CN108752460A

CN108752460A - The fusion protein and its pharmaceutical composition and purposes of a kind of PD-1 film outskirt mutant of high-affinity

Info

Publication number: CN108752460A
Application number: CN201810582025.5A
Authority: CN
Inventors: 赵健; 魏化伟; 黄亚杰
Original assignee: Jiangsu East Against Biological Medicine Technology Co Ltd
Current assignee: Jiangsu East Against Biological Medicine Technology Co Ltd
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2018-11-06
Anticipated expiration: 2038-06-07
Also published as: CN114181297B; CN114031682B; CN108752460B; CN114181297A; CN114181296A; CN114031682A; CN114181296B

Abstract

The invention discloses a kind of PD-1 film outskirt mutant of high-affinity, the PD-1 films outskirt mutant includes the amino acid sequence selected from SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.Invention further provides the fusion protein of the PD-1 film outskirt mutant of the high-affinity and its applications.The PD-1 film outskirt mutant or its fusion protein for having synthesized high-affinity of the invention, the fusion protein affinity, effect of bigger with higher combination human PD-L 1 or PD-L2 compared with wild type PD-1.The fusion protein of the present invention provides more wide approach for the immunization therapy of tumour.

Description

A kind of fusion protein and its pharmaceutical composition of the PD-1 film outskirt mutant of high-affinity Object and purposes

Technical field

The present invention relates to oncotherapies and molecular immunology field, are related to a kind of PD-1 film outskirt mutant of high-affinity Fusion protein and its pharmaceutical composition and purposes.

Background technology

The immunization therapy of tumour is to excite and enhance the immune function of body, to reach control and kill tumour cell Purpose, tumour immunotherapy are one of hot spots in current cancer therapies field, and significant treatment effect is also achieved in clinical test Fruit.PD-1 and its ligand PD-L1/L2 has mediated the effect of inhibition " exhaustion " and inducing immune tolerance to T cell, tumour thin PD-L1/L2 and the PD-1 interaction of cellular surface height expression lead to the immunologic escape of tumour cell, therefore inhibit PD1~PD- L1/L2 signal paths, reactivating repressed immune system becomes the hot spot of nearest immunization therapy.

Programmed death receptor 1 (PD-1, also referred to as PDCD1 and CD279) helps (Honjo by Kyoto Univ Japan's sheet is multitudinous Tasuku) professor had found in 1992.PD-1 points are three extracellular region, transmembrane region and cytoplasmic region parts, contain 288 amino acid Albumen, molecular weight is about 50~55kD.Extracellular region contains an IgV spline structures domain, is and the ligand binding of PD-1 and lures in turn The region of immune response function is led, which contains 4 N glycosylation sites；Cytoplasmic region contains 1 ITIM (Immunoreceptor tyrosine-based inhibitory motif namely immunity receptor Tyrosine Inhibitory Motifs), (Immunoreceptor tyrosine-based switch motif namely immunity receptor tyrosine convert base with 1 ITSM Sequence), wherein the activation of ITSM and T effector cell immune response are closely related.PD-1 is mainly expressed in the T cell of activation, B On cell and myeloid cell, mainly plays a significant role in maintaining T cell to exhaust, there is the effector function for inhibiting T cell, because And chronic pathogen and tumour cell escape immune response using PD-1 accesses.

PD-L1 albumen：The flat professor of scientist's display of Chinese origin had found B7-H1 in 1999, and the albumen is then in quilt in 2000 Confirmation can specifically bind PD-1, and be named as PD-L1, i.e. (the programmed death of programmed death molecule ligand -1 Ligand 1, also referred to as CD274 and B7-H1)；PD-L1 points are three extracellular region, transmembrane region hydrophobic region and cytoplasmic region parts, by 290 A amino acid subunit composition；Wherein extracellular region includes two constant region for immunoglobulin IgC and IgV spline structure domains, and cytoplasmic region is It is made of 30 amino acid.PD-L1 is mainly expressed in ripe CD4⁺、CD8⁺The hematopoiesis such as T cell, B cell, Dendritic Cells are thin Born of the same parents；PD-L1 is usually high simultaneously is expressed in kinds of tumor cells surface, such as melanoma, non-small cell lung cancer, breast cancer, ovary Cancer, head and neck squamous cell carcinoma etc., meanwhile, tumour cell PD-L1 it is high express the prognosis poor with tumor patient, tumour is answered Send out related, and also related to tumor size, lymph node involvement, classification, Overall survival.

PD-L2 albumen：Another ligand PD-L2, that is, -2 (programmed of programmed death molecule ligand of PD-1 Death ligand2, also referred to as CD273 and B7-DC) be to be found for 2001, PD-L2 by 274 amino acid residues form across Memebrane protein, PD-L2 and PD-L1 have very high similitude, PD-L2 and PD-1 interactions that can inhibit proliferation, the cell of T cell The generation of the factor and T cell dissolution, but the affinity of PD-L2 and PD-1 is 2~6 times of PD-L1.PD-L2 is thin in macrophage Born of the same parents, Dendritic Cells and some B cell subclass Membrane surface expression, while a variety of methods detect PD-L2 in tumour cell Expression, and in some of sample and the expression of PD-L1 is not detected.Therefore PD-1~PD-L1 can be efficiently blocked simultaneously And the drug of PD-1~PD-L2 needs further opening.

2005, the small wild pharmacy of Japan and U.S.'s Medarex pharmacy cooperated and develop PD-1 antibody drugs Nivolumab, and put into the pocket by Bristol Myers Squibb in 2009.Mo Shadong purchases Schering Plough within 2009, obtains PD-1 The subsequent development of antibody drug Pembrolizumab is weighed.2014, the Nivolumab's and Mo Shadong of Bristol Myers Squibb Pembrolizumab obtains listing approval in succession.

2016, the PD-L1 antibody As tezolizumab of Genentech obtained FDA approval listings；Pfizers in 2017 with it is silent The PD-1 antibody drugs Bavencio in husky east and the PD-1 antibody drugs Imfinzi of AstraZeneca are listed in succession.More major companies This immunotherapy of tumors drug is considered as the hit product that the coming years base oneself upon immunotherapy of tumors field.

Although having the drug listing of multiple PD-1 and PD-L1 antibody, the drug of the fusion protein based on PD-1 at present It is rarely reported；And efficiently the drug of PD-1~PD-L1 and PD-1~PD-L2 can be blocked also urgently to develop simultaneously, with more Effectively block the signal path of PD-l.

PD-L1/L2 and the PD-1 interaction of tumor cell surface height expression lead to the immunologic escape of tumour cell, use The antibody blocking of the PD-1 or PD-L1 access can activating immune system, and then killing tumor cell.And due to related PD-L1/L2 Equally also have expression on immunocyte with PD-1, it is contemplated that if PD-1/PD-L1 antibody have too strong ADCC, CDC, ADCP isoreactivities can kill self immunocyte, thus PD-1/PD-L1 antibody is designed as the antibody of blocking property mostly, without Have the effects that ADCC, CDC.The Pembrolizumab PD-1 antibody of the Nivolumab and Mo Shadong of such as Bristol Myers Squibb It is all made of the IgG4 hypotypes of weak ADCC activity；The PD-L1 antibody As tezolizumab of Genentech, which is used, removes glycosylation site IgG1 hypotypes do not have ADCC activity substantially；The Imfinzi of AstraZeneca is then by being mutated three amino acid of IgG1 (L234F, L235E, P331S) removes ADCC activity.

And the Avelumab of Pfizer/Merck uses the IgG1 hypotypes with ADCC, CDC isoreactivity, because PD-L1 is swollen In the significantly high expression of oncocyte, the PD-L1 antibody of strong ADCC activity also has its advantage, that is, releases carcinoma cell immunization escape NK cell killing cancer cells are mediated it is also possible to be acted in conjunction with ADCC.

Invention content

The purpose of the present invention is to provide a kind of PD-1 film outskirt mutant of high-affinity and include the high-affinity PD-1 film outskirt mutant fusion protein.

The present invention also aims to provide the PD-1 film outskirt mutant comprising above-mentioned high-affinity or its fusion egg Compositions of baiyao and application thereof.

To achieve the above object, present invention firstly provides a kind of PD-1 film outskirt mutant of high-affinity, the PD-1 Film outskirt mutant includes the amino acid sequence selected from SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 Row.PD-1 films outskirt mutant is M2C5 in one embodiment, the amino acid sequence with SEQ ID NO.2；Implement one PD-1 film outskirt mutant is M3H6 in example, the amino acid sequence with SEQ ID NO.3；In one embodiment outside PD-1 films Region mutation body is M4B3, the amino acid sequence with SEQ ID NO.4；PD-1 films outskirt mutant is in one embodiment M5G8, the amino acid sequence with SEQ ID NO.5.

Further, described the present invention also provides a kind of fusion protein of the PD-1 film outskirt mutant of high-affinity Fusion protein includes the amino acid sequence selected from SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 Albumen can also include optionally the albumen with other functions.

Preferably, the fusion protein improves at least 50 times with the affinity of PD-L1 compared with wild type PD-1；It is real one It applies in example, fusion protein of the invention improves 1000 times or more with the affinity of PD-L1 compared with wild type PD-1；The fusion Albumen improves at least 10 times with the affinity of PD-L2 compared with wild type PD-1；In one embodiment, fusion protein of the invention 300 times or more are improved compared with wild type PD-1 with the affinity of PD-L2.

Preferably, the fusion protein further includes Fc segments, and the Fc segments are selected from the Fc of human IgG1 or IgG4 and its dash forward Modification, the wherein amino acid sequence of the N298A mutant of human IgG1 Fc as shown in SEQ ID NO.8, wherein human IgG 4Fc's The amino acid sequence of S228P mutant is as shown in SEQ ID NO.9.

Preferably, the fusion protein further includes 6 × His, the amino acid sequence such as SEQ ID NO.20 of the 6 × His.

Preferably, the PD-1 films outskirt mutant connects into fusion protein by Linker and Fc segments, 6 × His, institute The amino acid sequence of linker is stated as shown in SEQ ID NO.6.

Further, the present invention also provides a kind of biomaterial of the DNA sequence dna of encoding said fusion protein, the lifes Object material is carrier, host cell or kit, such as kit be used to detect PD-L1 or (and) presence of PD-L2 or its It is horizontal.

Further, the present invention also provides a kind of pharmaceutical compositions, it includes the PD-1 films of above-mentioned high-affinity outside Region mutation body or its fusion protein；Optionally, further include pharmaceutically acceptable carrier and/or excipient.

Carrier of the present invention is pharmaceutically acceptable carrier, is referred to：One or more biocompatible solids Or liquid filler or gelatinous mass.They are suitable for people's use and it is necessary to have enough purity and sufficiently low toxicity." phase In capacitive " referred to herein as composition the active constituent of each component energy and the present invention and they between mutually admix, and it is unknown The aobvious drug effect for reducing active constituent.

Preferably, the carrier includes but not limited to：Diluent, buffer, suspension, emulsion, granule, encapsulation agents, Excipient, adhesive, spray, cutaneous permeable agent, wetting agent, disintegrant, sorbefacient, surfactant, filler Toner, corrigent or absorption carrier.

Preferably, the drug can be made including but not limited to microinjection agent, the dosage form suitable for transfection, injection, Tablet, pulvis, granula, capsule.The drug of above-mentioned various dosage forms can be prepared according to the conventional method of pharmaceutical field.

Further, the present invention also provides the PD-1 film outskirt mutant of the high-affinity or its fusion protein to make Purposes in standby following drug:

(1) drug for blocking PD-1 to be combined with PD-L1；

(2) drug for blocking PD-1 to be combined with PD-L2；

(3) while the drug that blocks PD-1 to be combined with PD-L2 with PD-L1 and PD-1；

(4) PD-L1 or/and PD-L2 activity or horizontal drug are adjusted；

(5) drug that PD-1 inhibits immunity of organism is released；Or

(6) drug of IFN mono- γ and/or IL-2 expression in T lymphocytes is improved.

Further, the present invention also provides the PD-1 film outskirt mutant of the high-affinity or its fusion protein to exist Prepare the purposes in the drug for preventing and/or treating tumour.

Preferably, the tumour be selected from melanoma, lung cancer in non-cellule type, colorectal cancer, kidney neoplasms, the dirty cancer of wing, Gastrointestinal cancer, prostate cancer, liver cancer, oophoroma and leukaemia.

Further, the present invention also provides a kind of kits comprising the PD-1 film outskirts of above-mentioned high-affinity Mutant or its fusion protein, the kit be used to detect PD-L1 or (and) presence of PD-L2 or it is horizontal.

Advantageous effect

The PD-1 film outskirt mutant or its fusion protein for having synthesized high-affinity of the invention, the fusion egg Affinity, the effect of bigger with higher combination human PD-L 1 or PD-L2 compared with wild type PD-1 in vain.The present invention's melts Hop protein provides more wide approach for the immunization therapy of tumour.

Description of the drawings

PD-1 and its variant sequence thereof comparison diagram prepared by Fig. 1 present invention；

PD-1 and its mutant fusion protein structural schematic diagram prepared by Fig. 2 present invention；

PD-1 and its mutant fusion protein coding DNA structural schematic diagram prepared by Fig. 3 present invention；

The PTT5 Vector maps that Fig. 4 present invention applies；

PD-1 and its mutant-IgG1-Fc fusion proteins prepared by Fig. 5 present invention is measured with PD-L1 combinations ELISA；A: The combination activity of M2C5-IgG1-Fc fusion proteins and PD-L1；B:The combination of M3H6-IgG1-Fc fusion proteins and PD-L1 are lived Property；C:The combination activity of M4B3-IgG1-Fc fusion proteins and PD-L1；D:The knot of M5G8-IgG1-Fc fusion proteins and PD-L1 Close activity；

PD-1 and its mutant fusion protein prepared by Fig. 6 present invention is measured with PD-L2 combinations ELISA；A:M2C5- The combination activity of IgG1-Fc fusion proteins and PD-L2；B:The combination activity of M3H6-IgG1-Fc fusion proteins and PD-L2；C: The combination activity of M4B3-IgG1-Fc fusion proteins and PD-L2；D:The combination of M5G8-IgG1-Fc fusion proteins and PD-L2 are lived Property；

The activity of PD-1 variant fusion proteins prepared by Fig. 7 present invention and PD-1/Fc-Biotin competitive bindings PD-L1 is surveyed It is fixed；A:The activity of M2C5-IgG1-Fc fusion protein competitive bindings PD-L1；B:M3H6-IgG1-Fc fusion protein competitive bindings The activity of PD-L1；C:The activity of M4B3-IgG1-Fc fusion protein competitive bindings PD-L1；D:M5G8-IgG1-Fc fusion proteins The activity of competitive binding PD-L1；

Fig. 8 present invention prepares the determination of activity of PD-1 variant fusion proteins and PD-1/Fc-Biotin competitive bindings PD-L2； A:The activity of M2C5-IgG1-Fc fusion protein competitive bindings PD-L2；B:M3H6-IgG1-Fc fusion protein competitive bindings PD-L2 Activity；C:The activity of M4B3-IgG1-Fc fusion protein competitive bindings PD-L2；D:M5G8-IgG1-Fc fusion proteins competition knot Close the activity of PD-L2；

PD-1 variant fusion proteins activation PBMC activity prepared by Fig. 9 present invention.

Specific implementation mode

Embodiment of the present invention is described in detail below in conjunction with embodiment.Those skilled in the art will manage Solution, the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Technology or condition person, it is (yellow such as with reference to works such as J. Pehanorm Brookers according to technology described in document in the art or condition What training hall etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Examination used Production firm person is not specified in agent or instrument, and being can be with conventional products that are commercially available.

Abbreviation PD-1 film outskirts mutant mentioned in the present invention, PD-1 variants all refer to the PD-1 film outskirts of high-affinity Mutant；The variant fusion proteins of the present invention refer to the fusion protein of the PD-1 film outskirt mutant of high-affinity.

The extracellular domain sequence of PD-1 sequences Q15116 in database UniProtKB of the wild type of the present invention.

Embodiment 1：The expression vector establishment of PD-1 variant-Fc fusion proteins

The amino acid sequence of 4 mutant of PD-1 films outskirt of wild type PD-1 and its high-affinity prepared by the present invention and Mutational site is as shown in Figure 1, the amino acid sequence of PD-1 is SEQ ID NO.1；The amino acid sequence of variant M2C5 is SEQ ID NO.2；The sequence of variant M3H6 amino acid is SEQ ID NO.3；The sequence of variant M4B3 amino acid is SEQ ID NO.4；Variant The amino acid sequence of M5G8 is SEQ ID NO.5.

PD-1 and its variant pass through linker (amino acid sequences：SEQ ID NO.6) and IgG1Fc (amino acid sequences：SEQ ID NO.8), IgG4Fc (amino acid sequences：SEQ ID NO.9) or 6 × His (amino acid sequences：SEQ ID NO.20) etc. even It connects to form fusion protein, protein structure schematic diagram is as shown in Figure 2.

For secreting, expressing PD-1 and its variant in mammalian cell, need to be added at its N sections in construction of expression vector Signal peptide (the amino acid sequence of secreting, expressing：SEQ ID NO.7), coding DNA structure is as shown in Figure 3.Using pTT5 conducts Expression vector (Fig. 4) is respectively synthesized DNA sequence dna (SEQ ID NO.10~SEQ of PD-1 and its mutant fusion protein coding ID NO.22, as shown in table 1).EcoRI restriction enzyme sites are introduced at 5 ' ends by Standard PCR, NotI digestions position is introduced at 3 ' ends Point is inserted between the EcoRI and NotI of PTT5 carriers, and structure obtains carrier for expression of eukaryon.

The amino acid and DNA sequence dna table of 1 PD-1 of table and its variant fusion proteins

Embodiment 2：The expression and purifying of PD-1 and its variant fusion proteins

Recombination PD-1 and its variant fusion are expressed by the transient transfection of 293E cells (purchase of Invitrogen companies) Albumen.By the 293E cells in exponential phase with 4 × 10⁵/ mL density is inoculated in shaking flask, sets 37 DEG C, 5% CO₂Shaking table 125r/min is transfected after cultivating 24 hours.

293E cells per 100mL take the OPTI-MEM of 5mL that the plasmid of 200 μ g, concussion mixing incubation at room temperature is added 5min obtains plasmid solution；The another PEI for taking the OPTI-MEM of 5mL that 600 μ g are added, concussion mixing are incubated at room temperature 5min, obtain PEI solution；Plasmid and PEI solution are mixed, concussion mixing is incubated at room temperature 20min, and reaction mixture is added dropwise in cell, is set 37 DEG C, 5% CO₂Shaking table 125r/min cultures, the 4th, 6 day flow feeding, the 8th day harvest supernatant.30 points are centrifuged with 14000g Clock and the cell culture supernatant for including antibody by (0.22 μm) filtering harvest of sterile filters.Using AKTA (GE companies) into Row isolates and purifies, and the Fc fusion proteins of expression cross Protein A affinity columns (MabSelect SuRe), and pH is in 3.4-3.6 The eluent of range (being monitored with 280nm) adjusts pH value to 6.0, and the fusion of PD-1 and its mutant is obtained after ultrafiltration concentration Albumen；6 × His fusion proteins of expression cross nickel column (HisTrap HP), are eluted by 200mM imidazoles, and buffer solution is replaced in ultrafiltration And 6 × His the fusion proteins obtained after concentrating.

Embodiment 3：PD-1 and its variant fusion proteins and PD-L1 combination activity analysis

Coating buffer dilutes PD-L1/His antigens (purchase is in Sino Biological, article No. 10084-H08H) to 1 μ g/ ML, 100 μ L are added per hole in elisa plate, are placed in wet box and stay overnight for 4 DEG C.Board-washing machine cleans elisa plate 3 times, 1.5% casein envelope It closes, per 200 μ L of hole, 37 DEG C of closing 1h of wet box.With the fusion protein of 1 × PBS dilution PD-1 and its mutant to 15 μ g/mL, and It after 3 times of gradient dilutions, is added in elisa plate per hole with 100 μ L, sheep is added in 37 DEG C of reaction 1h in wet box, cleaning elisa plate 3 times Anti-human Fc-HRP secondary antibodies react at room temperature 45min, clean elisa plate 5 times and the colour developing of 100 μ L tmb substrates is added, reaction 3min is used in combination 100μL 2N H₂SO₄Terminate reaction, enzyme-linked immunosorbent assay instrument 450nm readings.And using antibody concentration as abscissa, OD values are vertical sit Mark and draw antibody processed-antigen binding curve.It is fitted quadruplex parameters curve using Graphpad analysis softwares, equation is y=(A- D)/(1+ (X/C) ^B)+D, wherein B represent slope, and C represents EC50.

As a result：PD-1 and its mutant are combined activity such as Fig. 5 institutes with the fusion protein of IgG1-Fc with the ELISA of PD-L1 Show, PD-1 variants (M2C5, M3H6, M4B3 and M5G8) combined with PD-L1 activity far above wild type PD-1 (>1000 times)； Compared with PD-L1 antibody As tezolizumab, in conjunction with activity in the same order of magnitude, the combination activity of wherein M3H6 and M4B3 are slightly higher In Atezolizumab.

Embodiment 4：PD-1 and its mutant fusion protein and PD-L2 combination activity analysis

Coating buffer dilutes PD-L2/His antigens (purchase is in Sino Biological, article No. 10292-H08H) to 1 μ g/ ML, 100 μ L are added per hole in elisa plate, are placed in wet box and stay overnight for 4 DEG C.Board-washing machine cleans elisa plate 3 times, 1.5% casein envelope It closes, per 200 μ L of hole, 37 DEG C of closing 1h of wet box.With 1 × PBS dilution PD-1 (463) to 15 μ g/mL, and with 3 times of gradient dilutions after, And be added in elisa plate per hole with 100 μ L, goat-anti people's Fc-HRP secondary antibodies are added in 37 DEG C of reaction 1h in wet box, cleaning elisa plate 3 times 45min is reacted at room temperature, simultaneously the colour developing of 100 μ L tmb substrates is added in cleaning elisa plate 5 times, reacts 3min and with 100 μ L 2N H₂SO₄ Terminate reaction, enzyme-linked immunosorbent assay instrument 450nm readings.And using antibody concentration as abscissa, OD values are that ordinate drafting antibody-is anti- Former binding curve.Using Graphpad analysis softwares be fitted quadruplex parameters curve, equation be y=(A-D)/(1+ (X/C) ^B)+ D, wherein B represent slope, and C represents EC50.

As a result：PD-1 and its variant combined with the fusion protein of IgG1-Fc with the ELISA of PD-L2 activity as shown in fig. 6, PD-1 variants (M2C5, M3H6, M4B3 and M5G8) combined with PD-L2 activity far above wild type PD-1 (>300 times)；And PD- L1 antibody As tezolizumab is not combined substantially with PD-L2.

The activity of 5 competitive binding PD-L1 of embodiment

Coating buffer dilutes PD-L1/Fc antigens (purchase is in Sino Biological, article No. 10084-H02H) to 1 μ g/mL, 100 μ L are added per hole in elisa plate, are placed in wet box and stay overnight for 4 DEG C.Board-washing machine cleans elisa plate 3 times, the closing of 1.5% casein, Per 200 μ L of hole, 37 DEG C of closing lh of wet box.With 1 × PBS dilute PD-1/Fc-Biotin to 1.25 μ g/mL, using above-mentioned solution as Diluted antibody carries out 2 times of dilutions and obtains 12 concentration gradients altogether to 100 μ g/mL of antibody concentration.100 μ L add per hole Enter in elisa plate 37 DEG C of reaction 1h in wet box, Peroxidase-Labeled Streptavidin are added in cleaning elisa plate 3 times 45min is reacted at room temperature, simultaneously the colour developing of 100 μ L tmb substrates is added in cleaning elisa plate 5 times, reacts 3min and with 100 μ L 2N H₂SO₄ Terminate reaction, enzyme-linked immunosorbent assay instrument 450nm readings.Using antibody concentration as abscissa, OD values are that ordinate draws binding curve.

As a result：The fusion protein of PD-1 and its variant and IgG1-Fc and PD-1/Fc-Biotin competitive binding PD-L1, Competitive ELISA combines activity as shown in fig. 7, the competition activity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) is far above wild The PD-1 of type.

The activity of 6 competitive binding PD-L2 of embodiment

Coating buffer dilutes PD-L2/Fc antigens (purchase is in Sino Biological, article No. 10292-H02H) to 1 μ g/mL, 100 μ L are added per hole in elisa plate, are placed in wet box and stay overnight for 4 DEG C.Board-washing machine cleans elisa plate 3 times, the closing of 1.5% casein, Per 200 μ L of hole, 37 DEG C of closing lh of wet box.PD-1/Fc-Biotin to 5 μ g/mL is diluted with 1 × PBS, using above-mentioned solution as dilute It releases liquid and dilutes antibody to 100 μ g/mL of antibody concentration, and carry out 2 times of dilutions and obtain 12 concentration gradients altogether.100 μ L are added per hole Peroxidase-Labeled Streptavidin (1 are added in 37 DEG C of reaction 1h in wet box, cleaning elisa plate 3 times in elisa plate: 4000) 45min is reacted at room temperature, simultaneously the colour developing of 100 μ L tmb substrates is added in cleaning elisa plate 5 times, reacts 3min and with 100 μ L 2N H₂SO₄Terminate reaction, enzyme-linked immunosorbent assay instrument 450nm readings.Using antibody concentration as abscissa, OD values are that ordinate draws combination Curve.

As a result：The fusion protein of PD-1 and its variant and IgG1-Fc and PD-1/Fc-Biotin competitive binding PD-L2, Competitive ELISA combines activity as shown in figure 8, the competition activity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) is far above wild The PD-1 of type.

7 affinity of embodiment detects

The combination power of antibody and PD-L1 and PD-L2 antigens is measured using surface plasma resonance biosensor And affinity.Unless otherwise indicated, all reagents and material can be bought from GE companies, and can be surveyed at 25 DEG C Amount.Affinity analysis on SPR (Biacore T200) instrument by being analyzed, in CM5 chips by way of amino coupled It is coupled the antibody of anti-human igg Fc, each test antibodies are flowed into the flow velocity of 30 μ L/min, it is anti-human on chip with being coupled to The antibody capture test antibodies of IgG Fc；After analyte (PD-L1 or PD-L2) gradient dilution (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and 0nM), it is flowed into the flow velocity of 30 μ L/min of flow velocity, test antibodies and analyte binding time 120s, Dissociation time 1200s；Entire experiment uses HBS-EP as running buffer, chip 10mM glycine HCl, pH 2.1 60 pulse per second (PPS) of solution is regenerated.Determination data is fitted to 1:1 binding model, to measure equilibrium dissociation constant KD.

As a result:The equilibrium dissociation constant KD measurement results of PD-1 and its variant see the table below 2, PD-1 variants (M2C5, M3H6, M4B3 and M5G8) all it is far above the PD-1 of wild type with the affinity of PD-L1 and PD-L2.

2 PD-1 variant affinity determination values of table

Embodiment 8PBMC activation experiments

Take the mixing of 5 healthy volunteer's whole bloods, using human lymphocyte separating liquid (purchase is in Solarbio | article No.： P8610 human peripheral blood mononuclear cell PBMC (Peripheral Blood Mononuclear Cells) cell, physiology salt) are detached Wash cell 2 times, 1640 culture mediums of 10%FBS are resuspended cell and count, with 1 × 10⁵/ hole is inoculated in 96 orifice plates, is connect per hole 50 μ L of kind.1640 culture mediums of 10%FBS configure most suitable antibody irritaiting concentration (the final concentration of 1ug/ml of anti-CD3/CD28) with And PD-L1 inhibition concentrations (final concentration of 10 μ g/ml) are added in corresponding hole.Prepare PD1 variants and the sample of Atezolizumab Corresponding reaction system is added to 0 μ g/mL, 0.625 μ g/mL, 2.5 μ g/mL, 10 μ g/mL, 40 μ g/mL in product final concentration, and 37 DEG C carefully Born of the same parents incubator culture 72h is harvested supernatant and is measured the expression feelings of IFN-γ in supernatant using people's IFN-γ ELISA detection kit Condition.

The result shows that as shown in figure 9, the activity of PD1 variants activation PBMC and release IFN-γ is better than or resists with PD-L1 Body Atezolizumab is almost the same.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

SEQUENCE LISTING

<110>Jiangsu east antibiont Pharmaceutical Technology Co., Ltd

<120>The fusion protein and its pharmaceutical composition and purposes of a kind of PD-1 film outskirt mutant of high-affinity

<130> 18040

<160> 22

<170> PatentIn version 3.5

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Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

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Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

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Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

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Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

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Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

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Leu Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

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Leu Ala Pro Lys Ser Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

100 105 110

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

115 120 125

Pro

<210> 3

<211> 129

<212> PRT

<213> M3H6

<400> 3

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

1 5 10 15

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

20 25 30

Leu Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

35 40 45

Ala Ala Phe Pro Glu Asp Tyr Asn Gln Pro Val Gln Asp Cys Arg Phe

50 55 60

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

65 70 75 80

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Ile Cys Gly Ala Ile Ser

85 90 95

Leu Gly Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

100 105 110

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

115 120 125

Pro

<210> 4

<211> 129

<212> PRT

<213> M4B3

<400> 4

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

1 5 10 15

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

20 25 30

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

35 40 45

Ala Ala Phe Pro Glu Asp Arg Asn Gln Pro Leu Gln Asp Cys Arg Phe

50 55 60

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

65 70 75 80

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Tyr

85 90 95

Leu Gly Pro Lys Val Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

100 105 110

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

115 120 125

Pro

<210> 5

<211> 129

<212> PRT

<213> M5G8

<400> 5

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

1 5 10 15

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

20 25 30

Leu Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

35 40 45

Ala Ala Phe Pro Glu Asp Lys Asn Gln Pro Leu Gln Asp Cys Arg Phe

50 55 60

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

65 70 75 80

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Tyr

85 90 95

Leu Gly Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

100 105 110

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

115 120 125

Pro

<210> 6

<211> 6

<212> PRT

<213> linker

<400> 6

Gly Gly Gly Gly Gly Ser

1 5

<210> 7

<211> 20

<212> PRT

<213> signal peptide

<400> 7

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 8

<211> 227

<212> PRT

<213> IgG1 Fc

<400> 8

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 9

<211> 229

<212> PRT

<213> IgG4 Fc

<400> 9

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 10

<211> 1146

<212> DNA

<213> PD-1-IgG1 Fc DNA

<400> 10

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcgtgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg accggagcca gcccggccag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatcagcct ggcccccaag 360

gcccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgacaa gacccacacc 480

tgccccccct gccccgcccc cgagctgctg ggcggcccca gcgtgttcct gttccccccc 540

aagcccaagg acaccctgat gatcagccgg acccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggaccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccgggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccaccccc cccgtgctgg acagcgacgg cagcttcttc 1020

ctgtacagca agctgaccgt ggacaagagc cggtggcagc agggcaacgt gttcagctgc 1080

agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgagcccc 1140

ggcaag 1146

<210> 11

<211> 1146

<212> DNA

<213> M2C5-IgG1 DNA

<400> 11

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcctgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg accggaacca gcccgcccag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac atctgcggcg ccatcagcct ggcccccaag 360

agccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgacaa gacccacacc 480

tgccccccct gccccgcccc cgagctgctg ggcggcccca gcgtgttcct gttccccccc 540

aagcccaagg acaccctgat gatcagccgg acccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggaccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccgggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccaccccc cccgtgctgg acagcgacgg cagcttcttc 1020

ctgtacagca agctgaccgt ggacaagagc cggtggcagc agggcaacgt gttcagctgc 1080

agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgagcccc 1140

ggcaag 1146

<210> 12

<211> 1146

<212> DNA

<213> M3H6-IgG1 DNA

<400> 12

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcctgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg actacaacca gcccgtgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac atctgcggcg ccatcagcct gggccccaag 360

atccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgacaa gacccacacc 480

tgccccccct gccccgcccc cgagctgctg ggcggcccca gcgtgttcct gttccccccc 540

aagcccaagg acaccctgat gatcagccgg acccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggaccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccgggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccaccccc cccgtgctgg acagcgacgg cagcttcttc 1020

ctgtacagca agctgaccgt ggacaagagc cggtggcagc agggcaacgt gttcagctgc 1080

agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgagcccc 1140

ggcaag 1146

<210> 13

<211> 1146

<212> DNA

<213> M4B3-IgG1 DNA

<400> 13

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcgtgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg accggaacca gcccctgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatctacct gggccccaag 360

gtgcagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgacaa gacccacacc 480

tgccccccct gccccgcccc cgagctgctg ggcggcccca gcgtgttcct gttccccccc 540

aagcccaagg acaccctgat gatcagccgg acccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggaccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccgggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccaccccc cccgtgctgg acagcgacgg cagcttcttc 1020

ctgtacagca agctgaccgt ggacaagagc cggtggcagc agggcaacgt gttcagctgc 1080

agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgagcccc 1140

ggcaag 1146

<210> 14

<211> 1146

<212> DNA

<213> M5G8-IgG1 DNA

<400> 14

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcctgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg acaagaacca gcccctgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatctacct gggccccaag 360

gcccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgacaa gacccacacc 480

tgccccccct gccccgcccc cgagctgctg ggcggcccca gcgtgttcct gttccccccc 540

aagcccaagg acaccctgat gatcagccgg acccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggaccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccgggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccaccccc cccgtgctgg acagcgacgg cagcttcttc 1020

ctgtacagca agctgaccgt ggacaagagc cggtggcagc agggcaacgt gttcagctgc 1080

agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgagcccc 1140

ggcaag 1146

<210> 15

<211> 1152

<212> DNA

<213> PD-1-IgG4

<400> 15

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcgtgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg accggagcca gcccggccag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatcagcct ggcccccaag 360

gcccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgagag caagtacggc 480

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gccccagcgt gttcctgttc 540

ccccccaagc ccaaggacac cctgatgatc agccggaccc ccgaggtgac ctgcgtggtg 600

gtggacgtga gccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 660

gtgcacaacg ccaagaccaa gccccgggag gagcagttca acagcaccta ccgggtggtg 720

agcgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 780

agcaacaagg gcctgcccag cagcatcgag aagaccatca gcaaggccaa gggccagccc 840

cgggagcccc aggtgtacac cctgcccccc agccaggagg agatgaccaa gaaccaggtg 900

agcctgacct gcctggtgaa gggcttctac cccagcgaca tcgccgtgga gtgggagagc 960

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggacag cgacggcagc 1020

ttcttcctgt acagccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttc 1080

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1140

agcctgggca ag 1152

<210> 16

<211> 1152

<212> DNA

<213> M2C5-IgG4

<400> 16

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcctgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg accggaacca gcccgcccag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac atctgcggcg ccatcagcct ggcccccaag 360

agccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgagag caagtacggc 480

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gccccagcgt gttcctgttc 540

ccccccaagc ccaaggacac cctgatgatc agccggaccc ccgaggtgac ctgcgtggtg 600

gtggacgtga gccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 660

gtgcacaacg ccaagaccaa gccccgggag gagcagttca acagcaccta ccgggtggtg 720

agcgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 780

agcaacaagg gcctgcccag cagcatcgag aagaccatca gcaaggccaa gggccagccc 840

cgggagcccc aggtgtacac cctgcccccc agccaggagg agatgaccaa gaaccaggtg 900

agcctgacct gcctggtgaa gggcttctac cccagcgaca tcgccgtgga gtgggagagc 960

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggacag cgacggcagc 1020

ttcttcctgt acagccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttc 1080

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1140

agcctgggca ag 1152

<210> 17

<211> 1152

<212> DNA

<213> M3H6-IgG4

<400> 17

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcctgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg actacaacca gcccgtgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac atctgcggcg ccatcagcct gggccccaag 360

atccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgagag caagtacggc 480

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gccccagcgt gttcctgttc 540

ccccccaagc ccaaggacac cctgatgatc agccggaccc ccgaggtgac ctgcgtggtg 600

gtggacgtga gccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 660

gtgcacaacg ccaagaccaa gccccgggag gagcagttca acagcaccta ccgggtggtg 720

agcgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 780

agcaacaagg gcctgcccag cagcatcgag aagaccatca gcaaggccaa gggccagccc 840

cgggagcccc aggtgtacac cctgcccccc agccaggagg agatgaccaa gaaccaggtg 900

agcctgacct gcctggtgaa gggcttctac cccagcgaca tcgccgtgga gtgggagagc 960

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggacag cgacggcagc 1020

ttcttcctgt acagccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttc 1080

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1140

agcctgggca ag 1152

<210> 18

<211> 1152

<212> DNA

<213> M4B3-IgG4

<400> 18

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcgtgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg accggaacca gcccctgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatctacct gggccccaag 360

gtgcagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgagag caagtacggc 480

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gccccagcgt gttcctgttc 540

ccccccaagc ccaaggacac cctgatgatc agccggaccc ccgaggtgac ctgcgtggtg 600

gtggacgtga gccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 660

gtgcacaacg ccaagaccaa gccccgggag gagcagttca acagcaccta ccgggtggtg 720

agcgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 780

agcaacaagg gcctgcccag cagcatcgag aagaccatca gcaaggccaa gggccagccc 840

cgggagcccc aggtgtacac cctgcccccc agccaggagg agatgaccaa gaaccaggtg 900

agcctgacct gcctggtgaa gggcttctac cccagcgaca tcgccgtgga gtgggagagc 960

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggacag cgacggcagc 1020

ttcttcctgt acagccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttc 1080

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1140

agcctgggca ag 1152

<210> 19

<211> 1152

<212> DNA

<213> M5G8-IgG4

<400> 19

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcctgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg acaagaacca gcccctgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatctacct gggccccaag 360

gcccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagcgagag caagtacggc 480

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gccccagcgt gttcctgttc 540

ccccccaagc ccaaggacac cctgatgatc agccggaccc ccgaggtgac ctgcgtggtg 600

gtggacgtga gccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 660

gtgcacaacg ccaagaccaa gccccgggag gagcagttca acagcaccta ccgggtggtg 720

agcgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 780

agcaacaagg gcctgcccag cagcatcgag aagaccatca gcaaggccaa gggccagccc 840

cgggagcccc aggtgtacac cctgcccccc agccaggagg agatgaccaa gaaccaggtg 900

agcctgacct gcctggtgaa gggcttctac cccagcgaca tcgccgtgga gtgggagagc 960

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggacag cgacggcagc 1020

ttcttcctgt acagccggct gaccgtggac aagagccggt ggcaggaggg caacgtgttc 1080

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1140

agcctgggca ag 1152

<210> 20

<211> 6

<212> PRT

<213> 6×His

<400> 20

His His His His His His

1 5

<210> 21

<211> 483

<212> DNA

<213> M4B3-6×His

<400> 21

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcgtgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg accggaacca gcccctgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatctacct gggccccaag 360

gtgcagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagccacca ccaccaccac 480

cac 483

<210> 22

<211> 483

<212> DNA

<213> M5G8-6×His

<400> 22

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60

tggaaccccc ccaccttcag ccccgccctg ctggtggtga ccgagggcga caacgccacc 120

ttcacctgca gcttcagcaa caccagcgag agcttcctgc tgaactggta ccggatgagc 180

cccagcaacc agaccgacaa gctggccgcc ttccccgagg acaagaacca gcccctgcag 240

gactgccggt tccgggtgac ccagctgccc aacggccggg acttccacat gagcgtggtg 300

cgggcccggc ggaacgacag cggcacctac ctgtgcggcg ccatctacct gggccccaag 360

gcccagatca aggagagcct gcgggccgag ctgcgggtga ccgagcggcg ggccgaggtg 420

cccaccgccc accccagccc cagccccggc ggcggcggcg gcagccacca ccaccaccac 480

cac 483

Claims

1. a kind of PD-1 film outskirt mutant of high-affinity, the PD-1 films outskirt mutant include selected from SEQ ID NO.2, The amino acid sequence of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

2. a kind of fusion protein of the PD-1 film outskirt mutant of high-affinity, the fusion protein includes being selected from SEQ ID The albumen of the amino acid sequence of NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 can also include optionally Albumen with other functions；The fusion protein improves at least 50 times with the affinity of PD-L1 compared with wild type PD-1；Institute It states fusion protein and improves at least 10 times compared with wild type PD-1 with the affinity of PD-L2.

3. fusion protein as claimed in claim 2, which is characterized in that the fusion protein further includes Fc segments, the Fc pieces Section is selected from the Fc and its saltant type of human IgG1 or IgG4, wherein the amino acid sequence such as SEQ of the N298A mutant of human IgG1 Fc Shown in ID NO.8, wherein the amino acid sequence of the S228P mutant of human IgG 4Fc is as shown in SEQ ID NO.9.

4. fusion protein as claimed in claim 2, which is characterized in that the fusion protein further includes 6 × His, described 6 × The amino acid sequence of His such as SEQ ID NO.20.

5. fusion protein as described in claim 3 or 4, which is characterized in that the fusion protein further includes Linker, described PD-1 film outskirt mutant connects into fusion protein, the amino acid sequence of the linker by Linker and Fc segments, 6 × His Row are as shown in SEQ ID NO.6.

6. a kind of biomaterial of any one of coding claim 2 to 5 DNA sequence dna of the fusion protein, which is characterized in that The biomaterial is carrier, host cell or kit.

7. a kind of pharmaceutical composition, it includes region mutations outside the PD-1 films of the high-affinity described in any one of claim 1 to 5 Body or its fusion protein；Optionally, further include pharmaceutically acceptable carrier and/or excipient.

8. PD-1 film outskirt mutant or its fusion protein comprising any one of claim 1 to 5 high-affinity are being made Purposes in standby following drug:

(1) drug for blocking PD-1 to be combined with PD-L1；

(2) drug for blocking PD-1 to be combined with PD-L2；

(4) PD-L1 or/and PD-L2 activity or horizontal drug are adjusted；

(5) drug that PD-1 inhibits immunity of organism is released；Or

(6) drug of IFN mono- γ and/or IL-2 expression in T lymphocytes is improved.

9. prepared by the PD-1 film outskirt mutant of high-affinity according to any one of claims 1 to 6 or its fusion protein Purposes in the drug of prevention and/or treatment tumour.

10. a kind of kit, which is characterized in that it includes the PD-1 films of high-affinity according to any one of claims 1 to 6 Outskirt mutant or its fusion protein, the kit be used to detect PD-L1 or (and) presence of PD-L2 or it is horizontal.