A pd1-il15 bifunctional fusion protein and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a PD1-IL15 bifunctional fusion protein and application thereof. The PD1-IL15 bifunctional fusion protein provided by the application is obtained by connecting an anti-PD-1 antibody mutant sequence after double mutation of H310Q and H433N of an Fc region of an anti-PD-1 antibody and an IL-15 mutant sequence after N72D mutation of an IL-15 molecule through a linker. The PD1-IL15 bifunctional fusion protein prepared by the application can not only shorten the half-life of the fusion protein, improve the affinity of the fusion protein to an antigen, enhance the anti-tumor activity of the fusion protein, but also reduce the toxicity of the fusion protein in the body, reduce side effects, and greatly improve the drug safety of the PD1-IL15 bifunctional fusion protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a PD1-IL15 bifunctional fusion protein and its applications. Background Technology

[0002] PD-1 (programmed death receptor 1, also known as CD279) is a member of the immunoglobulin superfamily, primarily expressed on the surface of activated T cells, B cells, NK cells, and macrophages. Its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) are widely distributed on the surface of tumor cells, antigen-presenting cells, and stromal cells, forming the "PD-1 / PD-L1" immunosuppressive pathway. PD-1 is also expressed on the surface of tumor-infiltrating lymphocytes (TILs), especially depleted CD8+ cells. + T cells (TEX) have an expression level that is positively correlated with tumor progression. Therefore, PD-1 can serve as a reliable target for cancer treatment.

[0003] IL-15 is a cytokine expressed by various cell types, including monocytes, macrophages, epidermal cells, and fibroblasts. Unlike many other cytokines, IL-15 is generally not secreted by cells to exert its effects. Instead, it combines with IL-15Rα and localizes to the cell membranes of NK and CD8+ T cells, thereby stimulating nearby effector cells. Currently, several IL-15-related molecules are under development for the treatment of malignant tumors. Among them, ALT-803 is the most advanced, and its molecule is a complex formed by IL-15 and IL-15Rαsusi-hFc1.

[0004] Patent document CN105189562A discloses an IL-15 heterodimeric protein and its uses. The IL-15 heterodimeric protein comprises protein (I) and protein (II), wherein protein (I) is a recombinant of IL-15 or a variant thereof and a first Fc variant; protein (II) is a second Fc variant, or IL-15Rα or a variant thereof and a second Fc variant; the first Fc variant and the second Fc variant are linked in a Knob-into-Hole manner. This IL-15 heterodimer exhibits significant antitumor activity, showing better biological activity and a prolonged in vivo half-life than a single IL-15 molecule.

[0005] Patent document CN106459219A discloses an interleukin-15 protein complex, which is composed of a soluble fusion protein (I) and a soluble fusion protein (II). The fusion protein (I) is an IL-15 polypeptide or a functional fragment thereof, and the fusion protein (II) is an IL-15Rα polypeptide or a functional fragment thereof. One or more amino acid sites on either the fusion protein (I) or the fusion protein (II) are mutated to Cys, which pair with the corresponding Cys amino acid sites on either the fusion protein (II) or the fusion protein (I) to form disulfide bonds. This IL-15 protein complex can be used for tumor treatment.

[0006] Patent document CN112574316A discloses a tumor-targeting fusion protein, said fusion protein comprising at least (i) IL 15 peptides, IL 15. Peptide variants or functional fragments thereof, (ii) IL 15Ra peptide, IL (iii) an Fc domain, an Fc variant, or a functional fragment thereof, and (iv) an RGD peptide or a variant thereof. This tumor-targeting fusion protein can not only enhance IL-15Rα, but also... It has an anti-tumor effect of 15% and can also target the tumor site, specifically acting on tumor cells.

[0007] However, current research on IL-15 combination drugs generally involves different modifications to Fc fusion proteins. Given the toxicity of IL-15-Fc fusion proteins, dosage is limited. Therefore, developing drugs with high tumor targeting, low toxicity, and high safety remains a pressing challenge. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a novel PD1-IL15 bifunctional fusion protein with high tumor targeting, low toxicity, and high safety. The PD1-IL15 bifunctional fusion protein prepared by this invention not only shortens the half-life of the fusion protein and increases its affinity for the antigen, enhancing its in vivo antitumor activity, but also significantly reduces toxicity and improves drug safety.

[0009] To achieve the above objectives, the present invention proposes the following approach to modifying the novel PD1-IL15 bifunctional fusion protein: This invention utilizes hybridoma technology to screen for an anti-PD-1 antibody with high affinity and binding activity. After humanization, this antibody is used as part of the fusion protein of this invention. IL-15 is then used as the other part of the fusion protein. Given the risk of cytokine storms from prolonged IL-15 residence in the human body, this invention employs a double mutation (H310Q and H433N) in the Fc region of the anti-PD-1 antibody to obtain an anti-PD-1 antibody mutant, shortening the half-life of the fusion protein. Simultaneously, this invention mutates IL-15 by N72D to obtain an IL-15 mutant, enhancing receptor affinity, signal transduction, and reducing side effects, while also exhibiting synergistic anti-tumor effects. Finally, this invention connects the C-terminus of the anti-PD-1 antibody mutant Fc region to the N-terminus of the IL-15 mutant using a (GGGGS)5 linker to construct a vector for expression.

[0010] To address this, the present invention constructs a PD1-IL15 bifunctional fusion protein, the fusion protein comprising an anti-PD-1 antibody mutant and an IL-15 mutant; the anti-PD-1 antibody mutant is formed by double mutation of H310Q and H433N in the Fc region of the anti-PD-1 antibody; the IL-15 mutant is formed by N72D mutation in the IL-15 molecule.

[0011] Furthermore, the anti-PD-1 antibody of the present invention can be converted into CD73 monoclonal antibody or CD276 monoclonal antibody.

[0012] Furthermore, the connection method between the anti-PD-1 antibody mutant and the IL-15 mutant is as follows: the C-terminus of the anti-PD-1 antibody mutant Fc is connected to the N-terminus of the IL-15 mutant using a (GGGGS)5 linker.

[0013] Furthermore, the mutated region of the anti-PD-1 antibody is a mutated human IgG1 constant region, and the amino acid sequence of the Fc mutated region (mutated human IgG1 constant region) of the anti-PD-1 antibody is shown in SEQ ID NO.7.

[0014] Furthermore, the amino acid sequence of the IL-15 mutant is shown in SEQ ID NO.17.

[0015] Furthermore, the heavy chain amino acid sequence of the PD1-IL15 bifunctional fusion protein is shown in SEQ ID NO.19, and the light chain amino acid sequence is shown in SEQ ID NO.13.

[0016] Furthermore, the present invention provides a polynucleotide encoding the above-mentioned PD1-IL15 bifunctional fusion protein.

[0017] Furthermore, the present invention also provides an expression vector containing the above-mentioned polynucleotides.

[0018] Furthermore, the present invention also provides a host cell containing the above-mentioned polynucleotide or the above-mentioned expression vector.

[0019] In addition, the present invention provides a pharmaceutical composition comprising the PD1-IL15 bifunctional fusion protein, the polynucleotide, the expression vector or the host cell as active ingredients, and a pharmaceutically acceptable carrier.

[0020] Furthermore, the present invention also provides the use of the PD1-IL15 bifunctional fusion protein, the polynucleotide, the expression vector, the host cell, or the pharmaceutical composition in the preparation of a drug for treating tumors.

[0021] The PD1-IL15 bifunctional fusion protein constructed in this invention not only enhances the antitumor activity of the fusion protein but also significantly reduces toxic side effects and improves drug safety. Experiments have shown that the transient transgenic expression level of the PD1-IL15 bifunctional fusion protein constructed in this invention is approximately 370 mg / L, with a purity >97%, which is beneficial for industrial production. Furthermore, the PD1-IL15 bifunctional fusion protein constructed in this invention exhibits a high affinity for the PD-1 antigen and can significantly stimulate NK92 proliferation, demonstrating high biological activity. Furthermore, the PD1-IL15 bifunctional fusion protein constructed in this invention can significantly reduce toxicity in mice, and can also significantly inhibit the growth of xenografts of mouse colorectal cancer cells MC38 and human lung cancer cells NCI H1975. This demonstrates that the PD1-IL15 bifunctional fusion protein constructed in this invention not only enhances the antitumor activity of the fusion protein in vivo, but also significantly reduces the toxic side effects of the fusion protein in vivo, greatly improving the drug safety of the PD1-IL15 bifunctional fusion protein, which is conducive to the promotion and application of this fusion protein.

[0022] Compared with existing technologies, the PD1-IL15 bifunctional fusion protein provided by this invention has the following advantages: (1) The PD1-IL15 bifunctional fusion protein provided by the present invention has the advantages of high expression level and high purity. The transient transgenic protein expression level of the PD1-IL15 bifunctional fusion protein constructed by the present invention is about 370 mg / L, and the purity is >97%. Compared with the transient transgenic protein expression level of 180 mg / L of the wild-type PD1-IL15 bifunctional fusion protein under the same culture conditions, it is more than 2 times higher, which is beneficial to subsequent industrial production.

[0023] (2) The PD1-IL15 bifunctional fusion protein provided by this invention has the advantages of high affinity and high biological activity. The PD1-IL15 bifunctional fusion protein constructed in this invention binds to the PD-1 antigen in EC... 50 The concentration was 0.139 ng / mL, while the wild-type PD1-IL15 bifunctional fusion protein bound to the PD-1 antigen in EC2 was 0.139 ng / mL. 50 It is 0.395 ng / mL. Simultaneously, this PD1-IL15 bifunctional fusion protein stimulates EC proliferation of NK92 cells. 50 The concentration was 0.509 ng / mL, while the wild-type PD1-IL15 bifunctional fusion protein stimulated NK92 proliferation in EC2 cells. 50 The concentration was 4.641 ng / mL. This demonstrates that the PD1-IL15 bifunctional fusion protein prepared in this invention has high affinity and high biological activity.

[0024] (3) The PD1-IL15 bifunctional fusion protein provided by this invention has the advantages of low toxicity and high safety. At a dose of 1.0 mg / kg, the survival rate of the PD1-IL15 bifunctional fusion protein constructed by this invention in mice was 100%, while the survival rate of the wild-type PD1-IL15 bifunctional fusion protein in mice was 0%. This demonstrates that the PD1-IL15 bifunctional fusion protein prepared by this invention can significantly reduce the toxicity of the fusion protein in vivo and greatly improve drug safety.

[0025] (4) The PD1-IL15 bifunctional fusion protein provided by this invention has the advantage of high anti-tumor activity. The PD1-IL15 bifunctional fusion protein constructed by this invention showed an inhibition rate of 85.3% against mouse colorectal cancer cells MC38 and an inhibition rate of 79.9% against human lung cancer cells NCI H1975. This demonstrates that the PD1-IL15 bifunctional fusion protein prepared by this invention has a significant therapeutic effect on solid tumors. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of PD1-IL15 constructed in Example 1.

[0027] Figure 2 The image shows the SDS-PAGE detection results of muPD1-IL15 and wtPD1-IL15.

[0028] Figure 3 The image shows the HPLC purity test results for mu[PD1-IL15].

[0029] Figure 4 The graph shows the HPLC purity test results for wt PD1-IL15.

[0030] Figure 5 The graph shows the results of the affinity test for PD-1 antigen binding between mu PD1-IL15 and wt PD1-IL15.

[0031] Figure 6 Figure showing the activity results of muPD1-IL15 and wtPD1-IL15 in promoting NK92 proliferation.

[0032] Figure 7 The image shows the results of muPD1-IL15 inhibiting the growth of MC38 colorectal cancer xenografts in mice.

[0033] Figure 8 The figure shows the results of muPD1-IL15 inhibiting the growth of human lung cancer cell NCI H1975 xenografts. Detailed Implementation

[0034] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0035] Example 1: Construction of PD1-IL15 bifunctional fusion protein 1. Structure of the PD1-IL15 bifunctional fusion protein: The PD1-IL15 bifunctional fusion protein provided by this invention includes an anti-PD-1 antibody mutant and an IL-15 mutant. This invention utilizes hybridoma technology to screen for an anti-PD-1 antibody. The Fc region of the screened anti-PD-1 antibody is double-mutated with H310Q and H433N to obtain the anti-PD-1 antibody. The IL-15 molecule is mutated with N72D to obtain an IL-15 mutant. The C-terminus of the Fc region of the anti-PD-1 antibody mutant and the N-terminus of the IL-15 mutant are linked using a (GGGGS)5 linker. The structure of the PD1-IL15 bifunctional fusion protein is Fab-Fc-[(GGGGS)5-IL15]2, as shown in the schematic diagram below. Figure 1 As shown.

[0036] 2. Construction of the PD1-IL15 bifunctional fusion protein: (1) The full-length light chain variable region (VL) gene sequence of the anti-PD-1 monoclonal antibody screened in this invention is 321 bp, encoding 107 amino acid residues. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The full-length heavy chain variable region (VH) sequence of the anti-PD-1 monoclonal antibody is 339 bp, encoding 113 amino acid residues. The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0037] (2) Using site-directed mutagenesis or gene synthesis, the 310th amino acid H (histidine) of the human IgG1 constant region (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6) is converted to Q (glutamine) and the 433rd amino acid H (histidine) is converted to N (asparagine), while the other amino acid sequences remain unchanged, to form a mutated human IgG1 constant region (amino acid sequence as shown in SEQ ID NO.7, nucleotide sequence as shown in SEQ ID NO.8).

[0038] (3) The VH of the anti-PD-1 monoclonal antibody is spliced ​​with the constant region of human IgG1 to form a humanized antibody heavy chain (amino acid sequence as shown in SEQ ID NO.11, nucleotide sequence as shown in SEQ ID NO.12); the VL of the anti-PD-1 monoclonal antibody is spliced ​​with the constant region of human kappa chain (amino acid sequence as shown in SEQ ID NO.9, nucleotide sequence as shown in SEQ ID NO.10) to form a humanized antibody light chain (amino acid sequence as shown in SEQ ID NO.13, nucleotide sequence as shown in SEQ ID NO.14).

[0039] (4) The 72nd amino acid N (asparagine) of the human IL-15 molecule (amino acid sequence as shown in SEQ ID NO.15, nucleotide sequence as shown in SEQ ID NO.16) is converted to D (aspartic acid), while the other amino acids remain unchanged, to form a mutant IL-15 (amino acid sequence as shown in SEQ ID NO.17, nucleotide sequence as shown in SEQ ID NO.18); the spliced ​​humanized antibody heavy chain sequence of the anti-PD-1 antibody (amino acid sequence as shown in SEQ ID NO.11, nucleotide sequence as shown in SEQ ID NO.12) is linked with the IL-15 mutant sequence through (GGGGS)5 to form the heavy chain of the PD1-IL15 bifunctional fusion protein (amino acid sequence as shown in SEQ ID NO.19, nucleotide sequence as shown in SEQ ID NO.20); the light chain is the spliced ​​humanized antibody light chain (amino acid sequence as shown in SEQ ID NO.13, nucleotide sequence as shown in SEQ ID NO.14).

[0040] (5) The heavy chain amino acid sequence of the PD1-IL15 bifunctional fusion protein provided by the present invention is shown in SEQ ID NO.19, and the light chain amino acid sequence is shown in SEQ ID NO.13.

[0041] Example 2: Expression of PD1-IL15 bifunctional fusion protein 1. Experimental Method: The heavy and light chain genes of the PD1-IL15 bifunctional fusion protein prepared in Example 1 and the unmutated wild-type PD1-IL15 bifunctional fusion protein were constructed into the pcDNA3.4 expression vector, respectively. They were transfected into Expi-293F cells and purified by Protein A to obtain the mutant muPD1-IL15 (the PD1-IL15 bifunctional fusion protein constructed in this invention) and the wild-type wtPD1-IL15 (the unmutated PD1-IL15 bifunctional fusion protein). The molecular weight of each expressed antibody was determined to be around 180 kD by SDS-PAGE electrophoresis and SEC-HPLC, and the antibody purity was >95%. The antibodies were quantified, aliquoted, and frozen at -80 °C for later use.

[0042] 2. Experimental Results: The experimental results are shown in Table 1 and Figures 2-4 As shown.

[0043] 2.1 The expression levels of the PD1-IL15 bifunctional fusion protein are shown in Table 1: Table 1. Expression levels of the PD1-IL15 bifunctional fusion protein

[0044] 2. The SDS-PAGE electrophoresis and SEC-HPLC results of the PD1-IL15 bifunctional fusion protein are as follows: Figures 2-4 As shown: Figure 2 The image shows the SDS-PAGE detection results for muPD1-IL15 and wtPD1-IL15. Figure 3 The image shows the HPLC purity test results for muPD1-IL15. Figure 4 The graph shows the HPLC purity test results for wtPD1-IL15.

[0045] From Table 1 and Figures 2-4It can be seen that the purity of the PD1-IL15 bifunctional fusion protein (muPD1-IL15) prepared by this invention is >97%, and the transient transgenic protein expression level is about 370 mg / L. Compared with the transient transgenic protein expression level of the wild-type PD1-IL15 bifunctional fusion protein under the same culture conditions, which is 180 mg / L, the expression level is increased by more than 2 times, which is convenient for subsequent scale-up production.

[0046] Example 3: Determination of the affinity of the PD1-IL15 bifunctional fusion protein for PD-1 antigen in vitro. 1. Experimental Method: The affinity activities of muPD1-IL15 and wtPD1-IL15 for PD-1 antigen were demonstrated using an ELISA assay. The method was as follows: ELISA plates were coated with 1 μg / mL PD-1 antigen, blocked with 5% BSA, and washed with PBST. Then, muPD1-IL15, wtPD1-IL15, and nivolumab (PD-1 monoclonal antibody) were serially diluted (starting at 1 μg, followed by 10-fold serial dilutions, for a total of 8 dilutions), with 100 µL added to each well and incubated at 37 °C for 1 h. After washing with PBST, HRP-labeled secondary antibody was added, and the plates were incubated at 37 °C for 1 h. After washing with PBST, 100 µL of TMB chromogenic solution was added, and the plates were incubated in the dark for 3-5 min. Finally, 50 µL of 1 mol / L sulfuric acid stop solution was added to each well, and the readings were taken at 450 nm using a microplate reader. The EC50 was calculated. 50 To determine the activity of the sample.

[0047] 2. Experimental Results: Experimental results are as follows Figure 5 As shown.

[0048] Figure 5 This image shows the results of the affinity assay for PD-1 antigen binding to muPD1-IL15 and wtPD1-IL15. Figure 5 It can be seen that the ECG of PD-1 antigen binds to muPD1-IL15. 50 It is 0.139 ng / mL, EC bound to wt% PD1-IL15 50 It is 0.395 ng / mL, the EC50 that binds to nivolumab (PD-1 monoclonal antibody). 50 It is 0.042 ng / mL. This demonstrates that the PD1-IL15 bifunctional fusion protein prepared in this invention has a high affinity for the PD-1 antigen, and the affinity is equal to that of commercially available antibodies.

[0049] Example 4: Determination of NK92 proliferation stimulated by PD1-IL15 bifunctional fusion protein in vitro 1. Experimental Method: The biological activities of muPD1-IL15 and wtPD1-IL15 were demonstrated by the proliferation of NK92 cells (purchased from Wuhan Pronosai) stimulated with IL-15. The method was as follows: NK92 cells in logarithmic growth phase were heated to 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in blank culture medium and then starved in a CO2 incubator for 4 h; cell counts were performed, and the cell concentration was controlled at 6 x 10⁻⁶ cells / mL. 5 / ml; Take 50 μL of the above cell suspension into a 96-well cell culture plate, and simultaneously add 50 μL of IL-2 standard dilution (starting 1000 IU / mL, 10-fold serial dilution, 8 gradients, 2 replicates per gradient) and test sample dilution (starting 100 ng / mL, 10-fold serial dilution, 8 gradients, 2 replicates per gradient) to the plate, and mix gently; incubate at 37 ℃, 5% CO2 for 72-96 h; carefully pipette 10 μL of CCK-8 solution into the plate, mix gently, being careful not to introduce air bubbles; incubate at 37 ℃ for 1-4 h; detect the reading at 450 nm wavelength on a microplate reader and calculate EC. 50 To determine the activity of the sample.

[0050] 2. Experimental Results: Experimental results are as follows Figure 6 As shown.

[0051] Figure 6 The graph shows the activity results of muPD1-IL15 and wtPD1-IL15 in promoting NK92 proliferation. Figure 6 It is known that both muPD1-IL15 and wtPD1-IL15 can stimulate NK92 proliferation, EC 50 The concentrations were 0.509 ng / mL and 4.641 ng / mL, respectively, indicating that muPD1-IL15 has higher biological activity than wtPD1-IL15. As a control, the EC50 of IL-12 (R&D) was... 50 The concentration was 0.009 ng / mL (IL-2 was used as the standard because there are currently no IL-15 products on the market).

[0052] Example 5: Toxicity assay of PD1-IL15 bifunctional fusion protein 1. Experimental Method: mu PD1-IL15 and wt PD1-IL15 were administered intraperitoneally to C57BL / 6 mice (Vital River Pharmaceuticals) twice on days 1 and 3, with an injection volume of 0.2 mL per injection. The doses were 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg and 2 mg / kg, respectively. The mortality of the experimental mice was observed daily. The control group was given the same volume of PBS.

[0053] 2. Experimental Results: The experimental results are shown in Table 2.

[0054] Table 2 Survival rate of experimental animals in each dosage group

[0055] Table 2 shows that on day 7 of the experiment, in the muPD1-IL15 group, all experimental animals survived (100% survival rate) at doses of 0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg, while 70% of the experimental animals died (30% survival rate) at a dose of 2.0 mg / kg. However, in the wtPD1-IL15 group, 100% of the experimental animals died (0% survival rate) at doses of 1.0 mg / kg and 2.0 mg / kg, 60% of the experimental animals died (40% survival rate) at a dose of 0.5 mg / kg, and no experimental animals died (100% survival rate) at a dose of 0.2 mg / kg. These results indicate that after site-directed mutagenesis, the toxicity of muPD1-IL15 to experimental mice was significantly reduced compared to wtPD1-IL15.

[0056] Example 6: Anti-colorectal cancer activity of PD1-IL15 bifunctional fusion protein 1. Experimental Method: The in vivo antitumor activity of muPD1-IL15 and wtPD1-IL15 was evaluated using a mouse MC38 colorectal cancer xenograft model. The method involved culturing mouse colon cancer MC38 cells in vitro and adjusting the cell suspension concentration to 2 × 10⁻⁶. 7 / ml. Under aseptic conditions, 100 μL of cell suspension was inoculated subcutaneously into the right rib area of ​​C57BL / 6 mice. The diameter of the subcutaneous xenograft in mice was measured with calipers. The tumors were allowed to grow to an average volume of 100-200 mm. 3 The animals were then randomly divided into groups of 6. Both mu PD1-IL15 and wt PD1-IL15 were administered at 0.2 mg / kg, while the control group received an equal volume of PBS. The drugs were administered intraperitoneally twice weekly, with a volume of 0.2 mL per injection, for two consecutive weeks. Throughout the experiment, the tumor diameter was measured three times weekly, and the mice were weighed simultaneously. The formula for calculating tumor volume (TV) is: TV = 1 / 2 × a × b² Where a and b represent length and width, respectively. The relative tumor volume (RTV) is calculated based on the measurement results using the formula: RTV = Vt / V0. Here, V0 is the tumor volume measured at the time of grouped drug administration (i.e., d0), and Vt is the tumor volume at each measurement. The evaluation index for antitumor activity is the relative tumor proliferation rate (T / C%), calculated using the following formula: T / C (%) = (TRTV / CRTV) × 100 Tumor inhibition rate TGI (%) = 100 - T / C (%) TRTV: RTV in the treatment group; CRTV: RTV in the negative control group.

[0057] 2. Experimental Results: Experimental results are as follows Figure 7 As shown.

[0058] Figure 7 This image shows the results of muPD1-IL15 inhibiting the growth of MC38 colorectal cancer xenografts in mice. Figure 7 It can be seen that both muPD1-IL15 and wtPD1-IL15 exhibited strong anti-tumor activity, with TGI of 85.3% and 84.0% respectively, which is not much different. This indicates that the site-directed mutation of PD1-IL15 molecules does not affect its anti-tumor activity in vivo.

[0059] Example 7: Anti-lung cancer activity of PD1-IL15 bifunctional fusion protein 1. Experimental Method: Human lung cancer NCI-H1975 cells (tumor cells with high levels of PD-L1 protein on their surface, sensitive to immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors), suitable for determining the therapeutic effect of anti-PD-1 antibodies) were cultured in vitro, and the cell suspension concentration was adjusted to 5 × 10⁻⁶. 7 / mL. Under aseptic conditions, 100 μL of cell suspension was inoculated subcutaneously into the right rib area of ​​nude mice. Once tumor cells formed a solid tumor subcutaneously, the diameter of the transplanted tumor was measured using calipers. The tumor was considered complete when the average tumor volume reached 50-100 mm. 3 The animals were then randomly assigned to groups. They were administered the medication intraperitoneally three times a week at doses of 0.2 mg / kg, 0.5 mg / kg, and 1 mg / kg, for a total of six weeks. Throughout the experiment, the diameter of the transplanted tumor was measured twice a week, and the mice were weighed simultaneously. The formula for calculating tumor volume (TV) is as follows: TV = 1 / 2 × a × b² Where a and b represent length and width, respectively. The relative tumor volume (RTV) is calculated based on the measurement results using the formula: RTV = Vt / V0. Here, V0 is the tumor volume measured at the time of grouped drug administration (i.e., d0), and Vt is the tumor volume at each measurement. The evaluation index for antitumor activity is the relative tumor proliferation rate (T / C%), calculated using the following formula: T / C (%) = (TRTV / CRTV) × 100 Tumor inhibition rate TG (%) = 100 - T / C (%) TRTV: RTV in the treatment group; CRTV: RTV in the negative control group.

[0060] 2. Experimental Results: Experimental results are as follows Figure 8 As shown.

[0061] Figure 8 This figure shows the results of muPD1-IL15 inhibiting the growth of human lung cancer cell NCI H1975 xenografts. Figure 8 It can be seen that muPD1-IL15 exhibits strong anti-tumor activity, with TGI of 79.9%, 72.4% and 70.6% at doses of 1.0 mg / kg, 0.5 mg / kg and 0.2 mg / kg, respectively.

[0062] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A PD1-IL15 bifunctional fusion protein, characterized in that, The fusion protein includes an anti-PD-1 antibody mutant and an IL-15 mutant; the anti-PD-1 antibody mutant is a double mutation of H310Q and H433N in the Fc region of the anti-PD-1 antibody; the IL-15 mutant is an N72D mutation in the IL-15 molecule.

2. The PD1-IL15 bifunctional fusion protein as described in claim 1, characterized in that, The connection method between the anti-PD-1 antibody mutant and the IL-15 mutant is as follows: the C-terminus of the anti-PD-1 antibody mutant Fc is connected to the N-terminus of the IL-15 mutant using the (GGGGS)5 linker.

3. The PD1-IL15 bifunctional fusion protein as described in claim 1, characterized in that, The amino acid sequence of the Fc mutation region of the anti-PD-1 antibody is shown in SEQ ID NO.

7.

4. The PD1-IL15 bifunctional fusion protein as described in claim 1, characterized in that, The amino acid sequence of the IL-15 mutant is shown in SEQ ID NO.

17.

5. The PD1-IL15 bifunctional fusion protein as described in claim 1, characterized in that, The heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO.19, and the light chain amino acid sequence is shown in SEQ ID NO.

13.

6. A polynucleotide, characterized in that, The polynucleotide encodes the PD1-IL15 bifunctional fusion protein as described in any one of claims 1 to 5.

7. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 6.

8. A host cell, characterized in that, The host cell contains the polynucleotide as described in claim 6 or the expression vector as described in claim 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an active ingredient consisting of the PD1-IL15 bifunctional fusion protein as described in any one of claims 1 to 5, the polynucleotide as described in claim 6, the expression vector as described in claim 7, or the host cell as described in claim 8, and a pharmaceutically acceptable carrier.

10. Use of the PD1-IL15 bifunctional fusion protein as described in any one of claims 1 to 5, the polynucleotide as described in claim 6, the expression vector as described in claim 7, the host cell as described in claim 8, or the pharmaceutical composition as described in claim 9 in the preparation of a drug for treating tumors.

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