CN107987153A - The soluble PD-1 molecules of high-affinity - Google Patents

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

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Publication number
CN107987153A
CN107987153A CN201610959248.XA CN201610959248A CN107987153A CN 107987153 A CN107987153 A CN 107987153A CN 201610959248 A CN201610959248 A CN 201610959248A CN 107987153 A CN107987153 A CN 107987153A
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molecules
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李懿
李艳艳
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Guangdong Xiangxue Precision Medical Technology Co Ltd
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Guangdong Xiangxue Precision Medical Technology Co Ltd
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Priority to CN201610959248.XA priority Critical patent/CN107987153A/en
Priority to PCT/CN2017/107659 priority patent/WO2018077189A1/en
Priority to CN201780074723.8A priority patent/CN110023333B/en
Publication of CN107987153A publication Critical patent/CN107987153A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—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

Abstract

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

Description

The soluble PD-1 molecules of high-affinity
Technical field
The present invention relates to biological technical field, relate more specifically to the soluble programmed death acceptor of high-affinity (Programmed Death-1, 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 and purposes of the molecule.
Background technology
PD-1 is the inhibitive ability of immunity acceptor expressed in the T cell and B cell of activation, its ligand is PDL-1 or PDL- 2.PD-1 belongs to B7 families, is the I type transmembrane glycoprotein of Ig superfamilies that size is 50-55kD；By extracellular IgV areas, transmembrane region, born of the same parents Inner region three parts form, and find that, due to lacking film near-end cysteine residues, PD-1 is deposited with monomer by structure and biochemical analysis At (Xuewu Zhang and Almo, Immunity, 2004,20,337-347).PD-1 and ligand PDL-1 interacts, Played an important role in terms of the negative regulation of immune response.The high expression PDL-1 molecules of many tumor cell lines and tumour cell (Konishi J et al., Clin.Cancer Res., 2004,10 (15):5094-5100), itself and lymphocytic cell surface After PD-1 molecules combine, weaken body anti-tumor immune response (Radziewicz H et al., 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 nearly half Tumor-infiltrated CD8+T cell expresses PD-1 molecules, after it is combined with the PDL-1 of tumor cells expression, may cause CTL cells 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, the PD-1 of lymphocytic cell surface and the PDL-1 of tumor cell surface are blocked Interaction can improve the immunity of lymphocyte, therefore, help to remove tumour cell by immune system.For this Problem, researcher have been carried out numerous studies.In the mouse model of aggressive cancer of pancreas, (the Clin.Cancer such as T.Nomi Res.2007,13：2151-2157) prove the therapeutic efficiency for blocking PD-1 and PDL-1 to interact.Those skilled in the art cause Power is in the interaction of research PDL-1 and PD-1, to find the effective way for improving Lymphocvte Killer ability.
The content of the invention
It is an object of the invention to provide a kind of PD-1 molecules to PDL-1 molecules with higher affinity.
Another object of the present invention is to provide a kind of Preparation method and use of the PD-1 molecules of above-mentioned high-affinity.
In the first aspect of the present invention, there is provided a kind of PD-1 molecules, the PD-1 molecules are shown in SEQ ID NO.1 Contain mutation in amino acid sequence.
In another preference, the amino acid sequence of the PD-1 molecules is based on SEQ ID NO.:Amino acid sequence shown in 1 Row, and the amino acid sequence shown in SEQ ID NO.1 is carried out one or more amino acid residues mutation or amino acid it is residual The insertion of base is so as to obtain the PD-1 molecules.
In another preference, amino acid sequence and the amino acid sequence shown in SEQ ID NO.1 of the PD-1 molecules Have at least 90% (preferably, at least 92%；It is highly preferred that sequence thereto at least 94%).
In another preference, the affinity of the PD-1 molecules and PDL-1 molecules is wild type PD-1 molecules 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 preference, the affinity of the PD-1 molecules and PDL-1 molecules is wild type PD-1 molecules and PDL-1 At least 500 times of the affinity of molecule；Preferably, at least 1000 times；It is highly preferred that at least 2000 times.
In another preference, the acid residues sites that are mutated in the PD-1 molecules for 30~60, and/or 85~ One or more of 105 amino acids residues, wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1.
In another preference, the acid residues sites that are mutated in the PD-1 molecules are 31~37,40~48,56, And/or 89~103 one or more of amino acids residues, wherein numbering amino acid residues are using shown in SEQ ID NO.1 Numbering.
In another preference, 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 preference, the acid residues sites that are mutated in the PD-1 molecules include 31V, 33N, 35Y, 37M, One in 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 91G, 92A, 93I, 95L, 97P, 98K, 99A, 100Q, 101I, 103E A or multiple, wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1.
In another preference, the acid residues sites being mutated in the PD-1 molecules include 91G, and wherein amino acid is residual Base numbering is using the numbering shown in SEQ ID NO.1.
In another preference, the acid residues sites being mutated in the PD-1 molecules include 99A, and wherein amino acid is residual Base numbering is using the numbering shown in SEQ ID NO.1.
In another preference, the acid residues sites that are mutated in the PD-1 molecules further include 41N, 42Q, 43T, 48A, 95L, 97P, 98K, and/or 100Q, wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1.
In another preference, the PD-1 molecules after mutation include one or more amino acid residues selected from the group below： 31T；33L；35N or 35M；37V, 37L or 37E；40A or 40T；41G or 41L；42N；43V or 43G；48G or 48S； 56L；89M；91A, 91S, 91V or 91T；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 using the numbering shown in SEQ ID NO.1.
In another preference, the PD-1 molecules after mutation include 91V or 91S.
In another preference, the PD-1 molecules after mutation further include 99I or 99P.
In another preference, the PD-1 molecules include：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 using the numbering shown in SEQ ID NO.1.
In another preference, the amino acid sequences of the PD-1 molecules be selected from SEQ ID NO.5,7,9,11,13,15, 17th, 19,21,23,25,27,29,31,33,35,37,39,41 or 43.
In another preference, the PD-1 molecules are solvable.
In another preference, the C or N-terminal of the PD-1 molecules are combined with conjugate.
In another preference, the conjugate combined with the PD-1 molecules is φt cell receptor, it is preferable that the T cell Acceptor is high-affinity φt cell receptor.
The second aspect of the present invention, there is provided a kind of fusion protein, the fusion protein include first aspect present invention institute The PD-1 molecules stated.
In another preference, the fusion protein further includes IgG4.
The third aspect of the present invention, there is provided a kind of multivalence PD-1 compounds, the multivalence PD-1 compounds include at least Two PD-1 molecules, and at least one PD-1 molecules therein are the PD-1 molecules described in first aspect present invention；Or institute State multivalence PD-1 compounds and include the fusion protein described at least one second aspect of the present invention.
The fourth aspect of the present invention, there is provided a kind of nucleic acid molecules, the nucleic acid molecules, which include, encodes first party of the present invention The fusion protein described in PD-1 molecules, second aspect of the present invention described in face or the multivalence PD-1 described in third aspect present invention The nucleotide sequence of compound or its complementary series.
Fifth aspect present invention, there is provided a kind of carrier, the carrier contain the nucleic acid described in fourth aspect present invention Molecule.
The sixth aspect of the present invention, there is provided a kind of host cell, the 5th side of the invention is contained in the host cell The nucleic acid molecules described in the fourth aspect present invention of external source are integrated with carrier or chromosome described in face.
The seventh aspect of the present invention, there is provided a kind of pharmaceutical composition, the composition contain pharmaceutically acceptable load Fusion protein described in PD-1 molecules or second aspect of the present invention or the present invention described in body and first aspect present invention the PD-1 compounds described in three aspects.
The eighth aspect of the present invention, there is provided a kind of method for treating disease, including apply and fit to object in need for the treatment of Fusion protein described in PD-1 molecules, second aspect of the present invention or third party of the present invention described in the first aspect present invention of amount The pharmaceutical composition described in PD-1 compounds or the seventh aspect of the present invention described in face.
In another preference, the disease is tumour.
The ninth aspect of the present invention, there is provided PD-1 molecules, second aspect of the present invention institute described in first aspect present invention The purposes for the PD-1 compounds described in fusion protein or third aspect present invention stated, is used to prepare the medicine for the treatment of tumour.
The tenth aspect of the present invention, there is provided a kind of method for preparing the PD-1 described in first aspect present invention, including step Suddenly：
(i) host cell described in sixth aspect present invention is cultivated, so as to express the PD-1 described in first aspect present invention Molecule；
(ii) isolated or purified goes out the PD-1 molecules.
In the first aspect of the present invention, there is provided a kind of PD-1 molecules, the PD-1 molecules are shown in SEQ ID NO.1 Contain mutation in amino acid sequence.
It is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical solution.As space is limited, exist This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 is SDS-PAGE glue the figure .M, molecular weight of albumen Mark. after wild type PD-1 protein purifications
Fig. 2 is the BIAcore collection of illustrative plates that wild type PDL-1 molecules are combined with PD-1 molecules.
Fig. 3 is the flow cytometer detection that PD-1, L5B7 identify H1299 cell surfaces PD-L1, and display L5B7 identifies H1299 cells The ability of surface PDL-1 is higher than PD-1.Note：A, anti-PDL-1 antibody (2.5ul/ samples) identification H1299 cell surfaces PDL-1；B, PD-1, L5B7 (concentration 0.02mg/ml, 0.04mg/ml, 0.08mg/ml) identification H1299 cells of various concentrations The flow cytometer detection figure of the PDL-1 on surface, the wherein dosage of SA-PE are 0.5ul/ samples；C is right when concentration is 0.08mg/ml According to group, the streaming block diagram of PD-1, L5B7 identification PDL-1.
Fig. 4 a be PD-1 mutant and its mutant eukaryotic expression schematic diagram (PD-1-IgG4 fusion protein schematic diagrames, Note：Dimer theoretical molecular is 80kD or so, and actual molecular weight is 125kD or so after glycosylation).
Supernatant 8%SDS-PAGE gel electrophoresis figures are expressed after when Fig. 4 b are 48h small.Note：M, molecular weight of albumen Mark.1st, 2, The Non-Reducing electrophoresis result figures of PD-1-IgG4, L5B7-IgG4；3rd, 4, the Reducing of PD-1-IgG4, L5B7-IgG4 Electrophoresis result figure.
Fig. 4 c are the functional verification figure for the killing for promoting ImmTAC-IG4 mediations.Show PD-1-IgG4, L5B7-IgG4 Promote LDH releases and CD25, CD107a flow cytometer detection of ImmTAC-IG4 mediation killings.Note：A, LDH discharge, and formula changes Calculate to kill ratio.B, when killing is than being 1:When 1, CD25, CD107a expression streaming of cd8 t cell in reaction system are killed Detection figure, the streaming antibody dosage of detection CD25, CD107a are 2.5ul/ samples.
Embodiment
The present invention is by extensive and in-depth study, it has unexpectedly been found that has the solubility of high-affinity to PDL-1 molecules PD-1 molecules can effectively improve the killing ability of lymphocyte.Therefore, the present invention provides a kind of affinity to PDL-1 It is soluble high-affinity PD-1 molecule of the wild type PD-1 molecules to the affinity at least twice of PDL-1.
Specifically, heretofore described PD-1 molecules contain mutation in the amino acid sequence shown in SEQ ID NO.1.More Specifically, the amino acid sequence of the PD-1 molecules has at least 90% sequence with the amino acid sequence shown in SEQ ID NO.1 The phase same sex.
Before describing the present invention, it should be understood that the invention is not restricted to the specific method and experiment condition, because this Class method and condition can change.It should also be understood that its purpose of term used herein is only that description specific embodiment, and And it is not intended to restricted, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein are respectively provided with such as fields of the present invention with scientific terminology The normally understood identical meanings of those of ordinary skill.
Although it can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention And material, herein place enumerate preferable method and material.
Term
Wild type PD-1 molecules：Heretofore described wild type PD-1 molecules refer to the extracellular of wild type PD-1 molecules Area, its amino acid sequence and nucleotide sequence are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2：
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.1, wild type PD-1)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGGTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA(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：
FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLG NAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDH QVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPEL(SEQ ID NO.3PDL-1)
TTTACGGTTACGGTTCCGAAAGACCTGTATGTGGTTGAATACGGCTCTAATATGACGATTGAATGCAAATTCCCGGT TGAAAAACAACTGGATCTGGCGGCCCTGATTGTGTATTGGGAAATGGAAGACAAAAACATCATCCAATTCGTGCATG GCGAAGAAGATCTGAAAGTTCAGCACAGCTCTTACCGTCAACGCGCACGTCTGCTGAAAGACCAGCTGAGCCTGGGC AATGCAGCTCTGCAGATCACGGATGTTAAACTGCAAGACGCCGGTGTCTATCGCTGCATGATTTCTTATGGCGGTGC AGACTACAAACGTATCACCGTCAAAGTGAACGCTCCGTACAACAAAATTAATCAGCGCATCCTGGTGGTTGATCCGG TTACGTCCGAACATGAACTGACCTGTCAAGCGGAAGGCTATCCGAAAGCCGAAGTCATTTGGACCAGTTCCGATCAC CAGGTGCTGTCAGGTAAAACCACGACCACGAACTCGAAACGCGAAGAAAAACTGTTTAATGTCACGAGCACCCTGCG TATTAACACCACGACCAATGAAATCTTCTACTGCACCTTTCGTCGTCTGGACCCGGAAGAAAATCATACGGCGGAAC TGGTTATCCCGGAACTG(SEQ ID NO.4)
PBMC：Peripheral blood mononuclear cells (PBMC) is the haemocyte with circular karyon, as lymphocyte or monokaryon are thin Born of the same parents.These haemocytes are that immune system fight infects and is adapted to the key component of invader.Lymphocyte population is by T cell (CD4 the and CD8 positives about 75%), B cell and NK cells (merging about 25%) composition.
High-affinity φt cell receptor：Refer to wild-type T cells acceptor more corresponding with the affinity of its ligand and its ligand parent The φt cell receptor improved with power.For example, screen to obtain stability-enhanced single-stranded autoreactivity mouse by Yeast selective system Source 2C TCR, its affinity to ligand improve 100 times or so (Holler, P.D.et compared with wild type (9nM) al.Natl.Acad.Sci.USA.2000.97,5387-5392)。
Tumour：Finger includes all types of growth of cancer cells or oncogenic process, metastatic tissue or malignant conversioning cell, group Knit or organ, no matter histological type or the stage infected.The embodiment of tumour includes without limitation：Solid tumor, soft-tissue tumor, And metastasis (metastases).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, epithelium are thin Born of the same parents), pharynx.Lung squamous cancer includes malignant tumour, for example, most colon cancers, the carcinoma of the rectum, clear-cell carcinoma, liver cancer, lung it is non-small Cell cancer, carcinoma of small intestine and cancer of the esophagus.Above-mentioned cancer metastasis venereal disease, which becomes, equally to be treated with the method and composition of the present invention And prevention.
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 bufferings.The example of physiology acceptable carriers include buffer such as phosphate, Citrate and other organic acids；Antioxidant, including ascorbic acid；Low molecular weight (being less than about 10 residues) polypeptide；Albumen Matter, such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, paddy Glutamine, asparagine, arginine or lysine；Monose, disaccharides and other carbohydrates, including glucose, mannose or dextrin；Network Mixture such as EDTA；Sugar alcohol such as mannitol or sorbierite；Into salt counter ion such as sodium；And/or nonionic surfactant is such as TWEENTM, polyethylene glycol (PEG) and PLURONICSTM.
Detailed description of the invention
PD-1 (Programmed Death-1) is the inhibitive ability of immunity acceptor expressed in the T cell and B cell of activation, PDL-1 is its ligand.PD-1 belongs to B7 families, is the I type transmembrane glycoprotein of Ig superfamilies that size is 50-55kD；By extracellular IgV Area, transmembrane region, intracellular region three parts composition, are found by structure and biochemical analysis, due to lacking film near-end cysteine residues, PD-1 exists with monomer.PD-1 and its ligand PDL-1 (Programmed Death Ligand-1) interacts, and is answered immune Played an important role in terms of the negative regulation answered.The present invention is by extensive and in-depth study, it has unexpectedly been found that to PDL-1 molecules Soluble PD-1 molecules with high-affinity can effectively improve the killing ability of lymphocyte.Therefore, the present invention provides A kind of affinity to PDL-1 is soluble high-affinity of the wild type PD-1 molecules to the affinity at least twice of PDL-1 PD-1 molecules.
The binding affinity that above-mentioned PD-1 molecules and PDL-1 can be measured by any suitable method is (normal with dissociation equilibrium Number KDIt is inversely proportional) and (it is expressed as T with reference to half-life period1/2).It will be appreciated that affinity is double will to cause KDHalve.T1/2It is calculated as In2 Divided by dissociation rate (Koff).Therefore, KoffIt is double to cause T1/2Halve.It is preferred that parent is combined using identical testing program detection With power or combine half-life period for several times, take the average value of result such as 3 times or more.In a preferred embodiment, using this hair Surface plasmon resonance (BIAcore) method in bright embodiment carries out these detections.
This method detects that wild type PD-1 molecules are to the Dissociation equilibrium constant K of PDL-1 molecules in the present inventionDFor 2.815E-06M its BIAcore combination collection of illustrative plates is as shown in Figure 2.It will cause K since affinity is doubleDHalve, if so detecting Dissociation equilibrium constant K of the high-affinity PD-1 molecules to PDL-1 moleculesDFor 1.408E-06M, then illustrate high-affinity PDL-1 Molecule is wild type PD-1 molecules to the affinity of PD-1 molecules to 2 times of the affinity of PDL-1.Those skilled in the art are known KDThe conversion relation being worth between unit, i.e., 1M=1000 μM, 1 μM=1000nM, 1nM=1000pM.
In the preference of the present invention, PD-1 of the present invention is measured using the mode of currently preferred measure affinity The affinity of molecule and PDL-1 molecules is at least 2 times of wild type PD-1 molecules and the affinity of PDL-1 molecules；Preferably, extremely It is 10 times few；It is highly preferred that at least 50 times；Most preferably, at least 100 times.
In another preference, the affinity of the PD-1 molecules and PDL-1 molecules is wild type PD-1 molecules and PDL-1 At least 500 times of the affinity of molecule；Preferably, at least 1000 times；It is highly preferred that at least 2000 times.
Specifically, the affinity K of high-affinity PD-1 molecules 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。
The high-affinity PD-1 molecules of the present invention contain one or more in the amino acid sequence shown in SEQ ID NO.1 Mutation.Specifically, the amino acid sequence of the PD-1 molecules has at least 90% with the amino acid sequence shown in SEQ ID NO.1 (preferably, at least 92%；It is highly preferred that sequence thereto at least 94%).
More specifically, the acid residues sites being mutated in high-affinity PD-1 molecules of the present invention include 31V, 33N, 35Y, In 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 91G, 92A, 93I, 95L, 97P, 98K, 99A, 100Q, 101I, 103E One or more, wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1.
In another preference, the PD-1 molecules after mutation include one or more amino acid residues selected from the group below： 31T；33L；35N、35M；37V, 37L or 37E；40A、40T；41G、41L；42N；43V、43G；48G、48S；56L；89M；91A、 91S, 91V or 91T；92V、92Y；93L；95W、95F；97G；98R, 98Y or 98P；99P, 99V, 99I or 99F；100S、100W； 101V；103D wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1.
In another preference, the amino acid sequences of the PD-1 molecules be selected from SEQ ID NO.5,7,9,11,13,15, 17th, 19,21,23,25,27,29,31,33,35,37,39,41 or 43；
Its coding nucleotide sequence corresponds respectively to：SEQ ID NO.6、8、10、12、14、16、18、20、22、24、26、 28th, 30,32,34,36,38,40,42 and 44：
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLLWNRVSPANQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCAAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.5, L1B2)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTTCTGCTGTGGAATCGTGTGAGCCCGGCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGCTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.6, L1B2)
PPTFSPALLVVTEGDNATFTCSFSNTSESFTLLWMRLSPTNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.7, L1D3)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTACGCTGTTGTGGATGCGTCTTAGCCCGACTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGGTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.8, L1D3)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLLWMRESPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.9, L1G2)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTTCTGTTGTGGATGCGTGAGAGCCCGTCGAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGGTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.10, L1G2)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSGQGDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCSAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.11, L2B 12)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTGGGCAGGGGGATAAGCTGGCGGCGTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTAGTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.12, L2B 12)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSGQVDKLAGFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCVAISLAPKPQIKESLRAELRVTERRAE (SEQ ID NO.13, L2F8)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTGGTCAGGTTGATAAGCTGGCGGGTTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGTTGCAATTTCACTGGCTCCGAAACCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.14, L2F8)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSLNGDKLASFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCVAISLAPKPQIKESLRAELRVTERRAE (SEQ ID NO.15, L2F10)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTCTTAATGGTGATAAGCTGGCGTCGTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGTTGCAATTTCACTGGCTCCGAAACCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.16, L2F10)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYMCVAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.17, L3A7)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACATGTGTGTTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.18, L3A7)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQLGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCTAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.19, L3E3)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCTGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTACGGCTATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.20, L3E3)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCAALSWAGKAQIKESLRAELRVTERRAE (SEQ ID NO.21, L4B3)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGCTGCGCTGTCATGGGCTGGTAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.22, L4B3)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCSAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.23, L4C6)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTTCGGCTATTTCATTGGCTCCTAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.24, L4C6)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCVAISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.25, L4C11)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGTTGCTATTTCACTGGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.26, L4C11)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCSVISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.27, L4D7)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTTCGGTTATTTCATTGGCTCCTAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.28, L4D7)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCTAISWAGKAQIKESLRAELRVTERRAE (SEQ ID NO.29, L4E1)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTACTGCTATTTCATGGGCTGGGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.30, L4E 1)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCTYISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.31, L4E8)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTACTTATATTTCACTGGCTCCTAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.32, L4E8)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCVYISLAPKAQIKESLRAELRVTERRAE (SEQ ID NO.33, L4F6)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGTGTATATTTCACTTGCTCCGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.34, L4F6)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCSVISFAGKAQIKESLRAELRVTERRAE (SEQ ID NO.35, L4H10)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTTCGGTGATTTCATTTGCTGGGAAAGCCCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.36, L4H10)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCGAISLAPRVSVKESLRAELRVTERRAE (SEQ ID NO.37, L5A4)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGGTGCAATTTCACTGGCTCCGCGTGTTAGTGTTAAAGA GTCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.38, L5A4)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCSAISLAPYIQIKESLRAELRVTERRAE (SEQ ID NO.39, L5B7)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTAGTGCAATTTCACTGGCTCCGTATATTCAGATTAAAGA GTCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.40, L5B7)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCGAISLAPPFWIKDSLRAELRVTERRAE (SEQ ID NO.41, L5D1)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGGTGCAATTTCACTGGCTCCGCCTTTTTGGATTAAAGA TTCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.42, L5D1)
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSV VRARRNDSGTYLCVAISLAPKIQIKESLRAELRVTERRAE (SEQ ID NO.43, L45)
CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATAC GAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCGTCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAG ATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTG GTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGTTGCAATTTCACTGGCTCCGAAAATTCAAATCAAAGA ATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA (SEQ ID NO.44, L45)
To obtain soluble high-affinity PD-1 molecules, the wild type PD-1 molecules used in the present invention are free of transmembrane region. Therefore, in the preference of the present invention, the PD-1 molecules are solvable.
It can be mutated using any suitable method, include but not limited to foundation PCR (PCR) that A bit, according to Restriction Enzyme clone or do not depend on clone (LIC) method of connection.Many standard molecular biology teaching materials detail These methods.PCR (PCR) mutagenesis and the more details according to the clone of Restriction Enzyme can be found in Sambrook With Russel l, (2001) Molecular Cloning-A Laboratory handbook (Molecular Cloning-A Laboratory Manual) (third edition) CSHL publishing houses.More information visible (Rashtchian, CurrOpinBiotechnol, 1995,6 of LIC methods (1):30-6)。
Producing the method for the high-affinity PD-1 molecules of the present invention can be but not limited to from the such PD-1 molecules of displaying Filtered out in the diverse libraries of phage particle to PD-1 have high-affinity PD-1, as document (Li, et al., Nature Biotech,2005,23(3):Described in 349-354).
It is to be understood that 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 Gene can be adopted to prepare template strand.Then the high-affinity PD-1 for producing the present invention is introduced in the DNA for encoding the template strand Required change.
The present invention PD-1 molecules can also multivalence complex form provide.The present invention multivalence PD-1 include two, Three, the four or more PD-1 molecules of the present invention polymer that is combined and is formed, such as can use FC section of IgG to prepare dimerization Body, or four dimerization domains of p53 produce the tetramer, or multiple PD-1 of the present invention and another molecule with reference to and formed compound Thing.
The high-affinity PD-1 molecules of the present invention can be used alone, also can be with conjugate with covalent or other modes knot Close, preferably combined with covalent manner.The conjugate is preferably φt cell receptor, it is highly preferred that the φt cell receptor is high parent With property φt cell receptor.
The high-affinity PD-1 molecules of the present invention can be also combined with other molecules, produce effective synergistic effect.Preferably, Other described molecules are ImmTAC or HATac.Two kinds of molecules can redirect T cell, so as to play killing target cell Effect.The ImmTAC molecules are soluble double-strand TCR molecules and AntiCD3 McAb containing artificial interchain disulfide bond between α β constant regions The fusion molecule of antibody, 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 points Son is high-affinity T cell activation core (High Affinity T-cell activation core), and one form of which can The soluble single-chain T CR molecules and the fusion molecule of anti-cd 3 antibodies that the α being mutated by hydrophobic core is formed by connecting with β chain variable domains, institute State soluble single-chain T CR molecules and for details, reference can be made to patent document WO2014/206304.
The invention further relates to the nucleic acid molecules for encoding PD-1 of the present invention.The present invention nucleic acid molecules can be DNA form or Rna form.DNA can be coding strand or noncoding strand.For example, the nucleotide sequence of coding TCR of the present invention can be attached with the present invention Nucleotide sequence shown in figure is identical or the variant of degeneracy.The implication of " variant of degeneracy " is illustrated, such as this paper institutes With, " variant of degeneracy " refer in the present invention coding with SEQ ID NO.1 protein sequence, but with SEQ ID NO.2 The differentiated nucleotide sequence of sequence.
The present invention nucleic acid molecules full length sequence or its fragment usually can with but be not limited to PCR amplification method, recombination method or Artificial synthesized method obtains.At present, it is already possible to obtain encoding PD-1 of the present invention (or its piece by chemical synthesis completely Section, or derivatives thereof) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art In (or such as carrier) and cell.
Passed through the present invention also relates to the carrier of the nucleic acid molecules comprising the present invention, and with the carrier or coded sequence of the present invention The host cell that genetic engineering produces.
The present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition contains pharmaceutically acceptable carrier and sheet Invent PD-1 or of the present invention PD-1 compounds.
Suitable present invention is applied present invention also offers a kind of method for treating disease, including to object in need for the treatment of The pharmaceutical composition of PD-1 or of the present invention PD-1 compounds or the present invention；Especially, PD-1 molecules of the invention and other points Son combination, it is preferable that other molecules are ImmTAC or HATac.
It is to be understood that amino acid name is identified using international single English alphabet herein, amino corresponding thereto Sour three English alphabet of title is write a Chinese character in simplified form：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, albumen will not usually be changed The function of matter.The structure and work(of protein will not generally also be changed by adding one or several amino acid in C-terminal and/or N-terminal Energy.
Present invention additionally comprises the PD-1 molecules after slightly modifying PD-1 of the present invention.Modify (not changing primary structure usually) Form includes：Chemically derived the form such as acetylation or carboxylated of PD-1 of the present invention.Modification further includes glycosylation, as those are at this Invent in the synthesis and processing of PD-1 or PD-1 molecules that are glycosylation modified and producing are carried out in further processing step.It is this to repair Decorations can be by the way that PD-1 be completed exposed to glycosylated enzyme (glycosylase or deglycosylating enzyme of such as mammal) is carried out. Modified forms further include the sequence with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Further include and be modified so as to improve its anti-proteolysis performance or optimize the PD-1 of solubility property.
PD-1 the or PD-1 compounds of the present invention can provide together with pharmaceutically acceptable carrier in pharmaceutical composition. PD-1, the multivalence PD-1 compounds of the present invention is provided usually as a part for sterile pharmaceutical composition, and the composition is usual Including pharmaceutically acceptable carrier.The pharmaceutical composition can be that any suitable form (depends on giving the required of patient Method).It can use unit dosage forms to provide, and usually be provided in the container of sealing, can be provided as a part for kit.This Class kit (but nonessential) includes the use of specification.It may include multiple unit dosage forms.In addition, the PD-1 of the present invention can With alone, it also can be combined or be coupled together use with other therapeutic agents (as prepared in same pharmaceutical composition).
Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to for controlling Treat the carrier of agent administration.The term refers to some such medicament carriers：Themselves do not induce produced to receiving said composition The antibody that body is harmful to, and there is no undue toxicity after being administered.These carriers are well known to those of ordinary skill in the art.In thunder Bright pharmaceutical science (Remington's Pharmaceutical Sciences, Mack Pub.Co., N.J.1991)) in can Find discussing fully on pharmaceutically acceptable excipient.This kind of carrier includes (but being not limited to)：Brine, buffer solution, Glucose, water, glycerine, ethanol, adjuvant, and combinations thereof.
Acceptable carrier can contain liquid in therapeutic composition Chinese pharmacology, such as water, brine, glycerine and ethanol.In addition, It there is likely to be complementary material, such as wetting agent or emulsifying agent, pH buffer substance in these carriers.In general, it will can treat Injectable agent, such as liquid solution or suspension is made in property composition；May also be fabricated which before the injection be adapted to supplying solution or suspension in, 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, intracutaneous or local administration are preferably parenteral including subcutaneous, muscle It is interior or intravenous.The object waited to prevent or treated can be animal；Especially people.
When the pharmaceutical composition of the present invention is used for actual therapeutic, various different dosage forms can be used according to service condition Pharmaceutical composition.It is preferred that what can be enumerated has injection, oral agents etc..These pharmaceutical compositions can lead to according to conventional methods Cross mixing, dilution or dissolving and prepared, and add suitable medicated premix once in a while, such as excipient, disintegrant, bonding It is agent, lubricant, diluent, buffer, isotonic agent (isotonicities), preservative, wetting agent, emulsifying agent, dispersant, steady Determine agent and cosolvent, and the process for preparation can be carried out according to formulation with usual way.
The pharmaceutical composition of the present invention can be administered with sustained release formulation.For example, PD-1 of the present invention can be impregnated in be sustained Polymer is in the pill or micro-capsule of carrier, and the pill or micro-capsule then are passed through implantation tissue to be treated of performing the operation.As slow The example of polymer is released, what can be enumerated has ethylene-vinylacetate copolymer, poly- hydroxyl-metacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid- Ethanol copolymer etc., what can preferably be enumerated is that biodegradable polymer such as lactic acid polymer and lactic acid-ethanol are total to Polymers.
When the pharmaceutical composition of the present invention is used for actual therapeutic, PD-1 of the present invention or PD-1 as active component is answered Compound, can according to the weight of each patient to be treated, the age, gender, symptom degree and reasonably determined, finally by curing Teacher determines rational dosage.
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 molecules of the invention can effectively improve the killing ability of lymphocyte.
Following specific embodiment, the present invention is further explained.It is to be understood that these embodiments be merely to illustrate the present invention and It is not used in and limits the scope of the invention.The experimental method of actual conditions is not specified in the following example, 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 houses) described in condition, or according to the condition proposed by manufacturer.Unless In addition illustrate, 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, will be carried wild The target gene of the extracellular sequence of type PD-1 is through I double digestion of Nco I and Not, with passing through I double digestion of Nco I and Not PET28a carriers (Novagen) carrier carries biotin tag labels after optimization) connection.Connection product convert to E.coli DH5 α (Vazyme), are coated with the LB tablets containing kanamycins, 37 DEG C of inversion overnight incubations, and picking positive colony carries out PCR is screened, and positive recombinant is sequenced, and determines that sequence correctly extracts recombinant plasmid transformed to E.coli Rosetta bacterium afterwards In strain (TIANGEN), for expressing.
The above-mentioned Rosetta colony inoculations containing recombinant plasmid pET28a-PD-1 are cultivated in the LB containing kanamycins In base, 37 DEG C of cultures to OD600 are 0.6-0.8, add IPTG to final concentration of 0.7mM, and 37 DEG C are continued to cultivate 4h.6000g from Heart 15min harvests cell pellet, with BugbusterMaster Mix (Merck) cell lysis sediment, 6000g centrifugations 15min recycles inclusion body, then is washed with Bugbuster (Merck) to remove cell fragment and membrane component, 6000g centrifugations 15min, collects inclusion body.By solubilization of inclusion bodies in buffer solution (50mMTris-HCl, 200mMNaCl, 2mM EDTA, 6M Guanidine HCl, pH 8.1) in, high speed centrifugation removes insoluble matter, and supernatant after BCA standard measures with being dispensed, in -80 DEG C save backup.
In the PD-1 inclusion body proteins dissolved to 7mg, 2mL buffer solutions (50mMTris-HCl, 200mMNaCl, 2mM are added EDTA, 6M guanidine HCl, pH 8.1), add DTT to final concentration of 20mM, 37 DEG C of processing 1h.To 100mL renaturation Buffer solution (50mM HEPES, pH 7.5,500mM L-arginine, 9mM glutathione, 1mM glutathione Disulfide, 24Mm NaCl, 1mMKCl) in treated PD-1 mixed liquors are added dropwise, 4 DEG C of stirring 30min then will be multiple Property liquid load interception be 3.5KDCellulose membrane bag filter, bag filter is placed in the water of 2L precoolings, and 4 DEG C are slowly stirred overnight. 24 it is small when after, dialyzate is changed to the buffer solutions (10mMTris-HCl pH 8.5) of 2L precoolings into, 4 DEG C are continued the 24h that dialyses, then By dialyzate change into identical fresh buffer continue dialysis 24 it is small when, sample is laggard through 0.45 μm of membrane filtration, vacuum outgas The 0- that sample is prepared to anion-exchange column (HiTrap Q HP, GE Healthcare) with 10mMTris-HCl pH 8.5 1MNaCl linear gradient elution liquid purifying proteins, the elution fraction of collection carry out SDS-PAGE analyses.According to analysis result, collect Further purified after the concentration of target PD-1 components with solvent resistant column (Superdex 75 10/300, GE Healthcare), mesh Mark component also carries out SDS-PAGE analyses, and the results are shown in Figure 1.
Embodiment 2 combines characterization
BIAcore is analyzed
Wild type PD-1 molecules and the combination activity of PDL-1 are detected using BIAcore T200 real-time analyzers.Will be anti- The antibody (GenScript) of Streptavidin adds coupling buffer (10mM sodium-acetate buffers, pH 4.77), then will be anti- Body flows through the CM5 chips activated in advance with EDC and NHS, antibody is fixed on chip surface, finally molten with the hydrochloric acid of monoethanolamine Unreacted activating surface is closed in fluid-tight, completes coupling process, coupling horizontal about 15,000RU.
The Streptavidin of low concentration is flowed through the chip surface of coated antibody, then flow through biotinylated PD-1 Sense channel, another passage flow through chip 2min as reference channel, then by the biotin of 0.05mM with the flow velocity of 10 μ L/min, Close the remaining binding site of Streptavidin.Its affinity is measured using single cycle dynamic analysis method, PD-1 is used HEPES-EP buffer solutions (10mM HEPES, 150mMNaCl, 3mM EDTA, 0.005%P20, pH 7.4) are diluted to several differences Concentration, with the flow velocity of 30 μ L/min, flow successively through chip surface, the binding time of each sample introduction is 120s, for the last time into It is allowed to dissociate 600s after sample.Each round uses the 10mMGly-HCl regeneration chips of pH 1.75 after measuring.Utilize BIAcore Evaluation software computational dynamics parameters.
The amino acid sequence of PDL-1 used and nucleotide sequence are respectively such as SEQ ID NO.3, SEQ ID in the present embodiment Shown in NO.4, its expression, renaturation and purge process and the expression of wild type PDL-1, renaturation and purge process phase in embodiment 1 Together.Its biotinylated process is as follows：
A. biotinylation
With Millipore super filter tubes by the PDL-1 molecular concentrations of purifying, while it is 10mMTris pH by buffer exchange 8.0, then add biotinylation reagent 0.05MBicine pH 8.3,10mM ATP, 10mMMgOAc, 50 μM of D-Biotin, 100 μ g/ml BirA enzymes (GST-BirA), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detection biotinylations are complete.
B. the compound after purifying biological elementization
PDL-1 molecular concentrations after biotinylation is marked with Millipore super filter tubes are to 500 μ l, using gel filtration The biotinylated PDL-1 of chromatographic purifying, first with filtered 7510/300 solvent resistant columns of PBS pre-equilibrations Superdex, (GE leads to With electric corporation), the concentrated biotinylation PDL-1 molecules of 500 μ l are reloaded, are then eluted with PBS with 1ml/min flow velocitys, The component being collected into is subjected to SDS-PAGE analyses, the component containing target protein is merged according to result, is surpassed with Millipore Chimney filter concentrates, and BCA methods (Thermo) measure protein concentration, -80 DEG C are stored in by the packing of biotinylated PDL-1 molecules.
The above process detects the K of wild type PD-1 molecules and the binding affinity of PDL-1 molecules through this embodimentDValue It is as shown in Figure 2 for 2.815E-06M, its BIAcore combination collection of illustrative plates.
The generation of 3 high-affinity PD-1 molecules of embodiment
Using the extracellular sequence of the wild type PD-1 described in embodiment 1 as template strand, according to Li et al. (2005) Nature Biotech 23(3):349-354) the phage display and screening technique of description, carries out the screening of high-affinity PD-1.By Phage library after a few wheel screenings is and PD-1 has stronger binding signal, therefrom picking monoclonal, and carry out sequence analysis.
According to the expression of method described in embodiment 1, renaturation and purifying high-affinity PD-1 molecules of the present invention, and press embodiment Method described in 2 measures its affinity with PDL-1 molecules.The high-affinity PD-1 molecules obtained in the present invention divide with PDL-1 The affinity of son is at least 2 times of the affinity of wild type PD-1 molecules and PDL-1 molecules, its amino acid sequence and its and PDL-1 The affinity numerical value of molecule is as shown in table 1 below.
1 high-affinity of table clones the BIAcore results to PDL-1 molecules
The ability of 4 L5B7 of embodiment identification H1299 cell surfaces PD-L1 is higher than PD-1
Biacore is but this affine the results show that obtained the PD-1 mutant of affinity raising really after screening The combination whether change of power can influence cell surface PDL-1 under itself and physiological condition still needs to experimental verification.Therefore, we select The positive H1299 cells of PDL-1 expression, add biotinylation PD-1, L5B7 albumen of various concentrations, flow cytometry The ability of PD-1, L5B7 identification cell surface PDL-1.
Fig. 3 is the results show that the rise of PD-1, L5B7 protein concentration with addition, its recognition capability to PDL-1 are gradual Enhancing；Under the conditions of same concentration, the ability of L5B7 albumen identification PDL-1 is higher than PD-1, and the change of this recognition capability may Be due to L5B7 affinity it is higher caused, it is consistent with chemical result.
5 high-affinity PD-1 molecule bivalents fusion protein of embodiment produces
A expression vector establishments
In order to increase the stability and potency of PD-1 and its high affine mutant in vivo, eukaryotic expression is used to be melted with IgG4 Close the form of expression.PD-1 albumen eukaryotic expression sequences are saved worry Bioisystech Co., Ltd's optimum synthesis by Suzhou, with After the completion of IgG4overlapPCR splicings, it is connected to by EcoR I, I sites of Nhe on pGZFUSE plasmid vectors, and changes and go to In 10 bacterial strains of Top (while the mutant clone of other compatibilities is obtained by being mutated).By 1:1000 are inoculated in 200ml LB trainings Support in base, after 37 degree are incubated overnight, bacterium was received in second day, plasmid is carried out and largely extracts.OD260/0D280 surveys plasmid concentration, adjusts Whole is 1mg/ml, is preserved after packing with -20 degree.Big upgrading grain is spare.Amalgamation and expression albumen schematic diagram such as Fig. 4 a.
B protein expressions
Carrier for expression of eukaryon is built according to a, after being sequenced correctly, expressing fusion protein uses 293T attached cells expression system System.The day before transfection, paving exponential phase 293T cells are in 10cm culture dishes, and second day with plasmid：Lipo 2000=1:2 The condition transfectional cell of (volume ratio), replaces fresh Freestyle after 4hTM293 culture mediums, take after 48h a small amount of supernatant to run SDS-PAGE identification expressions such as Fig. 4 b.72 it is small when after receive supernatant, after 0.22 μm of membrane filtration, be diluted to 20 times of bodies In the 10mM Tris-Hcl of product precooling in advance (PH=8.5), anion and molecular sieve purification are carried out.PAGE gel electrophoresis The results show：The fusion protein purity of 293T attached cells expression is very high.The purpose that we are further purified has two, first, putting Change solution residing for albumen, prevent during expression or culture medium existing Cucumber influences subsequent experimental in itself；It is second, dense Pix protein, and further increase purity, avoids subsequent experimental due to adding a large amount of albumen, or there are some foreign proteins to be subject to shadow Ring.
C fusion protein Function Identifications
ImmTACs molecules can redirect T cell specific killing tumour cell and be reported in more research (Jakobsen,2013；Oates et al.,2015).Its basic principle is that ImmTACs can simulate T cell activation performance effect The key signal of function is answered, on the one hand by the MHC- peptide complexes on its high affine specificity TCR tumor cell surface, On the other hand the downstream signaling pathway of T cell activation is activated by its anti-CD3 antibody end, is killed so as to orient T cell specificity Hinder tumour cell.Therefore, can the function of evaluating PD-1/L5B7-IgG4 fusion proteins in our current research be promoted by the albumen Killing processes of the PBMC numerator mediated into ImmTAC-IG4 to Mel624 tumour cells is come Fig. 4 c for evaluating.
It turns out that when the concentration of ImmTAC is 10-9M, E:T=5：1 and 1:1 (PBMC is effector cell, and Mel62 is target Cell) when, PD-1-IgG4, L5B7-IgG4 fusion protein can promote ImmTAC molecular orientation PBMC killing tumor cells LDH discharges, and L5B7-IgG4 fusion proteins promote LDH releases to be higher than PD-1-IgG4 groups；Killing is collected than being 1:Reactant when 1 Cell in system carries out CD25, CD107a that flow cytometer detection finds PD-1-IgG4, L5B7-IgG4 fusion protein group cd8 t cell Expression more not plus protein groups raise, and L5B7-IgG4 groups up-regulation ratio higher than add PD-1-IgG4 groups, CD25 from 13.2%, CD107a is adjusted on 10.9% 19.8% is adjusted to from 16.9%, further demonstrated LDH and discharge increased result. Illustrate that PD-1-IgG4, L5B7-IgG4 fusion protein can promote the numerator mediated PBMC of ImmTAC to Mel624 tumour cells Killing, it is consistent with the result of study that solubility PD-1 in other documents can promote tumor specific T cells to kill, increase its parent Further strengthen with the facilitation after property.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited Enclose.
Sequence table
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>The soluble PD-1 molecules of high-affinity
<130> P2016-1601
<160> 44
<170> PatentIn version 3.5
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 2
<211> 351
<212> DNA
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
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Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu
210
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tttacggtta cggttccgaa agacctgtat gtggttgaat acggctctaa tatgacgatt 60
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atggaagaca aaaacatcat ccaattcgtg catggcgaag aagatctgaa agttcagcac 180
agctcttacc gtcaacgcgc acgtctgctg aaagaccagc tgagcctggg caatgcagct 240
ctgcagatca cggatgttaa actgcaagac gccggtgtct atcgctgcat gatttcttat 300
ggcggtgcag actacaaacg tatcaccgtc aaagtgaacg ctccgtacaa caaaattaat 360
cagcgcatcc tggtggttga tccggttacg tccgaacatg aactgacctg tcaagcggaa 420
ggctatccga aagccgaagt catttggacc agttccgatc accaggtgct gtcaggtaaa 480
accacgacca cgaactcgaa acgcgaagaa aaactgttta atgtcacgag caccctgcgt 540
attaacacca cgaccaatga aatcttctac tgcacctttc gtcgtctgga cccggaagaa 600
aatcatacgg cggaactggt tatcccggaa ctg 633
<210> 5
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Leu Trp Asn Arg Val Ser Pro Ala Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ala Ala Ile Ser Leu Ala
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Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
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aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
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<210> 7
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Thr Leu
20 25 30
Leu Trp Met Arg Leu Ser Pro Thr Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
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Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 8
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
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aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gaaagcccaa 300
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<210> 9
<211> 117
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Leu Trp Met Arg Glu Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
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aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 11
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Gly Gln Gly Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 12
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
gggcaggggg ataagctggc ggcgttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt agtgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 13
<211> 117
<212> PRT
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<400> 13
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Gly Gln Val Asp Lys Leu Ala Gly
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Pro Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 14
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
ggtcaggttg ataagctggc gggtttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgcaattt cactggctcc gaaaccccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 15
<211> 117
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Leu Asn Gly Asp Lys Leu Ala Ser
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Pro Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 16
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
cttaatggtg ataagctggc gtcgttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgcaattt cactggctcc gaaaccccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 17
<211> 117
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Met Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 18
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacatgtgt gttgcaattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 19
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Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Leu Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Thr Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 20
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cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagctggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt acggctattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 21
<211> 117
<212> PRT
<213>Artificial sequence
<400> 21
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ala Ala Leu Ser Trp Ala
85 90 95
Gly Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 22
<211> 351
<212> DNA
<213>Artificial sequence
<400> 22
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gctgcgctgt catgggctgg taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 23
<211> 117
<212> PRT
<213>Artificial sequence
<400> 23
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 24
<211> 351
<212> DNA
<213>Artificial sequence
<400> 24
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt tcggctattt cattggctcc taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 25
<211> 117
<212> PRT
<213>Artificial sequence
<400> 25
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 26
<211> 351
<212> DNA
<213>Artificial sequence
<400> 26
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgctattt cactggctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 27
<211> 117
<212> PRT
<213>Artificial sequence
<400> 27
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Val Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 28
<211> 351
<212> DNA
<213>Artificial sequence
<400> 28
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt tcggttattt cattggctcc taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 29
<211> 117
<212> PRT
<213>Artificial sequence
<400> 29
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Thr Ala Ile Ser Trp Ala
85 90 95
Gly Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 30
<211> 351
<212> DNA
<213>Artificial sequence
<400> 30
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt actgctattt catgggctgg gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 31
<211> 117
<212> PRT
<213>Artificial sequence
<400> 31
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Thr Tyr Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 32
<211> 351
<212> DNA
<213>Artificial sequence
<400> 32
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt acttatattt cactggctcc taaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 33
<211> 117
<212> PRT
<213>Artificial sequence
<400> 33
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Tyr Ile Ser Leu Ala
85 90 95
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 34
<211> 351
<212> DNA
<213>Artificial sequence
<400> 34
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gtgtatattt cacttgctcc gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 35
<211> 117
<212> PRT
<213>Artificial sequence
<400> 35
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Val Ile Ser Phe Ala
85 90 95
Gly Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 36
<211> 351
<212> DNA
<213>Artificial sequence
<400> 36
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt tcggtgattt catttgctgg gaaagcccaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 37
<211> 117
<212> PRT
<213>Artificial sequence
<400> 37
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Arg Val Ser Val Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 38
<211> 351
<212> DNA
<213>Artificial sequence
<400> 38
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gcgtgttagt 300
gttaaagagt cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 39
<211> 117
<212> PRT
<213>Artificial sequence
<400> 39
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Ser Ala Ile Ser Leu Ala
85 90 95
Pro Tyr Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 40
<211> 351
<212> DNA
<213>Artificial sequence
<400> 40
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt agtgcaattt cactggctcc gtatattcag 300
attaaagagt cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 41
<211> 117
<212> PRT
<213>Artificial sequence
<400> 41
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala
85 90 95
Pro Pro Phe Trp Ile Lys Asp Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 42
<211> 351
<212> DNA
<213>Artificial sequence
<400> 42
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt ggtgcaattt cactggctcc gcctttttgg 300
attaaagatt cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351
<210> 43
<211> 117
<212> PRT
<213>Artificial sequence
<400> 43
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn
1 5 10 15
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala
35 40 45
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val
50 55 60
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala
65 70 75 80
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Val Ala Ile Ser Leu Ala
85 90 95
Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr
100 105 110
Glu Arg Arg Ala Glu
115
<210> 44
<211> 351
<212> DNA
<213>Artificial sequence
<400> 44
cctcctacat tctccccggc actgctggtt gttaccgaag gcgataatgc gacctttacc 60
tgtagtttct ccaatacgag cgaatcgttt gtcctgaact ggtatcgtat gagcccgtct 120
aatcagaccg ataaactggc ggccttcccg gaagatcgct ctcagccggg ccaagacagc 180
cgttttcgcg ttacgcaact gccgaacggt cgtgatttcc atatgagtgt ggttcgcgcc 240
cgtcgcaatg actccggcac ctacctgtgt gttgcaattt cactggctcc gaaaattcaa 300
atcaaagaat cgctgcgtgc ggaactgcgt gttaccgaac gtcgtgccga a 351

Claims (13)

  1. A kind of 1. PD-1 molecules, it is characterised in that ammonia of the amino acid sequence of the PD-1 molecules shown in based on SEQ ID NO.1 Base acid sequence, and the amino acid sequence shown in SEQ ID NO.1 is carried out the mutation of one or more amino acid residues so as to Obtain the PD-1 molecules；The amino acid sequence of the PD-1 molecules has at least with the amino acid sequence shown in SEQ ID NO.1 90% sequence thereto；It is highly preferred that the affinity of the PD-1 molecules and PDL-1 molecules be wild type PD-1 molecules with At least 2 times of the affinity of PDL-1 molecules.
  2. 2. PD-1 molecules as claimed in claim 1, it is characterised in that the acid residues sites being mutated in the PD-1 molecules For one or more of the 30th~60, and/or 85~105 amino acids residues, wherein numbering amino acid residues use SEQ Numbering shown in ID NO.1.
  3. 3. PD-1 molecules as claimed in claim 1, it is characterised 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.
  4. 4. PD-1 molecules as claimed in claim 1, it is characterised in that the acid residues sites being mutated in the PD-1 molecules Including 31V, 33N, 35Y, 37M, 40S, 41N, 42Q, 43T, 48A, 56P, 89L, 91G, 92A, 93I, 95L, 97P, 98K, 99A, One or more of 100Q, 101I and 103E, wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1.
  5. 5. PD-1 molecules as claimed in claim 1, it is characterised in that the acid residues sites being mutated in the PD-1 molecules Including 91G, wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1；And/or
    The acid residues sites being mutated in the PD-1 molecules include 99A, and wherein numbering amino acid residues use SEQ ID Numbering shown in NO.1；
    Preferably, the acid residues sites being mutated in the PD-1 molecules further include 41N, 42Q, 43T, 48A, 95L, 97P, 98K, and/or 100Q, wherein numbering amino acid residues are using the numbering shown in SEQ ID NO.1.
    It is highly preferred that the PD-1 molecules include one or more amino acid residues selected from the group below：31T；33L；35N or 35M；37V, 37L or 37E；40A or 40T；41G or 41L；42N；43V or 43G；48G or 48S；56L；89M；91A、91S、 91V or 91T；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 using the numbering shown in SEQ ID NO.1；
    Most preferably, the amino acid sequence of the PD-1 molecules be selected from SEQ ID NO.5,7,9,11,13,15,17,19,21, 23rd, 25,27,29,31,33,35,37,39,41 or 43.
  6. 6. a kind of fusion protein, it is characterised in that the fusion protein includes the PD-1 molecules described in claim 1.
  7. A kind of 7. multivalence PD-1 compounds, it is characterised in that the multivalence PD-1 compounds include at least two PD-1 molecules, and And at least one PD-1 molecules therein are the PD-1 molecules described in claim 1；Or the multivalence PD-1 compounds include Fusion protein described at least one claim 6.
  8. 8. a kind of nucleic acid molecules, it is characterised in that the nucleic acid molecules include the PD-1 molecules described in coding claim 1, power Profit requires the nucleotide sequence or its complementary series of the multivalence PD-1 compounds described in fusion protein or claim 7 described in 6.
  9. 9. a kind of carrier, it is characterised in that the carrier contains the nucleic acid molecules described in claim 8.
  10. 10. a kind of host cell, it is characterised in that contain the carrier described in claim 9 or dyeing in the host cell The nucleic acid molecules described in the claim 8 of external source are integrated with body.
  11. 11. a kind of pharmaceutical composition, it is characterised in that the composition contains pharmaceutically acceptable carrier and claim The fusion protein described in PD-1 molecules or claim 6 described in 1 or the PD-1 compounds described in claim 7.
  12. 12. the fusion protein described in PD-1 molecules, claim 6 described in claim 1 or the PD-1 described in claim 7 The purposes of compound, it is characterised in that be used to prepare the medicine for the treatment of tumour.
  13. A kind of 13. method for preparing the PD-1 described in claim 1, it is characterised in that including step：
    (i) host cell described in claim 10 is cultivated, so as to express the PD-1 molecules described in claim 1；
    (ii) isolated or purified goes out the PD-1 molecules.
CN201610959248.XA 2016-10-27 2016-10-27 The soluble PD-1 molecules of high-affinity Pending CN107987153A (en)

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Cited By (4)

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CN108794619A (en) * 2018-05-31 2018-11-13 郑州大学 A kind of high affine PD-1 protein mutants
CN110478472A (en) * 2019-09-29 2019-11-22 北京鼎成肽源生物技术有限公司 PD-1 sealer and its application
CN111714618A (en) * 2019-03-22 2020-09-29 广东香雪精准医疗技术有限公司 Combination of T cells and high affinity PD-1 fusion proteins
WO2021051661A1 (en) * 2019-09-19 2021-03-25 北京伟杰信生物科技有限公司 Recombinant canine pd-1 fusion protein and preparation method therefor and application thereof

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Publication number Priority date Publication date Assignee Title
CN114181296B (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

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CN1309735C (en) * 2000-05-26 2007-04-11 布里斯托尔-迈尔斯斯奎布公司 Soluble CTL A4 mutant molecules and use thereof
JP5252635B2 (en) * 2005-07-01 2013-07-31 メダレックス インコーポレーティッド Human monoclonal antibody against 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

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CN108794619A (en) * 2018-05-31 2018-11-13 郑州大学 A kind of high affine PD-1 protein mutants
CN108794619B (en) * 2018-05-31 2021-09-17 郑州大学 High-affinity PD-1 protein mutant
CN111714618A (en) * 2019-03-22 2020-09-29 广东香雪精准医疗技术有限公司 Combination of T cells and high affinity PD-1 fusion proteins
WO2021051661A1 (en) * 2019-09-19 2021-03-25 北京伟杰信生物科技有限公司 Recombinant canine pd-1 fusion protein and preparation method therefor and application thereof
CN110478472A (en) * 2019-09-29 2019-11-22 北京鼎成肽源生物技术有限公司 PD-1 sealer and its application
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

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