-1 compound contains at least two PD-1 molecule, and at least one PD-1 molecule therein is PD-1 molecule described in first aspect present invention；Or the multivalence PD-1 compound includes fusion protein described at least one second aspect of the present invention.
The fourth aspect of the present invention, a kind of nucleic acid molecules are provided, the nucleic acid molecules include the nucleic acid sequence or its complementary series of PD-1 molecule described in coding first aspect present invention, multivalence PD-1 compound described in fusion protein or third aspect present invention described in second aspect of the present invention.
Fifth aspect present invention, provides a kind of carrier, and the carrier contains nucleic acid molecules described in fourth aspect present invention.
The sixth aspect of the present invention provides a kind of host cell, contains nucleic acid molecules described in the fourth aspect present invention for being integrated with external source in carrier or chromosome described in fifth aspect present invention in the host cell；Or
The host cell contains or expresses multivalence PD-1 compound described in fusion protein described in PD-1 molecule, second aspect of the present invention described in first aspect present invention or third aspect present invention.
The seventh aspect of the present invention, a kind of pharmaceutical composition is provided, the composition contains PD-1 compound described in fusion protein described in PD-1 molecule or second aspect of the present invention described in pharmaceutically acceptable carrier and first aspect present invention or third aspect present invention.
The eighth aspect of the present invention, a kind of method for treating disease is provided, including applying pharmaceutical composition described in PD-1 compound described in fusion protein described in PD-1 molecule, second aspect of the present invention described in suitable first aspect present invention or third aspect present invention or the seventh aspect of the present invention to object in need for the treatment of.
In another preferred example, the disease is tumour.
The ninth aspect of the present invention provides the purposes of PD-1 compound described in fusion protein described in PD-1 molecule, second aspect of the present invention described in first aspect present invention or third aspect present invention, is used to prepare the drug for the treatment of tumour.
The tenth aspect of the present invention provides a kind of method for preparing PD-1 described in first aspect present invention, comprising steps of
(i) host cell described in sixth aspect present invention is cultivated, to express PD-1 molecule described in first aspect present invention；
(ii) isolated or purified goes out the PD-1 molecule.
In the first aspect of the present invention, a kind of PD-1 molecule is provided, the PD-1 molecule contains mutation in the amino acid sequence shown in SEQ ID NO.1.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and it can be combined with each other between each technical characteristic specifically described in below (e.g. embodiment), to form a new or preferred technical solution.Due to space limitations, I will not repeat them here.
Detailed description of the invention
Fig. 1 is SDS-PAGE glue the figure .M, molecular weight of albumen Mark. after wild type PD-1 protein purification
Fig. 2 is BIAcore map of the wild type PDL-1 molecule in conjunction with PD-1 molecule.
Fig. 3 is the flow cytometer detection that PD-1, L5B7 identify H1299 cell surface PD-L1, and the ability of display L5B7 identification H1299 cell surface PDL-1 is higher than PD-1.Note: the PDL-1 of A, anti-PDL-1 antibody (2.5ul/ sample) identification H1299 cell surface；B, the flow cytometer detection figure of the PDL-1 of PD-1, L5B7 (concentration 0.02mg/ml, 0.04mg/ml, 0.08mg/ml) identification H1299 cell surface of various concentration, wherein the dosage of SA-PE is 0.5ul/ sample；C, when concentration is 0.08mg/ml, the streaming histogram of control group, PD-1, L5B7 identification PDL-1.
Fig. 4 a is the schematic diagram of PD-1 mutant and its mutant eukaryotic expression (PD-1-IgG4 fusion protein schematic diagram, note: dimer theoretical molecular weight is 80kD or so, and actual molecular weight is 125kD or so after glycosylation).
Fig. 4 b is that supernatant 8%SDS-PAGE gel electrophoresis figure is expressed after 48h hours.Note: M, molecular weight of albumen Mark.1,2, the Non-Reducing electrophoresis result figure of PD-1-IgG4, L5B7-IgG4；3,4, the Reducing electrophoresis result figure of PD-1-IgG4, L5B7-IgG4.
Fig. 4 c is the functional verification figure for the killing for promoting ImmTAC-IG4 to mediate.Show LDH release and CD25, CD107a flow cytometer detection that PD-1-IgG4, L5B7-IgG4 promote ImmTAC-IG4 to mediate killing.Note: A, LDH release, formula scales are killing ratio.B kills CD25, CD107a expression flow cytometer detection figure of cd8 t cell in reaction system when killing is than being 1:1, and the streaming antibody dosage of detection CD25, CD107a are 2.5ul/ sample.
Fig. 5 shows that high-affinity PD-1 mutant promotes the proliferative conditions of the PBMC of stimulation；Wherein the A-F in Fig. 5 a successively shows that high-affinity mutant L1B2, L2B12, L2F8, L2F10, L5B7, L45 promote the streaming figure of the PBMC proliferation of stimulation；5b is the statistical chart for the PBMC growth fraction that each high-affinity PD-1 mutant promotes stimulation.
Fig. 6 shows that high-affinity PD-1 mutant promotes the PBMC release IFN-γ of stimulation；Wherein Fig. 6 a shows inspection Survey the Elispot experimental result that high-affinity PD-1 mutation promotes the PBMC release IFN-γ of stimulation；6b is the statistical chart for the PBMC release IFN-γ ratio that each high-affinity PD-1 mutant promotes stimulation.
Specific embodiment
The present invention passes through extensive and in-depth research, it has unexpectedly been found that can effectively improve the killing ability of lymphocyte with the soluble PD-1 molecule of high-affinity to PDL-1 molecule.It therefore, is soluble high-affinity PD-1 molecule of the wild type PD-1 molecule to the affinity at least twice of PDL-1 the present invention provides the affinity of a kind of couple of PDL-1.
Specifically, mutation is contained in heretofore described PD-1 molecule amino acid sequence shown in SEQ ID NO.1.More specifically, amino acid sequence shown in the amino acid sequence of the PD-1 molecule and SEQ ID NO.1 has at least 90% sequence identity.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because such methods and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and it is not intended to restrictive, and the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as the normally understood identical meanings of those skilled in the art.
Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method and material
Material, place enumerates preferred method and material herein.
Term
Wild type PD-1 molecule: heretofore described wild type PD-1 molecule refers to the extracellular region of wild type PD-1 molecule, and amino acid sequence and nucleotide sequence are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2:
The amino acid sequence and nucleotide sequence of PDL-1 is respectively as shown in SEQ ID NO.3 and SEQ ID NO.4:
PBMC: peripheral blood mononuclear cells (PBMC) is the haemocyte with round karyon, such as lymphocyte or monocyte.These haemocytes are that immune system fight infects and is adapted to the key component of invader.Lymphocyte population is made of T cell (CD4 the and CD8 positive about 75%), B cell and NK cell (merging about 25%).
High-affinity T cell receptor: refer to the T cell receptor that wild-type T cells receptor more corresponding with the affinity of its ligand and its ligand affinity improve.Such as, it screens to obtain stability-enhanced single-stranded autoreactivity source of mouse 2C TCR by Yeast selective system, it improves 100 times or so (Holler compared with wild type (9nM) to the affinity of ligand, P.D.et al.Natl.Acad.Sci.USA.2000.97,5387-5392).
Tumour: referring to including all types of growth of cancer cells or oncogenic process, metastatic tissue or malignant conversioning cell, tissue or organ, regardless of histological type or the stage infected.The embodiment of tumour includes: solid tumor, soft-tissue tumor and metastasis (metastases) without limitation.The embodiment of solid tumor includes: the malignant tumour of Different Organs system, such as sarcoma, lung squamous cancer and cancer.Such as: the prostate of infection, lung, breast, lymph, stomach (such as: colon) and genitourinary tract (such as: kidney, epithelial cell), pharynx.Lung squamous cancer includes malignant tumour, for example, most colon cancers, the carcinoma of the rectum, clear-cell carcinoma, liver cancer, the non-small cell carcinoma of lung, carcinoma of small intestine and cancer of the esophagus.Above-mentioned cancer metastasis venereal disease change can be treated and prevented equally with method and composition of the invention.
Pharmaceutical carrier: the cell or individual that also referred to excipient or stabilizer, dosage and concentration expose it are nontoxic.Frequently, physiology acceptable carriers are the aqueous solutions of pH buffering.The example of physiology acceptable carriers includes buffer such as phosphate, citrate and other organic acids；Antioxidant, including ascorbic acid；Low molecular weight (being less than about 10 residues) polypeptide；Protein, such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, glutamine, asparagine, arginine or lysine；Monosaccharide, disaccharides and other carbohydrates, including glucose, mannose or dextrin；Complexing agent such as EDTA；Sugar alcohol such as mannitol or sorbierite；At salt counter ion such as sodium；And/or nonionic surfactant such as TWEENTM, polyethylene glycol (PEG) and PLURONICSTM.
Detailed description of the invention
PD-1 (Programmed Death-1) is the inhibitive ability of immunity receptor expressed in the T cell and B cell of activation, and PDL-1 is its ligand.PD-1 belongs to B7 family, is the I type transmembrane glycoprotein of Ig superfamily that size is 50-55kD；It is made of the extracellular area IgV, transmembrane region, intracellular region three parts, finds that, due to lacking film proximal end cysteine residues, PD-1 exists with monomer by structure and biochemical analysis.PD-1 and its ligand PDL-1 (Programmed Death Ligand-1) interact, and play an important role in terms of the negative regulation of immune response.The present invention passes through extensive and in-depth research, it has unexpectedly been found that can effectively improve the killing ability of lymphocyte with the soluble PD-1 molecule of high-affinity to PDL-1 molecule.It therefore, is soluble high-affinity of the wild type PD-1 molecule to the affinity at least twice of PDL-1 the present invention provides the affinity of a kind of couple of PDL-1 PD-1 molecule.
The binding affinity of above-mentioned PD-1 molecule and PDL-1 can be measured by any suitable method (with Dissociation equilibrium constant KDIt is inversely proportional) and half-life period is combined (to be expressed as T1/2).It will be appreciated that affinity is double to will lead to KDHalve.T1/2In2 is calculated as divided by dissociation rate (Koff).Therefore, KoffIt is double to will lead to T1/2Halve.It is preferred that using identical testing program detection binding affinity or combining half-life period for several times, such as 3 times or more, the average value of result is taken.In a preferred embodiment, these detections are carried out using surface plasmon resonance (BIAcore) method in the embodiment of the present invention.
This method detects that wild type PD-1 molecule is to the Dissociation equilibrium constant K of PDL-1 molecule in the present inventionDFor 2.815E-06M, BIAcore combination map is as shown in Figure 2.It will lead to K since affinity is doubleDHalve, so if detecting high-affinity PD-1 molecule to the Dissociation equilibrium constant K of PDL-1 moleculeDFor 1.408E-06M, then illustrate that high-affinity PDL-1 molecule is wild type PD-1 molecule to 2 times of the affinity of PDL-1 to the affinity of PD-1 molecule.The known K of those skilled in the artDThe conversion relation being worth between unit, i.e., 1M=1000 μM, 1 μM=1000nM, 1nM=1000pM.
In a preference of the invention, the affinity that PD-1 molecule of the present invention and PDL-1 molecule are measured in the way of currently preferred measurement affinity is at least 2 times of affinity of wild type PD-1 molecule and PDL-1 molecule；Preferably, at least 10 times；It is highly preferred that at least 50 times；Most preferably, at least 100 times.
In another preferred example, the affinity of the PD-1 molecule and PDL-1 molecule is at least 500 times of the affinity of wild type PD-1 molecule and PDL-1 molecule；Preferably, at least 1000 times；It is highly preferred that at least 2000 times.
Specifically, the affinity K of high-affinity PD-1 molecule and PDL-1 of the present inventionD≤1.408E-06M；Preferably, 1.0E-06M≤KD≤5.0E-06M；It is highly preferred that 1.0E-08M≤KD≤5.0E-07M；Most preferably, 1.0E-08M≤KD≤1.0E-11M。
High-affinity PD-1 molecule of the invention contains one or more mutation in the amino acid sequence shown in SEQ ID NO.1.Specifically, the amino acid sequence of the PD-1 molecule has at least 90% (preferably, at least 92% with amino acid sequence shown in SEQ ID NO.1；It is highly preferred that sequence identity at least 94%).
More specifically, the acid residues sites being mutated in high-affinity PD-1 molecule of the present invention include one or more of 91G, 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 93I, 95L, 97P, 98K, 99A, 100Q, 101I, 103E, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.Based on technology contents of the invention, especially disclosed sequence, those skilled in the art should understand that the single-letter in above-described acid residues sites is to represent the amino acid residue for being mutated the preceding site.Therefore, above-mentioned " acid residues sites " can also simply write the 91st, 31,33,35,37,40,41,42,43,48,56,89,92,93,95,97,98,99,100,101,103, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
In another preferred example, the PD-1 molecule after mutation includes one or more amino acid residues selected from the group below: 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 number using shown in SEQ ID NO.1 by numbering amino acid residues.
In another preferred example, the amino acid sequence of the PD-1 molecule is selected from SEQ ID NO.39,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,41 or 43；
Its coding nucleotide sequence corresponds respectively to: SEQ ID NO.40,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,42 and 44:
To obtain soluble high-affinity PD-1 molecule, wild type PD-1 molecule used in the present invention is free of transmembrane region.Therefore, in a preference of the invention, the PD-1 molecule is soluble.
Any suitable method can be used to be mutated, including but not limited to according to those of polymerase chain reaction (PCR), according to restriction enzyme clone or do not depend on clone (LIC) method of connection.Many standard molecular biology teaching materials detail these methods.The more details of polymerase chain reaction (PCR) mutagenesis and the clone according to restriction enzyme can be found in Sambrook and Russell, (2001) Molecular Cloning-A Laboratory handbook (Molecular Cloning-A Laboratory Manual) (third edition) CSHL publishing house.The more information of LIC method is visible (Rashtchian, CurrOpinBiotechnol, 1995,6 (1): 30-6).
The method for generating high-affinity PD-1 molecule of the invention can be but not limited to filter out the PD-1 for having high-affinity to PD-1 from the diverse libraries of phage particle for showing such PD-1 molecule, such as document (Li, et al., Nature Biotech, 2005,23 (3): 349-354) described in.
It should be understood that the gene for the wild type PD-1 of the present invention that the gene of expression wild type PD-1 of the present invention or expression are slightly modified can be adopted to prepare template strand.Then change needed for generating high-affinity PD-1 of the invention is introduced in the DNA for encoding the template strand.
PD-1 molecule of the invention can also be provided in the form of multivalence complex.Multivalence PD-1 of the invention includes the polymer that two, three, four, or more PD-1 molecule of the present invention is combined and formed, such as dimer can be prepared with FC sections of IgG, or the compound that four dimerization domains of p53 are formed to generate the tetramer or multiple PD-1 of the present invention in conjunction with another molecule.
High-affinity PD-1 molecule of the invention can be used alone, can also with conjugate with covalent or other modes in conjunction with, preferably combined with covalent manner.The conjugate is preferably T cell receptor, it is highly preferred that the T cell receptor is high-affinity T cell receptor.
High-affinity PD-1 molecule of the invention can also be combined with other molecules, generate effective synergistic effect.Preferably, other described molecules are ImmTAC or HATac.Two kinds of molecules can redirect T cell, to play the role of killing target cell.The ImmTAC molecule is the fusion molecule of soluble the double-strand TCR molecule and anti-cd 3 antibodies between α β constant region containing artificial interchain disulfide bond, for details, reference can be made to document (Joanne Oates, Bent K.Jakobsen, Novel bi-specific agents for targeted cancer thrapy.OncoImmunology, 2013,2:2, e22891).The HATac molecule is high-affinity T cell activation core (High Affinity T-cell activation core), the fusion molecule of soluble single-chain T CR molecule and anti-cd 3 antibodies that the α and β chain variable domain that one form of them can be mutated by hydrophobic core are formed by connecting, for details, reference can be made to patent document WO2014/206304 for the soluble single-chain T CR molecule.
The invention further relates to the nucleic acid molecules for encoding PD-1 of the present invention.Nucleic acid molecules of the invention can be DNA form or rna form.DNA can be coding strand or noncoding strand.For example, the nucleic acid sequence of coding TCR of the present invention can variant identical as present invention nucleic acid sequence shown in the drawings or degeneracy.The meaning of " variant of degeneracy " is illustrated, as used herein, " variant of degeneracy " refers to that coding has the protein sequence of SEQ ID NO.1, but the differentiated nucleic acid sequence of sequence with SEQ ID NO.2 in the present invention.
Nucleic acid molecules full length sequence or its segment of the invention usually can with but be not limited to PCR amplification method, recombination method or artificial synthesized method obtain.At present, it is already possible to obtain encoding the DNA sequence dna of PD-1 of the present invention (or its segment, or derivatives thereof) by chemical synthesis completely.Then the DNA sequence dna can be introduced into various existing DNA moleculars (or such as carrier) as known in the art and cell.
The present invention also relates to the carriers comprising nucleic acid molecules of the invention, and with the genetically engineered host cell of carrier or coded sequence of the invention.
The present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition contains pharmaceutically acceptable carrier and PD-1 of the present invention or PD-1 compound of the present invention.
The present invention also provides a kind of methods for treating disease, including applying suitable PD-1 of the present invention or PD-1 compound of the present invention or pharmaceutical composition of the invention to object in need for the treatment of；Especially, PD-1 molecule of the invention and other molecules are combined, it is preferable that other molecules are ImmTAC or HATac.
It should be understood that, amino acid name herein is identified using international single English alphabet, and three English alphabet of amino acid name corresponding thereto, which is write a Chinese character in simplified form, is respectively: 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 being substituted with similar nature or similar amino acid, the function of protein is not usually changed.The structure and function of protein will not be changed by adding one or several amino acid generally also in C-terminal and/or N-terminal.
The invention also includes the PD-1 molecules after slightly modifying PD-1 of the present invention.Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetylation or carboxylated of PD-1 of the present invention.Modification further includes glycosylation, carries out PD-1 molecule that is glycosylation modified and generating in the synthesis and processing of PD-1 of the present invention or in further processing step such as those.This modification is completed and can carrying out glycosylated enzyme (glycosylase or deglycosylation enzyme of such as mammal) by the way that PD-1 to be exposed to.Modified forms further include the sequence with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine).It further include being modified to improve its anti-proteolytic properties or optimize the PD-1 of solubility property.
PD-1 or PD-1 compound of the invention can provide in pharmaceutical composition together with pharmaceutically acceptable carrier.PD-1 of the invention, multivalence PD-1 compound are provided usually as a part of sterile pharmaceutical composition, and the composition generally includes pharmaceutically acceptable carrier.The pharmaceutical composition can be any suitable form (depending on giving the required method of patient).Unit dosage forms offer can be used in it, usually provides in the container of sealing, and a part that can be used as kit provides.Such kit (but nonessential) includes operation instructions.It may include multiple unit dosage forms.In addition, PD-1 of the invention can be applied alone, in conjunction with other therapeutic agents or can also be coupled together use (as prepared in the same pharmaceutical composition).
Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration.The term refers to medicament carriers some in this way: themselves not inducing and generates to receiving the harmful antibody of individual of the composition, and does not have excessive toxicity after being administered.These carriers are well known to those of ordinary skill in the art.At Remington pharmaceutical science (Remington's Pharmaceutical Sciences, Mack Pub.Co., N.J.1991)) in can find discussing fully about pharmaceutically acceptable excipient.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, adjuvant, and combinations thereof.
Acceptable carrier can contain liquid in therapeutic composition Chinese pharmacology, such as water, salt water, glycerol and ethyl alcohol.In addition, There is likely to be complementary substances, such as wetting agent or emulsifier, pH buffer substance in these carriers.In general, therapeutic composition can be made to injectable agent, such as liquid solution or suspension；It may also be fabricated which in suitable supplying solution before the injection or suspension, the solid form of liquid-carrier.Once being made into the composition of the present invention, it can be administered by conventional route, including (but being not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal or local administration, preferably parenteral includes subcutaneous, intramuscular or intravenous.Object to be prevented or to be treated can be animal；Especially people.
When pharmaceutical composition of the present invention is used for actual treatment, the pharmaceutical composition of various different dosage forms can be used according to service condition.Preferably, what can be enumerated has injection, oral agents etc..These pharmaceutical compositions can be prepared by mixing, diluting or dissolving according to conventional methods, and suitable medicated premix is added once in a while, such as excipient, disintegrating agent, adhesive, lubricant, diluent, buffer, isotonic agent (isotonicities), preservative, wetting agent, emulsifier, dispersing agent, stabilizer and cosolvent, and the process for preparation can be carried out with usual way according to dosage form.
Pharmaceutical composition of the invention can be administered with sustained release formulation.For example, PD-1 of the present invention can be impregnated in using release polymer as the pill or micro-capsule are then implanted into tissue to be treated by operation in the pill of carrier or micro-capsule.Example as release polymer, what can be enumerated has thylene-vinylacetate copolymer, poly- hydroxyl-metacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-ethanol copolymer etc., and preferably exemplifiable is biodegradable polymer such as lactic acid polymer and lactic acid-ethanol copolymer.
When pharmaceutical composition of the present invention is used for actual treatment, PD-1 or PD-1 compound of the present invention as active constituent, it can be reasonably determined according to the weight, age, gender, degree of symptoms of each patient to be treated, reasonable dosage is finally determined by doctor.
Main advantages of the present invention are:
(1) present invention obtains the PD-1 molecules to PDL-1 with high-affinity.
(2) high-affinity PD-1 molecule of the invention can effectively improve the killing ability of lymphocyte.
Following specific embodiment, the present invention is further explained.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as (Sambrook and Russell et al., molecular cloning: laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing house) described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
Expression, renaturation and the purifying of 1 wild type PD-1 of embodiment
The extracellular amino acid sequence and nucleotide sequence of wild type PD-1 is respectively SEQ ID NO.1 and 2, the target gene of the extracellular sequence of wild type PD-1 will be carried through I double digestion of Nco I and Not, have biotin tag label after optimization with by pET28a carrier (Novagen) carrier of I double digestion of Nco I and Not) it connect.Connection product is converted to E.coli DH5 α (Vazyme), it is coated with the LB plate containing kanamycins, 37 DEG C of inversion overnight incubations, picking positive colony carries out PCR screening, positive recombinant is sequenced, determines that sequence correctly extracts recombinant plasmid transformed to E.coli Rosetta bacterial strain afterwards (TIANGEN) in, for expressing.
By the above-mentioned Rosetta colony inoculation containing recombinant plasmid pET28a-PD-1 in LB culture medium containing kanamycin, 37 DEG C of cultures to OD600 are 0.6-0.8, and IPTG to final concentration of 0.7mM is added, and 37 DEG C are continued to cultivate 4h.6000g is centrifuged 15min and harvests cell precipitate, with BugbusterMaster Mix (Merck) lytic cell sediment, 6000g is centrifuged 15min and recycles inclusion body, it is washed again with Bugbuster (Merck) to remove cell fragment and membrane component, 6000g is centrifuged 15min, collects inclusion body.By solubilization of inclusion bodies in buffer (50mM Tris-HCl, 200mM NaCl, 2mM EDTA, 6M guanidine HCl, pH 8.1), high speed centrifugation removes insoluble matter, is dispensed after supernatant BCA standard measure, is saved backup in -80 DEG C.
In the PD-1 inclusion body protein dissolved to 7mg, it is added 2mL buffer (50mM Tris-HCl, 200mM NaCl, 2mM EDTA, 6M guanidine HCl, pH 8.1), adds DTT to final concentration of 20mM, 37 DEG C of processing 1h.To 100mL renaturation buffer (50mM HEPES, pH 7.5,500mM L-arginine, 9mM glutathione, 1mM glutathione disulfide, 24mM NaCl, 1mM KCl) in treated PD-1 mixed liquor is added dropwise, 4 DEG C of stirring 30min, it is 3.5K that renaturation solution, which is then packed into interception,DCellulose membrane bag filter, bag filter be placed in 2L pre-cooling water in, 4 DEG C be slowly stirred overnight.After 24 hours, dialyzate is changed into the buffer (10mMTris-HCl of 2L pre-cooling, pH 8.5), 4 DEG C are continued dialysis for 24 hours, then it changes dialyzate into identical fresh buffer and continues dialysis 24 hours, sample is through 0.45 μm of membrane filtration, and sample introduction is to anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgas.The elution fraction of the 0-1MNaCl linear gradient elution liquid purifying protein prepared with 10mMTris-HCl pH8.5, collection carries out SDS-PAGE analysis.Based on the analysis results, it is further purified with solvent resistant column (Superdex 75 10/300, GE Healthcare) after collecting the concentration of target PD-1 component, target components also carry out SDS-PAGE analysis, as a result as shown in Figure 1.
Embodiment 2 combines characterization
BIAcore analysis
Wild type PD-1 molecule and the combination activity of PDL-1 are detected using BIAcore T200 real-time analyzer.Coupling buffer (10mM sodium-acetate buffer is added in the antibody (GenScript) of anti-Streptavidin, pH 4.77), then antibody is flowed through to the CM5 chip activated in advance with EDC and NHS, antibody is set to be fixed on chip surface, finally unreacted activating surface is closed with the hydrochloric acid solution of ethanol amine, complete coupling process, coupling horizontal about 15,000RU.
The Streptavidin of low concentration is set to flow through the chip surface of coated antibody, then biotinylated PD-1 is flowed through into sense channel, chip 2min is flowed through as reference channel, then by the biotin of 0.05mM with the flow velocity of 10 μ L/min in another channel, closes the remaining binding site of Streptavidin.Its affinity is measured using single cycle dynamic analysis method, by PD-1 HEPES-EP buffer (10mM HEPES, 150mMNaCl, 3mM EDTA, 0.005%P20, pH 7.4) it is diluted to several different concentration, with the flow velocity of 30 μ L/min, chip surface is flowed successively through, the binding time of each sample introduction is 120s, it is allowed to dissociate 600s after last time sample introduction.Each round uses the 10mMGly-HCl regeneration chip of pH 1.75 after measuring.Utilize BIAcore Evaluation software computational dynamics parameter.
For the amino acid sequence and nucleotide sequence of PDL-1 used in the present embodiment respectively as shown in SEQ ID NO.3, SEQ ID NO.4, expression, renaturation and purification process are identical as the expression of wild type PDL-1, renaturation and purification process in embodiment 1.Its biotinylated process is as follows:
A. biotinylation
With Millipore super filter tube by the PDL-1 molecular concentration of purifying, it is simultaneously 10mMTris pH 8.0 by buffer exchange, then biotinylation reagent 0.05MBicine pH 8.3,10mM ATP, 10mMMgOAc, 50 μM of D-Biotin, 100 μ g/ml BirA enzymes (GST-BirA) is added, it is incubated at room temperature mixture to stay overnight, whether SDS-PAGE detects biotinylation complete.
B. the compound after purifying biological element
PDL-1 molecular concentration after being marked biotinylation with Millipore super filter tube is to 500 μ l, using the biotinylated PDL-1 of gel filtration chromatography, first 75 10/300 solvent resistant column of Superdex (GE General Electric Co. Limited) is pre-equilibrated with filtered PBS, reload the concentrated biotinylation PDL-1 molecule of 500 μ l, then with PBS with the elution of 1ml/min flow velocity, the component being collected into is subjected to SDS-PAGE analysis, merge the component containing target protein according to result, it is concentrated with Millipore super filter tube, BCA method (Thermo) measures protein concentration, the packing of biotinylated PDL-1 molecule is stored in -80 DEG C.
The above process detects the K of the binding affinity of wild type PD-1 molecule and PDL-1 molecule through this embodimentDValue is 2.815E-06M, and BIAcore combination map is as shown in Figure 2.
The generation of 3 high-affinity PD-1 molecule of embodiment
Using the extracellular sequence of wild type PD-1 described in embodiment 1 as template strand, according to Li et al., (2005) Nature Biotech 23 (3): the 349-354) phage display and screening technique described carries out the screening of high-affinity PD-1.Phage library after a few wheel screenings is and PD-1 has stronger binding signal, therefrom picking monoclonal, and carries out sequence analysis.
According to the expression of method described in embodiment 1, renaturation and high-affinity PD-1 molecule of the present invention is purified, and measures the affinity of itself and PDL-1 molecule by method described in embodiment 2.The affinity of the high-affinity PD-1 molecule that obtains in the present invention and PDL-1 molecule is at least 2 times of affinity of wild type PD-1 molecule and PDL-1 molecule, amino acid sequence and its as shown in table 1 below with the affinity numerical value of PDL-1 molecule.
1 high-affinity of table clones the BIAcore result to PDL-1 molecule
Embodiment 4L5B7 identifies that the ability of H1299 cell surface PD-L1 is higher than PD-1
Biacore the results show that obtained the PD-1 mutant of affinity raising, but whether the change of this affinity will affect the combination of cell surface PDL-1 under itself and physiological condition and still need to experimental verification really after screening.Therefore, the H1299 cell that we select PDL-1 expression positive, is added biotinylation PD-1, L5B7 albumen of various concentration, and flow cytometry PD-1, L5B7 identifies the ability of cell surface PDL-1.
Fig. 3 the results show that PD-1, L5B7 protein concentration with addition raising, the recognition capability of PDL-1 is gradually increased；Under the conditions of same concentration, L5B7 albumen identify PDL-1 ability be higher than PD-1, the change of this recognition capability may be due to the affinity of L5B7 it is higher caused by, it is consistent with chemical result.
5 high-affinity PD-1 molecule bivalent fusion protein of embodiment generates
A expression vector establishment
In order to increase the stability and potency of PD-1 and its high affine mutant in vivo, eukaryotic expression is using the form with IgG4 amalgamation and expression.PD-1 albumen eukaryotic expression sequence is saved worry Bioisystech Co., Ltd's optimum synthesis by Suzhou, after the completion of IgG4 overlapPCR splicing, it is connected on pGZFUSE plasmid vector by EcoR I, I site Nhe, and changes and go in 10 bacterial strain of Top (while the mutant clone for obtaining other compatibilities by mutation).It is inoculated in 200ml LB culture medium by 1:1000, after 37 degree are incubated overnight, in second day receipts bacterium, carry out plasmid and largely extract.OD260/0D280 surveys plasmid concentration, is adjusted to 1mg/ml, saves after packing with -20 degree.Big upgrading grain is spare.Amalgamation and expression albumen schematic diagram such as Fig. 4 a.
B protein expression
Carrier for expression of eukaryon is constructed according to a, after being sequenced correctly, expressing fusion protein uses 293T attached cell expression system.The day before transfection spreads logarithmic growth phase 293T cell in 10cm culture dish, and second day with plasmid: the condition of lipo 2000=1:2 (volume ratio) transfects cell, replaces fresh Freestyle after 4hTM293 culture mediums take after 48h a small amount of supernatant to run SDS-PAGE identification expression such as Fig. 4 b.Supernatant is received after 72 hours, after 0.22 μm of membrane filtration, is diluted in the 10mM Tris-Hcl that 20 times of volumes are pre-chilled in advance (pH=8.5), anion and molecular sieve purification are carried out.PAGE gel electrophoresis result is shown: the fusion protein purity of 293T attached cell expression is very high.There are two the purposes that we are further purified, first is that solution locating for replacement protein, prevents during expression or the existing Cucumber of culture medium itself influences subsequent experimental；Second is that protein concentrate, and purity is further increased, avoid subsequent experimental due to a large amount of albumen of addition, or there are some foreign proteins to be affected.
C fusion protein Function Identification
ImmTACs molecule can redirect T cell specific killing tumour cell more research in report (Jakobsen, 2013；Oates et al.,2015).The basic principle is that ImmTACs can simulate the key signal that T cell activation plays effector function, on the one hand pass through the MHC- peptide complexes on its high affine specificity TCR tumor cell surface, on the other hand the downstream signaling pathway that T cell activation is activated by its anti-CD3 antibody end, to orient T cell specific killing tumour cell.Therefore, can the function of evaluating PD-1/L5B7-IgG4 fusion protein in our current research be promoted by the albumen Fig. 4 c that ImmTAC-IG4 numerator mediated PBMC evaluates the killing process of Mel624 tumour cell.
As a result, it has been found that when the concentration of ImmTAC is 10-9M, (PBMC is effector cell by E:T=5:1 and 1:1, Mel62 is target cell) when, PD-1-IgG4, L5B7-IgG4 fusion protein can promote the LDH of ImmTAC molecular orientation PBMC killing tumor cell to discharge, and L5B7-IgG4 fusion protein promotes LDH release to be higher than PD-1-IgG4 group；Cell progress flow cytometer detection when collecting killing than being 1:1 in reaction system finds the expression of CD25, CD107a of PD-1-IgG4, L5B7-IgG4 fusion protein group cd8 t cell more not plus protein groups raise, and the ratio of L5B7-IgG4 group up-regulation is higher than plus PD-1-IgG4 group, CD25 is adjusted to 13.2% from 10.9%, CD107a is adjusted to 19.8% from 16.9%, further demonstrates LDH and discharges increased result.Illustrate the killing of PBMC that PD-1-IgG4, L5B7-IgG4 fusion protein can promote ImmTAC numerator mediated to Mel624 tumour cell, consistent with the result of study that solubility PD-1 in other documents can promote tumor specific T cells to kill, the facilitation further strengthens after increasing its compatibility.
6 high-affinity PD-1 molecule of embodiment promotes the proliferation experiment of the PBMC of activation
This experiment is carried out to verify high-affinity PD-1 molecule of the invention and can promote the PBMC increment of activation.
Fluorescein based dye CFSE, alternatively referred to as CFDA SE (5,6-carboxyfluorescein diacetate, succinimidyl ester), that is hydroxyl fluorescein diacetate succinimide rouge, be it is a kind of can penetrating cell film fluorescent dye, there is the succinimide aliphatic radical with cell-specific ining conjunction with to roll into a ball and the hydroxyl fluorescein diacetate group with non-enzymatic hydrolysis effect, this makes CFSE as a kind of good cell marker.When cell carries out division growth, the cytoplasmic protein with fluorescence, which is averaged, to be assigned in second generation cell, and in this way compared with first generation cell, fluorescence intensity will weaken to half；And so on, the fluorescence intensity of the third generation cell divided will weaken again than second generation cell.The case where this phenomenon can be analyzed under the exciting light of 488nm using flow cytomery, be reduced by detecting cell fluorescence intensity constantly, and further analysis obtains cell division proliferation.
In the present embodiment, by the peripheral blood mononuclear cells of fresh separated (PBMC) after final concentration of 1 μM of CFDA-SE dyeing, twice of termination CFDA-SE dyeing is washed with the RIPM-1640 containing 10%FBS.With 1.5*105A/hole is laid on the hole 96- flat underside, and 15 μ g/ml anti-CD3mAb and 7.5 μ g/ml anti-CD28mAb stimulation PBMC is added and is proliferated.The results show that high-affinity PD-1 molecule not only considerably reduces the quantity of non-proliferative cell；And from fluorescence shift amount, the group fluorescence left side of high-affinity PD-1 mutation is added more to offset, illustrates to be proliferated intensity more greatly (Fig. 5 a).It counts PD-1 albumen and high-affinity mutant promotes the ratio of proliferationIt was found that each high-affinity mutation physical efficiency promotes the PBMC proliferation of 18%~20% anti-CD3mAb and anti-CD28mAb mediation, but wild type PD-1 can not be obviously promoted the proliferation (Fig. 5 b) of PBMC.
The IFN-γ release experiment of 7 high-affinity PD-1 molecule of embodiment promotion PBMC
Anti-CD3mAb and anti-CD28mAb can not only promote PBMC to be proliferated, and PBMC can be promoted to discharge IFN-γ.In the present embodiment, PBMC is after 60 μ g/ml anti-CD3mAb and 30 μ g/ml anti-CD28mAb stimulation, it is tested by elisa (Elispot), it was found that PD-1 monomeric protein can not promote the PBMC release IFN-γ of stimulation, but after being separately added into 5 μ g/ml L1B2,5 μ g/ml L2B12,5 μ g/ml L2F8,5 μ g/ml L2F10,5 μ g/ml L5B7,5 μ g/ml L45 high-affinity mutant proteins, the spot of detection IFN-γ release obviously increases (figure 6a), and the increase of spot and affinity have apparent relationship.Count the ratio that different high-affinity mutant promote IFN-γ releaseIt is 20% or so that the L1B2 and L2B12 of low-affinity, which promote the ratio of IFN-γ release, further increases affinity, the ratio of IFN-γ release is promoted to increase to 40~50% (Fig. 6 b).
All references mentioned in the present invention is incorporated herein by reference, as if each reference was individually incorporated by reference.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can make various modifications or changes to the present invention, these equivalent forms also fall within the scope of the appended claims of the present application.

Claims (29)

  1. A kind of PD-1 molecule, it is characterized in that, the amino acid sequence of the PD-1 molecule is based on amino acid sequence shown in SEQ ID NO.1, and carries out the mutation of one or more amino acid residues to amino acid sequence shown in SEQ ID NO.1 to obtain the PD-1 molecule；Amino acid sequence shown in the amino acid sequence and SEQ ID NO.1 of the PD-1 molecule has at least 90% sequence identity；It is highly preferred that the affinity of the PD-1 molecule and PDL-1 molecule is at least 2 times of the affinity of wild type PD-1 molecule and PDL-1 molecule.
  2. PD-1 molecule as described in claim 1, which is characterized in that amino acid sequence shown in the amino acid sequence and SEQ ID NO.1 of the PD-1 molecule has 92%；Preferably, at least 94% sequence identity.
  3. PD-1 molecule as described in claim 1, which is characterized in that the affinity of the PD-1 molecule and PDL-1 molecule is at least 10 times of the affinity of wild type PD-1 molecule and PDL-1 molecule；Preferably, at least 100 times；It is highly preferred that at least 200 times.
  4. PD-1 molecule as described in claim 1, which is characterized in that the affinity of the PD-1 molecule and PDL-1 molecule is at least 500 times of the affinity of wild type PD-1 molecule and PDL-1 molecule；Preferably, at least 1000 times；It is highly preferred that at least 2000 times.
  5. PD-1 molecule as described in claim 1, it is characterized in that, the acid residues sites being mutated in the PD-1 molecule are one or more of the 30th~60 and/or 85~105 amino acids residues, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
  6. PD-1 molecule as claimed in claim 5, it is characterized in that, the acid residues sites being mutated in the PD-1 molecule are one or more of 31~37,40~48,56 and/or 89~103 amino acids residues, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
  7. PD-1 molecule as described in claim 1, which is characterized in that the quantity of the acid residues sites of mutation is n, wherein 1≤n≤15；Preferably, 2≤n≤11；It is highly preferred that 2≤n≤6, if n can be 1,2,3,4,5,6,7,8,9,10.
  8. PD-1 molecule as described in claim 1, it is characterized in that, the acid residues sites being mutated in the PD-1 molecule include one or more of 91G, 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 92A, 93I, 95L, 97P, 98K, 99A, 100Q, 101I and 103E, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
  9. PD-1 molecule as described in claim 1, which is characterized in that the acid residues sites being mutated in the PD-1 molecule include 91G, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1；And/or
    The acid residues sites being mutated in the PD-1 molecule include 99A, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1；
    Preferably, the acid residues sites being mutated in the PD-1 molecule further include 41N, 42Q, 43T, 48A, 95L, 97P, 98K and/or 100Q, and wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
  10. PD-1 molecule as described in claim 1, which is characterized in that the PD-1 molecule includes one or more amino acid residues selected from the group below: 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；100S or 100W；101V；And 103D；Wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
  11. PD-1 molecule as claimed in claim 10, which is characterized in that the PD-1 molecule includes 91V or 91S.
  12. PD-1 molecule as claimed in claim 10, which is characterized in that the PD-1 molecule further includes 99I or 99P.
  13. PD-1 molecule as claimed in claim 10, which is characterized in that the PD-1 molecule includes: 91V and 99I；Or
    91S, 98Y and 99I；Or
    41L, 42N, 43G, 48S, 91V and 99P；Or
    41G, 43V, 48G, 91V and 99P；
    Wherein numbering amino acid residues are numbered using shown in SEQ ID NO.1.
  14. PD-1 molecule as claimed in claim 10, which is characterized in that the amino acid sequence of the PD-1 molecule is selected from SEQ ID NO.39,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,41 or 43；
    Preferably, the amino acid sequence of the PD-1 molecule is selected from SEQ ID NO.39,5,11,13,15 or 43；
    Most preferably, the amino acid sequence of the PD-1 molecule is SEQ ID NO.39.
  15. PD-1 molecule as described in claim 1, which is characterized in that the PD-1 molecule is soluble.
  16. PD-1 molecule as described in claim 1, which is characterized in that the C or N-terminal of the PD-1 molecule are combined with conjugate.
  17. PD-1 molecule as claimed in claim 16, which is characterized in that the conjugate in conjunction with the PD-1 molecule is T cell receptor；Preferably, the T cell receptor is high-affinity T cell receptor.
  18. PD-1 molecule as described in claim 1, which is characterized in that the PBMC proliferation that the PD-1 molecule mediates anti-CD3mAb and anti-CD28mAb improves 15% or more, preferably 18%~20%；And/or
    The PD-1 molecule promotes the ratio about 20% or so of IFN-γ release；Preferably, promoting the ratio of IFN-γ release increases to 40~50%.
  19. A kind of fusion protein, which is characterized in that the fusion protein includes PD-1 molecule described in any one of claim 1-18.
  20. Fusion protein as claimed in claim 19, which is characterized in that the fusion protein further includes IgG4.
  21. A kind of multivalence PD-1 compound, which is characterized in that the multivalence PD-1 compound contains at least two PD-1 molecule, and at least one PD-1 molecule therein is PD-1 molecule described in any one of claim 1-18；Or the multivalence PD-1 compound includes fusion protein described at least one claim 19 or 20.
  22. A kind of nucleic acid molecules, it is characterized in that, the nucleic acid molecules include the nucleic acid sequence or its complementary series of PD-1 molecule described in any one of coding claim 1-18, multivalence PD-1 compound described in fusion protein or claim 21 described in claim 19 or 20.
  23. A kind of carrier, which is characterized in that the carrier contains nucleic acid molecules described in claim 22.
  24. A kind of host cell, which is characterized in that contain the carrier or dye described in claim 23 in the host cell Nucleic acid molecules described in the claim 22 of external source are integrated in colour solid；Or
    The host cell contains or expresses multivalence PD-1 compound described in PD-1 molecule described in any one of claim 1-18, fusion protein or claim 21 described in claim 19 or 20.
  25. A kind of pharmaceutical composition, which is characterized in that the composition contains PD-1 compound described in fusion protein described in PD-1 molecule described in any one of pharmaceutically acceptable carrier and claim 1-18 or claim 19 or 20 or claim 21.
  26. A method for the treatment of disease, it is characterised in that it includes applying pharmaceutical composition described in PD-1 compound described in PD-1 molecule described in any one of suitable claim 1-18, fusion protein or claim 21 described in claim 19 or 20 or claim 25 to object in need for the treatment of.
  27. Method as claimed in claim 26, which is characterized in that the disease is tumour.
  28. The purposes of PD-1 molecule described in any one of claim 1-18, PD-1 compound described in fusion protein or claim 21 described in claim 19 or 20 is used to prepare the drug for the treatment of tumour.
  29. A method of PD-1 described in any one of claim 1-18 is prepared, comprising steps of
    (i) host cell described in claim 24 is cultivated, to express PD-1 molecule described in any one of claim 1-18；
    (ii) isolated or purified goes out the PD-1 molecule.
CN201780074723.8A 2016-10-27 2017-10-25 High affinity soluble PD-1 molecules Active CN110023333B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN201610959248X 2016-10-27
CN201610959248.XA CN107987153A (en) 2016-10-27 2016-10-27 The soluble PD-1 molecules of high-affinity
PCT/CN2017/107659 WO2018077189A1 (en) 2016-10-27 2017-10-25 High-affinity soluble pd-1 molecule

Publications (2)

Publication Number Publication Date
CN110023333A true CN110023333A (en) 2019-07-16
CN110023333B CN110023333B (en) 2023-08-25

Family

ID=62024327

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201610959248.XA Pending CN107987153A (en) 2016-10-27 2016-10-27 The soluble PD-1 molecules of high-affinity
CN201780074723.8A Active CN110023333B (en) 2016-10-27 2017-10-25 High affinity soluble PD-1 molecules

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201610959248.XA Pending CN107987153A (en) 2016-10-27 2016-10-27 The soluble PD-1 molecules of high-affinity

Country Status (2)

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

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108794619B (en) * 2018-05-31 2021-09-17 郑州大学 High-affinity PD-1 protein mutant
CN114181297B (en) * 2018-06-07 2023-06-30 江苏东抗生物医药科技有限公司 Fusion protein of high-affinity PD-1 extracellular region mutant, and pharmaceutical composition and application thereof
CN111714618B (en) * 2019-03-22 2024-07-12 香雪生命科学技术（广东）有限公司 Combination of T cells and high affinity PD-1 fusion proteins
CN110590959B (en) * 2019-09-19 2021-01-05 北京伟杰信生物科技有限公司 Recombinant canine PD-1 fusion protein and preparation method and application thereof
CN110478472B (en) * 2019-09-29 2020-08-28 北京鼎成肽源生物技术有限公司 PD-1 sealant and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006265108A1 (en) * 2005-07-01 2007-01-11 E. R. Squibb & Sons, L.L.C. Human monoclonal antibodies to programmed death ligand 1 (PD-L1)
CN101255192A (en) * 2000-05-26 2008-09-03 布里斯托尔-迈尔斯斯奎布公司 Soluble CTLA4 mutant molecules and uses thereof
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
CN105985427A (en) * 2015-02-06 2016-10-05 广州市香雪制药股份有限公司 High-affinity NY-ESO T cell receptor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101255192A (en) * 2000-05-26 2008-09-03 布里斯托尔-迈尔斯斯奎布公司 Soluble CTLA4 mutant molecules and uses thereof
AU2006265108A1 (en) * 2005-07-01 2007-01-11 E. R. Squibb & Sons, L.L.C. Human monoclonal antibodies to programmed death ligand 1 (PD-L1)
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
CN105985427A (en) * 2015-02-06 2016-10-05 广州市香雪制药股份有限公司 High-affinity NY-ESO T cell receptor

Also Published As

Publication number Publication date
WO2018077189A1 (en) 2018-05-03
CN110023333B (en) 2023-08-25
CN107987153A (en) 2018-05-04

Similar Documents

Publication Publication Date Title
CN110023333A (en) The soluble PD-1 molecule of high-affinity
CN106565836A (en) High-affinity soluble PDL-1 molecule
CN109777784A (en) It is a kind of to enhance the Chimeric antigen receptor carrier construction method migrated to tumor locus and application
CN109879957A (en) For the high-affinity T cell receptor of PRAME
CN103501818A (en) Methods and reagents for efficient and targeted delivery of therapeutic molecules to cxcr4 cells
EP2957630B1 (en) Undifferentiated cell elimination method
US11680106B2 (en) Bispecific antigen-binding construct and preparation method and use thereof
CN106459178B (en) For the high-affinity T cell receptor of RHAMM antigen small peptides
CN108456254A (en) A kind of TCS- cell-penetrating peptides-oncoprotein zymolyte peptide fusion protein, preparation method and use
CN107108717A (en) A kind of solvable and stable heterogeneous dimerization TCR
CN104045715B (en) The preparation and application of Dimerized fusion protein
JP2023019098A (en) Nucleolus transitional carrier peptide fragment, and application of the same
TW202144403A (en) High-affinity TCR for recognizing hpv16
CN106432475B (en) high affinity NY-ESO T cell receptor
CN109867725A (en) PD-1-Fc fusion protein and its preparation method and application
CN103360497A (en) Novel antitumor fusion protein vaccine, and preparation method and application thereof
CN102391377B (en) Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein
US11339225B2 (en) Bispecific antigen-binding construct and preparation method and use thereof
CN106589061B (en) The candidate peptide aptamer of Sox2-CDP binding structural domain
CN109970869A (en) It is a kind of target human TNF related apoptosis-inducing ligand death receptor Chimerical receptor ligand and its application
CN101092452A (en) Preparation method for both of micromolecule polypeptide of tumor chalone for anti angiogenesis, and fusion protein
KR20240057442A (en) Bispecific NK cell agonist, manufacturing method and application
CN110526981A (en) A kind of AntiCD3 McAb and EpCAM bispecific antibody and its application in treatment lung cancer
CN110156889A (en) High-affinity HBs T cell receptor
EP3216801B1 (en) Soluble heterodimeric t cell receptor, and preparation method and use thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right

Effective date of registration: 20210422

Address after: 519031 2715 office building, no.3000 Huandao East Road, Hengqin New District, Zhuhai City, Guangdong Province

Applicant after: Xiangxue Life Science Technology (Guangdong) Co.,Ltd.

Address before: 510663 Jin Feng Park Road, Guangzhou, Guangdong high tech Industrial Development Zone, Guangzhou (first self compiled)

Applicant before: GUANGDONG XIANGXUE LIFE SCIENCES, Ltd.

TA01 Transfer of patent application right
GR01 Patent grant
GR01 Patent grant