Fusion antibody and use thereof

Abstract

A fusion antibody and a use thereof. The fusion antibody comprises: a) IL-15; b) IL-15Ra; and c) an antibody for targeted therapy against associated cell surface antigens, wherein the antibody comprises a heavy chain variable region VH, a heavy chain constant region CH1, a light chain variable region VL and a light chain constant region CL; IL-15 / IL-15Ra is located between a C terminal of the heavy chain variable region VH of the antibody and a N terminal of the heavy chain constant region CH1 of the antibody, and IL-15 / IL-15Ra is located between a C terminal of the light chain variable region VL of the antibody and a N terminal of the light chain constant region CL of the antibody. The fusion antibody can prolong a half-life period thereof and can use the targeting property of the antibody to specifically reach the site of action to exert the effect thereof, conferring specific cell targeting to IL-15. IL-15 can only exert the receptor activation effect thereof in an environment with high receptor concentrations of IL-2βγ, thereby reducing the systemic toxicity of an IL-15 / IL-15Ra complex.

Description

Fusion antibodies and their applications

[0001] This application is based on the Chinese application with CN application number 202311839194.X and application date December 28, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of genetic engineering, and in particular to a fusion antibody and applications thereof. Background Art

[0003] Cytokines are a class of proteins that function as signal transducers in the body. Produced by a variety of cell types, cytokines act on neighboring cells or the entire system, playing a crucial role in both innate and adaptive immunity.

[0004] Research has found that many cytokines can stimulate immune effector cells and enhance cytotoxicity. This property has led to the application of cytokines in tumor therapy. Interleukin-15 (IL-15) is a cytokine protein from the same family as interleukin-12 (IL-2) and was discovered in 1994. IL-15 and IL-2 share many similar biological activities, including stimulating the proliferation and activation of T cells and NK cells, inducing B cell immunoglobulin synthesis, and supporting the differentiation of cytotoxic effector cells.

[0005] IL-15 has a similar structure to IL-2 and shares the IL2Rβ and IL2Rγ receptors with IL-2. IL-15Ra, a transmembrane protein with high affinity for IL-15, forms a complex with IL-15 (IL-15 superagonist) that can present IL-15 to the surface of cells expressing IL-2Rβγ receptors, thereby activating the proliferation and activation of NK cells and T cells. Unlike IL-2, IL-15 does not lead to the activation of regulatory T cells (Tregs) or mediate activation-induced cell death (AICD), but rather inhibits IL-2-induced AICD. This suggests that IL-15 may be a better choice for tumor treatment than IL-2.

[0006] The anti-tumor effect of IL-15 is primarily through promoting the proliferation and activation of CD8+ T cells and NK cells. In various experimental animal tumor models (LA795 lung adenocarcinoma, melanoma (B16, B78-H1), MC38 colon cancer, liver cancer, and lymphoma), IL-15 treatment has been shown to promote tumor regression, reduce tumor metastasis, and improve survival rates. However, due to IL-15's low molecular weight and short half-life, which results in high renal clearance, multiple daily injections are extremely inconvenient. Therefore, the sole use of recombinant IL-15 is also limited in tumor treatment.

[0007] Currently, most clinically investigated drugs utilize PEGylation or Fc fusion to enhance the half-life of cytokines. While this half-life is prolonged, toxicity issues stemming from poor targeting persist. N-803 is the most rapidly advancing IL-15 fusion protein in clinical development, but its poor tumor targeting and ultra-potent agonist nature pose significant limitations in both indications and administration.

[0008] Therefore, it is very necessary to develop an IL-15 fusion protein with a long half-life, specific tumor treatment effect and low immunotoxicity in the application of IL-15 in tumor treatment. Summary of the Invention

[0009] The main purpose of the present invention is to provide a fusion antibody and its application to solve the problem in the prior art that IL-15 fusion protein lacks tumor cell or tumor microenvironment targeting.

[0010] To achieve the above objectives, according to the first aspect of the present invention, a fusion antibody is provided, comprising: a) IL-15; b) IL-15Ra; and c) an antibody targeting a therapeutically relevant cell surface antigen, the antibody comprising a heavy chain variable region VH, a heavy chain constant region CH1, a light chain variable region VL, and a light chain constant region CL; wherein IL-15 is located between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody, and IL-15Ra is located between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody; or IL-15 is located between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody, and IL-15Ra is located between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody.

[0011] Furthermore, IL-15 is fused directly or via a linker peptide between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody, and IL-15Ra is fused directly or via a linker peptide between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody; or IL-15 is fused directly or via a linker peptide between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody, and IL-15Ra is fused directly or via a linker peptide between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody.

[0012] Furthermore, the C-terminus of the heavy chain constant region CH1 contains an Fc fragment, and the C-terminus of the light chain constant region CL contains or does not contain an Fc fragment.

[0013] Furthermore, IL-15 has the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 81, or an amino acid sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 81.

[0014] Furthermore, IL-15Ra is full-length IL-15Ra or the Sushi domain of IL-15Ra; preferably, IL-15Ra has the amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more identity to the amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0015] Furthermore, the therapeutically relevant cell surface antigen is an immune checkpoint protein or a tumor antigen.

[0016] Furthermore, therapeutically relevant cell surface antigens include, but are not limited to, any one of the following: PD1, PDL1, B7H3, PSMA, Nectin-4, CD19, BCMA, CD22, CD20, GPCR5D, CD21, CD81, CD40, CD79, CD80, CD86, ICAM-1 (CD54), CD11a, CD18, CD45, GPC3, HER2, EGFR, GCN4, Tim3, CLL1, Trop2, Claudin18.2, Claudin6, Muc1, Muc16, or GIST.

[0017] Furthermore, antibodies for therapeutically relevant cell surface antigens include antibodies targeting PDL1, which have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs:15-17, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs:18-20; or have a VH sequence as shown in SEQ ID NO:21, and a VL sequence as shown in SEQ ID NO:22; or have a heavy chain amino acid sequence as shown in SEQ ID NO:23, and a light chain amino acid sequence as shown in SEQ ID NO:24.

[0018] Further, antibodies for therapeutically relevant cell surface antigens include antibodies targeting PD1, which have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 120-122, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 123-125; or have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 126-128, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 129-131; or have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 132-134, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 135-137; or have a VH sequence as shown in SEQ ID NO: 138, and a VH sequence as shown in SEQ ID NO: 139. NO: 139; or a heavy chain amino acid sequence as shown in SEQ ID NO: 140, and a light chain amino acid sequence as shown in SEQ ID NO: 141; or a heavy chain amino acid sequence as shown in SEQ ID NO: 142, and a light chain amino acid sequence as shown in SEQ ID NO: 143.

[0019] Furthermore, antibodies for therapeutically relevant cell surface antigens include antibodies targeting B7H3, which have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs:25-27, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs:28-30; or have a VH sequence as shown in SEQ ID NO:31, and a VL sequence as shown in SEQ ID NO:32; or have a heavy chain amino acid sequence as shown in SEQ ID NO:33, and a light chain amino acid sequence as shown in SEQ ID NO:34; or have a VH sequence as shown in SEQ ID NO:118, and a VL sequence as shown in SEQ ID NO:119.

[0020] Preferably, antibodies targeting therapeutically relevant cell surface antigens include antibodies targeting PSMA, having heavy chain complementary determining region HCDR1, HCDR2, and HCRD3 sequences as shown in SEQ ID NOs:35-37, and light chain complementary determining region LCDR1, LCDR2, and LCRD3 sequences as shown in SEQ ID NOs:38-40; or having a VH sequence as shown in SEQ ID NO:41, and a VL sequence as shown in SEQ ID NO:42; or having a heavy chain amino acid sequence as shown in SEQ ID NO:43, and a light chain amino acid sequence as shown in SEQ ID NO:44.

[0021] Furthermore, the antibody against the therapeutically relevant cell surface antigen contains an Fc fragment; preferably, the Fc fragment is selected from the Fc fragment of human IgG1, IgG2, IgG3 or IgG4.

[0022] Further, the connecting peptide is a cleavable connecting peptide or a non-cleavable connecting peptide; preferably, each connecting peptide is independently selected from [GGGGS]n, where n is selected from 1 to 6, EAAAK (SEQ ID NO: 79), EAAAKEAAAK (SEQ ID NO: 80), GGGGSIPVSLRSGGGGGSG (SEQ ID NO: 65) or GGGGSIPVSLRSGGGSG (SEQ ID NO: 62), GGGGSG (SEQ ID NO: 145), GGGGSGGGGSG (SEQ ID NO: 146), GGGGSGGGGSGGGGSG (SEQ ID NO: 64); more preferably, the connecting peptide is GGGGS (SEQ ID NO: 144), GGGGSG (SEQ ID NO: 145), GGGGSGGGGS (SEQ ID NO: 63), GGGGSGGGGSG (SEQ ID NO: 146), GGGGSGGGGSGGGGSG (SEQ ID NO: NO:64), GGGGSIPVSLRSGGGGGSG (SEQ ID NO:65) or GGGGSIPVSLRSGGGSG (SEQ ID NO:62).

[0023] Further, the fusion antibody comprises a heavy chain and a light chain having any of the following amino acid sequences: 1) SEQ ID NO: 1 and SEQ ID NO: 2; 2) SEQ ID NO: 3 and SEQ ID NO: 4; 3) SEQ ID NO: 1 and SEQ ID NO: 5; 4) SEQ ID NO: 1 and SEQ ID NO: 6; 5) SEQ ID NO: 7 and SEQ ID NO: 8; 6) SEQ ID NO: 9 and SEQ ID NO: 10; 7) SEQ ID NO: 5 and SEQ ID NO: 11; 8) SEQ ID NO: 45 and SEQ ID NO: 46; 9) SEQ ID NO: 57 and SEQ ID NO: 58; 10) SEQ ID NO: 59 and SEQ ID NO: 60; 11) SEQ ID NO: 57 and SEQ ID NO: 60; 12) SEQ ID NO: 67 and SEQ ID NO: 68; 13) SEQ ID NO: 69 and SEQ ID NO: 70; 14) SEQ ID NO: 71 and SEQ ID NO: 72; 15) SEQ ID NO: NO:73 and SEQ ID NO:74; 16) SEQ ID NO:75 and SEQ ID NO:76; 17) SEQ ID NO:77 and SEQ ID NO:78; 18) SEQ ID NO:82 and SEQ ID NO:8; 19) SEQ ID NO:83 and SEQ ID NO:84; 20) SEQ ID NO:85 and SEQ ID NO:84; 21) SEQ ID NO:83 and SEQ ID NO:86; 22) SEQ ID NO:85 and SEQ ID NO:86; 23) SEQ ID NO:88 and SEQ ID NO:89; 24) SEQ ID NO:90 and SEQ ID NO:91; 25) SEQ ID NO:92 and SEQ ID NO:93; 26) SEQ ID NO:94 and SEQ ID NO:95; 27) SEQ ID NO:96 and SEQ ID NO:97; 28) SEQ ID NO:98 and SEQ ID NO:99; 29) SEQ ID NO:102 and SEQ ID NO:103; NO: 103; 30) SEQ ID NO: 106 and SEQ ID NO: 107; 31) SEQ ID NO: 108 and SEQ ID NO: 107; 32) SEQ ID NO: 112 and SEQ ID NO: 113; 33) SEQ ID NO: 112 and SEQ ID NO: 114;34) SEQ ID NO: 115 and SEQ ID NO: 116; 35) SEQ ID NO: 115 and SEQ ID NO: 117.

[0024] In order to achieve the above object, according to a second aspect of the present invention, a DNA molecule is provided, which encodes the above fusion antibody.

[0025] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0026] In order to achieve the above object, according to a fourth aspect of the present invention, a host cell is provided, into which the above recombinant plasmid is transformed.

[0027] Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0028] In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a use of the above fusion antibody in the preparation of a medicament for preventing and / or treating cancer.

[0029] Furthermore, the cancer includes any one of the following: lung cancer, melanoma, colon cancer, rectal cancer, liver cancer, breast cancer, lymphoma, prostate cancer or blood tumor.

[0030] In order to achieve the above object, according to the sixth aspect of the present invention, a drug is provided, comprising the above fusion antibody.

[0031] Furthermore, the drug also includes an immune checkpoint inhibitor used in combination with the fusion antibody, and the immune checkpoint inhibitor includes any one or more of the following: aPDL1, aCTLA4, a41BB or aLAG3.

[0032] Using the technical solution of the present invention, the IL-15 domain or IL-15Ra domain is fused to the VH-CH1 or VL-CL of the targeting antibody, respectively, via a connecting peptide, to form a targeted fusion antibody pairing the IL-15 and IL-15Ra complex. This specific fusion method 1) fully exposes the N-terminus of the targeting antibody, fully leveraging the antibody's targeting function to specifically enrich IL15 at the target site; 2) imparts IL15 with a half-life consistent with that of the targeting antibody; and 3) attenuates the binding activity of IL-15 to IL-2Rβγ, reducing IL15's systemic toxicity while maintaining IL15's receptor activation function in a high-density IL-2Rβγ environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0034] FIG1 shows an SDS-PAGE gel image of the fusion antibody purified in Example 2 of the present application;

[0035] FIG2 shows the SEC analysis chart of the fusion antibody purified in Example 2 of the present application;

[0036] FIG3 shows a graph showing the thermodynamic stability of fusion antibodies FuAb1, FuAb5, FuAb6, FuAb17, FuAb18, and FuAb19 in Example 4 of the present application;

[0037] FIG4 shows the results of ELISA detection of binding of different fusion antibodies to the antigen PDL1 in Example 5 of the present application;

[0038] FIG5 shows the results of ELISA detection of binding of different fusion antibodies to the antigen PDL1 in Example 5 of the present application, wherein FuAb-C represents the fusion antibody after enzyme cleavage;

[0039] FIG6 shows the results of ELISA tests for binding of different fusion antibodies to antigens B7H3, PSMA, IL-15Ra, and IL-2Rβγ in Example 5 of the present application;

[0040] FIG7 shows the results of ELISA detection of binding of different fusion antibodies to antigens B7H3, IL-15Ra, and IL-2Rβγ in Example 5 of the present application;

[0041] FIG8 shows the results of ELISA detection of binding of different fusion antibodies to antigens B7H3 and IL-2Rβγ in Example 5 of the present application;

[0042] FIG9 shows the results of ELISA detection of binding of different fusion antibodies to antigen PD1 in Example 5 of the present application;

[0043] FIG10 shows the results of ELISA detection of binding of different fusion antibodies to the antigen IL-2Rβγ in Example 5 of the present application;

[0044] FIG11 shows the results of binding activity detection of different fusion antibodies binding to cells carrying different antigens in Example 5 of the present application;

[0045] FIG12 shows the binding activity test results of different fusion antibodies binding to cells carrying different antigens in Example 5 of the present application, wherein FuAb-C represents the fusion antibody after enzyme cleavage;

[0046] FIG13 shows the results of binding activity detection of different fusion antibodies binding to Mo7e cells in Example 5 of the present application;

[0047] Figure 14 shows the proliferation-promoting activity of different fusion antibodies on CTLL2 cells in Example 6 of the present application;

[0048] FIG15 shows the proliferation-promoting activity of different fusion antibodies on CTLL2 cells in Example 6 of the present application;

[0049] Figure 16 shows the proliferation-promoting activity of different fusion antibodies on Mo7e cells in Example 6 of the present application;

[0050] Figure 17 shows the proliferation-promoting activity of different fusion antibodies on Mo7e cells in Example 6 of the present application;

[0051] FIG18 shows the proliferation-promoting activity of different fusion antibodies on PBMC cells in Example 6 of the present application;

[0052] FIG19 shows the proliferation-promoting activity of different fusion antibodies on NK92 cells in Example 6 of the present application;

[0053] FIG20 shows the effects of different fusion antibody treatments on tumor volume and mouse body weight changes in the MC38 mouse tumor model in Example 7 of the present application;

[0054] FIG21 shows the effect of the fusion antibody targeting PD1 in Example 7 of the present application on tumor volume and mouse body weight changes in the MC38 mouse tumor model;

[0055] FIG22 shows the effect of the fusion antibody targeting PD1 on the changes in cell populations in the MC38 mouse tumor model in Example 7 of the present application;

[0056] FIG23 shows the effects of different doses of the fusion antibody FuAb1 on tumor volume and mouse body weight changes in the MC38 mouse tumor model in Example 8 of the present application;

[0057] FIG24 shows the flow cytometry analysis of cell populations in peripheral blood samples from the MC38 mouse tumor model treated with the fusion antibody in Example 9 of the present application;

[0058] FIG25 shows the flow cytometry analysis of cell populations in spleen samples from the MC38 mouse tumor model treated with the fusion antibody in Example 9 of the present application;

[0059] FIG26 shows the flow cytometry analysis of cell populations in tumor tissue samples from the MC38 mouse tumor model treated with the fusion antibody in Example 9 of the present application;

[0060] FIG27 shows the effects of different doses of the fusion antibody in Example 10 of the present application on tumor volume and mouse body weight changes in the MC38-hB7H3 mouse tumor model;

[0061] FIG28 shows the effects of different doses of the fusion antibody FuAb6 alone or in combination with aCTLA4 on tumor volume and mouse body weight changes in a mouse tumor model in Example 10 of the present application;

[0062] FIG29 shows the effects of different doses of the fusion antibody on changes in mouse tumor volume in the MC38-hB7H3 mouse tumor model in Example 11 of the present application;

[0063] FIG30 shows the effects of different doses of the fusion antibody on changes in mouse body weight in the MC38-hB7H3 mouse tumor model in Example 11 of the present application;

[0064] Figure 31 shows a schematic structural diagram of the fusion antibody obtained by fusing IL-15 to the heavy chain (first chain) of an antibody, and IL-15Ra to the light chain (second chain) of an antibody or a light chain with a heavy chain Fc region at the C-terminus;

[0065] Figure 32 shows a schematic structural diagram of the fusion antibody obtained by fusing IL-15 with the light chain (second chain) of an antibody or a light chain with a heavy chain Fc region at the C-terminus, and IL-15Ra with the heavy chain (first chain) of an antibody in the present application. DETAILED DESCRIPTION

[0066] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0067] As mentioned in the background art, the IL-15 fusion proteins in the prior art have the following problems: 1. Lack of targeting of tumor cells or tumor microenvironment; 2. Relatively short half-life in pharmacokinetic performance, which cannot match that of therapeutic antibodies; 3. Strong peripheral side effects, and the safe dose cannot match that of therapeutic antibodies.

[0068] Although the IL-15 fusion protein can form a complex with IL-15Ra and bind to the IL-2Rβγ receptor, thereby promoting the proliferation and activation of NK cells and T cells, and exerting a tumor-killing effect. However, due to the short half-life of IL-15, the cell targeting is low, and the superagonist activity of the IL-15 / IL-15Ra complex is prone to induce systemic immunotoxicity. In the process of using IL-15 for anti-tumor treatment, there is still the problem of requiring multiple administrations and difficulty in avoiding immunotoxicity. The present application intends to perform an embedded fusion of IL-15 and IL-15Ra with an antibody to obtain a fusion antibody with higher cell targeting for the prevention and treatment of tumors.

[0069] In a first typical embodiment of the present application, a fusion antibody is provided, comprising: a) IL-15; b) IL-15Ra; and c) an antibody targeting a therapeutically relevant cell surface antigen, the antibody comprising a heavy chain variable region VH, a heavy chain constant region CH1, a light chain variable region VL, and a light chain constant region CL; wherein IL-15 is located between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody, and IL-15Ra is located between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody; or IL-15 is located between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody, and IL-5Ra is located between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody.

[0070] The complex formed by IL-15 and IL-15Ra can bind to the IL-2Rβγ receptor, thereby activating the proliferation and activation of NK cells and T cells for anti-tumor treatment. The present application embeds IL-15 and IL-15Ra between the antibody VH-CH1 or VL-CL respectively, and through the targeting of the antibody to the cell surface antigen, the IL-15 / IL-15Ra complex can be specifically enriched in the tumor microenvironment, without affecting the antibody's specific recognition of the antigen, thereby achieving the purpose of specific tumor killing and avoiding the immunotoxicity induced by systemic over-activation of NK cells. At the same time, the embedding of IL-15 and IL-15Ra into the antibody prolongs the half-life of the IL-15 / IL-15Ra complex, providing the possibility of popularization and promotion for the practical application of IL-15 as a drug.

[0071] IL-15 and IL-15Ra can be embedded between the light chain VL-CL or the heavy chain VH-CH1, and the connection between IL-15, IL-15Ra and the antibody can be directly connected or connected using a connecting peptide, wherein the flexibility of the connecting peptide is utilized to enable the molecules connected at both ends of the connecting peptide to maintain their respective inherent biological activities.

[0072] As shown in Figure 19, in a preferred embodiment, IL-15 is directly linked between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody, and IL-15Ra is directly linked between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody.

[0073] In a preferred embodiment, IL-15Ra is linked between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody via a linker peptide; IL-15Ra is linked between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody via a linker peptide.

[0074] In a preferred embodiment, IL-15 is directly linked between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody, and IL-15Ra is linked between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody via a connecting peptide.

[0075] In a preferred embodiment, IL-15Ra is linked between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody via a linker peptide; IL-15Ra is directly linked between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody.

[0076] As shown in Figure 20, in a preferred embodiment, IL-15 is directly linked between the C-terminus of the antibody's light chain variable region VL and the N-terminus of the antibody's light chain constant region CL, and IL-15Ra is directly linked between the C-terminus of the antibody's heavy chain variable region VH and the N-terminus of the antibody's heavy chain constant region CH1.

[0077] In a preferred embodiment, IL-15 is linked between the C-terminus of the antibody's light chain variable region VL and the N-terminus of the antibody's light chain constant region CL via a linker peptide, and IL-15Ra is linked between the C-terminus of the antibody's heavy chain variable region VH and the N-terminus of the antibody's heavy chain constant region CH1 via a linker peptide.

[0078] In a preferred embodiment, IL-15 is directly linked between the C-terminus of the antibody's light chain variable region VL and the N-terminus of the antibody's light chain constant region CL, and IL-15Ra is linked between the C-terminus of the antibody's heavy chain variable region VH and the N-terminus of the antibody's heavy chain constant region CH1 via a connecting peptide.

[0079] In a preferred embodiment, IL-15 is linked between the C-terminus of the antibody's light chain variable region VL and the N-terminus of the antibody's light chain constant region CL via a linker peptide, and IL-15Ra is directly linked between the C-terminus of the antibody's heavy chain variable region VH and the N-terminus of the antibody's heavy chain constant region CH1.

[0080] Any light chain with a VL-CL structure or heavy chain with a VH-CH1 structure is applicable to the present application. In a preferred embodiment, the C-terminus of the heavy chain constant region CH1 contains Fc, and the C-terminus of the light chain constant region CL contains or does not contain Fc.

[0081] Any IL-15 with anti-tumor function that promotes the proliferation and activation of CD8+ T cells and NK cells is suitable for the present application. In a preferred embodiment, IL-15 has an amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 81, or an amino acid sequence having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with the amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 81.

[0082] SEQ ID NO: 12: hIL15

[0083] SEQ ID NO:81:hIL15 WT

[0084] Any IL-15Ra capable of binding to IL-15 to form an anti-tumor effect is suitable for use in this application. In a preferred embodiment, the IL-15Ra is full-length IL-15Ra or the sushi domain of IL-15Ra. The sushi domain of IL-15Ra is a domain with a specific structure located at the N-terminus of IL-15Ra and composed of a series of repeated sequences of approximately 60 amino acids, which plays an important role in cell signaling and immune response. Preferably, IL-15Ra has the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having greater than 80%, more preferably greater than 90%, and even more preferably greater than 95% homology to the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0085] SEQ ID NO: 13: hIL15Ra

[0086] SEQ ID NO: 14: hIL15RaSushi

[0087] Targeted therapy-related cell surface antigens are antigens on the cell surface that can be targeted for IL-15-mediated disease treatment. In a preferred embodiment, the targeted therapy-related cell surface antigen is an immune checkpoint protein or a tumor antigen. In a preferred embodiment, the targeted therapy-related cell surface antigen is selected from but not limited to any one of the following: PD-1, PDL1, B7H3, PSMA, Nectin-4, CD19, BCMA, CD22, CD20, GPCR5D, CD21, CD81, CD40, CD79, CD80, CD86, ICAM-1 (CD54), CD11a, CD18, CD45, GPC3, HER2, EGFR, Claudin18.2, PD-1, GIST, CLL1 or Tim3; preferably, the targeted therapy-related cell surface antigen is selected from any one of the following: PDL1, PD1, B7H3 or PSMA.

[0088] Any antibody that has binding activity with an antigen for the treatment of IL-15-mediated diseases and can embed IL-15 or IL-15Ra between VH-CH1 or VL-CL is suitable for this application, including but not limited to monoclonal antibodies and engineered antibodies. Among them, antibodies contain heavy and light chains, each containing a variable region and a constant region structure, and the heavy and light chains each have three complementary determining regions (CDRs) within the variable region sequence. CDRs determine the specificity and affinity of antibody binding to antigens, and their amino acid sequences can vary greatly in different antibody molecules.

[0089] Different antigens correspond to antibodies with different variable regions. In a preferred embodiment, antibodies targeting therapeutically relevant cell surface antigens include antibodies targeting PDL1, which have heavy chain complementary determining region HCDR1, HCDR2, and HCRD3 sequences as shown in SEQ ID NOs:15-17, and light chain complementary determining region LCDR1, LCDR2, and LCRD3 sequences as shown in SEQ ID NOs:18-20; or have a VH sequence as shown in SEQ ID NO:21, and a VL sequence as shown in SEQ ID NO:22; or have a heavy chain amino acid sequence as shown in SEQ ID NO:23, and a light chain amino acid sequence as shown in SEQ ID NO:24.

[0090] The aPDL1 sequences are as follows:

[0091] HCDR1:DSWIH (SEQ ID NO:15).

[0092] HCDR2: WISPYGGSTYYADSVKG (SEQ ID NO: 16).

[0093] HCDR3: RHWPGGFDY (SEQ ID NO: 17).

[0094] LCDR1: RASQDVSTAVA (SEQ ID NO: 18).

[0095] LCDR2:SASFLYS (SEQ ID NO:19).

[0096] LCDR3: QQYLYHPAT (SEQ ID NO: 20).

[0097] aPDL1 VH:

[0098] aPDL1 VL:

[0099] aPDL1 heavy chain:

[0100] aPDL1 light chain:

[0101] In a preferred embodiment, antibodies for therapeutically relevant cell surface antigens include antibodies targeting PD1, which have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 120-122, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 123-125; or have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 126-128, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 129-131; or have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 132-134, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 135-137; or have a VH sequence as shown in SEQ ID NO: 138, and a VH sequence as shown in SEQ ID NO: 139. NO: 139; or a heavy chain amino acid sequence as set forth in SEQ ID NO: 140, and a light chain amino acid sequence as set forth in SEQ ID NO: 141; or a heavy chain amino acid sequence as set forth in SEQ ID NO: 142, and a light chain amino acid sequence as set forth in SEQ ID NO: 143;

[0102] The aPD1-related sequences are as follows:

[0103] HCDR1:AQYMH(SEQ ID NO:120)。

[0104] HCDR2:IINPSGGETGYAQKFQG(SEQ ID NO:121)。

[0105] HCDR3:EGVADGYGLVDV(SEQ ID NO:122)。

[0106] LCDR1:RASQSVSSYLA(SEQ ID NO:123)。

[0107] LCDR2:DASKRAT(SEQ ID NO:124)。

[0108] LCDR3:DQRNNWPLT(SEQ ID NO:125)。

[0109] aPD1 VH:

[0110] aPD1 VL:

[0111] aPD1-2:

[0112] HCDR1:NYYMY(SEQ ID NO:132)。

[0113] HCDR2:GINPSNGGTNFNEKFKN(SEQ ID NO:133)。

[0114] HCDR3:RDYRFDMGFDY(SEQ ID NO:134)。

[0115] LCDR1:RASKGVSTSGYSYLH(SEQ ID NO:135)。

[0116] LCDR2:LASYLES(SEQ ID NO:136)。

[0117] LCDR3:QHSRDLPLT(SEQ ID NO:137)。

[0118] aPD1-2 VH:

[0119] aPD1-2 VL:

[0120] aPD1-3:

[0121] HCDR1:NSGMH(SEQ ID NO:126)。

[0122] HCDR2: VIWYDGSKRYYADSVKG (SEQ ID NO: 127).

[0123] HCDR3: NDDY (SEQ ID NO: 128).

[0124] LCDR1: RASQSVSSYLA (SEQ ID NO: 129).

[0125] LCDR2: DASNRAT (SEQ ID NO: 130).

[0126] LCDR3: QQSSNWPRT (SEQ ID NO: 131).

[0127] aPD1-3 VH:

[0128] aPD1-3 VL:

[0129] In a preferred embodiment, antibodies targeting therapeutically relevant cell surface antigens include antibodies targeting B7H3, which have heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs:25-27, and light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs:28-30; or have a VH sequence as shown in SEQ ID NO:31, and a VL sequence as shown in SEQ ID NO:32; or have a heavy chain amino acid sequence as shown in SEQ ID NO:33, and a light chain amino acid sequence as shown in SEQ ID NO:34.

[0130] The aB7H3 sequences are as follows:

[0131] HCDR1: NYDIN (SEQ ID NO: 25).

[0132] HCDR2: WIFPGDGSTQYNEKFKG (SEQ ID NO: 26).

[0133] HCDR3: QTTATWFAY (SEQ ID NO:27).

[0134] LCDR1: RASQSISDYLH (SEQ ID NO: 28).

[0135] LCDR2: YASQSIS (SEQ ID NO: 29).

[0136] LCDR3: QNGHSFPLT (SEQ ID NO:30).

[0137] aB7H3 VH:

[0138] aB7H3 VL:

[0139] aB7H3 heavy chain:

[0140] aB7H3 light chain:

[0141] aB7H3-2 VH:

[0142] aB7H3-2 VL:

[0143] In a preferred embodiment, antibodies targeting therapeutically relevant cell surface antigens include antibodies targeting PSMA, wherein the antibodies have the heavy chain complementary determining region HCDR1, HCDR2, and HCRD3 sequences shown in SEQ ID NOs:35-37, and the light chain complementary determining region LCDR1, LCDR2, and LCRD3 sequences shown in SEQ ID NOs:38-40; or have the VH sequence shown in SEQ ID NO:41, and the VL sequence shown in SEQ ID NO:42; or have the heavy chain amino acid sequence shown in SEQ ID NO:43, and the light chain amino acid sequence shown in SEQ ID NO:44.

[0144] The aPSMA sequences are as follows:

[0145] HCDR1:EYTIH (SEQ ID NO:35).

[0146] HCDR2: NINPNNGGTTYNQKFED (SEQ ID NO: 36).

[0147] HCDR3:GWNFDY (SEQ ID NO:37).

[0148] LCDR1: KASQDVGTAVD (SEQ ID NO: 38).

[0149] LCDR2: WASTRHT (SEQ ID NO:39).

[0150] LCDR3: QQYNSYPLT (SEQ ID NO: 40).

[0151] aPSMA VH:

[0152] aPSMA VL:

[0153] aPSMA heavy chain:

[0154] aPSMA light chain:

[0155] Antibodies contain the constant region Fc of the heavy chain and / or the constant region Fc of the light chain, located at the tail of the antibody. These regions bind to receptors on immune cells, thereby mediating various immune responses. The heavy chain Fc and light chain Fc can be the same or different. Fc can be derived from different antibody subtypes, such as IgG (IgG1, IgG2, IgG3, or IgG4), IgM, IgA, IgD, and IgE. In a specific embodiment, the Fc of the heavy chain and / or light chain can be modified accordingly as needed, such as amino acid substitutions at the N297 site to reduce glycosylation modification, amino acid substitutions that enhance or reduce Fc binding to Fc receptors and / or effector functions (such as antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)) (e.g., amino acid substitutions at one or more positions of Fc 228, 234, 235, 236, 243, 297, 298, 330, 331, 329, 333, 334, 239, 332, 330, 292, 300, 305, 396, 268, 270, 318, 320, 322, 252, 254, 256 [see Liu R et al., 2011; ... al.Antibodies (Basel) 2020, 9, 64; US5624821; US5648260; CN108350076B; CN106661120B]. In a specific embodiment, amino acid mutations can be performed on Fc as needed to promote the formation of heterologous pairing between the heavy chain Fc and the light chain Fc. Including but not limited to, for example, Fc with knob and hole structures can be used, or Fc substituted with charged amino acids, as long as it can promote the formation of heterologous pairing between the heavy chain Fc and the light chain Fc [see US7695936, US20030078385, US2020040075A1]. Since IgG is relatively stable, has a longer half-life, and has a higher affinity for Fc receptors, it can better bind to Fc receptors. In a preferred embodiment, the antibody targeting the surface antigen of the therapeutically relevant cell contains an Fc fragment; preferably, the Fc fragment is selected from the Fc fragment of human IgG1, IgG2, IgG3 or IgG4.

[0156] A connecting peptide is used to connect the two molecules to be fused, ensuring sufficient spatial distance between the two molecules to maintain their respective spatial configurations and biological activities. Specifically, a connecting peptide can be reasonably selected from existing connecting peptides as needed, or it can be screened based on actual results. In a preferred embodiment, the connecting peptide is a cleavable connecting peptide or a non-cleavable connecting peptide. Among them, the cleavable connecting peptide is a connecting peptide that can be self-cleaved, enzymatically cleaved, or chemically cleaved. Enzymatic cleavage includes the action of endopeptidases or exopeptidases. Depending on the type of amino acids to be connected, a connecting peptide with better connection effect is selected accordingly. In a preferred embodiment, each connecting peptide is independently selected from [GGGGS]n (when n=6, the connecting peptide is as shown in SEQ ID NO:61: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS), and n is selected from 1 to 6, EAAAK (SEQ ID NO:79), EAAAKEAAAK (SEQ ID NO:80), GGGGSIPVSLRSGGGGGSG (SEQ ID NO:65) or GGGGSIPVSLRSGGGSG (SEQ ID NO:62), GGGGSG (SEQ ID NO:145), GGGGSGGGGSG (SEQ ID NO:146), GGGGSGGGGSGGGGSG (SEQ ID NO:64). More preferred are GGGGS (SEQ ID NO: 144), GGGGSG (SEQ ID NO: 145), GGGGSGGGGS (SEQ ID NO: 63), GGGGSGGGGSG (SEQ ID NO: 146), GGGGSGGGGSGGGGSG (SEQ ID NO: 64), GGGGSIPVSLRSGGGGGSG (SEQ ID NO: 65) or GGGGSIPVSLRSGGGGSG (SEQ ID NO: 65). NO:62).

[0157] In practical applications, suitable IL-15 and IL-15Ra amino acid sequences can be embedded and fused with different antibodies according to specific effects. In a preferred embodiment, the sequence fusion antibody comprises a heavy chain and a light chain having any of the following amino acid sequences: 1) SEQ ID NO: 1 and SEQ ID NO: 2; 2) SEQ ID NO: 3 and SEQ ID NO: 4; 3) SEQ ID NO: 1 and SEQ ID NO: 5; 4) SEQ ID NO: 1 and SEQ ID NO: 6; 5) SEQ ID NO: 7 and SEQ ID NO: 8; 6) SEQ ID NO: 9 and SEQ ID NO: 10; 7) SEQ ID NO: 5 and SEQ ID NO: 11; 8) SEQ ID NO: 45 and SEQ ID NO: 46; 9) SEQ ID NO: 57 and SEQ ID NO: 58; 10) SEQ ID NO: 59 and SEQ ID NO: 60; 11) SEQ ID NO: 57 and SEQ ID NO: 60; 12) SEQ ID NO: 67 and SEQ ID NO: 68; 13) SEQ ID NO: 69 and SEQ ID NO: 70; 14) SEQ ID NO: 71 and SEQ ID NO: 72; 15) SEQ ID NO: NO:73 and SEQ ID NO:74; 16) SEQ ID NO:75 and SEQ ID NO:76; 17) SEQ ID NO:77 and SEQ ID NO:78; 18) SEQ ID NO:82 and SEQ ID NO:8; 19) SEQ ID NO:83 and SEQ ID NO:84; 20) SEQ ID NO:85 and SEQ ID NO:84; 21) SEQ ID NO:83 and SEQ ID NO:86; 22) SEQ ID NO:85 and SEQ ID NO:86; 23) SEQ ID NO:88 and SEQ ID NO:89; 24) SEQ ID NO:90 and SEQ ID NO:91; 25) SEQ ID NO:92 and SEQ ID NO:93; 26) SEQ ID NO:94 and SEQ ID NO:95; 27) SEQ ID NO:96 and SEQ ID NO:97; 28) SEQ ID NO:98 and SEQ ID NO:99; 29) SEQ ID NO:102 and SEQ ID NO:103; NO: 103; 30) SEQ ID NO: 106 and SEQ ID NO: 107; 31) SEQ ID NO: 108 and SEQ ID NO: 107;32) SEQ ID NO: 112 and SEQ ID NO: 113; 33) SEQ ID NO: 112 and SEQ ID NO: 114; 34) SEQ ID NO: 115 and SEQ ID NO: 116; 35) SEQ ID NO: 115 and SEQ ID NO: 117.

[0158] Among them, as needed, null mutations are performed on Fc to weaken ADCC / CDC function (relative to the wild type, the null mutation sites of Fc are E233P, L234V, del235L, G236A, A327G, A330S, and P331S); in some specific embodiments, Fc adopts a knob-into-hole form, and the mutation sites of Fc (knob) are S354C and T366W; the mutation sites of Fc (hole) are Y349C, T366S, L368A, and Y407V) (Kabat EU index numbering method).

[0159] SEQ ID NO:1: aPDL1 VH-hIL15-CH1-Fc

[0160] SEQ ID NO:2: aPDL1 VL-hIL15RaSushi-CL

[0161] SEQ ID NO:3: aPDL1 VH-hIL15RaSushi-CH1-Fc

[0162] SEQ ID NO:4: aPDL1 VL-hIL15-CL

[0163] SEQ ID NO:5: aPDL1 VL-MP2GS-hIL15RaSushi-CL

[0164] SEQ ID NO:6: aPDL1 VL-MP2-hIL15RaSushi-CL

[0165] SEQ ID NO:7:J591 VH-hIL15-CH1-Fc

[0166] SEQ ID NO:8:J591 VL-hIL15RaSushi-CL

[0167] SEQ ID NO:9:aB7H3 VH-hIL15-CH1-Fc

[0168] SEQ ID NO:10:aB7H3 VL-hIL15RaSushi-CL

[0169] SEQ ID NO:11:aPDL1 VH-MP2GS-hIL15-CH1-Fc

[0170] SEQ ID NO:45:aPDL1 VH-hIL15-CH1-Fc(hole)

[0171] SEQ ID NO:46:aPDL1 VL-hIL15RaSushi-CL-Fc(knob)

[0172] SEQ ID NO:47:hIL15-aPDL1-VH-CH1-Fc

[0173] SEQ ID NO:48:hIL15RaSushi-aPDL1-VL-CH1

[0174] SEQ ID NO:49:hIL15-aB7H3-VH-CH1-Fc

[0175] SEQ ID NO:50:hIL15RaSushi-aB7H3-VL-CL

[0176] SEQ ID NO:51:hIL15-aPSMA-VH-CH1-Fc

[0177] SEQ ID NO:52:hIL15RaSushi-aPSMA-VL-CL

[0178] SEQ ID NO:53:hIL15-aPDL1-VH-CH1-Fc(hole)

[0179] SEQ ID NO:54:hIL15RaSushi-aPDL1-VL-CL-Fc(knob)

[0180] SEQ ID NO:55:hIL15-aEGFR-VH-CH1-Fc

[0181] SEQ ID NO:56:hIL15RaSushi-aEGFR-VL-CL

[0182] SEQ ID NO:57:aPDL1 VH-MP2GS-hIL15RaSushi-CH1-Fc(hole)

[0183] SEQ ID NO:58:aPDL1 VL-hIL15-CL-Fc(knob)

[0184] SEQ ID NO:59:aPDL1 VH-hIL15RaSushi-CH1-Fc(hole)

[0185] SEQ ID NO:60:aPDL1 VL-MP2GS-hIL15-CL-Fc(knob)

[0186] SEQ ID NO:67:aPSMA VH-10GS-hIL15-10GS-CH1-Fc

[0187] SEQ ID NO:68:aPSMA VL-10GS-hIL15RaSushi-10GS-CL

[0188] SEQ ID NO:69:aPSMA VH-5GS-hIL15-10GS-CH1-Fc

[0189] SEQ ID NO:70:aPSMA VL-5GS-hIL15RaSushi-10GS-CL

[0190] SEQ ID NO:71:aPSMA VH-5GS-hIL15-5GS-CH1-Fc

[0191] SEQ ID NO:72:aPSMA VL-5GS-hIL15RaSushi-5GS-CL

[0192] SEQ ID NO:73:aPSMA VH-(EAAAK)2-hIL15-(EAAAK)2-CH1-Fc

[0193] SEQ ID NO:74:aPSMA VL-(EAAAK)2-hIL15RaSushi-(EAAAK)2-CL

[0194] SEQ ID NO:75:aPSMA VH-EAAAK-hIL15-(EAAAK)2-CH1-Fc

[0195] SEQ ID NO:76:aPSMA VL-EAAAK-hIL15RaSushi-(EAAAK)2-CL

[0196] SEQ ID NO:77:aPSMA VH-EAAAK-hIL15-EAAAK-CH1-Fc

[0197] SEQ ID NO:78:aPSMA VL-EAAAK-hIL15RaSushi-EAAAK-CL

[0198] SEQ ID NO:82:aPSMA VH-WT hIL15-CH1-Fc(null)

[0199] SEQ ID NO:83:aB7H3-2 VH-IL15-CH1-Fc(hIgG1,wt)

[0200] SEQ ID NO:84:aB7H3-2 VL-hIL15RaSUSHI-CL

[0201] SEQ ID NO:85:aB7H3-2 VH-IL15WT-CH1-Fc(hIgG1,wt)

[0202] SEQ ID NO:86:aB7H3-2-LC

[0203] SEQ ID NO:87:aB7H3-2-HC(hIgG1,wt)

[0204] SEQ ID NO:88:aPD1-2-16GS-IL15RaSUSHI-10GS-LC

[0205] SEQ ID NO:89:aPD1-2-16GS-IL15WT-10GS-HC-IgG4

[0206] SEQ ID NO:90:aPD1-2-11GS-IL15RaSUSHI-10GS-LC

[0207] SEQ ID NO:91:aPD1-2-11GS-IL15WT-10GS-HC-IgG4

[0208] SEQ ID NO:92:aPD1-2-6GS-IL15RaSUSHI-5GS-LC

[0209] SEQ ID NO:93:aPD1-2-6GS-IL15WT-5GS-HC-IgG4

[0210] SEQ ID NO:94:aPD1-16GS-IL15RaSUSHI-10GS-CL

[0211] SEQ ID NO:95:aPD1-16GS-IL15WT-10GS-CH1-Fc(null)

[0212] SEQ ID NO:96:aPD1-11GS-IL15RaSUSHI-10GS-CL

[0213] SEQ ID NO:97:aPD1-11GS-IL15WT-10GS-CH1-Fc(null)

[0214] SEQ ID NO:98:aPD1-6GS-IL15RaSUSHI-5GS-CL

[0215] SEQ ID NO:99:aPD1-6GS-IL15WT-5GS-CH1-Fc(null)

[0216] SEQ ID NO:100:aPD1-2-HC-IgG4

[0217] SEQ ID NO:101:aPD1-2-LC

[0218] SEQ ID NO:102:aPD1-2 VH-hIL15-HC-IgG4

[0219] SEQ ID NO:103:aPD1-2 VL-hIL15RaSushi-CL

[0220] SEQ ID NO:104:aPD1-HC

[0221] SEQ ID NO:105:aPD1-LC

[0222] SEQ ID NO:106:aB7H3-2 VH 5GS-IL15WT-5GS-HC(hIgG1,wt)

[0223] SEQ ID NO:107:aB7H3-2 VL-5GS-hIL15RaSUSHI-5GS-LC(hKappa)

[0224] SEQ ID NO:108:aB7H3-2 VH-5GS-IL15WT-5GS-HC(hIgG1,Null)

[0225] SEQ ID NO:109:aPD1-3-kLC

[0226] SEQ ID NO:110:aPD1-3-HC-IgG4

[0227] SEQ ID NO:111:aPD1-3 HC-IgG1(null)

[0228] SEQ ID NO:112: aPD1-3 VL-16GS-IL15RaSUSHI-10GS-LC

[0229] SEQ ID NO:113: aPD1-3 VH-16GS-IL15WT-10GS-HC-IgG4

[0230] SEQ ID NO:114: aPD1-3 VH-16GS-IL15WT-10GS-HC-IgG1(null)

[0231] SEQ ID NO:115: aPD1-3 VL-16GS-IL15WT-10GS-LC

[0232] SEQ ID NO:116: aPD1-3 VH-16GS-IL15RaSushi-10GS-HC-IgG4

[0233] SEQ ID NO:117:aPD1-3 VL-16GS-IL15RaSushi-10GS-HC-IgG1(null)

[0234] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned fusion antibody.

[0235] In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0236] In a fourth exemplary embodiment of the present application, a host cell is provided, wherein the host cell is transformed with the aforementioned recombinant plasmid. In a preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell may be Escherichia coli; the eukaryotic cell may be a yeast cell, a mammalian cell, an insect cell, or the like.

[0237] In a fifth exemplary embodiment of the present application, a use of the above-mentioned fusion antibody in the preparation of a medicament for preventing and / or treating cancer is provided. The above-mentioned fusion antibody can effectively target the tumor microenvironment and specifically kill tumors while avoiding systemic immunotoxicity induced by overactivation of NK cells. Furthermore, the fusion antibody has a long half-life, achieving better results in future tumor prevention and treatment processes.

[0238] In a preferred embodiment, the cancer includes any one of the following: lung cancer, melanoma, colon cancer, rectal cancer, liver cancer, lymphoma, breast cancer, prostate cancer or blood tumor.

[0239] In a sixth typical embodiment of the present application, a drug is provided, comprising the above-mentioned fusion antibody.

[0240] Since the fusion antibody of the present application has strong cell targeting and a long half-life, its combined use with immune checkpoint inhibitors can achieve better synergistic therapeutic effects. In a preferred embodiment, the drug also includes an immune checkpoint inhibitor used in combination with the fusion antibody, and the immune checkpoint inhibitor includes any one or more of the following: aPDL1 (PDL1 antibody), aCTLA4 (CTLA4 antibody), a41BB (41BB antibody) or aLAG3 (LAG3 antibody).

[0241] In the seventh typical embodiment of the present application, a method for treating cancer is provided, comprising: administering an effective amount of the above-mentioned fusion antibody or the above-mentioned drug to a subject. The above-mentioned fusion antibody or drug can effectively target the tumor microenvironment, specifically kill tumors, avoid systemic over-activation of T cells or NK cells-induced immunotoxicity, and have a long half-life, which can achieve better results in future tumor prevention and treatment processes. The combination with other drugs will enhance the killing function of tumor killer cells in the tumor microenvironment, promote the activation and proliferation of tumor killer cells, so that the combined drugs can exert better functional activity in the tumor microenvironment and achieve better synergistic therapeutic effects.

[0242] In a preferred embodiment, the cancer includes any one of the following: lung cancer, melanoma, colon cancer, rectal cancer, liver cancer, lymphoma, breast cancer, prostate cancer or blood tumor.

[0243] The present invention is further described in detail below with reference to specific examples, which should not be construed as limiting the scope of protection claimed in the present invention. Unless otherwise specified, the reagents or consumables used in the following examples were purchased from commercially available products.

[0244] Example 1 Construction of eukaryotic expression vector for cytokine fusion antibody

[0245] The VL and VH gene fragments of EGFR, PDL1, PSMA, B7H3 and PD1 antibodies, the gene fragment of IL-15, the sushi domain gene fragment of IL-15Ra, and the gene fragments of the constant regions of antibodies targeting PDL1, PSMA and B7H3 were synthesized respectively.

[0246] The above gene fragments are amplified separately by PCR. The amplified VH gene fragment is ligated with the IL-15 gene fragment or the IL-15Ra sushi domain gene fragment, and the antibody constant region gene fragment via a linker by overlap PCR to obtain a VH-IL-15-CH1-Fc fragment or a VH-IL-15Ra-CH1-Fc fragment as the first chain (having the same structure as the above-mentioned heavy chain but with a different description). The amplified VL gene fragment is ligated with the IL-15Ra gene fragment or the IL-15 gene fragment, and the antibody constant region gene fragment via a linker to obtain a VL-IL-15Ra-CL fragment, a VL-IL-15-CL-Fc fragment, or a VL-IL-15-CL fragment, a VL-IL-15Ra-CL-Fc fragment as the second chain (having the same structure as the above-mentioned light chain but with a different description). The above fragments were further ligated into the pFuse vector (InvivoGen, CA) for eukaryotic expression via homologous recombination and transformed into competent E. coli DH5α cells. Antibiotic selection was performed on LB plates. Positive clones were selected and plasmids were extracted using an endotoxin-free plasmid extraction kit. The extracted plasmid sequences were verified by sequencing. The nucleotide and amino acid sequences of each construct are shown in the sequence listing.

[0247] Table 1 Fusion protein sequence number

[0248] Example 2 Expression and Purification of Cytokine Fusion Antibodies

[0249] The eukaryotic expression vector plasmids carrying the light and heavy chains of the cytokine fusion antibody constructed in Example 1 were co-transfected into FreeStyle HEK293 cells and cultured at 125 rpm, 37°C, and 5% CO for 5-6 days. The cell culture supernatant was collected by centrifugation, filtered through a 0.22 μm filter, and purified using Protein A Resin (Genscript) according to the manufacturer's instructions. The concentration was determined using A280 and BCA assays (Pierce).

[0250] The fusion antibody purified from Protein A resin was further separated and purified using a GE AKTA chromatography system and a Superdex 200Increase 10 / 300GL gel exclusion chromatography column in PBS buffer (pH 7.4). The purified sample was stored in PBS buffer (pH 7.4). The composition and purity of the cytokine fusion antibody were determined by SDS-PAGE under reducing and non-reducing conditions. The monomeric components of the cytokine fusion antibody were analyzed by gel exclusion chromatography (SEC) in a saline solution environment.

[0251] The results are shown in Figure 1, where R represents the band after reduction with a reducing agent, i.e., the first and second chains, and NR represents the band without the addition of a reducing agent, i.e., the fusion antibody. The size of the fusion antibody, as indicated in the NR column, was consistent with expectations. As shown in Figure 2, in SEC analysis, the fusion antibody monomer peak accounted for greater than 95%, with no apparent aggregates or fragments, demonstrating similar expression and purification advantages to antibodies.

[0252] Example 3 Mass Spectrometry Analysis of Cytokine Fusion Antibodies

[0253] The samples purified in Example 2 (FuAb2, FuAb1, FuAb7, FuAb6, FuAb5, FuAb8, FuAb23, FuAb17, FuAb18, and FuAb19) were incubated with PNGase F (NEB) at a concentration of 1 mg / ml overnight at 37°C. The deglycosylated samples were reduced with 10 mM DTT and injected onto a 300SB-C8, 2.1 x 50 mm column on an HPLC-Q-TOF-MS (Agilent, USA) for MS analysis. The results are shown in Table 2. The theoretically predicted molecular weights of the two chains of the different cytokine fusion antibodies were generally consistent with those determined by mass spectrometry.

[0254] Table 2 MS analysis

[0255] Example 4 Thermodynamic stability test

[0256] Combine the 1mg / ml concentration of fusion antibody with freshly prepared thermal shift dye and shift buffer (Protein Thermal Shift TM Dye Kit, ThermoFisher Scientific, Cat. 4461146) were mixed according to the manufacturer's recommended ratio and the ViiA TMThe thermal scan was performed in the range of 25-99°C using a real-time PCR system at a heating rate of 0.05°C / s. The thermal melting temperature (Tm) was calculated using the "Area under curve (AUC)" analysis model of GraphPad Prism 7 software.

[0257] The results are shown in Figure 3. The melting temperatures of the fusion antibodies FuAb1, FuAb5, FuAb6, FuAb17, FuAb18 and FuAb19 samples fused with three different antibodies (PDL1, PSMA, B7H3) are in the range of 65-75°C, which are all higher than the first melting temperature Tm1 = 60.79°C of the parent antibody, indicating that the cytokine fusion antibodies have good thermodynamic stability.

[0258] Example 5 Binding Activity Detection

[0259] 5.1 ELISA test

[0260] Target antigen (100 ng / 100 μl / well) was coated on a 96-well ELISA plate and incubated overnight at 4° C. The plate was blocked with PBST containing 2% skim milk powder (0.5% Tween-20 in PBS) for 1 hour at room temperature and then washed with PBST. Cytokine fusion antibody samples or cytokine fusion antibodies cleaved with MMP-2 (where MMP-2 recognizes and cleaves the amino acid sequence in the linker peptide of SEQ ID NO: 62 or 65, between the S and L of IPVSLRSG (as shown in SEQ ID NO: 66)) (specific cleavage conditions are as follows: replace the fusion protein containing the cleavage site with a buffer containing 50 mM Tris, 10 mM CaCl2, 100 mM NaCl, pH 7.4, mix mMMP-2 and each fusion protein at a ratio of 1:40, and cleave at 37°C for 24 hours) (the suffix -C after the fusion antibody indicates the cleaved sample) were diluted to 50 nM in blocking buffer. From this starting concentration, 5-fold serial dilutions were performed for a total of 8 dilutions. 100 μl of the diluted cytokine fusion antibody sample was added to each well and incubated at room temperature for 2 hours. After washing three times with PBST, add 100 μl of HRP-labeled goat anti-human Fc monoclonal antibody diluted 1:500 according to the instructions to each well. Incubate at room temperature for 1 hour and then wash five times with PBST. Add 100 μl of freshly prepared TMB colorimetric reagent (BioLegend, Cat. 421101) to the washed ELISA wells and incubate at room temperature in the dark for 5-30 minutes. After color development, read the plate at 650 nm on a microplate reader.

[0261] Antigen-targeting monoclonal antibodies aPDL1 (wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 23 and 24), aB7H3 (wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 33 and 34), aB7H3-2 (wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 118 and 119), aPD1 (wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 138 and 139), aPD1-2 (wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 142 and 143), aPD1-3 (wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 140 and 141), or aPDL1 mono (compared to the bivalent binding of aPDL1, aPDL1 mono binds monovalently to the antigen, wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 23 and SEQ ID NOs: 24), are used. The EC values ​​were calculated using the "log (agonist) vs. response--Variable slope (four parameters)" analysis model of GraphPad Prism7 software. 50 The results are shown in Figure 4-6.

[0262] As shown in Figure 4, the ELISA binding activity of FuAb1 and FuAb8 for the PDL1 antigen was similar to that of aPDL1 and was stronger than that of FuAb9 and FuAb13, fusion proteins containing IL-15 and IL-15Ra at the N-terminus of the heavy and light chain variable regions, respectively. Furthermore, as shown in Figure 6, the ELISA binding activity of FuAb6 for the B7H3 antigen was consistent with that of aB7H3 and stronger than that of FuAb10, fusion protein containing IL-15 and IL-15Ra at the N-terminus of the heavy and light chain variable regions, respectively. The ELISA binding activity of FuAb17-22 for the PSMA antigen was consistent with that of FuAb5 and stronger than that of the control FuAb12. This indicates that the embedded fusion method has no effect on the antigen-binding activity of the N-terminal antigen-binding domain. Furthermore, the binding activity of FuAb5 and FuAb17-22 for IL15Ra and IL2Rβγ was significantly reduced compared to that of FuAb12.

[0263] As shown in Figure 5, the ELISA binding curves for FuAb1, a fusion protein without a cleavage site, and FuAb8-C, a sample digested with FuAb8, showed no significant changes in PD-L1 binding activity compared to the curves for FuAb1 and FuAb8 samples. This indicates that the ELISA antigen-binding activity of fusion proteins without a cleavage site is unaffected by proteases. The ELISA binding curves for fusion proteins with a cleavage site on one side (e.g., FuAb3, FuAb4, FuAb14, and FuAb15) shifted to the right after digestion, suggesting that the binding activity of the antigen-binding domain at the N-terminus is reduced after single digestion. Fusion proteins with cleavage sites on both sides (e.g., FuAb7 and FuAb16) showed almost no ELISA binding after digestion.

[0264] As shown in Figure 7, the ELISA binding activity of the chimeric fusion proteins FuAb24 and 25 to the B7H3 antigen was basically consistent with that of the aB7H3 monoclonal antibody, and their binding ability was stronger than that of FuAb26 and 27, which were unilaterally chimerically fused to the heavy and light chains of IL-15. In terms of IL-2Rβγ binding ability, the chimeric fusion proteins FuAb24, 25, 26, and 27 were all lower than the positive control sample FuAb12, in which IL-15 and IL-15Ra were fused to the N-termini of the antibody heavy chain and light chain, respectively, with EC50 differences of 10 times or more. In terms of IL15Ra binding ability, the chimeric fusion proteins were all lower than FuAb26 and 27, which were unilaterally chimerically fused to the heavy and light chains of IL-15, with EC50 differences of 50 times or more.

[0265] As shown in Figure 8 , the ELISA binding activities of FuAb38 and 39 to B7H3 antigen were basically consistent with that of the aB7H3-2 monoclonal antibody, and their IL-2Rβγ binding abilities were also lower than that of FuAb12, with EC50 differences of 40 times or more.

[0266] As shown in Figure 9 , in terms of PD1 binding ability, the ELISA binding activity of the chimeric fusion proteins FuAb29-34 and FuAb44-47 to the PD1 antigen was lower than that of the aPD1 monoclonal antibody (M-PD1-His is the mouse PD1 antigen). As shown in Figure 10 , the binding ability to IL-2Rβγ was lower than that of the positive control sample FuAb12, in which IL-15 and IL-15Ra were fused to the N-termini of the antibody heavy chain and light chain, respectively, and the EC50 difference was 4-25 times.

[0267] 5.2 Cell surface receptor binding activity detection

[0268] A431 cells (carrying natural human PDL1 antigen on the cell surface), MC38-hB7H3, MC38-hPSMA, and MC38 cells (carrying natural mouse PDL1 antigen on the cell surface) were cultured. MC38-hB7H3 and MC38-hPSMA are MC38 cells genetically modified to carry human B7H3 and PSMA antigens, respectively. All cells were cultured in DMEM medium containing 10% FBS, then trypsinized and used. 2x10 e were aliquoted into each flow cytometry well. 5 The cells were blocked with pre-cooled 2% FBS-PBS blocking solution for 30 minutes. The cytokine fusion antibody sample (enzyme-cleaved product or uncleaved) was diluted to 50nM with blocking solution, and with this as the starting concentration, a 5-fold gradient dilution was performed, with a total of 8 dilution gradients. The 0nM concentration was set as the staining background. 100ul of diluted cytokine fusion antibody sample was added to each well and incubated at 4°C for 30 minutes. After washing 3 times with PBS, 100ul of APC-labeled anti-human Fc monoclonal antibody diluted according to the instructions was added to each well, incubated at 4°C for 30 minutes, and then washed 3 times with PBS. 200ul of blocking solution was added to the washed flow tube, and the resuspended cell suspension was used for flow analysis. EC was calculated using the "log(ag onist) vs.response--Variable slope(four parameters)" analysis model of GraphPad Prism7 software. 50 value.

[0269] Monoclonal antibodies targeting antigens aPDL1, aB7H3, aPDL1 mono, or aPSMA were used as control samples.

[0270] The results are shown in Figure 11. The binding activity of the fusion proteins (such as FuAb1, FuAb5, FuAb6, and FuAb8) in which IL15 and IL15Ra are located between the variable and constant regions of the antibody, respectively, to cell surface receptors is consistent with that of the parent monoclonal antibodies targeting the antigen (aPDL1, aPSMA, and aB7H3), and is stronger than that of the fusion proteins (such as FuAb9, FuAb11, FuAb10, and FuAb13) in which IL15 and IL15Ra are located at the N-terminus of the antibody variable region, indicating that the fusion of IL-15 / IL-15Ra in the antibody of the present application (hereinafter referred to as "embedded fusion") does not affect the binding activity of the VH / VL antigen-binding domain located at the N-terminus to cell surface receptors.

[0271] As shown in Figure 12, the flow cytometric binding curve for FuAb1-C, a sample of MC38 cell binding activity after digestion of the fusion protein FuAb1 without a cleavage site, showed no significant change compared to the curve for the FuAb1 sample. However, the flow cytometric binding curve for fusion proteins with a cleavage site on one side (such as FuAb4 and FuAb3) shifted to the right after digestion, indicating decreased binding activity. Fusion proteins with cleavage sites on both sides (such as FuAb7) showed no cell binding activity after digestion.

[0272] 5.3 Mo7e surface receptor binding activity assay

[0273] Culture suspension human giant cell leukemia cells Mo7e (Mo7e is a human IL-2Rβγ receptor positive cell). Aliquot 2x10e into each flow cytometry staining well. 5 The cells were blocked with pre-cooled 2% FBS-PBS blocking solution for 30 minutes. The cytokine fusion antibody sample was diluted to 100nM with blocking solution, and a 5-fold gradient dilution was performed with this as the starting concentration, with a total of 8 dilution gradients. The 0nM concentration was set as the staining background. 100ul of diluted cytokine fusion antibody sample was added to each well and incubated at 4°C for 30 minutes. After washing with PBS three times, 100ul of APC-labeled anti-human Fc monoclonal antibody diluted according to the instructions was added to each well, incubated at 4°C for 30 minutes, and then washed with PBS three times. 200ul of blocking solution was added to the washed flow tube, and the resuspended cell suspension was used for flow cytometry analysis. EC was calculated using the "log (agonist) vs. response--Variable slope (four parameters)" analysis model of GraphPad Prism7 software. 50 value.

[0274] The results are shown in Figure 13. FuAb5 has very weak binding activity to Mo7e, while FuAb6 exhibits stronger binding activity to Mo7e, which is closely related to the expression of B7H3 receptor on Mo7e (commercial PE-labeled anti-human B7H3 antibody stains Mo7e cells positively, the results are not shown).

[0275] Example 6 Cell proliferation activity detection

[0276] Culture suspensions of human giant cell leukemia cells Mo7e and mouse T lymphocytes CTLL2 (CTLL2 is a cell expressing mouse IL2αβγ receptor). Take the cultured CTLL2 cells and centrifuge them, wash them three times with RPMI1640 medium, and resuspend them in 10% FBS-RPMI1640 medium at a density of 2x10e 5cells / ml. Dilute the fusion antibody sample 5-fold, take 10ul and add it to a 96-well culture plate with a black transparent bottom, then add 90ul to resuspend the cells and gently tap to mix. Place it in the incubator and incubate for 24h. Use an equal volume of CellTiter-Glo for lysis reading according to the manufacturer's instructions. Centrifuge the cultured Mo7e cells, wash them 3 times with RPMI1640 medium, and resuspend them in 10% FBS-RPMI1640 medium at a density of 2x10e 5 / ml. Take 10ul of 5-fold gradient dilution of fusion antibody sample (enzyme-cleaved and non-enzyme-cleaved) and add it to a 96-well culture plate with a black transparent bottom, then add 90ul of resuspended cells and gently tap to mix. Place it in an incubator and incubate for 72h. Use an equal volume of CellTiter-Glo for lysis reading according to the manufacturer's instructions. Use the "log (agonist) vs. response--Variable slope (four parameters)" analysis model of GraphPad Prism7 software to calculate EC 50 value.

[0277] PBMC cells co-activated with anti-human CD3 and anti-human CD28 antibodies were collected, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cells were washed three times with RPMI1640 medium and resuspended in 10% FBS-RPMI1640 medium at a density of 2x10e 5 / ml. Take 10ul of gradient diluted fusion antibody sample and add it to a 96-well culture plate with a black transparent bottom, then add 90ul of resuspended cells and gently tap to mix. Place it in an incubator and incubate for 72h. Use an equal volume of CellTiter-Glo for lysis reading according to the manufacturer's instructions. Use the "log (agonist) vs. response--Variable slope (four parameters)" analysis model of GraphPad Prism7 software to calculate EC 50 value.

[0278] The results are shown in Figures 14-15. Compared with the fusion proteins in which IL15 and IL15Ra are located at the N-terminus of the antibody variable region (such as FuAb9, FuAb10, FuAb11 and FuAb12), the fusion proteins in which IL15 and IL15Ra are located between the antibody variable region and the constant region (such as FuAb2, FuAb5, FuAb6, FuAb17-22, FuAb24-25, FuAb29-32, FuAb36 and FuAb44-47) have no obvious proliferative activity on CTLL2 cells. It can be seen that the embedded fusion method can greatly weaken the downstream proliferative activity after the interaction of the IL-15 / IL-15Ra complex with its corresponding receptor.

[0279] As shown in Figure 16, for Mo7e, compared to fusion proteins with IL15 and IL15Ra located at the N-terminus of the antibody variable region (such as FuAb9, FuAb10, FuAb11, and FuAb12), the three embedded fusion antibodies FuAb1, FuAb5, and FuAb6 had a 5-10-fold reduced proliferative activity on Mo7e cells, which corresponds to the weak binding activity to the IL-2βγ receptor demonstrated by flow cytometry. In addition, FuAb6's pro-proliferative ability was significantly stronger than that of FuAb1 and FuAb5, presumably related to the expression of B7H3 antigen on the Mo7e cell surface (flow cytometry results not shown), suggesting that the binding of FuAb6 to the human B7H3 receptor on the Mo7e cell surface has a synergistic effect on the proliferation signal of the cytokine complex.

[0280] As shown in Figure 17, the proliferative activity of embedded fusion proteins (FuAb44-47) fused to aPD1, aPD1-2, and aPD1-3 antibodies on Mo7e cells was reduced by more than 20-100 times compared to FuAb12. Furthermore, compared to the positive control sample (FuAb12) containing IL-15 and IL-15Ra fused to the N-termini of the heavy and light chains, respectively, the proliferative activity of FuAb24, 25, 26, and 27 on Mo7e cells was increased by 5-10 times, indicating that the binding of FuAb6 to the human B7H3 receptor on the Mo7e cell surface has a synergistic effect on the proliferation signal of the cytokine complex.

[0281] As shown in Figure 16, in terms of promoting the proliferation activity of Mo7e, the fusion proteins without enzyme cleavage sites (such as FuAb1, FuAb8 and FuAb17-22), the fusion proteins carrying enzyme cleavage sites on one side (such as FuAb3, FuAb4, FuAb14 and FuAb15), and the fusion protein samples carrying enzyme cleavage sites on both sides (such as FuAb7 and FuAb16) have increasing proliferation activities of Mo7e. Among them, the proliferation curves of the fusion proteins without cleavage sites after cleavage were similar to those without cleavage. The proliferation curves of the fusion proteins with cleavage sites (such as FuAb4, FuAb3, FuAb14, FuAb15, FuAb7, and FuAb16) shifted to the left after cleavage, and the recovery of proliferation activity was consistent with that of FuAb12. This may be due to the enzymatic cleavage caused by the MMP enzymes naturally released by giant cells incubated with human giant cell leukemia cells [Blood 2011, 118(7): 1903-1911], which released the activity of the chimeric IL15-IL15Ra.

[0282] In terms of promoting PBMC proliferation, the results show that the backbone antibody aPSMA had no effect on cell proliferation, as shown in Figure 18. Compared to the rhIL15 molecule alone and the fusion protein (FuAb12) with IL15 and IL15Ra located at the N-terminus of the antibody variable region, the five embedded fusion antibodies (FuAb5, FuAb17, FuAb18, FuAb19, and FuAb23) reduced their PBMC proliferation activity by 10-150 times.

[0283] As shown in Figure 19, in the NK92 proliferation experiment, the chimeric fusion proteins (FuAb6, FuAb24-27, FuAb29-31, FuAb36, and FuAb44-47) all showed weak NK92 proliferation-promoting ability. 50 It is 5-20 times higher than the positive control sample FuAb12, which is an antibody in which IL-15 and IL-15Ra are fused to the N-terminus of the heavy chain and light chain, respectively.

[0284] Example 7 Evaluation of drug efficacy and toxicity in MC38 mouse tumor model

[0285] 1. Fusion antibodies targeting PDL1

[0286] A tumor model was established using mouse colon cancer cells MC38 to evaluate the tumor growth inhibitory activity and weight-related drug toxicity of cytokine fusion antibodies. Cell lines with antibody-targeted antigens on their surfaces were cultured. MC38 cells carry the natural mouse PDL1 antigen on their surface and the MMP-2 enzyme is naturally present in mice. The cells were cultured in DMEM medium containing 10% FBS and then digested with trypsin. Six- to eight-week-old C57BL6J mice were taken and 5x10e 5 MC38 cells were inoculated subcutaneously on the dorsal side. When the tumor size reached 50-100 mm 3 After the volume of the mouse tumor was larger than 1000 mm, 2 mg / kg was administered intravenously by tail vein for a total of two doses. The changes in the volume of the mouse tumor were recorded every two days. When the volume of the mouse tumor exceeded 1000 mm 3 When , the mice were euthanized.

[0287] As shown in Figure 20, fusion antibodies with IL15 and IL15Ra located between the variable and constant regions (FuAb2 and FuAb8) exhibited similar tumor inhibitory activity as fusion antibodies with IL15 and IL15Ra located at the N-terminus of the variable region (FuAb9 and FuAb12). Fusion proteins harboring cleavage sites (e.g., FuAb4, FuAb3, and FuAb7) all exhibited superior tumor inhibitory activity compared to aPDL1 mAb, with similar tumor inhibitory activity to FuAb12. Furthermore, fusion proteins harboring cleavage sites demonstrated superior weight maintenance compared to FuAb12. This demonstrates that chimeric fusions can reduce IL15 activity, relying on enrichment of the N-terminal antigen-binding target and release of IL15 activity through cleavage. This combination resulted in superior tumor inhibitory activity for fusion proteins harboring cleavage sites and significantly mitigated weight-related systemic toxicity. Fusion proteins without cleavage sites, relying on strong N-terminal antigen-binding target enrichment and weak IL15 activity, also exhibited good tumor inhibitory activity without a significant weight loss trend. The tumor inhibitory activities of aPDL1 monoclonal antibody, fusion protein without enzyme cleavage site, and fusion protein with enzyme cleavage site increased in sequence, which is consistent with the mechanism that enzyme cleavage releases the activity of chimeric IL15-IL15Ra.

[0288] 2. Fusion antibodies targeting PD1

[0289] A tumor model was established using mouse colon cancer cells MC38 to evaluate the tumor growth inhibitory activity and weight-related drug toxicity of cytokine fusion antibodies. Cell lines with antibody-targeted antigens on their surfaces were cultured. MC38 cells carried the natural mouse PDL1 antigen on their surface. They were cultured in DMEM medium containing 10% FBS and then harvested after trypsin digestion. Six- to eight-week-old C57BL6J mice were taken and 5x10e 5 MC38 cells were inoculated in the axilla. When the tumor size reached 50-100 mm 3 After the volume of the mouse tumor exceeded 1000 mm, different doses of the drug were administered intravenously. The changes in the tumor volume of the mice were recorded every two days. 3 The mice were euthanized and spleen samples were collected for flow cytometry analysis of cell populations. + CD3 + Mark T cell populations using CD45 + CD3 + CD4 + CD4 marker + T cell population, using CD45 + CD3 + CD8 + CD8 marker +T cell populations, using PD1 + PD1-positive cell populations were marked, and Foxp3-positive cell populations were marked.

[0290] As shown in Figure 21, FuAb32 exhibited superior tumor suppression compared to the aPD1 mAb. FuAb36 exhibited only weak tumor suppression activity, suggesting that IL15 fusion alone, without PD1 targeting, does not result in robust tumor suppression. FuAb29 combined with an aPD1 mAb exhibited similar tumor suppression effects to aPD1 and FuAb32, suggesting that IL15 fusion alone and aPD1 mAb alone do not result in robust synergistic tumor suppression when used in combination.

[0291] Body weight was not affected by drug administration throughout the observation period, no health-related discomfort was observed, and no mouse deaths were found.

[0292] The flow cytometry staining results are shown in Figure 22. None of the samples in the 0.2 mg / kg dose group showed obvious changes in cell populations, suggesting that each sample had little effect on the mobilization of immune cells in the mouse spleen system, indicating that there was no toxicity risk in the mouse system.

[0293] Example 8 Effect of drug dosage on efficacy and toxicity in MC38 mouse tumor model

[0294] A tumor model was established using mouse colon cancer cells MC38. 6-8 week old C57BL6J mice were randomly divided into 5x10 5 MC38 cells were inoculated subcutaneously on the dorsal side. When the tumor size reached 50-100 mm 3 After the volume of the mouse tumor was increased, 2mg / kg, 5mg / kg, and 10mg / kg were administered intravenously by tail vein for a total of one dose. The changes in the volume of the mouse tumor were recorded every two days. When the volume of the mouse tumor exceeded 1000mm 3 When , the mice were euthanized.

[0295] As shown in Figure 23, compared to the DPBS group, FuAb1 at single doses of 2 mg / kg, 5 mg / kg, and 10 mg / kg all exhibited significant tumor growth inhibition activity. In the 10 mg / kg group, mouse body weight decreased significantly two days after administration, while the 2 mg / kg and 5 mg / kg groups showed no effect of dosing throughout the observation period, suggesting that the fusion antibody possesses a good safe and effective dose window.

[0296] Example 9 Mechanism of drug action in MC38 mouse tumor model

[0297] A tumor model was established using mouse colon cancer cells MC38. 6-8 week old C57BL6J mice were randomly divided into 5x105 MC38 cells were inoculated subcutaneously on the dorsal side. When the tumor size reached 300-500 mm 3 After the volume was 2 mg / kg, the drug was administered intravenously through the tail vein once. On the fourth day after administration, the mice were euthanized, and peripheral blood samples, spleen samples, and tumor tissue samples were collected for flow cytometry analysis of cell populations.

[0298] Flow cytometry staining uses CD45+CD3+ to mark T cell populations, CD45+CD3+CD4+ to mark CD4+ T cell populations, CD45+CD3+CD8+ to mark CD8+ T cell populations, CD45+CD19+ to mark B cell populations, and CD45+CD335+ to mark NK cell populations.

[0299] The results are shown in Figures 24-26. Analysis of peripheral blood samples, spleen samples, and tumor-infiltrating lymphocytes (TILs) can illustrate the expression of cell populations associated with tumor suppression and systemic toxicity.

[0300] As shown in Figures 24 and 25, FuAb12 / FuAb9 exhibited severe body weight-related systemic toxicity in Example 7. In the cell population analysis, a large amount of NK / T lymphocyte proliferation was observed in the peripheral blood and spleen (unrelated to the tumor). Compared with FuAb12 / FuAb9, the chimeric fusion protein FuAb2 / FuAb8 showed less change in cell populations in the peripheral blood and spleen (unrelated to the tumor), and the proportion of each cell was similar to that of the PDL1 mAb group. It can be seen that the chimeric fusion protein can maintain the stability of cell populations in the peripheral blood and spleen, effectively avoiding systemic toxicity caused by lymphocyte proliferation, which is consistent with the weight changes shown in Example 7.

[0301] As shown in Figure 26, in the cell population analysis within the tumor environment, compared with the aPDL1 administration group, the fusion protein (FuAb12, FuAb9, FuAb2 and FuAb8) administration groups all showed a significant increase in the proportion of cytotoxic lymphocytes (CD8+ T cells and NK cells). It can be seen that the chimeric fusion protein can achieve a tumor suppression therapeutic effect by specifically amplifying cytotoxic lymphocytes within the tumor environment. This is consistent with the tumor suppression results shown in Example 7.

[0302] In summary, the chimeric fusion protein FuAb2 / FuAb8 alone was able to specifically increase the proportion of CD8+ T cells and NK cells in the tumor environment without affecting peripheral blood and spleen cell populations. The chimeric fusion protein FuAb2 / FuAb8 alone had the ability to reduce systemic cytotoxicity, exert biological effects within the tumor microenvironment, enhance activation of NK cells and CD8+ T cells in the tumor tissue region, and achieve tumor growth inhibition.

[0303] Example 10 Effect of drug dosage on efficacy and toxicity in the MC38-hB7H3 mouse tumor model

[0304] A tumor model was established using mouse colon cancer cells MC38-hB7H3 to evaluate the tumor growth inhibitory activity and weight-related drug toxicity of cytokine fusion antibodies. MC38-hB7H3 cells were cultured adherently in DMEM medium containing 10% FBS and harvested after trypsinization. 6-8 week old C57BL6J mice were selected and 5x10e 5 MC38-hB7H3 cells were inoculated subcutaneously on the dorsal side. When the tumor size reached 50-100 mm 3 After the volume of the mouse tumor was increased, 2mg / kg, 5mg / kg, and 10mg / kg were administered intravenously by tail vein for a total of two times. The changes in the volume of the mouse tumor were recorded every two days. When the volume of the mouse tumor exceeded 1000mm 3 When , the mice were euthanized.

[0305] The results are shown in Figure 27. 10-15 days after inoculation, the tumor volume in the DPBS-treated group exceeded 1000 mm 3 Compared to the PBS group, the antigen-targeting monoclonal antibody aB7H3 showed no significant tumor growth inhibitory activity at a 2 mg / kg dose. FuAb10, a fusion protein containing IL15 and IL15Ra located at the N-terminus of the antibody variable region, exhibited significant tumor inhibitory activity while also experiencing a greater than 20% decrease in body weight over 10-15 days. FuAb6 exhibited significant tumor growth inhibitory activity at doses of 2 mg / kg, 5 mg / kg, and 10 mg / kg, and mouse body weight was minimally affected by dosing across the entire observation range. The weight loss trend in mice treated with 10 mg / kg FuAb6 was significantly improved compared to the 2 mg / kg FuAb10 dose, suggesting that FuAb6 possesses a well-defined safe and effective dosing window.

[0306] As shown in Figure 28, the above conclusion can also be drawn when used in combination with the immune checkpoint inhibitor aCTLA-4: the fusion antibody in which IL15 and IL15Ra are located between the variable region and the constant region has good efficacy and safety, and a large safe and effective dose window (2-10 mg / kg is safe and effective).

[0307] Example 11 Pharmacokinetic evaluation in mice

[0308] 6-8 week old C57BL6J mice were administered 2 mg / kg or 5 mg / kg via tail vein. Eye blood was collected at different time points. The concentration of intact fusion antibody in serum samples at each time point was determined by ELISA, and the data were processed using GraphPad Prism.

[0309] The results are shown in Table 3 below. FuAb2, FuAb6, FuAb17, FuAb19, and FuAb23 all have pharmacokinetic performance similar to that of antibody molecules, indicating that the embedded fusion method enables the fusion antibody molecules to have a good blood drug half-life.

[0310] Table 3 PK parameters of fusion antibodies

[0311] Example 12: Evaluation of drug efficacy and toxicity in the MC38-hPSMA mouse tumor model

[0312] A tumor model was established using mouse colon cancer cells MC38-human PSMA to evaluate the tumor growth inhibitory activity and weight-related drug toxicity of cytokine fusion antibodies. Cell lines with antibody-targeted antigens on their surfaces were cultured. MC38-human PSMA cells carried natural mouse PDL1 antigen and heterologously expressed human PSMA antigen on their surfaces. They were cultured in DMEM medium containing 10% FBS and then harvested after trypsin digestion. Six- to eight-week-old C57BL6J mice were taken and 5x10e 5 MMC38-human PSMA cells were inoculated in the armpit. When the tumor size reached 50-100 mm 3 After the volume of the mouse tumor exceeded 1000 mm, different doses of the drug were administered intravenously. The changes in the tumor volume of the mice were recorded every two days. 3 The mice were euthanized. As shown in Figures 29-30, 17-19 days after inoculation, the tumor volume of the DPBS-treated group exceeded 1000 mm 3 , and FuAb17-19 showed good tumor inhibitory activity and safety.

[0313] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: utilizing the symmetrical Y-shaped structure of natural antibodies, with a VH-IL-15-CH1-Fc fragment or a VH-IL-15Ra-CH1-Fc fragment as the first chain; and with a VL-IL-15Ra-CL fragment, a VL-IL-15-CL-Fc fragment or a VL-IL-15-CL fragment, a VL-IL-15Ra-CL-Fc fragment as the second chain, the fusion antibody formed has similar stability and half-life to natural antibodies; and significantly reduces the activity of the IL15 / IL15Ra complex, greatly reducing systemic toxicity. The targeting of the antibody can specifically act in the tumor environment, thereby improving the cell targeting of the IL15 / IL15Ra complex. In addition, since no exogenous sequence is introduced, the risk of immunogenicity is minimized, and the productivity and drugability of the molecular structure itself are improved.

[0314] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A fusion antibody, characterized in that, Comprising: a) IL-15; b) IL-15Ra; and c) an antibody targeting a cell surface antigen related to targeted therapy, said antibody comprising a heavy chain variable region VH, a heavy chain constant region CH1, a light chain variable region VL, and a light chain constant region CL; wherein, the IL-15 is located between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody, and the IL-15Ra is located between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody; or the IL-15 is located between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody, and the IL-15Ra is located between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody.

2. The fusion antibody according to claim 1, wherein The IL-15 is directly or fused via a linker peptide between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody, and the IL-15Ra is directly or fused via a linker peptide between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody; or the IL-15 is directly or fused via a linker peptide between the C-terminus of the light chain variable region VL of the antibody and the N-terminus of the light chain constant region CL of the antibody, and the IL-15Ra is directly or fused via a linker peptide between the C-terminus of the heavy chain variable region VH of the antibody and the N-terminus of the heavy chain constant region CH1 of the antibody.

3. The fusion antibody according to claim 1 or 2, characterized in that, The C-terminus of the heavy chain constant region CH1 contains an Fc fragment, and the C-terminus of the light chain constant region CL contains or does not contain an Fc fragment.

4. The fusion antibody according to claim 1, characterized in that, The IL-15 has an amino acid sequence as shown in SEQ ID NO:12 or SEQ ID NO:81, or an amino acid sequence having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:

81.

5. The fusion antibody according to claim 1, wherein The IL-15Ra is full-length IL-15Ra or the Sushi domain of IL-15Ra; Preferably, the IL-15Ra has an amino acid sequence as shown in SEQ ID NO:13 or SEQ ID NO:14, or an amino acid sequence having more than 80%, more preferably more than 90%, and further preferably more than 95% homology with the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:

14.

6. The fusion antibody according to any one of claims 1-5, characterized in that, The cell surface antigen related to the treatment is an immune checkpoint protein or a tumor antigen.

7. The fusion antibody according to claim 6, wherein The treatment-related cell surface antigens include any one of the following: PD1, PDL1, B7H3, PSMA, Nectin-4, CD19, BCMA, CD22, CD20, GPCR5D, CD21, CD81, CD40, CD79, CD80, CD86, ICAM-1 (CD54), CD11a, CD18, CD45, GPC3, HER2, EGFR, GCN4, Tim3, CLL1, Trop2, Claudin18.2, Claudin6, Muc1, Muc16 or GIST.

8. The fusion antibody according to claim 7, wherein The antibodies against the treatment-related cell surface antigens include antibodies targeting PDL1, which have heavy chain complementary determining regions HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 15-17, and light chain complementary determining regions LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 18-20; or have a VH sequence as shown in SEQ ID NO: 21, and a VL sequence as shown in SEQ ID NO: 22; or have a heavy chain amino acid sequence as shown in SEQ ID NO: 23, and a light chain amino acid sequence as shown in SEQ ID NO: 24; Preferably, the antibodies against the treatment-related cell surface antigens include antibodies targeting B7H3, which have heavy chain complementary determining regions HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 25-27, and light chain complementary determining regions LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 28-30; or have a VH sequence as shown in SEQ ID NO: 31 or 118, and a VL sequence as shown in SEQ ID NO: 32 or 119; or have a heavy chain amino acid sequence as shown in SEQ ID NO: 33, and a light chain amino acid sequence as shown in SEQ ID NO: 34; Preferably, the antibodies against the treatment-related cell surface antigens include antibodies targeting PSMA, which have heavy chain complementary determining regions HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs: 35-37, and light chain complementary determining regions LCDR1, LCDR2 and LCRD3 sequences as shown in SEQ ID NOs: 38-40; or have a VH sequence as shown in SEQ ID NO: 41, and a VL sequence as shown in SEQ ID NO: 42; or have a heavy chain amino acid sequence as shown in SEQ ID NO: 43, and a light chain amino acid sequence as shown in SEQ ID NO:

44. Preferably, the antibody against the therapeutically relevant cell surface antigen includes an antibody targeting PD1, which has heavy chain complementary determining regions HCDR1, HCDR2, and HCRD3 sequences as shown in SEQ ID NOs: 120 - 122, SEQ ID NOs: 126 - 128, or SEQ ID NOs: 132 - 134, and light chain complementary determining regions LCDR1, LCDR2, and LCRD3 sequences as shown in SEQ ID NOs: 123 - 125, SEQ ID NOs: 129 - 131, or SEQ ID NOs: 135 - 137; or has a VH sequence as shown in SEQ ID NO: 138 and a VL sequence as shown in SEQ ID NO: 139; or has a heavy chain amino acid sequence as shown in SEQ ID NO: 140 or 142 and a light chain amino acid sequence as shown in SEQ ID NO: 141 or 143.

9. The fusion antibody according to claim 7 or 8, characterized in that, The antibody against the therapeutically relevant cell surface antigen contains an Fc fragment; Preferably, the Fc fragment is selected from the Fc fragments of human IgG1, IgG2, IgG3, or IgG4.

10. The fusion antibody according to claim 2, wherein The linker peptide is a cleavable linker peptide or a non - cleavable linker peptide; Preferably, each of the linker peptides is independently selected from [GGGGS]n, where n is selected from 1 - 6, EAAAK (SEQ ID NO: 79), EAAAKEAAAK (SEQ ID NO: 80), GGGGSIPVSLRSGGGGGSG (SEQ ID NO: 65), or GGGGSIPVSLRSGGGSG (SEQ ID NO: 62), GGGGSG (SEQ ID NO: 145), GGGGSGGGGSG (SEQ ID NO: 146), GGGGSGGGGSGGGGSG (SEQ ID NO: 64); More preferably, the linker peptide is GGGGS (SEQ ID NO: 144), GGGGSG (SEQ ID NO: 145), GGGGSGGGGS (SEQ ID NO: 63), GGGGSGGGGSG (SEQ ID NO: 146), GGGGSGGGGSGGGGSG (SEQ ID NO: 64), GGGGSIPVSLRSGGGGGSG (SEQ ID NO: 65), or GGGGSIPVSLRSGGGSG (SEQ ID NO: 62).

11. The fusion antibody according to any one of claims 1-5, characterized in that, The fusion antibody comprises a heavy chain and a light chain respectively having any one of the following sets of amino acid sequences: 1) SEQ ID NO:1 and SEQ ID NO:2; 2) SEQ ID NO:3 and SEQ ID NO:4; 3) SEQ ID NO:1 and SEQ ID NO:5; 4) SEQ ID NO:1 and SEQ ID NO:6; 5) SEQ ID NO:7 and SEQ ID NO:8; 6) SEQ ID NO:9 and SEQ ID NO:10; 7) SEQ ID NO:5 and SEQ ID NO:11; 8) SEQ ID NO:45 and SEQ ID NO:46; 9) SEQ ID NO:57 and SEQ ID NO:58; 10) SEQ ID NO:59 and SEQ ID NO:60; 11) SEQ ID NO:57 and SEQ ID NO:60; 12) SEQ ID NO:67 and SEQ ID NO:68; 13) SEQ ID NO:69 and SEQ ID NO:70; 14) SEQ ID NO:71 and SEQ ID NO:72; 15) SEQ ID NO:73 and SEQ ID NO:74; 16) SEQ ID NO:75 and SEQ ID NO:76; 17) SEQ ID NO:77 and SEQ ID NO:78; 18) SEQ ID NO:82 and SEQ ID NO:8; 19) SEQ ID NO:83 and SEQ ID NO:84; 20) SEQ ID NO:85 and SEQ ID NO:84; 21) SEQ ID NO:83 and SEQ ID NO:86; 22) SEQ ID NO:85 and SEQ ID NO:86; 23) SEQ ID NO:88 and SEQ ID NO:89; 24) SEQ ID NO:90 and SEQ ID NO:91; 25) SEQ ID NO:92 and SEQ ID NO:93; 26) SEQ ID NO:94 and SEQ ID NO:95; 27) SEQ ID NO:96 and SEQ ID NO:97; 28) SEQ ID NO:98 and SEQ ID NO:99; 29) SEQ ID NO:102 and SEQ ID NO:103; 30) SEQ ID NO:106 and SEQ ID NO:107; 31) SEQ ID NO:108 and SEQ ID NO:107; 32) SEQ ID NO:112 and SEQ ID NO:113; 33) SEQ ID NO:112 and SEQ ID NO:114;34) SEQ ID NO: 115 and SEQ ID NO: 116; 35) SEQ ID NO: 115 and SEQ ID NO: 117.; 12. A DNA molecule, characterized in that, The DNA molecule encodes the fusion antibody according to any one of claims 1 - 11.

13. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the DNA molecule according to claim 12.

14. A host cell, characterized in that, The host cell is transformed with the recombinant plasmid according to claim 13.

15. The host cell according to claim 14, wherein, The host cell includes a prokaryotic cell or a eukaryotic cell.

16. Use of the fusion antibody according to any one of claims 1 - 11 in the preparation of a drug for preventing and / or treating cancer.

17. The use according to claim 16, characterized in that, The cancer includes any one of the following: lung cancer, melanoma, colon cancer, rectal cancer, liver cancer, breast cancer, lymphoma, prostate cancer or hematological tumor.

18. A drug, characterized in that, It includes the fusion antibody according to any one of claims 1-11.

19. The drug according to claim 18, wherein, The drug further includes an immune checkpoint inhibitor used in combination with the fusion antibody, and the immune checkpoint inhibitor includes any one or more of the following: aPDL1, aCTLA4, a41BB or aLAG3.

Images