CN114181297B

CN114181297B - Fusion protein of high-affinity PD-1 extracellular region mutant, and pharmaceutical composition and application thereof

Info

Publication number: CN114181297B
Application number: CN202111470830.7A
Authority: CN
Inventors: 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: 2023-06-30
Anticipated expiration: 2038-06-07
Also published as: CN114031682A; CN114031682B; CN114181297A; CN114181296B; CN108752460A; CN114181296A; CN108752460B

Abstract

The invention discloses a high-affinity PD-1 extracellular domain mutant, which consists of an amino acid sequence selected from SEQ ID NO. 4. The invention further provides fusion proteins of the high-affinity PD-1 extracellular domain mutants and application thereof. The invention creatively synthesizes a high-affinity PD-1 extracellular region mutant or fusion protein thereof, wherein 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 provides a wider approach for the immunotherapy of tumors.

Description

Fusion protein of high-affinity PD-1 extracellular region 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 fusion protein of a high-affinity PD-1 extracellular domain mutant, a pharmaceutical composition and application thereof.

Background

The tumor immunotherapy is one of the hot spots in the current tumor treatment field, and has obvious treatment effect in clinical experiments. PD-1 and its ligand PD-L1/L2 mediate the inhibition of T cells "exhaustion" and the induction of immune tolerance, and the interaction of PD-L1/L2 highly expressed on the surface of tumor cells with PD-1 leads to the immune escape of tumor cells, thus inhibiting the PD 1-PD-L1/L2 signal pathway, and reactivating the inhibited immune system as a hot spot of recent immunotherapy.

Programmed death receptor 1 (PD-1, also called PDCD1 and CD 279) was discovered in 1992 by the teachings of this science and bleb (Honjo Tasuku) of the university of Kyoto, japan. PD-1 is divided into three parts of extracellular region, transmembrane region and cytoplasmic region, contains 288 amino acids, and has a molecular weight of about 50-55 kD. The extracellular domain contains an IgV-like domain, which is a region that binds to the ligand of PD-1 and thereby induces an immune response, and which contains 4N-glycosylation sites; the cytoplasmic region contains 1 ITIM (Immunoreceptor tyrosine-based inhibitory motif, i.e., the immunoreceptor tyrosine repression motif), and 1 ITSM (Immunoreceptor tyrosine-based switch motif, i.e., the immunoreceptor tyrosine conversion motif), wherein activation of ITSM is closely related to an effector T cell immune response. PD-1 is expressed predominantly on activated T cells, B cells and myeloid cells, and plays an important role in maintaining T cell depletion, with the effector function of inhibiting T cells, thus chronic pathogens and tumor cells evade immune responses using the PD-1 pathway.

PD-L1 protein: the professor of the chinese scientist's display in 1999 found B7-H1, which was subsequently demonstrated to specifically bind PD-1 in 2000 and was designated PD-L1, the programmed death molecule ligand-1 (programmed death ligand 1, also known as CD274 and B7-H1); PD-L1 is divided into three parts of extracellular region, transmembrane region hydrophobic region and 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 predominantly expressed in mature CD4 ⁺ 、CD8 ⁺ Hematopoietic cells such as T cells, B cells, and dendritic cells; meanwhile, PD-L1 is often highly expressed on the surfaces 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 PD-L1 of the tumor cells and the poor tumor patientsPrognosis, recurrence of tumor, and also tumor size, lymph node involvement, classification, total survival.

PD-L2 protein: another ligand of PD-1, PD-L2 (programmed death ligand2, also called CD273 and B7-DC), was found in 2001 to be a transmembrane protein consisting of 274 amino acid residues in PD-L2, and PD-L2 has a high similarity to PD-L1, and PD-L2 interactions with PD-1 inhibit T cell proliferation, cytokine production and T cell lysis, but PD-L2 has 2 to 6 times the affinity of PD-L1. PD-L2 is expressed on the membrane surface of macrophages, dendritic cells and some B cell subsets, while various methods detect PD-L2 expression in tumor cells and no PD-L1 expression in some of the samples. Therefore, drugs capable of simultaneously and efficiently blocking PD-1 to PD-L1 and PD-1 to PD-L2 are still to be further opened.

In 2005, the drug of PD-1 antibody Nivolumab was developed by co-operation of japan small field pharmacy and usa Medarex pharmacy, and was collected in the capsule by bai-time meishi precious in 2009. The early-handicapped in the samsard of 2009 obtains the subsequent development rights of the PD-1 antibody drug Pembrolizumab. In 2014, nivolumab from precious, bai-Shi-Mei-Shi-Guibao and Pembrolizumab from moxadong successively received marketing approval.

In 2016, the PD-L1 antibody Atezolizumab, geneva, was FDA approved for market; the PD-1 antibody drug Bavencio and the PD-1 antibody drug Imfinzi of Aldrikang of the best in 2017 were marketed sequentially. Many companies consider this tumor immunotherapy drug as a heavy-weight product that stands for the tumor immunotherapy field for the next few years.

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

The interaction of the PD-L1/L2 with high expression on the surface of the tumor cells and the PD-1 can cause the immune escape of the tumor cells, and blocking the passage by using the PD-1 or the PD-L1 antibody can activate the immune system and further kill the tumor cells. However, since the related PD-L1/L2 and PD-1 are also expressed on immune cells, the PD-1/PD-L1 antibody is mostly designed as blocking antibody without ADCC, CDC and the like, considering that the PD-1/PD-L1 antibody kills autologous immune cells if having too strong ADCC, CDC, ADCP and the like activity. PD-1 antibodies such as Nivolumab and Pembrolizumab of moxadong of Beatles use the IgG4 subtype with weak ADCC activity; the gene Taike PD-L1 antibody Atezolizumab adopts the method of removing the glycosylation site IgG1 subtype and basically has no ADCC activity; imfinzi of the aslicon removes ADCC activity by mutating three amino acids (L234F, L235E, P331S) of IgG 1.

While the Avelumab of the fei/merck adopts the IgG1 subtype with the activities of ADCC, CDC and the like, the PD-L1 antibody with strong ADCC activity has the advantages on the remarkable high expression of the PD-L1 in tumor cells, that is, the cancer cells can be killed by combining with ADCC action while immune escape of the cancer cells is relieved.

Disclosure of Invention

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

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

To achieve the above object, the present invention first provides a high affinity PD-1 extracellular region mutant consisting of an amino acid sequence selected from SEQ ID NO. 4. In one embodiment the PD-1 extracellular region mutant is M2C5 having the amino acid sequence of SEQ ID NO.2; in one embodiment the PD-1 extracellular region mutant is M3H6 having the amino acid sequence of SEQ ID NO.3; in one embodiment the PD-1 extracellular region mutant is M4B3 having the amino acid sequence of SEQ ID NO.4; in one embodiment the PD-1 extracellular region mutant is M5G8, which has the amino acid sequence of SEQ ID NO.5.

Further, the invention also provides a fusion protein of the high-affinity PD-1 extracellular region mutant, wherein the fusion protein comprises the PD-1 extracellular region mutant and an Fc fragment/6 XHis, and the amino acid sequence of the PD-1 extracellular region mutant is shown as SEQ ID NO. 4.

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 increased by at least 10-fold compared to 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 the group consisting of the Fc of human IgG1 or IgG4 and mutants thereof, wherein the amino acid sequence of the N298A mutant of human IgG1 Fc is shown in SEQ ID NO.8, and wherein the amino acid sequence of the S228P mutant of human IgG4Fc is shown in SEQ ID NO. 9.

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

Preferably, the PD-1 extracellular region 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 as SEQ ID NO. 6.

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

Further, the invention also provides a pharmaceutical composition comprising the high affinity PD-1 extracellular domain mutant or 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 is characterized in that: one or more compatible solid or liquid filler or gel materials. They are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients.

Preferably, the carrier includes, but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulates, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

Preferably, the medicament can be formulated into dosage forms including, but not limited to, microinjection, dosage forms suitable for transfection, injection, tablet, powder, granule, capsule. The medicaments of the various formulations can be prepared according to the conventional method in the pharmaceutical field.

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

(1) Agents that block PD-1 from binding to PD-L1;

(2) Agents that block PD-1 from binding 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 PD-L1 or/and PD-L2 activity or levels;

(5) A medicament for relieving immunosuppression of PD-1 on an organism; or alternatively

(6) Drugs that increase IFN-gamma and/or IL-2 expression in T lymphocytes.

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

Preferably, the tumor is selected from the group consisting of melanoma, non-small cell lung cancer, colorectal cancer, renal tumor, cancer of the wings, cancer of the gastrointestinal tract, 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 a fusion protein thereof as 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 region mutant or fusion protein thereof, wherein 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 provides a wider approach for the immunotherapy of tumors.

Drawings

FIG. 1 is a schematic diagram showing alignment of PD-1 and its variants prepared according to the present invention;

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

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

FIG. 4 is a PTT5 carrier map for use with the present invention;

FIG. 5 PD-1 and mutant-IgG 1-Fc fusion proteins prepared according to the invention were assayed by ELISA for binding to PD-L1; 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 PD-1 and mutant fusion proteins prepared according to the invention are assayed by ELISA binding to 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 activity assay of PD-1 variant fusion proteins prepared according to the invention 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 an activity assay of the invention for preparing PD-1 variant fusion proteins to compete with PD-1/Fc-Biotin for binding to PD-L2; M2C5-IgG1-Fc fusion protein competes for binding to PD-L2 activity; 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 according to the invention activate PBMC activity.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The PD-1 extracellular region mutants for short and the PD-1 variants refer to the PD-1 extracellular region mutants with high affinity; the variant fusion proteins of the present invention refer to fusion proteins of high affinity PD-1 extracellular domain mutants.

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

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

The amino acid sequence and mutation sites of the 4 mutants of the wild type PD-1 and the high affinity PD-1 extracellular region 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 variant M4B3 amino acid is SEQ ID NO.4; the amino acid sequence of variant M5G8 is SEQ ID NO.5.

PD-1 and its variants are linked to IgG1 Fc (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) to form fusion proteins by linker (amino acid sequence: SEQ ID NO. 6) and the like, the protein structure of which is schematically shown in FIG. 2.

In order to secrete PD-1 and its variant in mammalian cells, a secretory signal peptide (amino acid sequence: SEQ ID NO. 7) is added to the N-segment of the expression vector for constructing the expression vector, and the encoding DNA structure is shown in FIG. 3. The DNA sequences (SEQ ID NO. 10-SEQ ID NO.22, as shown in Table 1) encoding PD-1 and its mutant fusion proteins were synthesized, respectively, using pTT5 as an expression vector (FIG. 4). An EcoRI restriction site is introduced at the 5 'end and a NotI restriction site is introduced at the 3' end by conventional PCR, and the mixture is inserted between EcoRI and NotI of the PTT5 vector, so that a eukaryotic expression vector is constructed.

Table 1 PD-1 and amino acid and DNA sequence listing of variant fusion proteins thereof

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 by Invitrogen). 293E cells in logarithmic growth phase were grown in 4X 10 ⁵ Inoculating in shake flask at 37deg.C and 5% CO ₂ Transfection was performed after incubation for 24 hours on shaker 125 r/min.

Adding 200 mug of plasmid into 5mL of OPTI-MEM (OPTI-MEM) per 100mL of 293E cells, shaking and uniformly mixing, and incubating at room temperature for 5min to obtain a plasmid solution; adding 600 mug of PEI into 5mL of OPTI-MEM, shaking and uniformly mixing, and incubating for 5min at room temperature to obtain a PEI solution; mixing plasmid and PEI solution, shaking, incubating at room temperature for 20min, adding dropwise the reaction mixture into cells, and standing at 37deg.C with 5% CO ₂ Shaking table 125r/min culture, feeding material for 4 th and 6 th days, and harvesting supernatant for 8 th days. Cell culture supernatants containing antibodies were harvested by centrifugation at 14000g for 30 minutes and filtration through a sterile filter (0.22 μm). Separating and purifying by AKTA (GE company), subjecting the expressed Fc fusion Protein to Protein A affinity chromatography column (MabSelect SuRe), adjusting pH to 6.0 with eluent with pH in the range of 3.4-3.6 (monitored by 280 nm), and ultrafiltering and concentrating to obtain PD-1 and its mutant fusion Protein; expressed 6 XHis fusion protein was subjected to a nickel column (HisTrap HP), eluted by 200mM imidazole, the buffer was replaced by ultrafiltration and concentrated to obtain a 6 XHis fusionAnd (5) synthesizing protein.

Example 3: PD-1 and variant fusion proteins thereof and PD-L1 binding Activity assay

The coating solution was diluted to 1. Mu.g/mL of PD-L1/His antigen (purchased from Sino Biological under the trade designation 10084-H08H), 100. Mu.L per well was added to the ELISA plate and placed in a wet box at 4℃overnight. The plate washer was cleaned 3 times, 1.5% casein blocked, 200. Mu.L per well, and the wet box blocked at 37℃for 1h. Diluting the fusion protein of PD-1 and its mutant with 1 XPBS to 15. Mu.g/mL, adding 100. Mu.L of each well after 3-fold gradient dilution to the ELISA plate, reacting at 37℃for 1h in a wet box, washing the ELISA plate 3 times, adding goat anti-human Fc-HRP secondary antibody to react at room temperature for 45min, washing the ELISA plate 5 times and adding 100. Mu.L of TMB substrate for color development, reacting for 3min and adding 100. Mu.L of 2N H ₂ SO ₄ The reaction was stopped and the ELISA was read at 450 nm. And drawing an antibody-antigen binding curve with the antibody concentration as an abscissa and the OD value as an ordinate. Four parameter equation curves were fitted using Graphpad analysis software, with the equation y= (a-D)/(1+ (X/C)/(B) +d, where B represents the slope and C represents the EC50.

Results: ELISA binding activity of fusion proteins of PD-1 and mutants thereof and IgG1-Fc and PD-L1 is shown in FIG. 5, and PD-1 variants (M2C 5, M3H6, M4B3 and M5G 8) have much higher binding activity with PD-L1 than wild-type PD-1 (> 1000 times); the binding activity was on the same order of magnitude as compared to the PD-L1 antibody Atezolizumab, with M3H6 and M4B3 binding activities slightly higher than for Atezolizumab.

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

The coating solution was diluted to 1. Mu.g/mL with PD-L2/His antigen (purchased from Sino Biological under the trade designation 10292-H08H), 100. Mu.L per well was added to the ELISA plate and placed in a wet box at 4℃overnight. The plate washer was cleaned 3 times, 1.5% casein blocked, 200. Mu.L per well, and the wet box blocked at 37℃for 1h. PD-1 (463) was diluted to 15. Mu.g/mL with 1 XPBS and diluted with a 3-fold gradient and added to the plate at 100. Mu.L per well, reacted in a wet box at 37℃for 1h, washed 3 times, reacted at room temperature for 45min with goat anti-human Fc-HRP secondary antibody, washed 5 times and developed with 100. Mu.L TMB substrate, reacted for 3min and reacted with 100. Mu.L 2N H ₂ SO ₄ Termination of the reaction, enzyme-linked immunosorbent assayThe detector reads at 450 nm. And drawing an antibody-antigen binding curve with the antibody concentration as an abscissa and the OD value as an ordinate. Four parameter equation curves were fitted using Graphpad analysis software, with the equation y= (a-D)/(1+ (X/C)/(B) +d, where B represents the slope and C represents the EC50.

Results: ELISA binding activity of fusion proteins of PD-1 and variants thereof and IgG1-Fc with PD-L2 is shown in FIG. 6, and PD-1 variants (M2C 5, M3H6, M4B3 and M5G 8) have much higher binding activity with PD-L2 than wild-type PD-1 (> 300-fold); whereas the PD-L1 antibody Atezolizumab does not bind substantially to PD-L2.

Example 5 Activity to compete for binding to PD-L1

The coating solution was diluted to 1. Mu.g/mL of PD-L1/Fc antigen (purchased from Sino Biological, cat. No. 10084-H02H), 100. Mu.L per well was added to the ELISA plate and placed in a wet box at 4℃overnight. The plate washer was cleaned 3 times, 1.5% casein blocked, 200. Mu.L per well, wet box blocked at 37℃lh. PD-1/Fc-Biotin was diluted to 1.25. Mu.g/mL with 1 XPBS, the antibody was diluted to an antibody concentration of 100. Mu.g/mL with the above solution as a diluent, and 2-fold dilution was performed to obtain 12 concentration gradients in total. 100. Mu.L of each well was put into a wet box in an ELISA plate and reacted at 37℃for 1 hour, the ELISA plate was washed 3 times, peroxidase-Labeled Streptavidin was added and reacted at room temperature for 45 minutes, the ELISA plate was washed 5 times and 100. Mu.L of TMB substrate was added and developed, and reacted for 3 minutes and then reacted with 100. Mu.L of 2N H ₂ SO ₄ The reaction was stopped and the ELISA was read at 450 nm. Binding curves were plotted with antibody concentration on the abscissa and OD on the ordinate.

Results: the fusion proteins of PD-1 and its variants with IgG1-Fc compete with PD-1/Fc-Biotin for binding to PD-L1, as shown in FIG. 7, and the competition ELISA binding activity of the PD-1 variants (M2C 5, M3H6, M4B3 and M5G 8) is much higher than that of wild-type PD-1.

Example 6 Activity to compete for binding to PD-L2

The coating solution was diluted to 1. Mu.g/mL of PD-L2/Fc antigen (purchased from Sino Biological, cat. No. 10292-H02H), 100. Mu.L per well was added to the ELISA plate and placed in a wet box at 4℃overnight. The plate washer was cleaned 3 times, 1.5% casein blocked, 200. Mu.L per well, wet box blocked at 37℃lh. PD-1/Fc-Biotin was diluted to 5. Mu.g/mL with 1 XPBS, and the above solution was used as a diluentThe antibody was diluted to an antibody concentration of 100. Mu.g/mL and 2-fold dilution was performed to obtain 12 concentration gradients in total. 100. Mu.L of each well was added to a wet box in a ELISA plate and reacted at 37℃for 1h, the ELISA plate was washed 3 times, peroxidase-Labeled Streptavidin (1:4000) was added and reacted at room temperature for 45min, the ELISA plate was washed 5 times and 100. Mu.L of TMB substrate was added and developed, and the reaction was carried out for 3min and 100. Mu.L of 2N H was used ₂ SO ₄ The reaction was stopped and the ELISA was read at 450 nm. Binding curves were plotted with antibody concentration on the abscissa and OD on the ordinate.

Results: the fusion proteins of PD-1 and its variants with IgG1-Fc compete with PD-1/Fc-Biotin for binding to PD-L2, as shown in FIG. 8, and the competition ELISA binding activity of the PD-1 variants (M2C 5, M3H6, M4B3 and M5G 8) is much higher than that of wild-type PD-1.

Example 7 affinity detection

Surface plasmon resonance biosensors were used to measure the binding kinetics and avidity of antibodies to PD-L1 and PD-L2 antigens. All reagents and materials can be purchased from GE and can be measured at 25 ℃, unless otherwise indicated. Affinity analysis was performed by SPR (Biacore T200) instrument, antibodies against human IgG Fc were coupled to CM5 chips by amino coupling, each antibody to be tested was flowed in at a flow rate of 30 μl/min, and the antibody coupled to the anti human IgG Fc on the chip was used to capture the antibody to be tested; after gradient dilution of the analyte (PD-L1 or PD-L2) (100 nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and 0 nM), the antibody to be tested was flowed in at a flow rate of 30. Mu.L/min for a binding time of 120s with the analyte and a dissociation time of 1200s; the whole experiment was performed using HBS-EP as running buffer and the chip was regenerated with a 60 second pulse of 10mM glycine HCl, pH 2.1 solution. The assay data were fit to a 1:1 binding model to determine the equilibrium dissociation constant KD.

As a result, PD-1 and its variants were assayed for equilibrium dissociation constants KD as shown in Table 2 below, and PD-1 variants (M2C 5, M3H6, M4B3 and M5G 8) had significantly higher affinity for both PD-L1 and PD-L2 than wild-type PD-1.

Table 2 PD-1 variant affinity assay values

EXAMPLE 8PBMC activation assay

Whole blood from 5 healthy volunteers was mixed, human peripheral blood mononuclear cells PBMC (Peripheral Blood Mononuclear Cells) were isolated using human lymphocyte separation liquid (purchased from Solarbio|cat# P8610), the cells were washed 2 times with physiological saline, and 10% FBS 1640 medium was used to resuspend the cells and counted 1X 10 ⁵ Wells were seeded in 96-well plates at 50 μl per well. Optimal antibody stimulation concentrations (1 ug/ml final anti-CD3/CD 28) and PD-L1 inhibition concentrations (10 ug/ml final) were added to the corresponding wells in 10% FBS 1640 medium. PD1 variant and Atezolizumab are prepared, the final concentration of the sample reaches 0 mug/mL, 0.625 mug/mL, 2.5 mug/mL, 10 mug/mL and 40 mug/mL, the corresponding reaction system is added, the cell culture is carried out for 72 hours at 37 ℃, the supernatant is obtained, and the expression of IFN-gamma in the supernatant is measured by using a human IFN-gamma ELISA detection kit.

The results show that the activity of the PD1 variant to activate PBMC and release IFN-gamma is superior or substantially identical to that of the PD-L1 antibody Atezolizumab, as shown in FIG. 9.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Jiangsu Dong anti-biological medicine technology Co., ltd

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

<130> P210259

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<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

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 outer membrane region mutant, said PD-1 outer membrane region mutant consisting of an amino acid sequence selected from the group consisting of SEQ ID No. 4.

2. A fusion protein of a high affinity PD-1 outer membrane region mutant, said fusion protein comprising a PD-1 outer membrane region mutant and an Fc fragment, or said fusion protein comprising a PD-1 outer membrane region mutant and 6 xhis, said PD-1 outer membrane region mutant having an amino acid sequence as set forth in SEQ ID No. 4.

3. The fusion protein of claim 2, wherein the Fc fragment is selected from the group consisting of human IgG1 or IgG4Fc and mutants thereof, wherein the amino acid sequence of the N298A mutant of human IgG1 Fc is shown in SEQ ID No.8, and wherein the amino acid sequence of the S228P mutant of human IgG4Fc is shown in SEQ ID No. 9.

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

5. The fusion protein of claim 3 or 4, further comprising a Linker, wherein the PD-1 extracellular domain mutant is linked to the Fc fragment and 6 xhis via the Linker to form the fusion protein, and wherein the Linker has the amino acid sequence shown in SEQ ID No. 6.

6. A biological material encoding a DNA sequence of the fusion protein of any one of claims 2 to 5, wherein the biological material is a vector or a host cell.

7. A pharmaceutical composition comprising the high affinity PD-1 extracellular domain mutant of claim 1 or the fusion protein of any one of claims 2 to 5; it also includes pharmaceutically acceptable carriers and/or excipients.

8. Use of a high affinity PD-1 extracellular domain mutant according to claim 1 or a fusion protein according to any one of claims 2 to 5 for the manufacture of a medicament for the prevention and/or treatment of a tumor.

9. The use of claim 8, wherein the mechanism of action of the PD-1 extracellular region mutant or fusion protein is:

(1) Blocking the binding of PD-1 to PD-L1;

(2) Blocking the binding of PD-1 to PD-L2;

(3) Simultaneously blocking the combination of PD-1 and PD-L1 and the combination of PD-1 and PD-L2;

(4) Modulating PD-L1 or/and PD-L2 activity or level;

(5) Relieving immunosuppression of PD-1 on organism; or alternatively

(6) Increasing IFN-gamma and/or IL-2 expression in T lymphocytes.

10. Kit comprising the high affinity PD-1 extracellular domain mutant of claim 1 or the fusion protein of any one of claims 2 to 5 for detecting the presence or level of PD-L1 or/and PD-L2.