Dual fusion protein and use thereof

Abstract

A PD-1 targeted IL15Rα sushi-IL-15 bispecific recombinant protein and a use thereof.

Description

A double fusion protein and its use Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a PD-1-targeted IL15RαSUSHI-IL15 bispecific recombinant protein and its use. Background Art

[0002] Two very promising approaches in cancer immunotherapy include cytokine-based treatment and blockade of immune checkpoint proteins such as PD-1.

[0003] Cytokines such as interleukin-2 (IL2) and interleukin-15 (IL15) contribute to the proliferation and differentiation of B cells, T cells, and natural killer (NK) cells. Both cytokines exert their cell signaling function by binding to a trimeric complex composed of two shared receptors: the common γ chain (γc; CD132) and the IL2 receptor β chain (IL2Rβ; CD122), as well as an α-chain receptor unique to each cytokine: the IL2 receptor α (IL2Rα; CD25) or the IL15 receptor α (IL15Rα; CD215). Both cytokines are considered potentially valuable therapeutic agents in oncology, and IL2 has been approved for the treatment of patients with metastatic renal cell carcinoma and malignant melanoma. Although several recombinant IL15s are underway in clinical trials, no recombinant IL15s are currently approved for marketing. However, as potential drugs, both cytokines have very rapid clearance rates, with half-lives of several minutes. Due to its short half-life, IL2 immunotherapy can cause systemic toxicity when high doses are administered to ensure efficacy. This systemic toxicity has also been reported in recent clinical trials of IL15 immunotherapy (Guo et al., J Immunol, 2015, 195(5): 2353-64).

[0004] After T cell activation, immune checkpoint proteins (such as PD-1) are upregulated to regulate autoimmunity by depleting activated T cells by binding to immune checkpoint ligands (such as PD L1). However, immune checkpoint proteins are also upregulated in tumor-infiltrating lymphocytes (TILs), and immune checkpoint ligands are overexpressed on tumor cells, thereby contributing to the immune escape of tumor cells. Blocking the interaction between immune checkpoint proteins and ligands by drugs such as nivolumab and pembrolizumab eliminates the inhibitory effect on TILs, making it highly effective for cancer treatment. Although immune checkpoint blockade therapies such as nivolumab and pembrolizumab are promising, many patients still do not benefit from immune checkpoint blockade alone.

[0005] Despite years of application in tumor immunotherapy, cytokines are often hindered by systemic side effects, such as systemic toxicity, which hinder their full potential in tumor treatment and address unmet clinical needs. Recent studies have demonstrated that "targeted cytokines," such as antibody-cytokine fusion proteins, can deliver cytokines to desired cell types while minimizing peripheral exposure and resulting toxicity. Therefore, the development of therapeutic agents based on targeted cytokines will be of great value in treating various diseases, such as cancer.

[0006] WO2019166946A1 discloses variants related to human IL15 and fusion proteins comprising the same. It has been confirmed that, in the first aspect, compared to wild-type human IL15 polypeptide or wild-type IL15 receptor α-IL15 fusion polypeptide, the IL15 variants of the application have reduced binding to IL15 receptor α (CD215) or no binding activity, and / or the interaction between IL15 and its signaling receptor (composed of IL2 receptor β (CD122) and common γ chain (CD132)) is reduced. In the second aspect, these IL15 variants with reduced affinity (when presented as antibody cytokine fusion proteins) selectively target the desired cell types (those expressing the antibody target). Compared to those cells expressing the two components, cell types expressing the IL15 receptor complex (but not expressing the antibody target) are less activated or not activated. Therefore, the IL15 variants and IL15 fusion proteins disclosed in WO2019166946A1 selectively regulate the activation of cell subpopulations to effectively and safely promote biological activities, such as anti-tumor activity.

[0007] However, data from the IL15 variants and IL15 fusion proteins disclosed in WO2019166946A1 show that the fusion protein of this application still shows obvious cytokine release. As shown in Figure 14 of WO2019166946A1, after the PD-1-targeted IL15 fusion protein was administered to mice, obvious IL-6 and IFNγ release was still detected in the serum outside the tumor microenvironment. Elevated IL-6 levels in serum are a sign of a cytokine storm. It can be seen that even if the targeting effect of the immune checkpoint is increased, IL15 will not only activate tumor-infiltrating lymphocytes (TIL cells), but also activate immune cells in non-tumor tissues that do not express immune checkpoints, release cytokines, and induce potential immune safety risks.

[0008] Clinical data have shown that the combination of anti-PD-1 antibodies and IL15 protein has clinical benefits (Lancet Oncol. 2018 May; 19(5): 694-704), indicating that the combination of anti-PD-1 antibodies and IL15 protein has an anti-tumor synergistic effect. However, the combination of anti-PD-1 antibodies and IL15 protein has the following drawbacks: the combination of anti-PD-1 antibodies and IL15 is limited by the toxic side effects of IL15. The most common adverse reactions are injection site reactions (90%, 19 / 21) and flu-like symptoms (71%); two patients experienced the most common grade 3 adverse reactions, namely lymphopenia and fatigue, and one patient suffered a grade 3 myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1. SDS-PAGE electrophoresis of LC01-7-054-M5 after protein A purification.

[0010] Figure 2. SDS-PAGE electrophoresis of LC01-7-054-M5 after purification (non-reducing gel).

[0011] Figure 3. Proliferative activity of the bispecific recombinant protein on PD-1-negative non-target cells M-07e.

[0012] Figure 4. Proliferation activity detection of LC01-7-054-M5 in the 1-100 nM range on PD-1 negative non-target target cells M-07e.

[0013] Figure 5. Detection of the proliferation activity of the bispecific recombinant protein on the PD-1 positive target cells M-07e-PD-1.

[0014] Figure 6. PD-1 expression on the surface of CD8+ cell population in unstimulated PBMCs.

[0015] Figure 7. PD-1 expression on the surface of CD8+ cell population in OKT3-activated PBMCs.

[0016] Figure 8. Cell surface PD-1 expression abundance diagram of different cell subsets.

[0017] Figure 9. Proliferation activity of various cell subsets on the eighth day after treatment with different drug groups.

[0018] Figure 10 shows the proliferation activity of CD8+ cell population on the eighth day after treatment with different drug-dosing groups.

[0019] Figure 11. Proliferative activity of various cell subsets in cynomolgus monkeys at different time points after a single dose of LC01-7-054-M5.

[0020] Figure 12. Tumor volume (mm) on day 35 after treatment of tumor-bearing mice in different drug groups 3 ).

[0021] Figure 13. Schematic diagram of the effect of LC01-7-054-M5 on the expansion of tumor-infiltrating immune cells (TILs).

[0022] Figure 14. IFN-γ release profile of PBMCs treated with LC01-7-054-M5 for 72 hours.

[0023] Figure 15. IL-6 release profile of PBMCs treated with LC01-7-054-M5 for 72 hours.

[0024] Figure 16. TNF-α release profile of PBMCs treated with LC01-7-054-M5 for 72 hours.

[0025] Figure 17. LC01-7-054-M5 block diagram.

[0026] Figure 18. Pharmacodynamic evaluation of different dosing groups in a subcutaneous transplant tumor model of human liver cancer Huh-7 cells mixed with PBMCs.

[0027] Figure 19. Pharmacodynamic evaluation of different dosing groups in a subcutaneous transplant tumor model of human liver cancer Huh-7 cells mixed with PBMCs.

[0028] Detailed Description of the Invention

[0029] This application superimposes cytokine therapy with immune checkpoint blockade by designing a highly effective and low-toxic bispecific recombinant protein, preferably an antibody-cytokine fusion protein, to increase the effective concentration of cytokines in the tumor microenvironment while reducing the potential systemic toxicity of cytokines caused by activation of non-target cells.

[0030] In a first aspect, the present application provides a bispecific recombinant protein, wherein the bispecific recombinant protein comprises two parts: an immune checkpoint blocker and a cytokine receptor-cytokine, wherein the cytokine is an IL15 variant, and compared with wild-type IL15, the IL15 variant has a weakened affinity for IL2Rβ.

[0031] In some embodiments, the immune checkpoint blocker is a PD-1 blocker, and the cytokine receptor-cytokine is IL15R-IL15; wherein IL15 is an IL15 variant with reduced affinity for IL2Rβ compared to wild-type IL15 as shown in SEQ ID NO:18; the PD-1 blocker is an anti-PD-1 antibody or an antigen-binding fragment thereof; the N-terminus of the IL15R is connected to the C-terminus of the light chain of the anti-PD-1 antibody or its antigen-binding fragment via a linker; and the N-terminus of the IL15 variant is connected to the C-terminus of the IL15R via a linker.

[0032] In some embodiments, the IL15 variant comprises one or more point mutations relative to wild-type IL15 selected from positions N1, N4, S7, D8, K10, K11, S29, D30, V31, H32, D61, E64, N65, I68, L69, N72, Q108, M109, and I111.

[0033] In some embodiments, the IL15 variant comprises one or more point mutations selected from positions N1, N4, and D61 relative to wild-type IL15.

[0034] In some embodiments, the IL15 variant comprises one or more point mutations at positions selected from N1D, N1A, N1K, N1R, N1E, N1Q, N1G, N4A, N4D, N4E, N4Q, N4G, N4L, N4Y, and D61A relative to wild-type IL15.

[0035] In some embodiments, the IL15 variant comprises one or more point mutations at positions selected from N1D, N4A, N4D, and D61A relative to wild-type IL15.

[0036] In some embodiments, the IL15 variant comprises a point mutation of D61A relative to wild-type IL15.

[0037] In some embodiments, the linker connecting the IL15R and IL15 variant is (GGGGS) n , wherein n is selected from an integer of 1-10, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the linker is (GGGGS)5.

[0038] In some embodiments, the IL15R is a truncated IL15RαSUSHI; preferably, the sequence of the IL15RαSUSHI comprises SEQ ID NO: 17 or is as shown in SEQ ID NO: 17, and the sequence of the IL15 variant comprises any one of SEQ ID NOs: 19-22 or is as shown in any one of SEQ ID NOs: 19-22.

[0039] In some embodiments, the C-terminus of the IL15 variant is linked to the N-terminus of the Fc region directly or through a linker.

[0040] In some embodiments, the linker is (GGGGS) n , wherein n is selected from an integer of 1-10, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0041] In some embodiments, the C-terminus of the IL15 variant is directly linked to the N-terminus of the Fc region.

[0042] In some embodiments, the Fc region sequence is an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0043] In some embodiments, the Fc region sequence is an IgG1 Fc region.

[0044] In some embodiments, the Fc region sequence is an IgG1 Fc variant.

[0045] In some embodiments, the IgG1 Fc variant comprises mutations that reduce Fc effects, mutations that extend half-life, and / or knob-into-hole mutations that facilitate purification.

[0046] In some embodiments, the mutation that reduces the Fc effect comprises one or more point mutations selected from L234A and L235A; preferably, the mutation that reduces the Fc effect comprises a combination of L234A-L235A mutations.

[0047] In some embodiments, the half-life-extending mutation comprises one or more point mutations selected from M252Y, S254T, and T256E; preferably, the half-life-extending mutation comprises a combination of M252Y-S254T-T256E mutations.

[0048] In some embodiments, in the knob-into-hole mutation that facilitates purification, the knob mutation is T366W, and the hole mutation is T366S-L368A-Y407V.

[0049] The numbering of amino acids in the Fc variants is based on the EU index.

[0050] In some embodiments, the Fc region sequence comprises any one of SEQ ID NOs: 11-15 or is shown as any one of SEQ ID NOs: 11-15; when the Fc region is an Fc wild type, the Fc region sequence is shown as SEQ ID NO: 11; when the Fc region is an Fc variant, it is composed of an FcA region and an FcB region, when the FcA region sequence is SEQ ID NO: 12, the FcB region sequence is SEQ ID NO: 13, and when the FcA region sequence is SEQ ID NO: 14, the FcB region sequence is SEQ ID NO: 15; preferably, the Fc region sequence is shown as SEQ ID NO: 13 or SEQ ID NO: 15.

[0051] In some embodiments, the immune checkpoint blocker is an agent that blocks the interaction between PD-1 and PD-L1.

[0052] In some embodiments, the immune checkpoint blocker is a PD-1 blocker, preferably, the PD-1 blocker is an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0053] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from Nivolumab and Pembrolizumab or an antigen-binding fragment thereof.

[0054] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain; in some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 half antibody.

[0055] In some embodiments, the C-terminus of the anti-PD-1 half-antibody light chain is connected to the cytokine receptor-cytokine via a linker; the linker is (GGGGS) n , wherein n is selected from an integer of 1-10, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the cytokine receptor-cytokine is IL15R-IL15.

[0056] In some embodiments, the heavy chain and light chain of the anti-PD-1 half antibody comprise a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3; wherein the heavy chain CDR1 sequence comprises SEQ ID NO: 1 or is as shown in SEQ ID NO: 1, the heavy chain CDR2 sequence comprises SEQ ID NO: 2 or is as shown in SEQ ID NO: 2, and the heavy chain CDR3 sequence comprises SEQ ID NO: 3 or is as shown in SEQ ID NO: 3; the light chain CDR1 sequence comprises SEQ ID NO: 4 or is as shown in SEQ ID NO: 4, the light chain CDR2 sequence comprises SEQ ID NO: 5 or is as shown in SEQ ID NO: 5, and the light chain CDR3 sequence comprises SEQ ID NO: 6 or is as shown in SEQ ID NO: 6, and the CDR regions are determined using Kabat numbering.

[0057] In some embodiments, the heavy chain variable region sequence of the anti-PD-1 half antibody comprises SEQ ID NO: 7 or is set forth as SEQ ID NO: 7, and the light chain variable region sequence comprises SEQ ID NO: 8 or is set forth as SEQ ID NO: 8.

[0058] In some embodiments, the Fc region sequence of the anti-PD-1 half antibody is an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0059] In some embodiments, the Fc region sequence of the anti-PD-1 half antibody is an IgG1 Fc region.

[0060] In some embodiments, the Fc region sequence of the anti-PD-1 half-antibody is an IgG1 Fc variant.

[0061] In some embodiments, the IgG1 Fc variant comprises mutations that reduce Fc effects, mutations that extend half-life, and / or knob-into-hole mutations that facilitate purification.

[0062] In some embodiments, the mutation that reduces the Fc effect comprises one or more point mutations selected from L234A and L235A; preferably, the mutation that reduces the Fc effect comprises a combination of L234A-L235A mutations.

[0063] In some embodiments, the half-life-extending mutation comprises one or more point mutations selected from M252Y, S254T, and T256E; preferably, the half-life-extending mutation comprises a combination of M252Y-S254T-T256E mutations.

[0064] In some embodiments, in the knob-into-hole mutation that facilitates purification, the knob mutation is T366W, and the hole mutation is T366S-L368A-Y407V.

[0065] In some embodiments, the Fc region sequence of the anti-PD-1 half antibody is as shown in any one of SEQ ID NOs: 11-15; when the Fc region is Fc wild-type, the Fc region sequence is as shown in SEQ ID NO: 11; when the Fc region is an Fc variant, the Fc region sequence of the anti-PD-1 half antibody is as shown in any one of SEQ ID NOs: 12-15; preferably, the Fc region sequence of the anti-PD-1 half antibody is as shown in SEQ ID NO: 12 or SEQ ID NO: 14. In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody and an IL15RαSUSHI-(GGGGS)5-IL15 variant, wherein the anti-PD-1 half antibody is composed of a heavy chain and a light chain, and the heavy chain and light chain comprise a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3; wherein the heavy chain CDR1 sequence comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1, the heavy chain CDR2 sequence comprises SEQ ID NO: 2 or as shown in SEQ ID NO: 2, and the heavy chain CDR3 sequence comprises SEQ ID NO: 3 or as shown in SEQ ID NO: 3; the light chain CDR1 sequence comprises SEQ ID NO: 4 or as shown in SEQ ID NO: 4, the light chain CDR2 sequence comprises SEQ ID NO: 5 or as shown in SEQ ID NO: 5, and the light chain CDR3 sequence comprises SEQ ID NO: 6 or as shown in SEQ ID NO: 7. NO:6; the CDR regions are determined using the Kabat numbering method; the IL15 variant comprises one or more point mutations selected from N1D, N4A, N4D, and D61A relative to the wild-type IL15 as shown in SEQ ID NO:18, wherein the light chain C-terminus of the anti-PD-1 half antibody is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a linker.

[0066] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain, and an IL15RαSUSHI-(GGGGS)5-IL15 variant; wherein the N-terminus of the IL15R is connected to the C-terminus of the light chain of the anti-PD-1 antibody via a linker; the heavy and light chains of the anti-PD-1 half antibody comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence comprises SEQ ID NO:7 or is as shown in SEQ ID NO:7, and the light chain variable region sequence comprises SEQ ID NO:8 or is as shown in SEQ ID NO:8; the IL15 variant comprises one or more point mutations selected from N1D, N4A, N4D, and D61A relative to wild-type IL15 as shown in SEQ ID NO:18, wherein the C-terminus of the light chain of the anti-PD-1 half antibody is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a linker.

[0067] In some embodiments, the bispecific recombinant protein is further connected to the N-terminus of the Fc region at the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant; preferably, the connection is directly or through a linker to the N-terminus of the Fc region.

[0068] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain and an IL15RαSUSHI-(GGGGS)5-IL15 variant, wherein the heavy chain and light chain of the anti-PD-1 half antibody comprise a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3; wherein the heavy chain CDR1 sequence comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1, the heavy chain CDR2 sequence comprises SEQ ID NO: 2 or as shown in SEQ ID NO: 2, and the heavy chain CDR3 sequence comprises SEQ ID NO: 3 or as shown in SEQ ID NO: 3; the light chain CDR1 sequence comprises SEQ ID NO: 4 or as shown in SEQ ID NO: 4, the light chain CDR2 sequence comprises SEQ ID NO: 5 or as shown in SEQ ID NO: 5, and the light chain CDR3 sequence comprises SEQ ID NO: 6 or as shown in SEQ ID NO: 7. NO:6; the CDR region is determined using the Kabat numbering method; the IL15 variant comprises a point mutation of D61A relative to the wild-type IL15 shown in SEQ ID NO:18; wherein the light chain C-terminus of the anti-PD-1 half antibody is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a linker.

[0069] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain, and an IL15RαSUSHI-(GGGGS)5-IL15 variant, wherein the heavy chain and light chain of the anti-PD-1 half antibody comprise a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region sequence comprises SEQ ID NO:7 or is as shown in SEQ ID NO:7, and the light chain variable region sequence comprises SEQ ID NO:8 or is as shown in SEQ ID NO:8; the IL15 variant comprises a point mutation of D61A relative to wild-type IL15 as shown in SEQ ID NO:18; wherein the C-terminus of the light chain of the anti-PD-1 half antibody is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a linker.

[0070] In some embodiments, the bispecific recombinant protein is further connected to the N-terminus of the Fc region at the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant; preferably, the connection is directly connected or connected to the N-terminus of the Fc region through a linker; more preferably, the connection is through (GGGGS) n The linker is attached to the N-terminus of the Fc region, wherein n is selected from an integer of 1-10, such as n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0071] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain, and an IL15RαSUSHI-(GGGGS)5-IL15 variant, wherein the heavy chain and light chain of the anti-PD-1 half antibody comprise a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region sequence comprises SEQ ID NO:7 or is as shown in SEQ ID NO:7, and the light chain variable region sequence comprises SEQ ID NO:8 or is as shown in SEQ ID NO:8; the IL15 variant comprises a point mutation, D61A, relative to wild-type IL15 as shown in SEQ ID NO:18; wherein the C-terminus of the anti-PD-1 half antibody light chain is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a linker; and wherein the heavy chain constant region of the anti-PD-1 half antibody comprises a mutation that reduces Fc effect, a mutation that increases half-life, and / or a knob-into-hole mutation that facilitates purification.

[0072] In some embodiments, the Fc region sequence of the anti-PD-1 half antibody is an IgG1 Fc region.

[0073] In some embodiments, the Fc region sequence of the anti-PD-1 half-antibody is an IgG1 Fc variant.

[0074] In some embodiments, the IgG1 Fc variant comprises mutations that reduce Fc effects, mutations that extend half-life, and / or knob-into-hole mutations that facilitate purification.

[0075] In some embodiments, the mutation that reduces the Fc effect comprises one or more point mutations selected from L234A and L235A; preferably, the mutation that reduces the Fc effect comprises a combination of L234A-L235A mutations.

[0076] In some embodiments, the half-life-extending mutation comprises one or more point mutations selected from M252Y, S254T, and T256E; preferably, the half-life-extending mutation comprises a combination of M252Y-S254T-T256E mutations.

[0077] In some embodiments, in the knob-into-hole mutation that facilitates purification, the knob mutation is T366W, and the hole mutation is T366S-L368A-Y407V.

[0078] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain, and an IL15RαSUSHI-(GGGGS)5-IL15 variant, wherein the heavy chain and light chain of the anti-PD-1 half antibody comprise a heavy chain and a light chain; wherein the heavy chain sequence comprises SEQ ID NO:23 or is as shown in SEQ ID NO:23, and the light chain sequence comprises SEQ ID NO:10 or is as shown in SEQ ID NO:10; the IL15RαSUSHI-(GGGGS)5-IL15 variant sequence comprises SEQ ID NO:24 or is as shown in SEQ ID NO:24; wherein the C-terminus of the anti-PD-1 half antibody light chain is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a linker; and the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant is connected to the N-terminus of the Fc region directly or via a linker.

[0079] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain, and an IL15RαSUSHI-(GGGGS)5-IL15 variant, wherein the heavy chain sequence of the anti-PD-1 half antibody comprises SEQ ID NO: 23 or is as shown in SEQ ID NO: 23, and the light chain sequence comprises SEQ ID NO: 10 or is as shown in SEQ ID NO: 10; the IL15RαSUSHI-(GGGGS)5-IL15 variant sequence comprises SEQ ID NO: 24 or is as shown in SEQ ID NO: 24; wherein the C-terminus of the anti-PD-1 half antibody light chain is connected by (GGGGS)5-IL15. n The linker is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant, and the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant is directly connected to the N-terminus of the Fc region, and n is selected from an integer of 1-10, such as n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0080] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain, and an IL15RαSUSHI-(GGGGS)5-IL15 variant; wherein the heavy chain sequence of the anti-PD-1 half antibody comprises SEQ ID NO:23 or is as shown in SEQ ID NO:23, and the light chain sequence comprises SEQ ID NO:10 or is as shown in SEQ ID NO:10; the sequence of the IL15RαSUSHI-(GGGGS)5-IL15 variant comprises SEQ ID NO:24 or is as shown in SEQ ID NO:24; wherein the C-terminus of the anti-PD-1 half antibody light chain is linked to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a (GGGGS)5 linker, and the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant is directly linked to the N-terminus of the Fc region.

[0081] In some embodiments, the bispecific recombinant protein comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain, and an IL15RαSUSHI-(GGGGS)5-IL15 variant; wherein the heavy chain sequence of the anti-PD-1 half antibody comprises SEQ ID NO:23 or is as shown in SEQ ID NO:23, and the light chain sequence comprises SEQ ID NO:10 or is as shown in SEQ ID NO:10; the IL15RαSUSHI-(GGGGS)5-IL15 variant sequence comprises SEQ ID NO:24 or is as shown in SEQ ID NO:24; wherein the C-terminus of the anti-PD-1 half antibody light chain is linked to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a (GGGGS)5 linker, and the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant is directly linked to the N-terminus of the Fc region, and the Fc region sequence comprises SEQ ID NO:15 or is as shown in SEQ ID NO:15.

[0082] In some embodiments, the bispecific recombinant protein is LC01-7-032-M1, LC01-7-032-M3, LC01-7-032-M4, LC01-7-032-M5, or LC01-7-054-M5; wherein the left arm structure of LC01-7-032-M1 is Nivolumab (H) -Fc1, and the right arm structure is Nivolumab (L) -(GGGGS) 5-I The left arm structure of L15RαSUSHI-(GGGGS)5-IL15-N1D-Fc2; LC01-7-032-M3 is: Nivolumab(H)-Fc1, and the right arm structure is Nivolumab(L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-N4A-Fc2; the left arm structure of LC01-7-032-M4 is: Nivolumab The left arm structure of LC01-7-032-M5 is: Nivolumab (H) -Fc1, and the right arm structure is Nivolumab (L) -(GGGGS) 5-IL15Rα SUSHI-(GGGGS) 5-IL15-N4D-Fc2; the left arm structure of LC01-7-032-M5 is: Nivolumab (H) -Fc1, and the right arm structure is Nivolumab (L) -(GGGGS) 5-IL15Rα SUSHI-(GGGGS) 5-IL15-D61A-Fc2; the left arm structure of LC01-7-054-M5 is: Nivolumab (H) -Fc3, and the right arm structure is Nivolumab (L) -(GGGGS) 5-IL15Rα SUSHI-(GGGGS) 5-IL15-D61A-Fc4; wherein the Nivolumab (H) refers to the heavy chain VH and CH1 domains, and its amino acid sequence is as shown in SEQ ID NO:9; Nivolumab (L) refers to the light chain VL and CL domains, the amino acid sequence of which is shown in SEQ ID NO:10; the amino acid sequence of Fc1 is shown in SEQ ID NO:12; the amino acid sequence of Fc2 is shown in SEQ ID NO:13; the amino acid sequence of Fc3 is shown in SEQ ID NO:14; the amino acid sequence of Fc4 is shown in SEQ ID NO:15; the amino acid sequence of (GGGGS)5 is shown in SEQ ID NO:16; the amino acid sequence of IL15RαSUSHI is shown in SEQ ID NO:17; the amino acid sequence of IL15-N1D is shown in SEQ ID NO:19; the amino acid sequence of IL15-N4A is shown in SEQ ID NO:20; the amino acid sequence of IL15-N4D is shown in SEQ ID NO:21; and the amino acid sequence of IL15-D61A is shown in SEQ ID NO:22.

[0083] In a second aspect, the present application provides a nucleic acid molecule encoding the aforementioned bispecific recombinant protein.

[0084] In a third aspect, the present application provides a vector comprising a nucleic acid molecule encoding the aforementioned bispecific recombinant protein.

[0085] In a fourth aspect, the present application provides a host cell comprising the above-mentioned vector.

[0086] In a fifth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned bispecific recombinant protein.

[0087] In a sixth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned bispecific recombinant protein and a pharmaceutically acceptable carrier.

[0088] In a seventh aspect, the present application provides the use of the above-mentioned bispecific recombinant protein in the preparation of a drug for preventing and / or treating cancer.

[0089] In some embodiments, the present application provides the above-mentioned bispecific recombinant protein for use in preventing and / or treating cancer.

[0090] In some embodiments, the present application provides a method for preventing and / or treating cancer, comprising administering the aforementioned dual-property recombinant protein or the pharmaceutical composition to a subject in need thereof.

[0091] In some embodiments, the cancer is selected from a solid tumor and a hematological tumor; preferably, the cancer is a solid tumor; more preferably, the cancer is melanoma.

[0092] definition

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to in Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0094] As used herein, the terms "host," "subject," or "individual" refer to mammals, such as humans, but may also be other animals, such as wild animals, livestock, or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

[0095] Intact antibodies / full-length antibodies are glycoproteins comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.

[0096] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as PD-1). It has been shown that the antigen-binding function of an antibody can be achieved by certain fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment consisting of two Fab' fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the single-arm VL and VH domains of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 241: 544-546); and (vi) CDRs. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be recombinantly linked together by a synthetic linker to form a single, linked chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody.

[0097] The terms "Fc fragment", "Fc region", "Fc domain", "Fc portion" or similar terms in this application refer to a portion of the constant region of the heavy chain of an antibody, including a portion of the CH1 fragment, the hinge region, the CH2 fragment and the CH3 fragment. The Fc region can be engineered or modified, including modifications related to effector functions, mutations that extend half-life and / or knob-into-hole mutations that facilitate purification, such as reducing or eliminating antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can be achieved by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody. Figure 17 of the specification of this application is only a brief structural diagram. In fact, the Fc region described in this application does not only contain CH2 and CH3 fragments. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0098] As used herein, the term "half-antibody" refers to a heterodimeric glycoprotein composed of a light chain (L) and a heavy chain (H) of an antibody, which is the basic structure of the immunoglobulin molecule; its molecular weight is half that of the corresponding antibody, approximately 75,000 daltons, in which the light chain is connected to the heavy chain by a covalent disulfide bond. The heavy and light chains also have regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL) and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0099] The term "IL15" as used herein refers to the interleukin-15 protein. The wild-type amino acid sequence of IL15 is shown in SEQ ID NO:18, and the amino acid sequences of IL15 mutants are shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22. The term "IL2" as used herein refers to the interleukin-2 protein. The M-07e cells used herein are a human cytomegalovirus leukemia cell line purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0100] OKT3 used in this article is a monoclonal antibody against human mature T cell common differentiation antigen CD3, which can stimulate T cell activation.

[0101] As used herein, the term "Fc effector" refers to the function contributed by the Fc effector domain(s) of IgG (e.g., the Fc region of an immunoglobulin). This function can be achieved, for example, by binding of the Fc effector domain(s) to Fc receptors on immune cells with phagocytic or cytolytic activity, or by binding of the Fc effector domain(s) to components of the complement system. Examples of typical effector functions include: C1q-mediated complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0102] The term "Kabat numbering" and similar terms are generally recognized in the art and refer to a system for numbering the amino acid residues in the heavy chain and light chain variable regions of an antibody or its antigen-binding fragment. In some aspects, CDR can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391; and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH publication number 91-3242). Using the Kabat numbering system, the CDR in the antibody heavy chain molecule is typically present at amino acid positions 31 to 35, and the CDR optionally can include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1) after position 35; amino acid positions 50 to 65 (CDR2); and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs in the antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0103] When describing certain embodiments involving amino acid or nucleic acid sequence mutations, this application uses the definition of "XaaaY" (e.g., L234A, etc.), where "aaa" represents the sequence position of the amino acid or base (when a specific reference sequence exists, "aaa" represents the sequential position of the residue in the reference sequence; or the position numbering method commonly used in the art, such as the EU numbering system, etc.), "X" represents the original amino acid or base at "aaa", and "Y" represents the changed amino acid or base at "aaa". As an example, when describing the L234A mutation in the human heavy chain constant region, it means that according to the EU numbering system of the human heavy chain constant region, the leucine (L) at position 234 is mutated to an alanine (A).

[0104] The term "recombinant protein," as used herein, generally refers to a protein composed of at least two domains that are not naturally associated and are encoded by separate genes that are linked and transcribed and translated as a whole to produce a single protein. In the technical context of this application, a "fusion protein" comprising an antibody or antigen-binding fragment refers to a product obtained by fusing an antibody or antigen-binding fragment with another biologically active protein using genetic engineering techniques. This type of antibody fusion protein combines the antigen-binding ability of an antibody with the unique biological properties of the biologically active protein to which it is fused.

[0105] The term "pharmaceutically acceptable" refers to non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Example

[0106] The present application is further described below with reference to specific examples. It should be understood that these examples are intended only to illustrate the present application and are not intended to limit its scope. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer.

[0107] Example 1. Preparation of bispecific recombinant protein samples

[0108] The PD-1-targeted IL15RαSUSHI-IL15 bispecific recombinant protein specifically recognizes PD-1-positive T cells in the tumor microenvironment through an anti-PD-1 half-antibody, and specifically amplifies and activates these potentially cytotoxic T cells through IL15. The heavy chain variable region sequence of the anti-PD-1 half-antibody used in this application is shown in SEQ ID NO: 7, and the light chain variable region sequence is shown in SEQ ID NO: 8. The C-terminus of the anti-PD-1 half-antibody light chain was linked to the N-terminus of the IL15RαSUSHI domain via (GGGGS)5, and the C-terminus of the IL15RαSUSHI domain was linked to IL15 via (GGGGS)5. The light chain-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15 complex was linked to the N-terminus of the IgG1 Fc to form the right arm of the PD-1-targeted IL15RαSUSHI-IL15 bispecific recombinant protein. The left arm is the anti-PD-1 half-antibody heavy chain. A schematic diagram of its structure is shown in Figure 17. Point mutations were performed on the IL15 functional fragment of the PD-1-targeted IL15RαSUSHI-IL15 bispecific recombinant protein, with single or combined mutations at the N1, N4, D61, N64, and N65 sites.

[0109] The nucleic acid encoding the PD-1-targeted IL15RαSUSHI-IL15 bispecific recombinant protein was directly synthesized by GENEWIZ based on the amino acid sequence (N-terminal signal peptide, signal peptide sequence as shown in SEQ ID NO: 25), linked to the pCDNA3.4 plasmid, and sequenced. Different expression plasmids were mixed and paired to transfect the expression cells ExpiCHO-S (Thermo Fisher) to obtain the PD-1-targeted IL15RαSUSHI-IL15 bispecific recombinant protein (see Table 1). Subsequent experimental materials were extracted after transfection of expression cells with this series of plasmids.

[0110] Table 1: Molecular structure of PD-1-targeted IL15RαSUSHI-IL15 bispecific recombinant protein

[0111] In Table 1, Nivolumab (H) refers to the domain consisting of the heavy chain VH and CH1, the amino acid sequence of which is shown in SEQ ID NO: 9; Nivolumab (L) refers to the domain consisting of the light chain VL and CL, the amino acid sequence of which is shown in SEQ ID NO: 10; the amino acid sequence of Fc1 is shown in SEQ ID NO: 12, the amino acid sequence of Fc2 is shown in SEQ ID NO: 13, the amino acid sequence of Fc3 is shown in SEQ ID NO: 14, and the amino acid sequence of Fc4 is shown in SEQ ID NO: 15; the amino acid sequence of (GGGGS)5 is shown in SEQ ID NO: 16; the amino acid sequence of IL15RαSUSHI is shown in SEQ ID NO: 17; the amino acid sequence of IL15 is shown in SEQ ID NO: 18; the amino acid sequence of IL15-N1D is shown in SEQ ID NO: 19; the amino acid sequence of IL15-N4A is shown in SEQ ID NO: 20; the amino acid sequence of IL15-N4D is shown in SEQ ID NO: 21; the amino acid sequence of IL15-D61A is shown in SEQ ID NO: NO:22 shown.

[0112] Table 2: Serial numbers and corresponding sequences

[0113] Table 3: Control sample information

[0114] The protein expression and purification steps are as follows:

[0115] 1. Cell transfection and protein expression:

[0116] After filtering the obtained expression plasmid with a 0.22 μm filter membrane, 50 μg of plasmid (wherein the mass ratio of the left arm and right arm expression plasmids is 2:1 or 3:1) was drawn into 2 mL of OptiPRO SFM Medium (GIBCO, product number 12309050) and mixed. 160 μL of transfection reagent ExpiFectamine CHO Reagent (GIBCO, product number A29129) was drawn into 2 mL of OptiPRO SFM Medium and mixed. The obtained transfection reagent mixed solution was added to the mixed solution containing the plasmid and mixed. The mixture of plasmid and transfection reagent was slowly and evenly added to a 50 mL volume and a cell density of 6×10 6The cells were suspended in a suspension of host cells ExpiCHO-S (Thermo Fisher, Catalog No. A29127) at 10 viable cells / mL and cultured in a 37°C, 8% CO2 incubator. On day 1 (18-22 hours later), 300 μL of ExpiCHO Enhancer (GIBCO, Catalog No. A29129) and 8 mL of ExpiCHO Feed (GIBCO, Catalog No. A29129) were added, and the culture temperature was lowered to 32°C. On day 5, a second feeding was performed with 8 mL of ExpiCHO Feed, and the cells were harvested after 12 days of culture. The cell suspension was centrifuged at 8000 rpm for 15 minutes, and the supernatant obtained from the centrifugation, i.e., the cell culture harvest, was used for purification of the target protein.

[0117] 2. Protein purification:

[0118] (1) Sample capture (Protein A affinity capture): The cell culture harvest was centrifuged at 10,000 rpm for 30 min to remove cells and debris. The sample was then loaded onto a Protein A affinity column (GE Healthcare) using Mabselect Sure (GE, Cat. No. 17543804) as the filler, and the target protein was eluted and harvested. Protein purity was determined by SDS-PAGE. The Protein A affinity capture method is a conventional protein purification method well known to those skilled in the art. For specific experimental methods, please refer to the GE Healthcare Protein A product instructions and the GE Antibody Purification Manual.

[0119] (2) Sample purification: The target protein obtained by affinity capture of the bispecific recombinant protein LC01-7-054-M5 by protein A was used to remove aggregates and other impurities in the sample using Superdex 200 pg filler (Cytiva). The experimental process is as follows:

[0120] a) Equilibration: Equilibrate the column with equilibration solution (50 mM NaAC-HAC, pH 5.5) until the UV detection line is stable;

[0121] b) Sample loading: Use a sample pump to load the sample, with a retention time of 5 min and a loading capacity of ≤50 mg / mL;

[0122] c) Re-equilibration: flush the column with equilibration solution (50 mM NaAC-HAC, pH 5.5) for 5 column volumes;

[0123] d) Elution: The target protein was eluted using an eluent (50 mM NaAC-HAC, 250 mM, pH 5.5), and the protein purity was detected by SDS-PAGE.

[0124] After two purification steps, the sample reached over 99% purity as determined by SEC. Reducing SDS-PAGE revealed two target bands at 50 and 75 kD, demonstrating no protein fragmentation or degradation. The results of the one-step purification (Protein A affinity capture) and the refined SDS-PAGE analysis of LC01-7-054-M5 are shown in Figures 1 and 2.

[0125] Example 2. Effects of different bispecific recombinant proteins on the proliferation activity of M-07e cells

[0126] The proliferation activity of different mutants was detected on PD-1 positive and PD-1 negative immune cells. The PD-1 overexpressing M-07e cell line was constructed by lentiviral infection of M-07e cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.). The PD-1 overexpressing cell line is hereinafter referred to as M-07e-PD1. The more common method for constructing cell lines through lentiviral infection is referenced in Elegheert J, et al. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Nat Protoc. 2018 December; 13(12): 2991-3017.

[0127] The specific experimental steps for proliferation activity detection are as follows:

[0128] 1. Collect M-07e cells and M-07e-PD1 cells by centrifugation, and resuspend the cells in RPMI1640 + 20% FBS medium to a density of 2×10 5 / mL.

[0129] 2. Add 50 μL / well of the cell suspension to a black 96-well plate (Corning Cat. #3904) and incubate in a 37°C 5% CO2 incubator for 4 hours (without adding GM-CSF).

[0130] 3. Prepare samples of LC01-7-032 and different mutants described in Table 1 with an initial concentration of 666 nM (2x), 3-fold serial dilution, for a total of 8 concentration points, with final concentrations of 333, 111, 37, 12.3, 4.1, 1.37, 0.457, and 0.152 nM, respectively.

[0131] 4. Prepare a 3-fold gradient dilution of the positive control IL15RA / IL15-Fc (purchased from novoprotein, product number C15Y) with an initial concentration of 20 nM (2x), for a total of 9 points, with final concentrations of 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, and 0.0015 nM, respectively.

[0132] 5. Add serially diluted samples or positive controls to a black 96-well plate at 50 μL / well and mix well.

[0133] 6. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 72 hours.

[0134] 7. After 3 days, add 40 μL of CellTiter-Glo solution (Promega Cat.#G7573) to each well, mix well, and detect with a microplate reader after 10 minutes.

[0135] As shown in Figure 3, compared with the bispecific recombinant protein LC01-7-032 fused with the wild-type IL15, the bispecific fusion proteins LC01-7-032-M1 (N1D), LC01-7-032-M3 (N4A), LC01-7-032-M4 (N4D), and LC01-7-032-M5 (D61A) fused with mutant IL15 (mutants with reduced affinity for IL2Rβ receptor) had a greater effect on the proliferation of PD-1 negative non-target cells M-07e. The activity of both drugs decreased, and compared to mutants with weaker affinity for the IL15 receptor (IL2Rβ or IL15Rα), such as N4A (see Table 2 of patent US11059876B2), the inventors unexpectedly discovered that LC01-7-032-M5 (D61A) (D61A is not the IL15 mutant with the weakest affinity for the IL15 receptor (IL2Rβ or IL15Rα)) has the best safety (weakest cell proliferation activity on non-target cells) against PD-1-negative M-07e cells. As shown in Figures 3 and 4, LC01-7-032-M5 (D61A) and LC01-7-054-M5 (D61A) had almost no cell proliferation effect on PD-1-negative non-target M-07e cells in the 1-100 nM range. As for the PD-1 positive target cells M-07e-PD1 cells, LC01-7-054-M5 fully demonstrated cell proliferation activity against PD-1 positive M-07e-PD1 cells in the range of anti-PD-1 antibody concentration (1-100nM) (Cancer Immunol Res (2014) 2(9):846-856) (as shown in Figure 5). It can be seen that in the range of anti-PD-1 antibody (such as Nivolumab) concentration (1-100nM), LC01-7-054-M5 had almost no cell proliferation activity against non-target cells that do not express PD-1 (PD-1 negative cells), but could fully demonstrate its cell proliferation activity against target cells that express PD-1 (PD-1 positive cells), which unexpectedly demonstrated the balance and perfect match between safety and efficacy of LC01-7-054-M5. At the same time, it could fully exert its effect of blocking PD-1 and PD-L1 on target cells.

[0136] Example 3. Detection of PD-1+ cell expression in human PBMC in vitro

[0137] According to literature reports, IL15 upregulates PD-1 expression on the surface of CD8+ T cells and activates them. To test the effect of the bispecific recombinant protein LC01-7-054-M5 on PD-1 expression on the surface of CD8+ T cells, the inventors used flow cytometry to measure PD-1 expression in vitro after co-incubation of human PBMC with the sample. The specific experimental steps are as follows:

[0138] 1. Collect PBMC cells by centrifugation and resuspend the cells in RPMI1640 + 10% FBS medium to a density of 4 × 10 6 The cells were plated into 6-well plates (Corning 3516) at 3 mL / well. The cells were split into two plates. One plate was directly incubated at 37°C overnight, and the other plate was added with OKT3 (Invitrogen 16-0037-85) at a final concentration of 5 ng / mL and incubated at 37°C for 48 h.

[0139] 2. Collect the cells by centrifugation for 5 minutes (the centrifuge speed is set at 300g), and resuspend the cells in RPMI1640 + 10% FBS medium to a density of 1×10 5 / mL, add 100uL / well to a 96-well plate (Corning 3799).

[0140] 3. Prepare LC01-7-054-M5 with an initial concentration of 400 nM (2x) and a 5-fold serial dilution, for a total of 8 points, with final concentrations of 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.00256 nM, respectively.

[0141] 4. Prepare a 5-fold serial dilution of the positive control IL15RA / IL15-Fc with an initial concentration of 100 nM (2x), for a total of 9 points, with final concentrations of 50, 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064, and 0.000128 nM, respectively.

[0142] 5. Add 100 μL / well of serially diluted LC01-7-054-M5 or IL15RA / IL15-Fc (purchased from Novoprotein, Cat. No. C15Y) to a 96-well plate and mix thoroughly. Incubate at 37°C in a 5% CO2 incubator for 96 hours.

[0143] 6. After the incubation time is over, collect the cells by centrifugation for 5 minutes (the centrifuge speed is set to 500g), discard the supernatant, and add 200uL / well FACS buffer (2% BSA / PBS) to wash twice.

[0144] 7. Add 200 nM control sample anti-PD-1 antibody Nivolumab (purchased from Bristol-Myers Squibb, trade name OPDIVO) to a 96-well plate at a volume of 100 uL / well and incubate at 4°C for 40 minutes.

[0145] 8. Wash twice with FACS buffer, discard the supernatant, add 100uL anti-Human IgG Fc, FITC Conjugate (Jackson 109-095-098, used at 1:200) to each well, and incubate at 4°C for 40 minutes.

[0146] 9. After washing twice with FACS buffer, resuspend the cells in PBS and analyze them using the FACS marker PerCP-Cyanine5.5 anti-human CD8 (eBioscience, 45-0088-42).

[0147] Figures 6 and 7 show the PD-1 detection results for CD8+ cell populations in unstimulated PBMC (resting peripheral blood mononuclear cells, PD-1 negative, Rest PBMC) and OKT3 (a monoclonal antibody against the human mature T cell common differentiation antigen CD3, which stimulates T cell activation) activated PBMC cells (PD-1 positive). The results showed that the control sample IL15RA / IL15-Fc can upregulate PD-1 expression levels in CD8+ T cells of Rest PBMC (PD-1 negative) and CD8+ T cells of OKT3 activated PBMC (PD-1 positive). However, within the effective dose range (1-100M) of anti-PD-1 antibodies (such as Nivolumab), LC01-7-054-M5 had no significant upregulation effect on PD-1 on the CD8+ T cell subset of Rest PBMC (unstimulated PBMC) (PD-1 negative). At the same time, LC01-7-054-M5 also showed downregulation of PD-1 on the CD8+ T cells of OKT3-activated PBMC cells (PD-1 positive). The above results also revealed that the bispecific recombinant protein LC01-7-054-M5 of the present application had almost no effect on the expression level of PD-1 on the CD8+ T cell subset in unactivated PBMC (Rest PBMC) within the effective dose range of anti-PD-1 antibodies (1-100M); while it significantly downregulated the expression of PD-1 on the CD8+ T cell subset that had been activated by OKT3, thereby further relieving the immunosuppression caused by the high expression of PD-L1 in tumor cells and binding to the activated CD8+ T cell subset that highly expressed PD-1 in the tumor microenvironment. The inventors also conducted activation experiments on Rest PBMCs using bispecific recombinant proteins and positive control samples. The cell activation trend was consistent with the results of the PD-1 upregulation experiment (results not shown), that is, in the effective dose range of anti-PD-1 antibodies of 1-100nM, LC01-7-054-M5 did not cause activation of CD8+ T cells in Rest PBMCs, while IL15RA / IL15-Fc could significantly activate CD8+ T cells in Rest PBMCs.

[0148] The above results indicate that within the effective dose range of 1-100 nM for anti-PD-1 antibodies, LC01-7-054-M5 hardly upregulates the PD-1 expression level of CD8+ T cells in resting peripheral blood monocytes. That is, LC01-7-054-M5 does not cause systemic toxicity caused by activation of CD8+ T cells in resting peripheral blood monocytes within the effective dose range for anti-PD-1 antibodies, while the positive control sample has a significant activation effect on CD8+ T cells within this dose range. This indicates that the bispecific recombinant protein LC01-7-054-M5 of the present application has excellent safety within the effective dose range of anti-PD-1 antibodies. Furthermore, within the effective dose range of 1-100 nM of anti-PD-1 antibodies, for PD-1-positive CD8+ T cells in the tumor microenvironment, LC01-7-054-M5 not only does not upregulate the expression level of PD-1 on CD8+ T cells as reported in the positive control sample or other literature, but can also downregulate the expression level of PD-1 on CD8+ T cells. While the anti-PD-1 half-antibody exerts its immunosuppression-relieving effect, it further relieves the immunosuppression caused by the binding of PD-1 and PD-L1 in the tumor microenvironment by downregulating the expression level of PD-1.

[0149] Example 4. Effect of LC01-7-054-M5 on the expansion of immune cells in a mouse model reconstituted with human hematopoietic stem cells

[0150] To demonstrate that LC01-7-054-M5 achieves the same efficacy in preclinical animal models, the expansion of PD-1-negative and PD-1-positive immune cells was evaluated in a mouse model reconstituted with human hematopoietic stem cells. The specific experimental procedures were as follows: NCG mice were transplanted with human CD34+ hematopoietic stem cells via the tail vein to establish hematopoietic stem cell-reconstituted mice. When the mean percentage of viable hCD45+ and hCD3+ cells in peripheral blood reached 21.82%, mice were randomly divided into groups based on body weight. The mean percentage refers to the average percentage of viable hCD45+ and hCD3+ cells across all mice. On the day of grouping (D0), mice were administered intravenously at a volume of 10 μL / g x mouse body weight (g). Peripheral blood was collected on day 8 (D8) for flow cytometry to detect the proportions of Ki67, a proliferation marker of different cell subsets, including CD4+T (CCR7± and CD45RA±), CD8+T (CCR7± and CD45RA±), γδT (TCRγδT) and NK (CD56+).

[0151] As shown in Figure 8, in the mouse model reconstituted with human hematopoietic stem cells, the abundance of PD-1 expression was high on human CD8 (hCD8) cells and human TCRγδT cells, while the abundance of PD-1 expression on NK cells (CD56+) was low or negative. In human CD8 (hCD8) cells, the abundance of PD-1 expression was high on CD8 effector cells (hCD8 Effector, CD8+CD45RA+CCR7-) and CD8 effector memory cells (hCD8Effector Memory, CD8+CD45RA-CCR7-), while hCD8 The cells (CD8+CD45RA+CCR7+) expressed low abundance or negative PD-1.

[0152] In a mouse model reconstituted with human hematopoietic stem cells, LC01-7-054-M5 significantly expanded hCD8+ cells and hTCRγδT cells, which express high abundance of PD-1. As shown in Figure 9, on day 8 after administration, LC01-7-054-M5 at different doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) significantly increased the Ki67 ratio of hCD8+ cells and hTCRγδT cells. LC01-7-054-M5 at a dose of 0.3 mg / kg (the dose required for complete PD-1 occupancy by anti-PD-1 antibodies) significantly maintained the expression of the proliferation marker Ki67 on hCD8+ cells (maintaining a high Ki67 ratio on day 8), while the control sample IL15RA / IL15-Fc had no significant proliferative activity on day 8. Moreover, on day 8 after administration, LC01-7-054-M5 showed no proliferative activity against NK cells (CD56+) with low PD-1 expression abundance. As shown in Figure 10, on day 8 after administration, LC01-7-054-M5 was used to detect the proliferative activity of different PD-1 expression abundance subpopulations in CD8+ cells at different doses (0.3 mg / kg, 1 mg / kg, 3 mg / kg). The results showed that LC01-7-054-M5 had no effect on NK cells with low PD-1 expression abundance. While no proliferation was observed on PD-1-positive CD8+ effector cells (hCD8 Effector), LC01-7-054-M5 had a highly significant proliferation effect on PD-1-positive CD8+ effector cells. These results demonstrate that LC01-7-054-M5 exhibited almost no cell proliferation activity on non-target cells that do not express PD-1 (PD-1-negative cells) in an in vivo animal model, while it exhibited substantial cell proliferation activity on target cells that express PD-1 (PD-1-positive cells), and this activity was sustained.

[0153] Example 5. Effect of LC01-7-054-M5 on the expansion of immune cells in cynomolgus monkey model

[0154] To validate the activity of LC01-7-054-M5 in expanding PD-1-negative and PD-1-positive immune cells in a cynomolgus macaque model, the following experimental protocol was used:

[0155] Cynomolgus monkeys were given a single dose of 0.2 mg / kg, and peripheral blood was collected before dosing, on Day 1-4h (i.e., 1-4 hours after dosing, with dosing time counted as Day 0), Day 5, Day 7, Day 10, Day 15, Day 22, and Day 29. The samples were tested for cell proliferation of CD45+CD3+ (total T cells), CD45+CD3+CD4+ (CD4+T), CD45+CD3+CD8+ (CD8+T), CD45+CD3-CD56+ (NK), CD45+CD3+CD56+ (NKT), and CD45+CD3+CD4-CD8- (γδT) cell subsets. The flow cytometry antibodies used were: FITC Mouse Anti-NHP CD45 (BD Biosciences 557803), Percp Mouse Anti-Human CD3 (BD ​​552851), BUV395 Mouse Anti-Human CD4 (BD 564107), BV605 Mouse Anti-Human CD8 (BD 564116), BV421 Mouse Anti-Human CD56 (Biolegend 318328), and PE-eFluor 610 Ki67 (eBioscience 61-5698-82).

[0156] The results are shown in Figure 11. In the cynomolgus macaque model, LC01-7-054-M5 significantly expanded PD-1-positive (high PD-1 expression) γδT cells (CD45+CD3+CD4-CD8-), and the effective dose (0.2 mg / kg) matched the clinical dosage of the anti-PD-1 antibody Nivolumab. At this dose concentration, LC01-7-054-M5 had a very weak expansion effect on other types of immune cells (T cells, NK cells, NKT cells) that were PD-1-negative or had low PD-1 expression.

[0157] Example 6. Pharmacodynamic evaluation in a human melanoma A375 cell mixed PBMC subcutaneous transplant tumor model

[0158] To further verify the tumor inhibitory effect of LC01-7-054-M5, the anti-tumor effect of LC01-7-054-M5 was evaluated in a humanized mouse A375 tumor model. The specific experimental plan is as follows:

[0159] 1. A375 cells (human malignant melanoma cells, obtained from the Chinese Academy of Sciences Cell Bank, Catalog No. SCSP-533) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). A375 cells were harvested during the logarithmic growth phase, resuspended in HBSS, and used for subcutaneous tumor inoculation in NCG mice.

[0160] 2. The revived frozen PBMCs were co-cultured with A375 cells for 5 days. PBMCs were collected after 5 days. 5 A375 cells 4×10 6 Each mouse was inoculated subcutaneously on the right side of the NCG mouse with 0.2 ml of PBS containing 50% Matrigel (a substance that promotes tumor formation). On the day of inoculation, the mice were randomly divided into 6 groups of 6 mice each based on their body weight. The day of group administration was designated as day 0.

[0161] 3. Detailed administration method, dosage and route of administration are shown in Table 4:

[0162] Table 4. Grouping and dosing Note: N: number of animals used; iv: tail vein injection; QW*4W: once a week for 4 weeks, a total of 4 times; Dosage volume: adjusted according to the weight of the tumor-bearing mice (0.1mL / 10g)

[0163] Tumor volume was measured twice a week using a vernier caliper. Tumors were collected at the end of the experiment for tumor-infiltrating lymphocyte (TILs) analysis using FACS markers including APC anti-human CD45 (BD Biosciences, Catalog No. 555485), FITC anti-human CD3 (BD ​​Biosciences, Catalog No. 555339), BV605 anti-human CD4 (Biolegend, Catalog No. 317438), and APC / Cy7 anti-human CD8 (Biolegend, Catalog No. 300926).

[0164] As shown in Figure 12, LC01-7-054-M5 exhibited significant anti-tumor effects at dose concentrations of 0.3 mg / kg (QW*4W), 1 mg / kg (QW*4W), and 1 mg / kg (single dose), with superior tumor inhibition compared to the anti-PD-1 antibodies Nivolumab and IL15RA / IL15-Fc. Furthermore, compared to the anti-PD-1 antibody Nivolumab, LC01-7-054-M5 significantly amplified tumor-infiltrating immune cells (Figure 13). These results demonstrate that LC01-7-054-M5 exhibits significant anti-tumor effects and amplifies tumor-infiltrating immune cells, with the effective dose matching the clinical dosage of anti-PD-1 antibodies.

[0165] Example 7. Safety evaluation of LC01-7-054-M5 in cynomolgus monkey model

[0166] To demonstrate the safety of LC01-7-054-M5 at an effective dose (1-100 nM) matching the dose of the anti-PD-1 antibody Nivolumab, the safety of LC01-7-054-M5 was evaluated in a cynomolgus monkey pre-toxicology model.

[0167] LC01-7-054-M5 was administered intravenously to cynomolgus monkeys at doses of 1 mg / kg and 5 mg / kg, once weekly for four weeks, for a total of five doses. The final dose was administered on the 29th day, and the monkeys were autopsied on the 30th day. During this period, the monkeys underwent clinical observation, body weight and food intake monitoring, and blood biochemistry, coagulation, and cytokine assays. At the end of the study, the monkeys were autopsied for gross examination and histopathological examination.

[0168] Table 5. Pre-toxicity results

[0169] In a 4-week toxicology study of LC01-7-054-M5 at repeated doses of 1 mg / kg and 5 mg / kg, cynomolgus monkeys remained in good condition, with no deaths or near-mortal states. The highest non-serious non-toxic dose (HNSTD) was 5 mg / kg. Specific parameters are shown in Table 5.

[0170] Example 8. In vitro human PBMC cytokine release experiment

[0171] To demonstrate the safety of LC01-7-054-M5 at an effective dose matching the dose of the anti-PD-1 antibody nivolumab, we evaluated the safety of LC01-7-054-M5 using an in vitro human PBMC cytokine release assay. The specific experimental steps are as follows:

[0172] 1. Collect PBMC cells by centrifugation and resuspend the cells in RPMI1640 + 10% FBS medium to a density of 1×10 6 / mL.

[0173] 2. Add 100 μL / well of cell suspension to a round-bottom 96-well plate (Corning Cat. #3799).

[0174] 3. Prepare 6 positive control samples of IL15RA / IL15-Fc and LC01-7-054-M5 with an initial concentration of 100 nM and 10-fold serial dilutions.

[0175] 4. Add 100 μL of serially diluted LC01-7-054-M5 or IL15RA / IL15-Fc to each well and mix well.

[0176] 5. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 72 hours.

[0177] 6. After 3 days, the supernatant was collected by centrifugation and IL-6, TNF-α, and IFN-γ in the supernatant were detected according to the HTRF (homogeneous time-resolved fluorescence) instructions (Cisbio, catalog numbers 62HIL06PEG, 62HTNFAPEH, and 62HIFNGPEH, respectively).

[0178] The results, shown in Figures 14-16, show that in a human PBMC (peripheral blood mononuclear cell) cytokine release assay, no IFN-γ release was detected in the PBMC culture supernatant at the effective dose concentrations (1-100 nM) of LC01-7-054-M5 for 72 hours, while minimal IL-6 and TNF-α release was detected. In contrast, significant IL-6 and IFN-γ release was detected with IL15RA / IL15-Fc at a concentration of 1 nM, and TNF-α release was detected at a concentration of 0.01 nM, with dose-dependent increases in cytokine release. These results demonstrate that LC01-7-054-M5 has a significantly better safety profile than IL15RA / IL15-Fc.

[0179] Example 9. Effect of high dose LC01-7-054-M (300 nM) on cytokine release from healthy human PBMCs.

[0180] method:

[0181] Peripheral blood mononuclear cells (PBMC) from three donors were cultured at a concentration of 2×10 5 LC01-7-054-M5 was seeded into 96-well plates at a concentration of 1 μg / well. PBMCs were incubated with LC01-7-054-M5 in duplicate using both solid-phase and liquid-phase methods. Positive controls contained 2 μg / mL CD3 (Thermo, Cat. No. 16-0037-85) and CD28 (BD, Cat. No. 555725) and 100 ng / mL LPS (Sigma, Cat. No. L2880). Negative controls contained PBMCs alone, and a 300 nM isotype antibody control well (HulgG1) was also included. After 24 or 48 hours of incubation in an incubator, supernatants were collected and assayed for IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and TNF-α using an MSD kit. Cell survival was assessed using CTG in liquid-phase assays.

[0182] result:

[0183] Under the current in vitro experimental conditions, compared with the negative control group and the isotype control HulgG1 group, the LPS and CD3 / CD28 positive control groups can significantly promote the release of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α in human PBMCs. LC01-7-054-M5 did not promote the release of various cytokines under both solid phase and liquid phase methods, and did not affect the survival of PBMC cells.

[0184] LC01-7-054-M5 has no risk of inducing CRS in vitro.

[0185] Example 10. Pharmacodynamic evaluation of the test drug in a subcutaneous transplant tumor model of human liver cancer Huh-7 cells mixed with PBMC

[0186] Cell line: Huh-7 cells were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (SIBS, catalog number SCSP-526).

[0187] Experimental animals: Female NCG mice, 6-8 weeks old, weighing approximately 20-22 g. Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Housed in an SPF-grade environment. Adaptation was allowed for at least 3 days before formal experiments.

[0188] Method 1:

[0189] PBMCs co-cultured with Huh-7 cells in vitro and freshly digested Huh-7 cells were used as the control. 5 Huh-7 cells 4×10 6 0.2 mL / mouse (containing 50% matrix gel) was inoculated subcutaneously on the right side of the back of NCG mice. After inoculation, the tumor was allowed to grow to 90-115 mm. 3 Around 6:00 p.m., 26 mice were randomly divided into four groups based on tumor size: a vehicle group (0.9% sodium chloride injection) with 8 mice in each group and other groups with 6 mice in each group. Day 0 was the day of group administration. The experimental group was divided into G1: vehicle control group; G2: LC01-7-054-M5 0.1 mg / kg group; G3: LC01-7-054-M5 0.3 mg / kg group; and G4: Keytruda (purchased from Merck) 10 mg / kg group. Groups G1-G4 were administered via tail vein injection starting on the day of group assignment (D0). Body weight and tumor volume were measured twice weekly during the dosing and observation period, and the measurements were recorded. After dosing ended on Day 18, observation continued, and the experiment ended on Day 22. Tumors were weighed, photographed, and the tumor growth inhibition rate (TGI) (%) was calculated.

[0190] Table 6. Grouping and Dosing Note: BIW: twice a week; QW: once a week.

[0191] result:

[0192] On day 21, the average tumor volume of the G1 solvent control group was 1081.80 ± 97.01 mm 3 The mean tumor volumes of the G2 (LC01-7-054-M5, 0.1 mg / kg) group, the G3 (LC01-7-054-M5, 0.3 mg / kg) group, and the G4 (Keytruda, 10 mg / kg) group were 790.04 ± 79.69 mm 3 、583.20±173.04mm 3 , and 960.57±137.34m 3 Compared with the G1 solvent control group, the tumor growth inhibition rates (TGI) of the G2-G4 groups were 29.82%, 50.91%, and 12.35%, respectively, as shown in FIG18 .

[0193] The experiment was terminated on day 22, and all mice were euthanized, their tumors removed, weighed, and photographed. The average tumor weight in the G1 vehicle control group was 1.140±0.098 g; the average tumor weights in the G2 (LC01-7-054-M, 0.1 mg / kg) group, the G3 (LC01-7-054-M, 0.3 mg / kg) group, and the G4 (Keytruda, 10 mg / kg) group were 0.839±0.093 g, 0.566±0.170 g, and 1.009±0.146 g, respectively.

[0194] LC01-7-054-M5 can effectively inhibit the growth of Huh-7 subcutaneous transplanted tumors in mice in a dose-dependent manner, with better efficacy than Keytruda.

[0195] Method 2:

[0196] PBMCs co-cultured with Huh-7 cells in vitro and freshly digested Huh-7 cells were used as the control. 5 Huh-7 cells 4×10 6 0.2 mL / mouse (containing 50% matrix gel) was inoculated subcutaneously on the right side of the back of NCG mice. After inoculation, the tumor was allowed to grow to 90-100 mm. 3Around 6:00 p.m., 18 mice were randomly divided into three groups of 6 mice each based on tumor size. Day 0 was designated as the day of group administration. The experimental group was divided into: G1: solvent control group; G2: LC01-7-054-M5, 1 mg / kg group; and G3: Camrelizumab (provided by Shanghai Lidi Biotechnology Co., Ltd.) 10 mg / kg group. Groups G1-G3 were administered via tail vein injection starting on the day of grouping (D0). Mouse body weight and tumor volume were measured twice weekly during the dosing and observation period, and the measured values ​​were recorded. After dosing ended on Day 16, observations continued. The experiment ended on Day 23. Tumor-bearing mice were euthanized, tumors were removed, weighed, photographed, and the tumor growth inhibition rate (TGI) (%) was calculated.

[0197] Table 7. Grouping and Dosing Note: BIW: twice a week; QW: once a week.

[0198] result:

[0199] On day 23, the mean tumor volume in the G1 vehicle control group was 684.26±102.60 mm³; the mean tumor volumes in the G2 (LC01-7-054-M5, 1 mg / kg) and G3 (Camrelizumab, 10 mg / kg) groups were 371.54±105.24 mm³ and 67.57±25.26 mm³, respectively. Compared with the G1 vehicle control group, the tumor growth inhibition rates (TGI) in the G2 and G3 groups were 105.65% and 53.58%, respectively. The TGI in the G2 and G3 groups increased by 52.07%, and tumor volume was significantly reduced (p<0.05), as shown in Figure 19.

[0200] The experiment was terminated on day 23, and all mice were euthanized. Their tumors were removed, weighed, and photographed. The average tumor weight in the G1 vehicle control group was 0.693±0.111 g; the average tumor weights in the G2 (LC01-7-054-M 1 mg / kg) and G3 (camrelizumab 10 mg / kg) groups were 0.066±0.024 g and 0.371±0.102 g, respectively. LC01-7-054-M5 1 mg / kg was significantly more effective than camrelizumab 10 mg / kg.

Claims

1. A bispecific recombinant protein comprising a PD-1 blocker and an IL15R-IL15 moiety, wherein the IL15 is an IL15 variant with reduced affinity for IL2Rβ compared to wild-type IL15 as shown in SEQ ID NO: 18, and the PD-1 blocker is an anti-PD-1 antibody or an antigen-binding fragment thereof; The N-terminus of the IL15R is connected to the C-terminus of the light chain of the anti-PD-1 antibody or its antigen-binding fragment via a linker; the N-terminus of the IL15 variant is connected to the C-terminus of the IL15R via a linker. 2 . The bispecific recombinant protein of claim 1 , wherein the IL15 variant comprises one or more point mutations selected from positions N1, N4, and D61 relative to wild-type IL15.

3. The bispecific recombinant protein of claim 2, wherein the IL15 variant comprises a point mutation at position D61 relative to wild-type IL15; preferably, the IL15 variant comprises a point mutation at position D61A relative to wild-type IL15.

4. The bispecific recombinant protein according to any one of claims 1 to 3, wherein the linker connecting the IL15R and IL15 variants is (GGGGS) n , wherein n is selected from an integer of 1-10, such as n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the linker is (GGGGS)5. 5 . The bispecific recombinant protein according to claim 1 , wherein the IL15R is a truncated IL15RαSUSHI; preferably, the IL15RαSUSHI comprises the amino acid sequence shown in SEQ ID NO:

17.

6. The bispecific recombinant protein of any one of claims 1 to 5, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises an anti-PD-1 half antibody consisting of one heavy chain and one light chain; wherein the heavy chain and light chain comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3; wherein the heavy chain CDR1 sequence comprises SEQ ID NO: 1 or is as set forth in SEQ ID NO: 1, the heavy chain CDR2 sequence comprises SEQ ID NO: 2 or is as set forth in SEQ ID NO: 2, and the heavy chain CDR3 sequence comprises SEQ ID NO: 3 or is as set forth in SEQ ID NO: 3; the light chain CDR1 sequence comprises SEQ ID NO: 4 or is as set forth in SEQ ID NO: 4, the light chain CDR2 sequence comprises SEQ ID NO: 5 or is as set forth in SEQ ID NO: 5, and the light chain CDR3 sequence comprises SEQ ID NO: 6 or is as set forth in SEQ ID NO: 6, and the CDR regions are determined using Kabat numbering.

7. The bispecific recombinant protein of claim 6, wherein the heavy chain variable region sequence comprises SEQ ID NO: 7 or is as shown in SEQ ID NO: 7, and the light chain variable region sequence comprises SEQ ID NO: 8 or is as shown in SEQ ID NO:

8.

8. The bispecific recombinant protein according to any one of claims 1 to 7, wherein the C-terminus of the IL15 variant is further connected to the N-terminus of the Fc sequence.

9. The bispecific recombinant protein of claim 8, wherein the Fc region of the anti-PD-1 antibody or antigen-binding fragment thereof and the Fc region of the IL15 variant are IgG1 Fc regions or IgG1 Fc variant sequences; preferably, the IgG1 The Fc variant comprises mutations that reduce Fc effect, mutations that extend half-life and / or knob-into-hole mutations that facilitate purification, wherein the mutations that reduce Fc effect comprise one or more point mutations of L234A and L235A; preferably, the mutations that reduce Fc effect comprise a combination of L234A-L235A mutations; the mutations that extend half-life comprise one or more point mutations of M252Y, S254T, and T256E; preferably, the mutations that extend half-life comprise a combination of M252Y-S254T-T256E mutations; in the knob-into-hole mutations that facilitate purification, the knob mutation is T366W and the hole mutation is T366S-L368A-Y407V; the amino acid numbering refers to the EU index numbering.

10. A bispecific recombinant protein comprising an anti-PD-1 half antibody and an IL15RαSUSHI-(GGGGS)5-IL15 variant; wherein the anti-PD-1 half antibody consists of a heavy chain and a light chain, and the N-terminus of the IL15R is connected to the C-terminus of the light chain of the anti-PD-1 antibody via a linker; The heavy chain and light chain of the anti-PD-1 half antibody comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3; wherein the heavy chain CDR1 sequence comprises SEQ ID NO: 1 or is set forth in SEQ ID NO: 1, the heavy chain CDR2 sequence comprises SEQ ID NO: 2 or is set forth in SEQ ID NO: 2, and the heavy chain CDR3 sequence comprises SEQ ID NO: 3 or is set forth in SEQ ID NO: 3; the light chain CDR1 sequence comprises SEQ ID NO: 4 or is set forth in SEQ ID NO: 4, the light chain CDR2 sequence comprises SEQ ID NO: 5 or is set forth in SEQ ID NO: 5, and the light chain CDR3 sequence comprises SEQ ID NO: 6 or is set forth in SEQ ID NO: 6, and the CDR regions are identified using the Kabat numbering system; The IL15 variant comprises one or more point mutations selected from N1D, N4A, N4D, and D61A relative to the wild-type IL15 as shown in SEQ ID NO: 18; wherein the light chain C-terminus of the anti-PD-1 half antibody is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via a linker; Preferably, the heavy chain variable region sequence is shown as SEQ ID NO: 7, and the light chain variable region sequence is shown as SEQ ID NO:

8.

11. The bispecific recombinant protein of claim 10, wherein the bispecific recombinant protein is further linked to an Fc region at the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant; The bispecific recombinant protein is linked to the N-terminus of the Fc region at the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant; preferably, the bispecific recombinant protein is directly linked to the N-terminus of the Fc region at the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant.

12. The bispecific recombinant protein according to claim 10 or 11, wherein the Fc region of the anti-PD-1 antibody or antigen-binding fragment thereof and the Fc region of the IL15 variant are IgG1 Fc regions or IgG1 Fc variant sequences; preferably, the IgG1 The Fc variant comprises mutations that reduce Fc effect, mutations that extend half-life and / or knob-into-hole mutations that facilitate purification, wherein the mutations that reduce Fc effect comprise one or more point mutations of L234A and L235A; preferably, the mutations that reduce Fc effect comprise a combination of L234A-L235A mutations; the mutations that extend half-life comprise one or more point mutations of M252Y, S254T, and T256E; preferably, the mutations that extend half-life comprise a combination of M252Y-S254T-T256E mutations; in the knob-into-hole mutations that facilitate purification, the knob mutation is T366W and the hole mutation is T366S-L368A-Y407V; the amino acid numbering refers to the EU index numbering.

13. The bispecific recombinant protein of claim 12, wherein the heavy chain sequence is as shown in SEQ ID NO: 23, and the light chain sequence is as shown in SEQ ID NO: 10; the IL15RαSUSHI-(GGGGS)5-IL15 variant sequence is as shown in SEQ ID NO: 24; wherein the C-terminus of the light chain of the anti-PD-1 half antibody is connected to the N-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant via (GGGGS)5, and the C-terminus of the IL15RαSUSHI-(GGGGS)5-IL15 variant is directly connected to the N-terminus of the Fc region, and the Fc region sequence is as shown in SEQ ID NO:

15.

14. A bispecific recombinant protein, which is: LC01-7-032-M1, LC01-7-032-M3, LC01-7-032-M4, LC01-7-032-M5 or LC01-7-054-M5; The left arm structure of LC01-7-032-M1 is Nivolumab(H)-Fc1, and the right arm structure is Nivolumab(L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-N1D-Fc2; The left arm structure of LC01-7-032-M3 is: Nivolumab(H)-Fc1, and the right arm structure is Nivolumab(L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-N4A-Fc2; The left arm structure of LC01-7-032-M4 is: Nivolumab(H)-Fc1, and the right arm structure is Nivolumab(L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-N4D-Fc2; The left arm structure of LC01-7-032-M5 is: Nivolumab(H)-Fc1, and the right arm structure is Nivolumab(L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-D61A-Fc2; The left arm structure of LC01-7-054-M5 is: Nivolumab(H)-Fc3, and the right arm structure is Nivolumab(L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-D61A-Fc4; Wherein, Nivolumab (H) refers to the heavy chain VH and CH1 domains, and its amino acid sequence is shown in SEQ ID NO:9; Nivolumab (L) refers to the light chain VL and CL domains, and its amino acid sequence is shown in SEQ ID NO:10; the amino acid sequence of Fc1 is shown in SEQ ID NO:12; the amino acid sequence of Fc2 is shown in SEQ ID NO:13; the amino acid sequence of Fc3 is shown in SEQ ID NO:14; the amino acid sequence of Fc4 is shown in SEQ ID NO:15; the amino acid sequence of (GGGGS)5 is shown in SEQ ID NO:16; the amino acid sequence of IL15RαSUSHI is shown in SEQ ID NO:17; the amino acid sequence of IL15-N1D is shown in SEQ ID NO:19; the amino acid sequence of IL15-N4A is shown in SEQ ID NO:20; the amino acid sequence of IL15-N4D is shown in SEQ ID NO:21; and the amino acid sequence of IL15-D61A is shown in SEQ ID NO:

22.

15. A nucleic acid molecule encoding the bispecific recombinant protein according to any one of claims 1 to 14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the nucleic acid molecule of claim 15 or the vector of claim 16.

18. A pharmaceutical composition comprising the bispecific recombinant protein according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

19. Use of the bispecific recombinant protein according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 18 in the preparation of a medicament for preventing and / or treating cancer.

20. The use according to claim 19, wherein the cancer is selected from solid tumors and hematological tumors, preferably, the cancer is a solid tumor, more preferably, the cancer is melanoma.

21. A method for preventing and / or treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the bispecific recombinant protein according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 18.

22. The method of claim 21, wherein the cancer is selected from a solid tumor and a hematological tumor; preferably, the cancer is a solid tumor; more preferably, the cancer is melanoma.

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