CN108752460B

CN108752460B - High-affinity fusion protein of PD-1 extracellular domain mutant and pharmaceutical composition and application thereof

Info

Publication number: CN108752460B
Application number: CN201810582025.5A
Authority: CN
Inventors: 赵健; 魏化伟; 黄亚杰
Original assignee: Jiangsu Dongkang Biomedical Technology Co ltd
Current assignee: Jiangsu Dongkang Biomedical Technology Co ltd
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2022-05-10
Anticipated expiration: 2038-06-07
Also published as: CN108752460A; CN114181297B; CN114031682A; CN114181296A; CN114181296B; CN114181297A; CN114031682B

Abstract

The invention discloses a high-affinity PD-1 extracellular domain mutant, which comprises an amino acid sequence selected from SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. The invention further provides a fusion protein of the high-affinity PD-1 extracellular domain mutant and application thereof. The invention creatively synthesizes a high-affinity PD-1 extracellular domain mutant or a fusion protein thereof, and the fusion protein has higher affinity and higher efficacy for binding human PD-L1 or PD-L2 compared with wild type PD-1. The fusion protein of the invention provides a wider approach for the immunotherapy of tumors.

Description

High-affinity fusion protein of PD-1 extracellular domain mutant and pharmaceutical composition and application thereof

Technical Field

The invention relates to the fields of tumor treatment and molecular immunology, and relates to a high-affinity fusion protein of a PD-1 extracellular domain mutant, a pharmaceutical composition and application thereof.

Background

The immunotherapy of tumor is to stimulate and enhance the immune function of the organism to achieve the purpose of controlling and killing tumor cells, the immunotherapy of tumor is one of the hot spots in the current tumor therapy field, and the clinical test also obtains obvious treatment effect. PD-1 and a ligand PD-L1/L2 thereof mediate the effects of inhibiting 'exhaustion' of T cells and inducing immune tolerance, and PD-L1/L2 highly expressed on the surface of tumor cells interacts with PD-1 to cause the immune escape of the tumor cells, so that a signal path from PD1 to PD-L1/L2 is inhibited, and the reactivated inhibited immune system becomes a hot spot of recent immunotherapy.

Programmed death receptor 1(PD-1, also known as PDCD1 and CD279) was discovered in 1992 by the professor "Canjo Tasuku" of Kyoto university, Kyoto, Japan. PD-1 is divided into three parts of an extracellular region, a transmembrane region and a cytoplasmic region, contains protein of 288 amino acids, and has a molecular weight of about 50-55 kD. The extracellular region contains an IgV-like structure domain, is a region which is combined with a ligand of PD-1 and further induces an immune response function, and contains 4N glycosylation sites; the cytoplasmic domain contains 1 ITIM (Immunoreceptor tyrosine-based inhibition motif), and 1 ITSM (Immunoreceptor tyrosine-based switch motif), wherein activation of ITSM is closely associated with an effector T cell immune response. PD-1 is mainly expressed on activated T cells, B cells and myeloid cells, plays an important role in maintaining the exhaustion of the T cells and has an effect of inhibiting the T cells, so that chronic pathogens and tumor cells use a PD-1 channel to escape immune response.

PD-L1 protein: the chinese scientist professor was found in 1999 to B7-H1, which was subsequently demonstrated to specifically bind PD-1 in 2000 and was named PD-L1, a programmed death molecule ligand-1 (also known as CD274 and B7-H1); PD-L1 is divided into three parts of an extracellular region, a hydrophobic region of a transmembrane region and a cytoplasmic region and consists of 290 amino acid subunits; wherein the extracellular region comprises two immunoglobulin constant regions IgC and IgV-like domains and the cytoplasmic region consists of 30 amino acids. PD-L1 is expressed primarily in mature CD4⁺、CD8⁺Hematopoietic cells such as T cells, B cells, and dendritic cells; meanwhile, PD-L1 is often highly expressed on the surface of various tumor cells, such as melanoma, non-small cell lung cancer, breast cancer, ovarian cancer, head and neck squamous cell carcinoma and the like, and meanwhile, the high expression of the tumor cells PD-L1 is related to the poor prognosis of tumor patients, the recurrence of tumors, and the correlation among the tumor size, the lymph node involvement, the classification and the overall survival period.

PD-L2 protein: another ligand PD-L2 of PD-1, namely programmed death molecule ligand-2 (also called CD273 and B7-DC), was discovered in 2001, PD-L2 is transmembrane protein consisting of 274 amino acid residues, PD-L2 and PD-L1 have high similarity, and the interaction between PD-L2 and PD-1 can inhibit the proliferation of T cells, the generation of cytokines and the lysis of T cells, but the affinity of PD-L2 and PD-1 is 2-6 times that of PD-L1. PD-L2 was expressed on the membrane surface of macrophages, dendritic cells and some B cell subsets, while various methods detected expression of PD-L2 in tumor cells, some of which did not detect expression of PD-L1. Therefore, the medicine capable of effectively blocking PD-1-PD-L1 and PD-1-PD-L2 needs to be further opened.

In 2005, the small wild drug of Japan and the American Metarx drug were used together to develop the PD-1 antibody drug Nivolumab, and were taken into the capsule by Michelia Baishigui in 2009. In 2009, Mr. Sa Dong bought Xilingbaya to obtain the subsequent development right of PD-1 antibody drug Pembrolizumab. In 2014, Nivolumab of BaishiGuibao and Pembrolizumab of Moshadong in Baishi were successively approved for marketing.

In 2016, PD-L1 antibody Atezolizumab, gene Tak, obtained FDA approval for marketing; PD-1 antibody drug Bavenciio of Perey and Moshadong and PD-1 antibody drug Imfinzi of Aslicon were marketed sequentially in 2017. This tumor immunotherapy drug is considered by several major companies as a heavy product in the field of tumor immunotherapy for the next few years.

Although many drugs of PD-1 and PD-L1 antibodies are currently on the market, drugs based on fusion proteins of PD-1 are rarely reported; and drugs capable of simultaneously and efficiently blocking PD-1-PD-L1 and PD-1-PD-L2 are also urgently needed to be developed so as to more effectively block the signal path of PD-L.

The interaction between PD-L1/L2 with high expression on the surface of the tumor cell and PD-1 leads to the immune escape of the tumor cell, and the blocking of the pathway by using the antibody of PD-1 or PD-L1 can activate the immune system and further kill the tumor cell. Since the PD-L1/L2 and PD-1 are expressed on immune cells in the same manner, the PD-1/PD-L1 antibody is often designed as a blocking antibody without ADCC, CDC or the like, considering that the PD-1/PD-L1 antibody kills autologous immune cells if it has too strong ADCC, CDC, ADCP or the like activity. PD-1 antibodies such as Nivolumab of Baishimaibao and Pembrolizumab of Moshadong adopt IgG4 subtype with weak ADCC activity; the PD-L1 antibody Atezolizumab of gene tek adopts IgG1 subtype without glycosylation site, and basically has no ADCC activity; afrikang's Imfinzi removes ADCC activity by mutating three amino acids (L234F, L235E, P331S) of IgG 1.

While the Avelumab of the pfeire/merck adopts IgG1 subtype with ADCC, CDC and other activities, because PD-L1 has obvious high expression in tumor cells, the PD-L1 antibody with strong ADCC activity also has the advantage that the immune escape of cancer cells is relieved, and simultaneously, the ADCC function can be combined to mediate NK cells to kill the cancer cells.

Disclosure of Invention

The invention aims to provide a high-affinity PD-1 extracellular domain mutant and a fusion protein containing the high-affinity PD-1 extracellular domain mutant.

The invention also aims to provide a pharmaceutical composition containing the high-affinity PD-1 extracellular domain mutant or the fusion protein medicine thereof and application thereof.

In order to achieve the above objects, the present invention provides a high affinity PD-1 extracellular domain mutant, wherein the PD-1 extracellular domain mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. In one embodiment the PD-1 extracellular domain mutant is M2C5 having the amino acid sequence of SEQ ID No. 2; in one embodiment the PD-1 extracellular domain mutant is M3H6 having the amino acid sequence of SEQ ID No. 3; in one embodiment the PD-1 extracellular domain mutant is M4B3 having the amino acid sequence of SEQ ID No. 4; in one embodiment the PD-1 extracellular domain mutant is M5G8 having the amino acid sequence of SEQ ID No. 5.

Further, the invention also provides a high-affinity fusion protein of the PD-1 extracellular domain mutant, which comprises a protein of an amino acid sequence selected from SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and optionally, can also comprise proteins with other functions.

Preferably, the affinity of the fusion protein to PD-L1 is increased by at least 50-fold compared to wild-type PD-1; in one embodiment, the affinity of the fusion protein of the invention to PD-L1 is increased by more than 1000-fold compared to wild-type PD-1; the affinity of the fusion protein to PD-L2 is improved by at least 10 times compared with wild type PD-1; in one embodiment, the affinity of the fusion protein of the invention for PD-L2 is increased by more than 300-fold compared to wild-type PD-1.

Preferably, the fusion protein further comprises an Fc fragment selected from Fc of human IgG1 or IgG4 and a mutant thereof, wherein the amino acid sequence of the N298A mutant of human IgG1Fc is shown as SEQ ID NO.8, and the amino acid sequence of the S228P mutant of human IgG4Fc is shown as SEQ ID NO. 9.

Preferably, the fusion protein further comprises 6 XHis, and the amino acid sequence of 6 XHis is shown in SEQ ID No. 20.

Preferably, the PD-1 extracellular domain mutant is connected with an Fc fragment and 6 XHis through a Linker to form a fusion protein, and the amino acid sequence of the Linker is shown in SEQ ID NO. 6.

Further, the invention also provides a biological material encoding the DNA sequence of the fusion protein, which is a vector, a host cell or a kit, such as for detecting the presence or level of PD-L1 or (and) PD-L2.

Further, the invention also provides a pharmaceutical composition, which comprises the high-affinity PD-1 extracellular domain mutant or the fusion protein thereof; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

The carrier provided by the invention is a pharmaceutically acceptable carrier, and refers to: one or more compatible solid or liquid fillers or gel substances. They are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient.

Preferably, the vectors include, but are not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavors, or adsorptive carriers.

Preferably, the medicament can be prepared into a dosage form including but not limited to microinjection, transfection-suitable dosage forms, injection, tablets, powder, granules and capsules. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

Further, the invention also provides an application of the high-affinity PD-1 extracellular domain mutant or the fusion protein thereof in preparing the following medicaments:

(1) (ii) an agent that blocks the binding of PD-1 to PD-L1;

(2) (ii) an agent that blocks the binding of PD-1 to PD-L2;

(3) drugs that simultaneously block the binding of PD-1 to PD-L1 and PD-1 to PD-L2;

(4) agents that modulate the activity or level of PD-L1 or/and PD-L2;

(5) medicine for relieving the immunosuppression of PD-1 on the organism; or

(6) An agent that increases the expression of IFN- γ and/or IL-2 in T lymphocytes.

Furthermore, the invention also provides application of the high-affinity PD-1 extracellular domain mutant or the fusion protein thereof in preparing a medicament for preventing and/or treating tumors.

Preferably, the tumor is selected from melanoma, non-small cell lung cancer, colorectal cancer, renal tumor, cancer of the bladder, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer and leukemia.

Still further, the present invention provides a kit comprising the high affinity PD-1 extracellular domain mutant or fusion protein thereof described above for detecting the presence or level of PD-L1 or (and) PD-L2.

Advantageous effects

The invention creatively synthesizes a high-affinity PD-1 extracellular domain mutant or a fusion protein thereof, and the fusion protein has higher affinity and higher efficacy for binding human PD-L1 or PD-L2 compared with wild type PD-1. The fusion protein of the invention provides a wider approach for the immunotherapy of tumors.

Drawings

FIG. 1 is a sequence alignment of PD-1 and variants thereof prepared according to the invention;

FIG. 2 is a schematic diagram of the structure of PD-1 and its mutant fusion protein prepared by the present invention;

FIG. 3 is a schematic diagram of the structure of the DNA encoding PD-1 and its mutant fusion protein prepared by the present invention;

FIG. 4 is a PTT5 carrier map used in the present invention;

FIG. 5 shows that PD-1 and its mutant-IgG 1-Fc fusion protein prepared by the invention is measured by combining with PD-L1 by ELISA; binding activity of M2C5-IgG1-Fc fusion protein to PD-L1; binding activity of M3H6-IgG1-Fc fusion protein to PD-L1; binding activity of M4B3-IgG1-Fc fusion protein to PD-L1; binding activity of M5G8-IgG1-Fc fusion protein to PD-L1;

FIG. 6 shows that PD-1 and its mutant fusion protein prepared by the present invention are tested by ELISA in combination with PD-L2; binding activity of M2C5-IgG1-Fc fusion protein to PD-L2; binding activity of M3H6-IgG1-Fc fusion protein to PD-L2; binding activity of M4B3-IgG1-Fc fusion protein to PD-L2; binding activity of M5G8-IgG1-Fc fusion protein to PD-L2;

FIG. 7 determination of the activity of PD-1 variant fusion proteins prepared according to the invention in competing with PD-1/Fc-Biotin for binding to PD-L1; M2C5-IgG1-Fc fusion protein competes for binding to PD-L1 activity; M3H6-IgG1-Fc fusion protein competes for binding to PD-L1 activity; M4B3-IgG1-Fc fusion protein competes for binding to PD-L1 activity; M5G8-IgG1-Fc fusion protein competes for binding to PD-L1 activity;

FIG. 8 Activity assay of PD-1 variant fusion proteins prepared in accordance with the present invention in competition with PD-1/Fc-Biotin for binding to PD-L2; M2C5-IgG1-Fc fusion protein competes for binding to the activity of PD-L2; M3H6-IgG1-Fc fusion protein competes for binding to PD-L2 activity; M4B3-IgG1-Fc fusion protein competes for binding to PD-L2 activity; M5G8-IgG1-Fc fusion protein competes for binding to PD-L2 activity;

FIG. 9 the PD-1 variant fusion proteins prepared in accordance with the invention activate PBMC activity.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The short-term PD-1 extracellular domain mutant and the PD-1 variant mentioned in the invention both refer to high-affinity PD-1 extracellular domain mutant; the variant fusion protein of the invention refers to a fusion protein of a high-affinity PD-1 extracellular domain mutant.

The wild-type PD-1 sequence used in the invention is derived from the extracellular region sequence of Q15116 in UniProtKB database.

Example 1: expression vector construction of PD-1 variant-Fc fusion protein

The amino acid sequences and mutation sites of the wild type PD-1 and 4 mutants of the PD-1 extracellular region with high affinity prepared by the invention are shown in figure 1, and the amino acid sequence of the PD-1 is SEQ ID NO. 1; the amino acid sequence of the variant M2C5 is SEQ ID NO. 2; the sequence of the variant M3H6 amino acid is SEQ ID NO. 3; the sequence of the amino acid of the variant M4B3 is SEQ ID NO. 4; the amino acid sequence of variant M5G8 is SEQ ID NO. 5.

PD-1 and its variant are connected with IgG1Fc (amino acid sequence: SEQ ID NO.8), IgG4Fc (amino acid sequence: SEQ ID NO.9) or 6 XHis (amino acid sequence: SEQ ID NO.20) by linker (amino acid sequence: SEQ ID NO.6) to form fusion protein, and the protein structure schematic diagram is shown in FIG. 2.

In order to realize secretory expression of PD-1 and the variant thereof in mammalian cells, a signal peptide (amino acid sequence: SEQ ID NO.7) for secretory expression is added to the N segment of the expression vector when the expression vector is constructed, and the structure of the coding DNA is shown in figure 3. The DNA sequences (SEQ ID NO. 10-SEQ ID NO.22, shown in Table 1) encoded by PD-1 and its mutant fusion proteins were synthesized separately using pTT5 as an expression vector (FIG. 4). EcoRI restriction site is introduced into the 5 'end by conventional PCR, NotI restriction site is introduced into the 3' end, and the restriction site is inserted between EcoRI and NotI of PTT5 vector to construct and obtain eukaryotic expression vector.

TABLE 1 amino acid and DNA sequence listing of PD-1 and its variant fusion proteins

Example 2: expression and purification of PD-1 and variant fusion proteins thereof

Recombinant PD-1 and its variant fusion proteins were expressed by transient transfection of 293E cells (purchased from Invitrogen). 293E cells in logarithmic growth phase at 4X 10⁵Inoculating in shake flask at a density of/mL, and placing in 5% CO at 37 deg.C₂After culturing for 24 hours at 125r/min by a shaker, transfection is carried out.

Every 100mL of 293E cells, 5mL of OPTI-MEM is added into 200 μ g of plasmid, and the mixture is shaken, mixed uniformly and incubated at room temperature for 5min to obtain a plasmid solution; adding another 5mL of OPTI-MEM into 600 mu g of PEI, shaking, uniformly mixing, and incubating at room temperature for 5min to obtain a PEI solution; mixing the plasmid and PEI solution, shaking, mixing, incubating at room temperature for 20min, adding dropwise the reaction mixture into cells, standing at 37 deg.C and 5% CO₂Culturing at 125r/min in a shaking table, feeding materials on days 4 and 6, and harvesting the supernatant on day 8. Cell culture supernatants containing the antibodies were harvested by centrifugation at 14000g for 30 minutes and filtration through sterile filters (0.22 μm). Separating and purifying by AKTA (GE company), passing the expressed Fc fusion Protein through Protein A affinity chromatography column (Mabselect SuRe), eluting with eluent with pH of 3.4-3.6 (monitored by 280 nm), adjusting pH to 6.0, ultrafiltering and concentrating to obtain PD-1 and its mutant fusion Protein; the expressed 6 XHis fusion protein was passed through a nickel column (HisTrap HP), eluted with 200mM imidazole, and the buffer was replaced by ultrafiltration and concentrated to obtain 6 XHis fusion protein.

Example 3: analysis of binding Activity of PD-1 and its variant fusion proteins to PD-L1

The coating diluted PD-L1/His antigen (purchased from Nano Biological, cat. 10084-H08H) to 1. mu.g/mL, 100. mu.L per well was added to the enzyme-linked plate and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, 1.5% casein block, 200 μ L per well, block for 1h at 37 ℃ in a wet box. Diluting PD-1 and its mutant fusion protein with 1 × PBS to 15 μ g/mL, diluting with 3 times of gradient, adding 100 μ L per well into the enzyme-linked plate, reacting at 37 deg.C for 1h in a wet box, washing the enzyme-linked plate 3 times, adding goat anti-human Fc-HRP secondary antibody chamberReacting at room temperature for 45min, washing enzyme linked plate for 5 times, adding 100 μ L TMB substrate for color development, reacting for 3min, and reacting with 100 μ L2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm. And an antibody-antigen binding curve is drawn by taking the antibody concentration as an abscissa and the OD value as an ordinate. A four parameter equation curve was fitted using Graphpad analysis software, the equation being y ═ a-D)/(1+ (X/C) ^ B) + D, where B represents slope and C represents EC 50.

As a result: the ELISA binding activity of the fusion protein of PD-1 and its mutant and IgG1-Fc and PD-L1 is shown in FIG. 5, and the binding activity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) and PD-L1 is much higher than that of wild type PD-1(>1000 times); compared with the PD-L1 antibody Atezolidumab, the binding activity is in the same order of magnitude, wherein the binding activity of M3H6 and M4B3 is slightly higher than that of Atezolidumab.

Example 4: analysis of binding activity of PD-1 and mutant fusion protein thereof to PD-L2

The coating diluted PD-L2/His antigen (purchased from Nano Biological, cat. No. 10292-H08H) to 1. mu.g/mL, 100. mu.L per well was added to the enzyme conjugate plate and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, 1.5% casein block, 200 μ L per well, block for 1h at 37 ℃ in a wet box. PD-1(463) was diluted to 15. mu.g/mL with 1 XPBS and diluted in a 3-fold gradient, and added to an enzyme-linked plate at 100. mu.L per well, reacted at 37 ℃ for 1h in a wet box, the enzyme-linked plate was washed 3 times, a goat anti-human Fc-HRP secondary antibody was added and reacted at room temperature for 45min, the enzyme-linked plate was washed 5 times and developed with 100. mu.L of TMB substrate, reacted for 3min and reacted with 100. mu.L of 2N H₂SO₄The reaction is stopped, and the enzyme linked immunosorbent assay instrument reads 450 nm. And an antibody-antigen binding curve is drawn by taking the antibody concentration as an abscissa and the OD value as an ordinate. A four parameter equation curve was fitted using Graphpad analysis software, the equation being y ═ a-D)/(1+ (X/C) ^ B) + D, where B represents slope and C represents EC 50.

As a result: ELISA binding activity of PD-1 and its variants and IgG1-Fc fusion protein with PD-L2 As shown in FIG. 6, PD-1 variants (M2C5, M3H6, M4B3 and M5G8) bind PD-L2 much more than wild-type PD-1(>300 fold); while the PD-L1 antibody Atezolizumab did not bind substantially to PD-L2.

Example 5 Activity of competitive binding to PD-L1

The coating solution diluted PD-L1/Fc antigen (purchased from Sino Biological, cat. No. 10084-H02H) to 1. mu.g/mL, 100. mu.L per well was added to the enzyme-linked plate and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, 1.5% casein block, 200. mu.L per well, wet box 37 ℃ block lh. PD-1/Fc-Biotin was diluted to 1.25. mu.g/mL with 1 XPBS, and the antibody was diluted to an antibody concentration of 100. mu.g/mL using the above solution as a diluent, and 2-fold dilution was performed to obtain 12 concentration gradients in total. Adding 100 μ L per well into wet box in enzyme-linked plate, reacting at 37 deg.C for 1h, washing enzyme-linked plate for 3 times, adding Peroxidase-Labeled Streptavidin, reacting at room temperature for 45min, washing enzyme-linked plate for 5 times, adding 100 μ L TMB substrate, developing, reacting for 3min, and reacting with 100 μ L2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm. The antibody concentration was plotted on the abscissa and the OD value on the ordinate to prepare a binding curve.

As a result: fusion proteins of PD-1 and its variants and IgG1-Fc compete for binding to PD-L1 with PD-1/Fc-Biotin, the competitive ELISA binding activity is shown in FIG. 7, and the competitive activity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) is much higher than that of wild-type PD-1.

Example 6 Activity of competitive binding to PD-L2

The coating solution diluted PD-L2/Fc antigen (purchased from Sino Biological, cat. No. 10292-H02H) to 1. mu.g/mL, 100. mu.L per well was added to the enzyme conjugate plate and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, 1.5% casein block, 200. mu.L per well, wet box 37 ℃ block lh. PD-1/Fc-Biotin was diluted to 5. mu.g/mL with 1 XPBS, and the antibody was diluted to an antibody concentration of 100. mu.g/mL using the above solution as a diluent, and 2-fold dilution was performed to obtain 12 concentration gradients in total. Adding 100 μ L per well into wet box in enzyme-linked plate, reacting at 37 deg.C for 1h, washing enzyme-linked plate for 3 times, adding Peroxidase-Labeled Streptavidin (1:4000), reacting at room temperature for 45min, washing enzyme-linked plate for 5 times, adding 100 μ L TMB substrate, developing, reacting for 3min, and reacting with 100 μ L2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm. The antibody concentration was plotted on the abscissa and the OD value on the ordinate to prepare a binding curve.

As a result: fusion proteins of PD-1 and its variants with IgG1-Fc compete with PD-1/Fc-Biotin for binding to PD-L2, the competitive ELISA binding activity is shown in FIG. 8, and the competitive activity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) is much higher than that of wild-type PD-1.

Example 7 affinity assays

Surface plasmon resonance biosensors were used to measure the binding kinetics and affinity of antibodies to PD-L1 and PD-L2 antigens. Unless otherwise indicated, all reagents and materials were purchased from GE corporation and measurements were made at 25 ℃. Affinity analysis was performed by SPR (Biacore T200) instrument, coupling anti-human IgG Fc antibody to CM5 chip by amino coupling, flowing each antibody to be tested at a flow rate of 30 μ L/min, and capturing the antibody to be tested with the anti-human IgG Fc antibody coupled to the chip; after dilution of the analyte (PD-L1 or PD-L2) in a gradient (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and 0nM), the analyte was introduced at a flow rate of 30. mu.L/min, with a binding time of 120s for the antibody to be tested and the analyte and a dissociation time of 1200 s; HBS-EP was used as the running buffer throughout the experiment and the chip was regenerated with a 60 second pulse of 10mM glycine HCl, pH 2.1. The data were fit to a 1:1 binding model to determine the equilibrium dissociation constant, KD.

Results equilibrium dissociation constant KD determination results for PD-1 and its variants are shown in Table 2 below, and the affinity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) to PD-L1 and PD-L2 are much higher than that of wild-type PD-1.

TABLE 2 affinity determinations for PD-1 variants

Example 8PBMC activation assay

Whole Blood of 5 healthy volunteers was mixed, and human Peripheral Blood Mononuclear Cells (PBMC) (peripheral Blood Mononular cells) were separated using human lymphocyte separation medium (purchased from solarbo |: Cat. No.: P8610), washed 2 times with physiological saline, resuspended in 10% FBS 1640 medium and counted, and 1X 10 cells were added⁵Perwell was plated in 96-well plates at 50. mu.L/well. The 10% FBS 1640 culture medium is provided with the optimal antibody stimulation concentration (anti-CD3/CD28 with the final concentration of 1ug/ml) and the PD-L1 inhibition concentration (with the final concentration of 1ug/ml)10. mu.g/ml) was added to the corresponding well. Preparing PD1 variant and samples of Atezolizumab to the final concentrations of 0 mug/mL, 0.625 mug/mL, 2.5 mug/mL, 10 mug/mL and 40 mug/mL, adding the samples into corresponding reaction systems, culturing the samples in a 37 ℃ cell culture box for 72 hours, harvesting supernatant, and determining the expression condition of IFN-gamma in the supernatant by using a human IFN-gamma ELISA detection kit.

The results show that the activity of PD1 variants to activate PBMCs and release IFN- γ is superior to or substantially consistent with the PD-L1 antibody Atezolizumab, as shown in figure 9.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> eastern Biocide science and technology Co., Ltd of Jiangsu

<120> high-affinity fusion protein of PD-1 extracellular domain mutant and pharmaceutical composition and application thereof

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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 high-affinity PD-1 extracellular domain mutant, wherein the PD-1 extracellular domain mutant consists of an amino acid sequence shown in SEQ ID NO. 2.

2. A high affinity fusion protein of a PD-1 extracellular domain mutant, the fusion protein comprising a PD-1 extracellular domain mutant and an Fc fragment; or the fusion protein comprises a PD-1 extracellular domain mutant and 6 XHis, and the amino acid sequence of the PD-1 extracellular domain mutant is shown in SEQ ID NO. 2.

3. The fusion protein of claim 2, wherein the Fc fragment is selected from the group consisting of Fc of human IgG1 or IgG4, and mutants thereof.

4. The fusion protein of claim 3, wherein the Fc mutant is an N298A mutant of human IgG1Fc or an S228P mutant of human IgG4Fc, wherein the amino acid sequence of the N298A mutant is shown as SEQ ID No.8, and the amino acid sequence of the S228P mutant is shown as SEQ ID No. 9.

5. The fusion protein of claim 2, wherein the amino acid sequence of 6 xhis is as set forth in SEQ ID No. 20.

6. The fusion protein of claim 2, further comprising a Linker, wherein the PD-1 extracellular domain mutant is connected with the Fc fragment or 6 XHis through the Linker to form the fusion protein, and the amino acid sequence of the Linker is shown in SEQ ID NO. 6.

7. A biological material comprising a DNA sequence encoding the fusion protein of any one of claims 2 to 6, wherein the biological material is a vector or a host cell.

8. A pharmaceutical composition comprising the high affinity PD-1 extracellular domain mutant or fusion protein thereof of any one of claims 1 to 6; it also comprises a pharmaceutically acceptable carrier and/or excipient.

9. Use of the high affinity PD-1 extracellular domain mutant or fusion protein thereof according to any one of claims 1 to 6 for the preparation of:

(1) (ii) an agent that blocks the binding of PD-1 to PD-L1;

(2) (ii) an agent that blocks the binding of PD-1 to PD-L2;

(4) agents that modulate the activity or level of PD-L1 or/and PD-L2;

(5) medicine for relieving the immunosuppression of PD-1 on the organism; or alternatively

(6) An agent that increases IFN- γ and/or IL-2 expression in T lymphocytes.

10. Use of the high affinity PD-1 extracellular domain mutant or fusion protein thereof according to any one of claims 1 to 6 for the preparation of a medicament for the prevention and/or treatment of tumors.

11. A kit comprising the high affinity PD-1 extracellular domain mutant or fusion protein thereof according to any one of claims 1 to 6, for detecting the presence or level of PD-L1 or/and PD-L2.