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

Abstract

The invention discloses a mutant of the extramembrane region of PD-1 with high affinity, and the mutant of the extramembrane region of PD-1 is composed of an amino acid sequence selected from SEQ ID NO.4. The present invention further provides the fusion protein of the high-affinity PD‑1 extramembrane region mutant and its application. The present invention creatively synthesizes a high-affinity PD‑1 extramembrane region mutant or its fusion protein, which has a higher binding affinity to human PD‑L1 or PD‑L2 than wild-type PD‑1, greater potency. The fusion protein of the invention provides a broader approach for tumor immunotherapy.

Description

A fusion protein of a high-affinity mutant of the extramembrane region of PD-1 and its drug combination substance and use

technical field

The invention relates to the fields of tumor therapy and molecular immunology, and relates to a high-affinity fusion protein of a mutant of the extramembrane region of PD-1 and its pharmaceutical composition and use.

Background technique

Tumor immunotherapy is to stimulate and enhance the immune function of the body to achieve the purpose of controlling and killing tumor cells. Tumor immunotherapy is one of the current hot spots in the field of tumor treatment, and clinical trials have also achieved remarkable therapeutic effects. PD-1 and its ligand PD-L1/L2 mediate the inhibitory "exhaustion" of T cells and the induction of immune tolerance. The highly expressed PD-L1/L2 on the surface of tumor cells interacts with PD-1 to cause tumor The immune escape of cells, therefore inhibiting the PD1-PD-L1/L2 signaling pathway and reactivating the suppressed immune system has become a hot spot in immunotherapy recently.

Programmed death receptor 1 (PD-1, also known as PDCD1 and CD279), was discovered in 1992 by Professor Honjo Tasuku of Kyoto University, Japan. PD-1 is divided into three parts: the extracellular region, the transmembrane region and the cytoplasmic region. It is a 288-amino acid protein with a molecular weight of about 50-55kD. The extracellular region contains an IgV-like domain, which is the region that binds to the ligand of PD-1 and induces an immune response function. This region contains 4 N-glycosylation sites; the cytoplasmic region contains 1 ITIM (Immunoreceptor tyrosine -based inhibitory motif, that is, immunoreceptor tyrosine-based switch motif), and 1 ITSM (Immunoreceptor tyrosine-based switch motif, that is, immunoreceptor tyrosine conversion motif), wherein, the activation and effect of ITSM closely related to T cell immune response. PD-1 is mainly expressed on activated T cells, B cells and myeloid cells. It mainly plays an important role in maintaining T cell exhaustion and has the effector function of inhibiting T cells. Therefore, chronic pathogens and tumor cells use the PD-1 pathway to escape immune response.

PD-L1 protein: Chinese scientist Professor Lieping Chen discovered B7-H1 in 1999, and the protein was subsequently confirmed to specifically bind PD-1 in 2000, and named PD-L1, namely programmed death ligand-1 (programmed deathligand 1, also known as CD274 and B7-H1); PD-L1 is divided into three parts: the extracellular region, the transmembrane hydrophobic region and the cytoplasmic region, and consists of 290 amino acid subunits; the extracellular region contains two An immunoglobulin constant region IgC and IgV-like domain, the cytoplasmic region is composed of 30 amino acids. PD-L1 is mainly expressed in mature CD4 ⁺ , CD8 ⁺ T cells, B cells, dendritic cells and other hematopoietic cells; at the same time, 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, squamous cell carcinoma of the head and neck, etc. At the same time, the high expression of PD-L1 in tumor cells is related to the poor prognosis of tumor patients, tumor recurrence, and is related to tumor size, lymph node involvement, grade, and overall survival. period is also relevant.

PD-L2 protein: PD-L2, another ligand of PD-1, is the programmed death molecule ligand-2 (programmed death ligand2, also known as CD273 and B7-DC), which was discovered in 2001. PD-L2 consists of 274 amino acids A transmembrane protein composed of residues, PD-L2 and PD-L1 have a high similarity, and the interaction between PD-L2 and PD-1 can inhibit the proliferation of T cells, the production of cytokines and the lysis of T cells, but PD The affinity between -L2 and PD-1 is 2-6 times that of PD-L1. PD-L2 is expressed on the membrane surface of macrophages, dendritic cells and some B cell subtypes, and PD-L2 expression in tumor cells was detected by various methods, while PD-L2 was not detected in some samples. Expression of L1. Therefore, drugs that can effectively block PD-1-PD-L1 and PD-1-PD-L2 at the same time need to be further developed.

In 2005, Ono Pharmaceuticals of Japan and Medarex Pharmaceuticals of the United States jointly developed the PD-1 antibody drug Nivolumab, which was acquired by Bristol-Myers Squibb in 2009. In 2009, Merck acquired Schering-Plough and obtained the subsequent development rights of the PD-1 antibody drug Pembrolizumab. In 2014, Bristol-Myers Squibb's Nivolumab and Merck's Pembrolizumab were approved for marketing.

In 2016, Genentech's PD-L1 antibody Atezolizumab was approved by the FDA; in 2017, Pfizer and Merck's PD-1 antibody drug Bavencio and AstraZeneca's PD-1 antibody drug Imfinzi were launched successively. Many large companies regard this tumor immunotherapy drug as a blockbuster product in the field of tumor immunotherapy in the next few years.

Although there are several anti-PD-1 and PD-L1 antibody drugs on the market, there are few reports on drugs based on fusion proteins of PD-1; and they can effectively block PD-1~PD-L1 and PD-1~ Drugs for PD-L2 are also urgently needed to be developed to more effectively block the signaling pathway of PD-1.

The highly expressed PD-L1/L2 on the surface of tumor cells interacts with PD-1 to cause immune escape of tumor cells. Blocking this pathway with antibodies to PD-1 or PD-L1 can activate the immune system and kill tumor cells. Since related PD-L1/L2 and PD-1 are also expressed on immune cells, considering that if PD-1/PD-L1 antibodies have too strong ADCC, CDC, ADCP and other activities, they will kill their own immune cells, so Most PD-1/PD-L1 antibodies are designed as blocking antibodies, without ADCC, CDC and other functions. For example, PD-1 antibodies such as Bristol-Myers Squibb’s Nivolumab and Merck’s Pembrolizumab use the IgG4 subtype with weak ADCC activity; Genentech’s PD-L1 antibody Atezolizumab uses the IgG1 subtype with glycosylation sites removed, and basically does not have ADCC activity ; AstraZeneca's Imfinzi removed ADCC activity by mutating three amino acids of IgG1 (L234F, L235E, P331S).

However, Pfizer/Merck’s Avelumab uses IgG1 subtypes with ADCC, CDC and other activities. Because PD-L1 is significantly highly expressed in tumor cells, PD-L1 antibodies with strong ADCC activity also have their advantages, that is, to relieve cancer cells. At the same time of immune escape, it can also mediate NK cells to kill cancer cells in combination with ADCC.

Contents of the invention

The purpose of the present invention is to provide a high-affinity PD-1 extramembrane mutant and a fusion protein comprising the high-affinity PD-1 extramembrane mutant.

The object of the present invention is also to provide a drug composition comprising the above-mentioned high-affinity PD-1 extramembrane region mutant or its fusion protein drug and its application.

To achieve the above purpose, the present invention firstly provides a high-affinity mutant of the extramembrane region of PD-1, the mutant of the extramembrane region of PD-1 consists of an amino acid sequence selected from SEQ ID NO.4. In one embodiment, the PD-1 extramembrane mutant is M2C5, which has the amino acid sequence of SEQ ID NO.2; in one embodiment, the PD-1 extramembrane mutant is M3H6, which has SEQ ID NO.3 The amino acid sequence of the PD-1 extramembrane region mutant is M4B3 in one embodiment, which has the amino acid sequence of SEQ ID NO.4; in one embodiment, the PD-1 extramembrane region mutant is M5G8, which has SEQ ID NO.4 Amino acid sequence of ID NO.5.

Further, the present invention also provides a high-affinity fusion protein of a PD-1 extramembrane region mutant, the fusion protein includes a PD-1 extramembrane region mutant and an Fc fragment/6×His, and the PD- 1 The amino acid sequence of the extramembrane mutant is shown in SEQ ID NO.4.

Preferably, the affinity of the fusion protein to PD-L1 is at least 50 times higher than that of wild-type PD-1; in one embodiment, the affinity of the fusion protein of the present invention to PD-L1 is comparable to that of wild-type PD-1 The ratio is increased by more than 1000 times; the affinity of the fusion protein to PD-L2 is at least 10 times higher than that of wild-type PD-1; in one embodiment, the affinity of the fusion protein of the present invention to PD-L2 is comparable to that of wild-type PD-1 Compared with -1, it is more than 300 times higher.

Preferably, the fusion protein further includes an Fc fragment selected from 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, wherein The amino acid sequence of the S228P mutant of human IgG4 Fc is shown in SEQ ID NO.9.

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

Preferably, the mutant of the extramembrane region of PD-1 is linked with the Fc fragment and 6×His 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 present invention also provides a biological material encoding the DNA sequence of the fusion protein, the biological material is a vector, a host cell or a kit, such as the kit is used to detect PD-L1 or (and) The presence or level of PD-L2.

Further, the present invention also provides a pharmaceutical composition, which comprises the above-mentioned high-affinity PD-1 extramembrane region mutant or its fusion protein; optionally, it also includes a pharmaceutically acceptable carrier and/or excipient.

The carrier described in the present invention is a pharmaceutically acceptable carrier, which 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 low toxicity. "Compatibility" here means that each component in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

Preferably, the carrier includes, but is not limited to: diluents, buffers, suspensions, emulsions, granules, capsules, excipients, fillers, adhesives, sprays, transdermal absorbents, moistening agent, disintegrant, absorption enhancer, surfactant, coloring agent, flavoring agent or adsorption carrier.

Preferably, the drug can be prepared including but not limited to microinjection, dosage form suitable for transfection, injection, tablet, powder, granule, capsule. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy.

Further, the present invention also provides the use of the high-affinity PD-1 extramembrane region mutant or its fusion protein in the preparation of the following medicines:

(1) Drugs that block the combination of PD-1 and PD-L1;

(2) Drugs that block the combination of PD-1 and PD-L2;

(3) Drugs that simultaneously block the combination of PD-1 and PD-L1 and PD-1 and PD-L2;

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

(5) Drugs that relieve the immune suppression of PD-1 on the body; or

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

Furthermore, the present invention also provides the use of the high-affinity PD-1 extramembrane region mutant or its fusion protein in the preparation of drugs 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 tumors, bladder cancer, gastrointestinal tract cancer, prostate cancer, liver cancer, ovarian cancer and leukemia.

Furthermore, the present invention also provides a kit, which includes the above-mentioned high-affinity PD-1 extramembrane region mutant or its fusion protein, and the kit is used to detect PD-L1 or/and PD-L2 presence or level.

Beneficial effect

The present invention creatively synthesizes a high-affinity mutant of the extramembrane region of PD-1 or its fusion protein, and the fusion protein has a higher binding affinity to human PD-L1 or PD-L2 than wild-type PD-1, greater potency. The fusion protein of the invention provides a broader approach for tumor immunotherapy.

Description of drawings

Figure 1 is a sequence comparison diagram of PD-1 and its variants prepared by the present invention;

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

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

The PTT5 carrier map of Fig. 4 application of the present invention;

Figure 5 PD-1 and its mutants prepared by the present invention-IgG1-Fc fusion protein and PD-L1 binding ELISA assay; A: M2C5-IgG1-Fc fusion protein and PD-L1 binding activity; B: M3H6-IgG1- Binding activity of Fc fusion protein to PD-L1; C: binding activity of M4B3-IgG1-Fc fusion protein to PD-L1; D: binding activity of M5G8-IgG1-Fc fusion protein to PD-L1;

Figure 6 PD-1 and its mutant fusion protein prepared by the present invention bind to PD-L2 by ELISA assay; A: the binding activity of M2C5-IgG1-Fc fusion protein to PD-L2; B: the binding activity of M3H6-IgG1-Fc fusion protein to PD-L2 PD-L2 binding activity; C: M4B3-IgG1-Fc fusion protein and PD-L2 binding activity; D: M5G8-IgG1-Fc fusion protein and PD-L2 binding activity;

Figure 7: The PD-1 variant fusion protein prepared by the present invention competes with PD-1/Fc-Biotin to bind to PD-L1; A: M2C5-IgG1-Fc fusion protein competes to bind to PD-L1; B: M3H6 -IgG1-Fc fusion protein competes for the activity of binding to PD-L1; C: M4B3-IgG1-Fc fusion protein competes for the activity of binding to PD-L1; D: M5G8-IgG1-Fc fusion protein competes for the activity of binding to PD-L1;

Figure 8: Determination of the activity of the PD-1 variant fusion protein prepared by the present invention to compete with PD-1/Fc-Biotin for binding to PD-L2; A: M2C5-IgG1-Fc fusion protein competes for the activity of binding to PD-L2; B: M3H6- IgG1-Fc fusion protein competes for the activity of binding to PD-L2; C: M4B3-IgG1-Fc fusion protein competes for the activity of binding to PD-L2; D: M5G8-IgG1-Fc fusion protein competes for the activity of binding to PD-L2;

Fig. 9 The PD-1 variant fusion protein prepared by the present invention activates PBMC activity.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Those who do not indicate specific techniques or conditions in the embodiments, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, the third edition, Science Press) or follow the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

The abbreviated PD-1 extramembrane region mutants and PD-1 variants mentioned in the present invention all refer to high-affinity PD-1 extramembrane region mutants; the variant fusion protein of the present invention refers to high-affinity PD-1 1 Fusion protein of extramembrane region mutants.

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

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 prepared in the present invention and its four high-affinity PD-1 extramembrane mutants are shown in Figure 1, and the amino acid sequence of PD-1 is SEQ ID NO.1; The amino acid sequence of the variant M2C5 is SEQ ID NO.2; the amino acid sequence of the variant M3H6 is SEQ ID NO.3; the amino acid sequence of the variant M4B3 is SEQ ID NO.4; the amino acid sequence of the variant M5G8 is SEQ ID NO.5 .

PD-1 and its variants are linked to IgG1 Fc (amino acid sequence: SEQ ID NO.8), IgG4 Fc (amino acid sequence: SEQ ID NO.9) or 6×His ( Amino acid sequence: SEQ ID NO.20) etc. are connected to form a fusion protein, and its protein structure schematic diagram is shown in FIG. 2 .

In order to secrete and express PD-1 and its variants in mammalian cells, a signal peptide (amino acid sequence: SEQ ID NO.7) for secreted expression must be added to the N segment of the expression vector when constructing the expression vector. The encoded DNA structure is shown in the figure 3. Using pTT5 as the expression vector (FIG. 4), the DNA sequences encoded by PD-1 and its mutant fusion proteins (SEQ ID NO.10-SEQ ID NO.22, as shown in Table 1) were synthesized respectively. The EcoRI restriction site was introduced at the 5' end and the NotI restriction site was introduced at the 3' end by conventional PCR, and inserted between EcoRI and NotI of the PTT5 vector to construct a eukaryotic expression vector.

Table 1 Amino acid and DNA sequence list of PD-1 and its variant fusion proteins

Example 2: Expression and purification of PD-1 and its variant fusion proteins

Recombinant PD-1 and its variant fusion proteins were expressed by transient transfection of 293E cells (purchased by Invitrogen). The 293E cells in the logarithmic growth phase were inoculated in shake flasks at a density of 4×10 ⁵ /mL, cultured at 37°C, 5% CO ₂ on a shaker at 125r/min for 24 hours, and then transfected.

For every 100 mL of 293E cells, take 5 mL of OPTI-MEM and add 200 μg of plasmid, shake and mix and incubate at room temperature for 5 minutes to obtain a plasmid solution; take another 5 mL of OPTI-MEM and add 600 μg of PEI, shake and mix for 5 minutes at room temperature to obtain a PEI solution ; Mix the plasmid and PEI solution, oscillate and incubate at room temperature for 20 minutes, add the reaction mixture dropwise to the cells, place in a 37°C, 5% CO ₂ shaker at 125r/min for culture, feed feeding on the 4th and 6th day, and Harvest the supernatant at 8 days. Cell culture supernatants containing antibodies were harvested by centrifugation at 14000 g for 30 minutes and filtration through sterile filters (0.22 μm). AKTA (GE company) was used for separation and purification, and the expressed Fc fusion protein was passed through a Protein A affinity chromatography column (MabSelect SuRe), and the eluate with a pH in the range of 3.4-3.6 (monitored with 280nm) was adjusted to a pH of 6.0 , the fusion protein of PD-1 and its mutants was obtained after ultrafiltration and concentration; the expressed 6×His fusion protein was passed through a nickel column (HisTrap HP), eluted by 200mM imidazole, and the 6× His fusion protein.

Example 3: Analysis of the binding activity of PD-1 and its variant fusion proteins to PD-L1

Dilute the PD-L1/His antigen (purchased from Sino Biological, Cat. No. 10084-H08H) in the coating solution to 1 μg/mL, add 100 μL to each well of the enzyme-linked plate, and place it in a humid box at 4°C overnight. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute the fusion protein of PD-1 and its mutants with 1×PBS to 15 μg/mL, and after 3-fold serial dilution, add 100 μL per well to the enzyme-linked plate, react in a wet box at 37°C for 1 hour, and wash the enzyme-linked plate 3 times, add goat anti-human Fc-HRP secondary antibody to react at room temperature for 45 minutes, wash the enzyme-linked plate 5 times and add 100 μL TMB substrate for color development, react for 3 minutes and stop the reaction with 100 μL 2N H ₂ SO ₄ , read at 450 nm on the enzyme-linked immunosorbent detector . And the antibody-antigen binding curve was drawn with the antibody concentration as the abscissa and the OD value as the ordinate. Using Graphpad analysis software to fit the four-parameter equation curve, the equation is y=(AD)/(1+(X/C)^B)+D, where B represents the slope and C represents EC50.

Results: The ELISA binding activity of the fusion protein of PD-1 and its mutants and IgG1-Fc to PD-L1 is shown in Figure 5, and the binding activity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) to PD-L1 Much higher than wild-type PD-1 (>1000 times); compared with PD-L1 antibody Atezolizumab, the binding activity is in the same order of magnitude, and the binding activity of M3H6 and M4B3 is slightly higher than that of Atezolizumab.

Example 4: Analysis of the binding activity of PD-1 and its mutant fusion proteins to PD-L2

Dilute the PD-L2/His antigen (purchased from Sino Biological, Cat. No. 10292-H08H) in the coating solution to 1 μg/mL, add 100 μL per well to the enzyme-linked plate, and place it in a humid box at 4°C overnight. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute PD-1(463) to 15 μg/mL with 1×PBS, and after 3-fold gradient dilution, add 100 μL per well to the enzyme-linked plate, react in a wet box at 37°C for 1 hour, wash the enzyme-linked plate 3 times, Add goat anti-human Fc-HRP secondary antibody to react at room temperature for 45 minutes, wash the enzyme-linked plate 5 times, add 100 μL TMB substrate for color development, react for 3 minutes and stop the reaction with 100 μL 2N H ₂ SO ₄ , and read at 450 nm with an enzyme-linked immunosorbent detector. And the antibody-antigen binding curve was drawn with the antibody concentration as the abscissa and the OD value as the ordinate. Using Graphpad analysis software to fit the four-parameter equation curve, the equation is y=(AD)/(1+(X/C)^B)+D, where B represents the slope and C represents EC50.

Results: The ELISA binding activity of the fusion protein of PD-1 and its variants and IgG1-Fc to PD-L2 is shown in Figure 6, and the binding activity of PD-1 variants (M2C5, M3H6, M4B3 and M5G8) to PD-L2 Much higher than wild-type PD-1 (>300 times); while the PD-L1 antibody Atezolizumab basically does not bind to PD-L2.

Example 5 Competing for the activity of binding to PD-L1

Dilute the PD-L1/Fc antigen (purchased from Sino Biological, Cat. No. 10084-H02H) in the coating solution to 1 μg/mL, add 100 μL per well to the enzyme-linked plate, and place it in a humid box overnight at 4°C. Wash the ELISA plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute PD-1/Fc-Biotin to 1.25 μg/mL with 1×PBS, dilute the antibody to an antibody concentration of 100 μg/mL with the above solution as the diluent, and perform 2-fold dilution to obtain a total of 12 concentration gradients. Add 100 μL to each well of the enzyme-linked plate and react in a wet box at 37°C for 1 hour, wash the enzyme-linked plate 3 times, add Peroxidase-Labeled Streptavidin to react at room temperature for 45 minutes, wash the enzyme-linked plate 5 times and add 100 μL TMB substrate for color development, react for 3 minutes and use 100 μL of 2N H ₂ SO ₄ was used to terminate the reaction, and the enzyme-linked immunosorbent assay was read at 450 nm. The binding curve was drawn with the antibody concentration as the abscissa and the OD value as the ordinate.

Results: The fusion protein of PD-1 and its variants and IgG1-Fc competes with PD-1/Fc-Biotin for binding to PD-L1, and its competitive ELISA binding activity is shown in Figure 7. PD-1 variants (M2C5, M3H6 , M4B3 and M5G8) were much more competitive than wild-type PD-1.

Example 6 Competing for the activity of binding to PD-L2

Dilute the PD-L2/Fc antigen (purchased from Sino Biological, Cat. No. 10292-H02H) to 1 μg/mL in the coating solution, add 100 μL to each well of the enzyme-linked plate, and place it in a humid box at 4°C overnight. Wash the ELISA plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute PD-1/Fc-Biotin with 1×PBS to 5 μg/mL, use the above solution as diluent to dilute the antibody to an antibody concentration of 100 μg/mL, and perform 2-fold dilution to obtain a total of 12 concentration gradients. Add 100 μL to each well of the enzyme-linked plate in a wet box at 37°C for 1 h, wash the enzyme-linked plate 3 times, add Peroxidase-Labeled Streptavidin (1:4000) to react at room temperature for 45 min, wash the enzyme-linked plate 5 times, and add 100 μL TMB substrate to display Color, react for 3 minutes and terminate the reaction with 100 μL 2NH ₂ SO ₄ , and read at 450 nm with an enzyme-linked immunosorbent detector. The binding curve was drawn with the antibody concentration as the abscissa and the OD value as the ordinate.

Results: The fusion protein of PD-1 and its variants and IgG1-Fc competes with PD-1/Fc-Biotin for binding to PD-L2, and its competitive ELISA binding activity is shown in Figure 8. PD-1 variants (M2C5, M3H6 , M4B3 and M5G8) were much more competitive than 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 were purchased from GE Corporation and were measured at 25 °C unless otherwise stated. Affinity analysis was performed on an SPR (Biacore T200) instrument, and an anti-human IgG Fc antibody was coupled to a CM5 chip through amino coupling. The anti-human IgG Fc antibody on the surface captures the antibody to be tested; the analyte (PD-L1 or PD-L2) is serially diluted (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and OnM), at a flow rate of 30μL/ The flow rate of min is inflow, the binding time of the antibody to be tested and the analyte is 120s, and the dissociation time is 1200s; the whole experiment uses HBS-EP as the running buffer, and the chip is regenerated with 10mM glycine HCl, pH 2.1 solution for 60 seconds. The assay data were fitted to a 1:1 binding model to determine the equilibrium dissociation constant KD.

Results: The results of the KD determination of the equilibrium dissociation constants of PD-1 and its variants are shown in Table 2 below. Wild-type PD-1.

Table 2 Affinity determination of PD-1 variants

Embodiment 8 PBMC activation experiment

The whole blood of 5 healthy volunteers was mixed, and the human peripheral blood mononuclear cells (PBMCs) were separated by human lymphocyte separation medium (purchased from Solarbio | product number: P8610), and the cells were washed twice with normal saline, 10% The cells were resuspended in FBS 1640 medium, counted, and inoculated in a 96-well plate at 1×10 ⁵ /well, 50 μL per well. 10% FBS 1640 medium was prepared with the optimal antibody stimulation concentration (anti-CD3/CD28 final concentration 1ug/ml) and PD-L1 inhibitory concentration (final concentration 10μg/ml) and added to the corresponding wells. Prepare the final concentrations of PD1 variants and Atezolizumab samples to 0 μg/mL, 0.625 μg/mL, 2.5 μg/mL, 10 μg/mL, 40 μg/mL and add to the corresponding reaction system, culture in a cell incubator at 37°C for 72 hours, harvest the supernatant and The expression of IFN-γ in the supernatant was determined by human IFN-γELISA detection kit.

The results showed that, as shown in Figure 9, the activity of PD1 variants in activating PBMCs and releasing IFN-γ was superior to or basically consistent with that of PD-L1 antibody Atezolizumab.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

SEQUENCE LISTING

<110> Jiangsu Dongkang Biomedical Technology Co., Ltd.

<120> A fusion protein of a high-affinity PD-1 extramembrane region mutant and its pharmaceutical composition and use

<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 accaccctgat gatcagccgg accccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggacccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccggggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccacccccc 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 accaccctgat gatcagccgg accccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggacccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccggggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccacccccc 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 accaccctgat gatcagccgg accccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggacccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccggggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccacccccc 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 accaccctgat gatcagccgg accccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggacccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccggggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccacccccc 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 accaccctgat gatcagccgg accccccgagg tgacctgcgt ggtggtggac 600

gtgagccacg aggacccccga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac 660

aacgccaaga ccaagccccg ggaggagcag tacgccagca cctaccgggt ggtgagcgtg 720

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 780

aaggccctgc ccgcccccat cgagaagacc atcagcaagg ccaagggcca gccccggggag 840

ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgagcctg 900

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 960

cagcccgaga acaactacaa gaccacccccc 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 gccccggggag 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 aacccactaca 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 gccccggggag 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 aacccactaca 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 gccccggggag 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 aacccactaca 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 gccccggggag 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 aacccactaca 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 gccccggggag 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 aacccactaca cccagaagag cctgagcctg 1140

agcctgggca ag 1152

<210> 20

<211> 6

<212> PRT

<213> 6×His

<400> 20

His His 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 (10)

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.

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