WO2025140627A1 - Fusion antibody and use thereof - Google Patents

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

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 public content of the CN application is again introduced as a whole into this application.

Technical Field

The present invention relates to the field of genetic engineering, and in particular to a fusion antibody and application thereof.

Background Art

Cytokines are a class of proteins that are used as signal transduction in organisms. Cytokines can be produced by a variety of cell types and act on neighboring cells or the entire system, playing an important role in innate and adaptive immunity.

Studies have found that many cytokines can stimulate immune effector cells and enhance cytotoxicity. This property makes cytokines used in tumor treatment. Interleukin 15 (IL-15) is a cytokine protein in the same family as interleukin 12 (IL-2) and was discovered in 1994. IL-15 has many similar biological activities to IL-2, 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.

IL-15 has a similar structure to IL-2, and shares the IL2Rβ and IL2Rγ receptors with IL-2. IL-15Ra, as a transmembrane protein with high affinity for IL-15, forms a complex with IL-15 (IL-15 superagonist) and is able to 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 (Treg), does not mediate activation-induced cell death (AICD), but inhibits IL-2-induced AICD. This also means that IL-15 may be a better choice than IL-2 in tumor treatment.

The anti-tumor effect of IL-15 is mainly through promoting the proliferation and activation of CD8+T cells and NK cells. In a variety of experimental animal tumor models (LA795 lung adenocarcinoma, melanoma (B16, B78-H1), MC38 colon cancer, liver cancer and lymphoma), IL-15 treatment can promote tumor regression, reduce tumor metastasis and improve survival rate. However, due to the low molecular weight and short half-life of IL-15, the renal clearance rate is high, and multiple injections per day are extremely inconvenient in terms of method. Therefore, the simple use of recombinant IL-15 is also limited in tumor treatment.

Currently, most clinical research drugs use PEG modification or Fc fusion to increase the half-life of cytokines. Although the half-life is extended, it still faces toxicity problems caused by poor targeting. In clinical development, N-803 is the IL-15 fusion protein with the fastest clinical progress, but in the treatment of tumors, due to its super-strong agonist properties with poor tumor targeting, it faces great limitations in both indications and administration methods.

Therefore, it is 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

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

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 light chain constant region CL of the antibody.

Further, IL-15 is fused directly or through a connecting 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 through a connecting 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 through a connecting 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 through a connecting 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.

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.

Furthermore, 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.

Furthermore, IL-15Ra is full-length IL-15Ra or the Sushi domain of IL-15Ra; preferably, 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 as shown in SEQ ID NO: 13 or SEQ ID NO: 14.

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

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.

Furthermore, antibodies for treating 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.

Further, antibodies for treating 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: 132-133. The heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences shown in SEQ ID NOs:132-134, and the light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequences shown in SEQ ID NOs:135-137; or has a VH sequence shown in SEQ ID NO:138, and a VL sequence shown in SEQ ID NO:139; or has a heavy chain amino acid sequence shown in SEQ ID NO:140, and a light chain amino acid sequence shown in SEQ ID NO:141; or has a heavy chain amino acid sequence shown in SEQ ID NO:142, and a light chain amino acid sequence shown in SEQ ID NO:143.

Further, antibodies for treating 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.

Preferably, antibodies for therapeutically relevant cell surface antigens include antibodies targeting PSMA, which have 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 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.

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.

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, 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), GGGGSGGGG SGGGGSG (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 (S EQ ID NO:146), GGGGSGGGGSGGGGSG (SEQ ID NO:64), GGGGSIPVSLRSGGGGGSG (SEQ ID NO:65) or GGGGSIPVSLRSGGGGSG (SEQ ID NO:62).

Further, the fusion antibody comprises a heavy chain and a light chain having any of the following groups of amino acid sequences, respectively: 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. 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; 1 4) 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 : 114) SEQ ID NO: 115 and SEQ ID NO: 116; 35) SEQ ID NO: 115 and SEQ ID NO: 117.

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

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.

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.

Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell.

In order to achieve the above object, according to the 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.

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.

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.

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.

By applying the technical solution of the present invention, the IL-15 domain or the IL-15Ra domain is fused to the VH-CH1 or VL-CL of the targeting antibody through a connecting peptide, respectively, to form a targeted fusion antibody that pairs the IL-15 with the IL-15Ra complex. This specific fusion method 1) fully exposes the N-terminus of the targeting antibody, fully utilizes the targeting function of the antibody, and specifically enriches IL15 to the targeting site; 2) gives IL15 a half-life consistent with that of the targeting antibody; 3) weakens the binding activity of IL-15 with IL-2Rβγ, reduces the systemic toxicity of IL15, and maintains the receptor activation function of IL15 in a high-density IL-2Rβγ environment.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

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

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

FIG3 shows a thermodynamic stability experimental diagram of fusion antibodies FuAb1, FuAb5, FuAb6, FuAb17, FuAb18 and FuAb19 in Example 4 of the present application;

FIG4 shows the result of ELISA detection of binding of different fusion antibodies to antigen PDL1 in Example 5 of the present application;

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

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

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;

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;

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

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

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;

FIG12 shows the results of the binding activity test 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;

FIG13 shows the results of the binding activity test of different fusion antibodies binding to Mo7e cells in Example 5 of the present application;

FIG14 shows the proliferation-promoting activity of different fusion antibodies on CTLL2 cells in Example 6 of the present application;

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

FIG16 shows the proliferation-promoting activity of different fusion antibodies on Mo7e cells in Example 6 of the present application;

FIG17 shows the proliferation-promoting activity of different fusion antibodies on Mo7e cells in Example 6 of the present application;

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

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

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;

FIG21 shows the effect of the fusion antibody targeting PD1 on the tumor volume and weight change of mice in the MC38 mouse tumor model in Example 7 of the present application;

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;

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

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

FIG25 shows the flow staining 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;

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

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

FIG28 shows the effects of different doses of 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;

FIG29 shows the effect of different doses of fusion antibody on the change of mouse tumor volume in the MC38-hB7H3 mouse tumor model in Example 11 of the present application;

FIG30 shows the effect of different doses of fusion antibody on the weight change of mice in the MC38-hB7H3 mouse tumor model in Example 11 of the present application;

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

Figure 32 shows a schematic diagram of the structure 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

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present 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.

As mentioned in the background technology, the IL-15 fusion protein in the prior art has the following problems: 1. Lack of targeting of tumor cells or tumor microenvironment; 2. The half-life in pharmacokinetic performance is relatively short and cannot match that of therapeutic antibodies; 3. The peripheral side effects are strong and the safe dose cannot match that of therapeutic antibodies.

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 low cell targeting, and the superagonist activity of the IL-15/IL-15Ra complex, it is easy to induce systemic immunotoxicity. In the process of using IL-15 for anti-tumor treatment, there is still the problem of multiple administrations and difficulty in avoiding immunotoxicity. The present application intends to embed IL-15 and IL-15Ra into an antibody to obtain a fusion antibody with high cell targeting for the prevention and treatment of tumors.

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.

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. In this application, IL-15 and IL-15Ra are respectively embedded between the antibody VH-CH1 or VL-CL, and the IL-15/IL-15Ra complex can be specifically enriched in the tumor microenvironment through the targeting of the antibody to the cell surface antigen, without affecting the antibody's specific recognition of the antigen, so as to achieve the purpose of specific tumor killing and avoid systemic over-activation of NK cells induced immunotoxicity. At the same time, the embedding of IL-15 and IL-15Ra into antibodies 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.

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 so that the molecules connected at both ends of the connecting peptide can maintain their respective inherent biological activities.

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.

In a preferred embodiment, IL-15Ra is connected 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 through a connecting peptide; IL-15Ra is connected 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 through a connecting peptide.

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.

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 through a connecting 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.

As shown in Figure 20, in a preferred embodiment, IL-15 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, and IL-15Ra 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.

In a preferred embodiment, IL-15 is connected 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 through a connecting peptide, and IL-15Ra is connected 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 through a connecting peptide.

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.

In a preferred embodiment, IL-15 is linked to 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 through a connecting peptide, and IL-15Ra is directly linked to 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.

Any light chain with a VL-CL structure or a heavy chain with a VH-CH1 structure is suitable for 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.

Any IL-15 with anti-tumor function of promoting 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.

SEQ ID NO:12: hIL15

SEQ ID NO:81:hIL15 WT

Any IL-15Ra that can bind to IL-15 to form an anti-tumor effect is suitable for the present application. In a preferred embodiment, IL-15Ra is full-length IL-15Ra or the Sushi domain of IL-15Ra. Among them, the Sushi domain of IL-15Ra is a domain with a specific structure, located at the N-terminus of IL-15Ra, and is composed of a series of repeated sequences of about 60 amino acids, which plays an important role in cell signal transduction and immune response. Preferably, IL-15Ra has an amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence with 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: 13 or SEQ ID NO: 14.

SEQ ID NO:13: hIL15Ra

SEQ ID NO:14: hIL15RaSushi

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, targeted therapy-related cell surface antigens are immune monitoring point proteins or tumor antigens. In a preferred embodiment, targeted therapy-related cell surface antigens are 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, targeted therapy-related cell surface antigens are selected from any one of the following: PDL1, PD1, B7H3 or PSMA.

Any antibody that has binding activity with antigens 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, the antibody contains a heavy chain and a light chain, which respectively contain a variable region and a constant region structure, and the heavy chain and the light chain have three complementary determining regions (CDRs) in the variable region sequence. CDR determines the specificity and affinity of the antibody to the antigen. In different antibody molecules, their amino acid sequences will vary greatly.

Different antigens correspond to antibodies with different variable regions. In a preferred embodiment, the antibody targeting the surface antigen of a therapeutically relevant cell includes an antibody targeting PDL1, which has 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 has a VH sequence as shown in SEQ ID NO: 21, and a VL sequence as shown in SEQ ID NO: 22; or has 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.

The aPDL1 sequences are as follows:

HCDR1:DSWIH(SEQ ID NO:15).

HCDR2:WISPYGGSTYYADSVKG(SEQ ID NO:16).

HCDR3:RHWPGGFDY(SEQ ID NO:17).

LCDR1:RASQDVSTAVA(SEQ ID NO:18).

LCDR2:SASFLYS(SEQ ID NO:19).

LCDR3: QQYLYHPAT(SEQ ID NO:20).

aPDL1 VH:

aPDL1 VL:

aPDL1 Heavy Chain:

aPDL1 light chain:

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: 132-133. N ID NOs:132-134, and the light chain complementary determining region HCDR1, HCDR2 and HCRD3 sequences as shown in SEQ ID NOs:135-137; or having the VH sequence as shown in SEQ ID NO:138, and the VL sequence as shown in SEQ ID NO:139; or having the heavy chain amino acid sequence as shown in SEQ ID NO:140, and the light chain amino acid sequence as shown in SEQ ID NO:141; or having the heavy chain amino acid sequence as shown in SEQ ID NO:142, and the light chain amino acid sequence as shown in SEQ ID NO:143;

The aPD1-related sequences are as follows:

HCDR1: AQYMH (SEQ ID NO: 120).

HCDR2: IINPSGGETGYAQKFQG (SEQ ID NO: 121).

HCDR3: EGVADGYGLVDV (SEQ ID NO: 122).

LCDR1:RASQSVSSYLA(SEQ ID NO:123).

LCDR2: DASKRAT (SEQ ID NO: 124).

LCDR3:DQRNNWPLT(SEQ ID NO:125).

aPD1 VH:

aPD1 VL:

aPD1-2:

HCDR1:NYYMY(SEQ ID NO:132).

HCDR2: GINPSNGGTNFNEKFKN (SEQ ID NO: 133).

HCDR3:RDYRFDMGFDY(SEQ ID NO:134).

LCDR1:RASKGVSTSGYSYLH(SEQ ID NO:135).

LCDR2: LASYLES(SEQ ID NO:136).

LCDR3: QHSRDLPLT(SEQ ID NO:137).

aPD1-2 VH:

aPD1-2 VL:

aPD1-3:

HCDR1: NSGMH (SEQ ID NO: 126).

HCDR2:VIWYDGSKRYYADSVKG(SEQ ID NO:127).

HCDR3:NDDY(SEQ ID NO:128).

LCDR1:RASQSVSSYLA(SEQ ID NO:129).

LCDR2:DASNRAT(SEQ ID NO:130).

LCDR3: QQSSNWPRT(SEQ ID NO:131).

aPD1-3 VH:

aPD1-3 VL:

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.

The aB7H3 sequences are as follows:

HCDR1: NYDIN (SEQ ID NO: 25).

HCDR2: WIFPGDGSTQYNEKFKG (SEQ ID NO: 26).

HCDR3: QTTATWFAY (SEQ ID NO:27).

LCDR1:RASQSISDYLH(SEQ ID NO:28).

LCDR2: YASQSIS (SEQ ID NO: 29).

LCDR3: QNGHSFPLT(SEQ ID NO:30).

aB7H3 VH:

aB7H3 VL:

aB7H3 Heavy Chain:

aB7H3 Light Chain:

aB7H3-2 VH:

aB7H3-2 VL:

In a preferred embodiment, antibodies targeting therapeutically relevant cell surface antigens include antibodies targeting PSMA, which have 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 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.

The aPSMA sequences are as follows:

HCDR1:EYTIH(SEQ ID NO:35).

HCDR2: NINPNNGGTTYNQKFED (SEQ ID NO: 36).

HCDR3:GWNFDY(SEQ ID NO:37).

LCDR1: KASQDVGTAVD(SEQ ID NO:38).

LCDR2: WASTRHT(SEQ ID NO:39).

LCDR3: QQYNSYPLT(SEQ ID NO:40).

aPSMA VH:

aPSMA VL:

aPSMA Heavy Chain:

aPSMA light chain:

The antibody includes the constant region Fc of the heavy chain and/or the constant region Fc of the light chain, which is located at the tail of the antibody and can bind to the receptors of immune cells, thereby mediating a variety of immune effects. The heavy chain Fc and the 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 isotypes. In a specific embodiment, the Fc of the heavy chain and/or light chain can be modified accordingly as needed, such as amino acid replacement at the N297 site to reduce glycosylation modification, amino acid replacement that enhances or reduces Fc binding to Fc receptors and/or effector functions (such as antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC)) (such as amino acid replacement 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. Antibodies (Basel) 2020, 9, 64; US5624821; US 5648260; CN108350076B; CN106661120B]. In a specific embodiment, amino acid mutations can be performed on Fc as needed to promote the formation of heterologous pairing between heavy chain Fc and 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 heavy chain Fc and 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 relevant cell contains an Fc fragment; preferably, the Fc fragment is selected from the Fc fragment of human IgG1, IgG2, IgG3 or IgG4.

The connecting peptide is used to connect two molecules to be fused so that there is enough space between the two molecules to be fused to maintain their respective spatial configurations and their biological activities. Specifically, it can be reasonably selected from existing connecting peptides as needed, or it can be obtained by self-screening according to actual effects. 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, and the enzymatic cleavage includes the action of endopeptidase or exopeptidase. 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 preferably GGGGS (SEQ ID NO: 144), GGGGSG (SEQ ID NO: 145), GGGGSGGGS (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).

In practical applications, the amino acid sequences of suitable IL-15 and IL-15Ra can be embedded and fused with different antibodies according to the specific effects. In a preferred embodiment, the sequence fusion antibody comprises a heavy chain and a light chain having any of the following groups 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: 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:79 and SEQ ID NO:80; NO:84; 26) SEQ ID NO:84 and SEQ ID NO:85; 27) SEQ ID NO:86 and SEQ ID NO:87; 28) SEQ ID NO:88 and SEQ ID NO:89; 29) SEQ ID NO:90 and SEQ ID NO:91; 30) SEQ ID NO:92 and SEQ ID NO:93; 31) SEQ ID NO:94 and SEQ ID NO:95; 32) SEQ ID NO:96 and SEQ ID NO:97; 33) SEQ ID NO:98 and SEQ ID NO:99; 34) SEQ ID NO:99 and SEQ ID NO:90; 35) SEQ ID NO:91 and SEQ ID NO:92; 36) SEQ ID NO:93 and SEQ ID NO:94; 37) SEQ ID NO:96 and SEQ ID NO:97; No.:114; 34) SEQ ID NO:115 and SEQ ID NO:116; 35) SEQ ID NO:115 and SEQ ID NO:117.

Among them, as needed, null mutations were 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 implementation schemes, 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).

SEQ ID NO:1: aPDL1 VH-hIL15-CH1-Fc

SEQ ID NO:2: aPDL1 VL-hIL15RaSushi-CL

SEQ ID NO:3: aPDL1 VH-hIL15RaSushi-CH1-Fc

SEQ ID NO:4: aPDL1 VL-hIL15-CL

SEQ ID NO:5: aPDL1 VL-MP2GS-hIL15RaSushi-CL

SEQ ID NO:6: aPDL1 VL-MP2-hIL15RaSushi-CL

SEQ ID NO:7：J591 VH-hIL15-CH1-Fc

SEQ ID NO:8：J591 VL-hIL15RaSushi-CL

SEQ ID NO:9: aB7H3 VH-hIL15-CH1-Fc

SEQ ID NO:10: aB7H3 VL-hIL15RaSushi-CL

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

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

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

SEQ ID NO:47: hIL15-aPDL1-VH-CH1-Fc

SEQ ID NO:48: hIL15RaSushi-aPDL1-VL-CH1

SEQ ID NO:49: hIL15-aB7H3-VH-CH1-Fc

SEQ ID NO:50: hIL15RaSushi-aB7H3-VL-CL

SEQ ID NO:51: hIL15-aPSMA-VH-CH1-Fc

SEQ ID NO:52: hIL15RaSushi-aPSMA-VL-CL

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

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

SEQ ID NO:55: hIL15-aEGFR-VH-CH1-Fc

SEQ ID NO:56: hIL15RaSushi-aEGFR-VL-CL

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:78: aPSMA VL-EAAAK-hIL15RaSushi-EAAAK-CL

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

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

SEQ ID NO:84: aB7H3-2 VL-hIL15RaSUSHI-CL

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

SEQ ID NO:86: aB7H3-2-LC

SEQ ID NO:87：aB7H3-2-HC(hIgG1,wt)

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

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

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

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

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

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

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

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

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

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

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

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

SEQ ID NO:100: aPD1-2-HC-IgG4

SEQ ID NO:101: aPD1-2-LC

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

SEQ ID NO:103: aPD1-2 VL-hIL15RaSushi-CL

SEQ ID NO: 104: aPD1-HC

SEQ ID NO:105: aPD1-LC

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

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

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

SEQ ID NO:109: aPD1-3-kLC

SEQ ID NO:110: aPD1-3-HC-IgG4

SEQ ID NO:111：aPD1-3 HC-IgG1(null)

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

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

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

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

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

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

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

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.

In a fourth typical embodiment of the present application, a host cell is provided, into which the above-mentioned recombinant plasmid is transformed. In a preferred embodiment, the above-mentioned host cell is a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be Escherichia coli; the eukaryotic cell can be a yeast cell, a mammalian cell, an insect cell, etc.

In the fifth typical embodiment of the present application, a use of the above-mentioned fusion antibody in the preparation of a drug 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 over-activation of NK cells-induced immunotoxicity, and has a long half-life, achieving better results in future tumor prevention and treatment processes.

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.

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

Since the fusion antibody of the present application has strong cell targeting and a long half-life, its combined use with an immune checkpoint inhibitor can achieve a better synergistic therapeutic effect. 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).

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 play a better functional activity in the tumor microenvironment and achieve a better synergistic therapeutic effect.

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.

The present application is further described in detail below in conjunction with specific examples, which should not be construed as limiting the scope of protection claimed in the present application. The reagents or consumables used in the following examples, unless otherwise specified, were purchased from commercially available products.

Example 1 Construction of eukaryotic expression vector of cytokine fusion antibody

The gene fragments of VL and VH 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.

The above gene fragments are amplified by PCR respectively, and the overlap PCR gene fragment obtained by amplifying the VH gene fragment is connected with the IL-15 gene fragment or the sushi domain gene fragment of IL-15Ra and the gene fragment of the antibody constant region through a linker to obtain the VH-IL-15-CH1-Fc fragment or VH-IL-15Ra-CH1-Fc fragment as the first chain (a different description of the same structure as the above-mentioned heavy chain); the amplified VL gene fragment is connected with the IL-15Ra gene fragment or the IL-15 gene fragment and the gene fragment of the antibody constant region through a linker to obtain the VL-IL-15Ra-CL fragment, VL-IL-15-CL-Fc fragment or VL-IL-15-CL fragment, VL-IL-15Ra-CL-Fc fragment as the second chain (a different description of the same structure as the above-mentioned light chain). The above fragments were further connected to the pFuse vector (InvivoGen, CA) used for eukaryotic expression by homologous recombination, and transformed into E. coli competent DH5α cells. Antibiotic screening was performed on LB plates. After positive clones were selected, plasmids were extracted using an endotoxin-free plasmid extraction kit, and the extracted plasmid sequences were sequenced and verified. The nucleotide and amino acid sequences of each construct are shown in the sequence table.

Table 1 Fusion protein sequence number

Example 2 Expression and purification of cytokine fusion antibodies

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., 5% CO ₂ for 5-6 days. The cell culture supernatant was collected by centrifugation, filtered through a 0.22 μm filter, and the fusion antibody was purified using Protein A Resin (Genscript) according to the manufacturer's instructions. Its concentration was determined by A280 and BCA (Pierce).

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

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 shown in the NR column is in line with expectations. As shown in Figure 2, in the SEC analysis, the fusion antibody monomer peak accounted for more than 95%, with no obvious aggregates or fragments, and had similar expression and purification advantages as antibodies.

Example 3 Mass spectrometry analysis of cytokine fusion antibodies

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

Table 2 MS analysis

Example 4 Thermodynamic stability test

The fusion antibody at a concentration of 1 mg/ml was mixed with freshly prepared thermal shift dye and shift buffer (Protein Thermal Shift ^™ Dye Kit, ThermoFisher Scientific, Cat. 4461146) according to the manufacturer's recommended ratio, and thermal scanning was performed at a heating rate of 0.05°C/s at 25-99°C using ViiA ^™ 7 Real-Time PCR System. The thermal melting temperature Tm value was calculated using the "Area under curve (AUC)" analysis model of GraphPad Prism7 software.

The results are shown in FIG3 . The melting temperatures of the fusion antibodies FuAb1, FuAb5, FuAb6, FuAb17, FuAb18 and FuAb19 samples fused with three different antibodies (PDL1, PSMA and B7H3) were in the range of 65-75° C., which were all higher than the first melting temperature Tm ₁ = 60.79° C. of the parent antibody, indicating that the cytokine fusion antibodies had good thermodynamic stability.

Example 5 Binding Activity Detection

5.1 ELISA test

The target antigen (100 ng/100 ul/well) was coated on a 96-well ELISA plate and incubated overnight at 4° C. After blocking with PBST containing 2% skim milk powder (0.5% Tween-20 in PBS) at room temperature for 1 hour, the plate was washed with PBST. Cytokine fusion antibody samples or cytokine fusion antibodies after MMP-2 digestion (wherein the MMP-2 enzyme can recognize and cut the amino acid sequence in the fusion antibody such as the digestion site in the connecting peptide shown in SEQ ID NO: 62 or 65: between S and L of IPVSLRSG (as shown in SEQ ID NO: 66)) (specific digestion conditions are as follows: replace the fusion protein containing the digestion site with a buffer containing 50mM Tris, 10mM CaCl ₂ , 100mM NaCl, pH 7.4, mix mMMP-2 and each fusion protein at a ratio of 1:40, and digest at 37°C for 24h) (the suffix -C after the fusion antibody indicates the digested sample) are diluted to 50nM with blocking solution, and this is used as the starting concentration, and a 5-fold gradient dilution is performed, with a total of 8 gradient dilutions. 100ul of the diluted cytokine fusion antibody sample is added to each well and incubated at room temperature for 2h. After washing 3 times with PBST, add 100ul of HRP-labeled goat anti-human Fc monoclonal antibody diluted 1:500 according to the instructions to each well, incubate at room temperature for 1h, and then wash 5 times with PBST. Add 100ul of freshly prepared TMB colorimetric reagent (BioLegend, Cat.421101) to the washed ELISA wells, incubate at room temperature in the dark for 5-30 minutes, and read at 650nm on an ELISA reader after color development.

The monoclonal antibodies targeting the antigen aPDL1 (wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO: 23 and SEQ ID NO: 24), aB7H3 (wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO: 33 and SEQ ID NO: 34), aB7H3-2 (wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO: 118 and SEQ ID NO: 119), aPD1 (wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO: 138 and SEQ ID NO: 139), aPD1-2 (wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO: 142 and SEQ ID NO: 143), aPD1-3 (wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO: 140 and SEQ ID NO: 141), or aPDL1 mono (compared to the bivalent aPDL1, aPDL1 mono binds monovalently to the antigen, wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO: 23 and SEQ ID NO: 24) are used. ID NO:24+Fc) was used as the control sample, and the _EC50 value was calculated using the "log(agonist)vs.response--Variable slope(four parameters)" analysis model of GraphPad Prism7 software. The results are shown in Figures 4-6.

As shown in Figure 4, the ELISA binding activity of FuAb1 and FuAb8 to PDL1 antigen is similar to that of aPDL1, and is stronger than the binding ability of FuAb9 and FuAb13, in which IL-15 and IL-15Ra are located at the N-terminus of the heavy chain variable region and the light chain variable region, respectively. In addition, as shown in Figure 6, the ELISA binding activity of FuAb6 to B7H3 antigen is consistent with that of aB7H3, and is stronger than the binding ability of FuAb10, in which IL-15 and IL-15Ra are located at the N-terminus of the heavy chain variable region and the light chain variable region, respectively; the ELISA binding activity of FuAb17-22 to PSMA antigen is consistent with that of FuAb5, and is stronger than the binding ability of the control FuAb12. It can be seen that the embedded fusion method has no effect on the binding activity of the antigen binding domain located at the N-terminus with the antigen. In addition, the binding ability of FuAb5 and FuAb17-22 to IL15Ra and IL2Rβγ is significantly reduced compared with that of FuAb12.

As shown in Figure 5, in terms of PD-L1 binding activity, the ELISA binding curves of the fusion protein FuAb1 without cleavage sites and the sample FuAb8-C after FuAb8 digestion did not change significantly compared with the curves of the FuAb1 and FuAb8 samples. It can be seen that the ELISA antigen binding activity of the fusion protein without cleavage sites is not affected by the protease; the ELISA binding curves of the fusion proteins with cleavage sites on one side (such as FuAb3, FuAb4, FuAb14, FuAb15) after digestion are all shifted to the right, indicating that the binding activity of the antigen binding domain at the N-terminus with the antigen is reduced after single digestion; the fusion proteins with cleavage sites on both sides (such as FuAb7, FuAb16) have almost no binding in ELISA after digestion.

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 the binding ability was stronger than that of FuAb26 and 27, which were unilaterally chimeric fused to the heavy chain and light chain 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-terminus of the heavy chain and light chain of the antibody, respectively, and the EC50 difference was 10 times or more; in terms of IL15Ra binding ability, the chimeric fusion proteins were all lower than FuAb26 and 27, which were unilaterally chimeric fused to the heavy chain and light chain of IL-15, and the EC50 difference was 50 times or more.

As shown in Figure 8 , the ELISA binding activity of FuAb38 and 39 to B7H3 antigen was basically consistent with that of aB7H3-2 monoclonal antibody, and their IL-2Rβγ binding ability was also lower than that of FuAb12, with an EC50 difference of 40 times or more.

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 (where 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.

5.2 Cell surface receptor binding activity detection

A431 (with natural human PDL1 antigen on the cell surface), MC38-hB7H3, MC38-hPSMA, and MC38 cells (with natural mouse PDL1 antigen on the cell surface) were cultured, where MC38-hB7H3 and MC38-hPSMA were MC38 cells genetically modified to carry human B7H3 and PSMA antigens, respectively. All of the above cells were cultured in DMEM medium containing 10% FBS, and were taken out after trypsin digestion. 2x10e ⁵ cells were dispensed into each flow staining well and blocked with pre-cooled 2% FBS-PBS blocking solution for 30 minutes. The cytokine fusion antibody sample (enzyme-cleaved product or unenzyme-cleaved) was diluted to 50nM with blocking solution, and this was used as the starting concentration for 5-fold gradient dilution, with a total of 8 gradient dilutions. Set 0nM concentration as the staining background. Add 100ul of diluted cytokine fusion antibody sample to each well and incubate at 4°C for 30 minutes. After washing 3 times with PBS, 100ul 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 blocking solution was added to the washed flow tube, and the resuspended cell suspension was used for flow analysis. The EC ₅₀ value was calculated using the "log(ag onist) vs. response--Variable slope (four parameters)" analysis model of GraphPad Prism7 software.

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

The results are shown in Figure 11. The binding activity of IL15 and IL15Ra fusion proteins located between the variable region and constant region of the antibody, respectively (such as FuAb1, FuAb5, FuAb6, FuAb8), to cell surface receptors is consistent with the parent monoclonal antibodies targeting the antigen (aPDL1, aPSMA, aB7H3), and is stronger than the fusion proteins of IL15 and IL15Ra located at the N-terminus of the antibody variable region (such as FuAb9, FuAb11, FuAb10 and FuAb13), 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 domains located at the N-terminus to cell surface receptors.

As shown in Figure 12, in terms of binding activity with MC38 cells, the flow cytometry binding curve of the sample FuAb1-C after digestion of the fusion protein FuAb1 without the cleavage site did not change significantly compared with the curve of the FuAb1 sample. However, the flow cytometry binding curve of the fusion protein with a cleavage site on one side (such as FuAb4 and FuAb3) shifted to the right after digestion, and the binding activity decreased, while the fusion protein with a cleavage site on both sides (such as FuAb7) had no cell binding activity after digestion.

5.3 Mo7e surface receptor binding activity detection

Culture human giant cell leukemia cells Mo7e (Mo7e is a human IL-2Rβγ receptor positive cell) in suspension. Each flow staining well was divided into ^2x10e5 cells and 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 this was used as the starting concentration, and a 5-fold gradient dilution was performed, with a total of 8 gradient dilutions. Set 0nM concentration as the staining background. Add 100ul of diluted cytokine fusion antibody sample to each well and incubate at 4℃ for 30 minutes. After washing with PBS 3 times, add 100ul of APC-labeled anti-human Fc monoclonal antibody diluted according to the instructions to each well, incubate at 4℃ for 30 minutes, and then wash with PBS 3 times. Add 200ul of blocking solution to the washed flow tube, and the resuspended cell suspension is used for flow analysis. The EC ₅₀ value was calculated using the "log(agonist)vs.response--Variable slope(four parameters)" analysis model of GraphPad Prism7 software.

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).

Example 6 Cell proliferation activity detection

Culture suspension 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 3 times with RPMI1640 medium, and resuspend them with 10% FBS-RPMI1640 medium at a density of 2x10e ⁵ cells/ml. Dilute the fusion antibody sample 5 times, 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 an incubator and incubate for 24h. Use an equal volume of CellTiter-Glo for lysis reading according to the manufacturer's instructions. Take the cultured Mo7e cells and centrifuge them, wash them 3 times with RPMI1640 medium, and resuspend them with 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. Calculate the EC ₅₀ value using the "log (agonist) vs. response--Variable slope (four parameters)" analysis model of GraphPad Prism7 software.

Collect PBMC cells after co-activation of anti-human CD3 antibody and anti-human CD28 antibody, centrifuge at 300g for 5 minutes, discard the supernatant, wash 3 times with RPMI1640 medium, resuspend with 10% FBS-RPMI1640 medium, density 2x10e ⁵ /ml. Take 10ul gradient dilution fusion antibody sample and add it to a 96-well culture plate with a black transparent bottom, then add 90ul 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. Calculate the EC ₅₀ value using the "log (agonist) vs. response--Variable slope (four parameters)" analysis model of GraphPad Prism7 software.

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.

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

As shown in Figure 17, the embedded fusion protein (FuAb44-47) obtained by fusion with aPD1, aPD1-2, and aPD1-3 antibodies has a 20- to 100-fold lower proliferative activity on Mo7e than FuAb12. In addition, compared with the positive control sample (FuAb12) in which IL-15 and IL-15Ra are fused to the N-terminus of the heavy chain and light chain of the antibody, respectively, the proliferation activity of FuAb24, 25, 26, and 27 on Mo7e cells increased by 5-10 times, indicating that the binding of FuAb6 to the human B7H3 receptor on the surface of Mo7e cells has a synergistic effect on the proliferation signal of the cytokine complex.

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

In terms of promoting the proliferation activity of PBMC, the results are shown in Figure 18. The backbone antibody aPSMA has no effect on cell proliferation. Compared with the single rhIL15 molecule and the fusion protein (FuAb12) in which IL15 and IL15Ra are located at the N-terminus of the antibody variable region, the five embedded fusion antibodies (FuAb5, FuAb17, FuAb18, FuAb19, FuAb23) have a 10-150-fold reduction in the proliferation activity of PBMC.

As shown in Figure 19, in the NK92 proliferation experiment, the chimeric fusion proteins (FuAb6, FuAb24-27, FuAb29-31, FuAb36, FuAb44-47) all showed weak NK92 proliferation-promoting ability, and their _EC50 was 5-20 times higher than that of FuAb12, a positive control sample in which IL-15 and IL-15Ra were fused to the N-termini of the heavy chain and light chain of the antibody, respectively.

Example 7 Evaluation of efficacy and toxicity in MC38 mouse tumor model

1. Fusion antibody targeting PDL1

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 the culture surface, in which MC38 cells carry natural mouse PDL1 antigens on the surface and MMP-2 enzymes naturally exist in mice, are cultured in DMEM medium containing 10% FBS and are taken after trypsin digestion. 6-8 week old C57BL6J mice were taken and 5x10e ⁵ MC38 cells were inoculated subcutaneously on the dorsal side. When the tumor size reached a volume of 50-100mm ³ , 2 mg/kg was administered intravenously through the tail vein for a total of two times. During this period, the changes in mouse tumor volume were recorded every two days, and the mice were euthanized when the mouse tumor volume exceeded 1000mm ³ .

As shown in Figure 20, the fusion antibodies (FuAb2 and FuAb8) in which IL15 and IL15Ra are located between the variable region and the constant region of the antibody and the fusion antibodies (FuAb9 and FuAb12) in which IL15 and IL15Ra are located at the N-terminus of the variable region of the antibody show similar tumor inhibitory activity. The fusion proteins carrying the restriction site (such as FuAb4, FuAb3, and FuAb7) all show tumor inhibitory activity superior to that of aPDL1mAb, and the tumor inhibitory activity is similar to that of FuAb12; at the same time, the fusion protein carrying the restriction site is superior to FuAb12 in weight maintenance. It can be seen that the chimeric fusion method can reduce IL15 activity, relying on the enrichment of the N-terminal antigen binding target and the release of IL15 activity by restriction cleavage. This combination method brings better tumor inhibitory activity to the fusion protein samples carrying the restriction site, and at the same time, greatly alleviates the weight-related systemic toxicity. The fusion protein without the restriction site relies on the strong enrichment of the N-terminal antigen binding target and the weak IL15 activity, and also shows good tumor inhibitory activity, and there is no obvious trend of weight loss. The inhibitory activity of aPDL1 monoclonal antibody, fusion protein without cleavage site, and fusion protein with cleavage site on tumors increased in sequence, which is consistent with the mechanism that enzymatic cleavage releases the activity of chimeric IL15-IL15Ra.

2. Fusion antibodies targeting PD1

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 the culture surface, among which MC38 cells carry natural mouse PDL1 antigens on the surface, are cultured in DMEM medium containing 10% FBS and are taken after trypsin digestion. 6-8 week-old C57BL6J mice were taken and 5x10e ⁵ MC38 cells were inoculated in the axilla. When the tumor size reached a volume of 50-100mm ³ , different doses were administered by tail vein administration. During this period, the changes in mouse tumor volume were recorded every two days, and when the mouse tumor volume exceeded 1000mm ³ , the mouse was euthanized. Mouse spleen samples were collected for flow staining analysis of cell populations. Flow staining used 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, PD1 ⁺ to mark PD1-positive cell populations, and Foxp3 to mark Foxp3-positive cell populations.

As shown in Figure 21, the tumor inhibition effect of FuAb32 is better than that of aPD1 monoclonal antibody. FuAb36 has only weak tumor inhibition activity, indicating that IL15 fusion alone cannot bring good tumor inhibition effect without PD1 targeting. The tumor inhibition effect of FuAb29 combined with aPD1 mAb is similar to that of aPD1 and FuAb32, indicating that IL15 fusion alone and aPD1 monoclonal antibody alone cannot bring good synergistic tumor inhibition effect when used in combination.

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

The flow 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 is no toxicity risk in the mouse system.

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

A tumor model was established using mouse colon cancer cells MC38. 6-8 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 , 2 mg/kg, 5 mg/kg, and 10 mg/kg were administered via the tail vein for a total of one administration. During this period, the changes in the tumor volume of the mice were recorded every two days. When the tumor volume of the mice exceeded 1000 mm ³ , the mice were euthanized.

As shown in Figure 23, compared with the DPBS group, at a single dosing frequency, 2 mg/kg, 5 mg/kg, and 10 mg/kg FuAb1 all showed good tumor growth inhibition activity. Among them, in the 10 mg/kg dosing group, the weight of mice decreased significantly two days after dosing, and the weight of mice in the 2 mg/kg and 5 mg/kg dose groups was not affected by dosing within the entire observation range, indicating that the fusion antibody has a good safe and effective dose window.

Example 9: Mechanism of drug action in MC38 mouse tumor model

A tumor model was established using mouse colon cancer cells MC38. 6-8 week old C57BL6J mice were taken and 5x10e ⁵ MC38 cells were inoculated subcutaneously on the dorsal side. When the tumor size reached a volume of 300-500mm ³ , 2 mg/kg was administered via the tail vein for a total of one administration. On the fourth day after administration, the mice were euthanized, and peripheral blood samples, spleen samples, and tumor tissue samples of the mice were collected for flow cytometry analysis of cell populations.

Flow staining used 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.

The results are shown in Figures 24-26. The analysis of peripheral blood samples, spleen samples and tumor infiltrating lymphocytes (TIL) can illustrate the expression of cell populations related to tumor suppression and systemic toxicity.

As shown in Figures 24 and 25, FuAb12/FuAb9 showed severe body weight-related systemic toxicity in Example 7, and in the cell population analysis, a large number of NK/T lymphocytes were proliferated in the peripheral blood and spleen (unrelated to tumors). Compared with FuAb12/FuAb9, the chimeric fusion protein FuAb2/FuAb8 showed less changes in cell populations in peripheral blood and spleen (unrelated to tumors), 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 peripheral blood and spleen, and effectively avoid systemic toxicity caused by lymphocyte proliferation, which is consistent with the weight changes shown in Example 7.

As shown in Figure 26, in the cell population analysis in 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 killing-related 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 the killing-related lymphocytes in the tumor environment. This is consistent with the tumor suppression results shown in Example 7.

In summary, only the chimeric fusion protein FuAb2/FuAb8 can specifically increase the proportion of CD8+T cells and NK cells in the tumor environment without affecting the peripheral blood and spleen cell populations. Only the chimeric fusion protein FuAb2/FuAb8 has the ability to reduce cytotoxicity in the systemic system, achieve biological effects in the tumor microenvironment, enhance the activation of NK cells and CD8+T cells in the tissue area of the tumor site, and achieve tumor growth inhibition activity.

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

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 in DMEM medium containing 10% FBS and taken after trypsin digestion. 6-8 week old C57BL6J mice were taken and 5x10e ⁵ MC38-hB7H3 cells were inoculated subcutaneously on the dorsal side. When the tumor size reached a volume of 50-100mm ³ , 2mg/kg, 5mg/kg, and 10mg/kg were administered through the tail vein for a total of two times. During this period, the changes in mouse tumor volume were recorded every two days, and when the mouse tumor volume exceeded 1000mm ³ , the mouse was euthanized.

The results are shown in Figure 27. 10-15 days after inoculation, the tumor volume of the DPBS group exceeded 1000 mm ³ ; compared with the PBS group, the antigen-targeting monoclonal antibody aB7H3 did not show significant tumor growth inhibition activity at a dose of 2 mg/kg, while the fusion protein FuAb10 of IL15 and IL15Ra located at the N-terminus of the variable region of the antibody showed significant tumor inhibition activity, and its body weight decreased by more than 20% in 10-15 days; while FuAb6 had significant tumor growth inhibition activity at doses of 2 mg/kg, 5 mg/kg, and 10 mg/kg, and the weight of mice was less affected by the administration in the entire observation range. The weight loss trend of mice in the FuAb6 group at 10 mg/kg was significantly improved compared with FuAb10 at a dose of 2 mg/kg, indicating that FuAb6 has a good safe and effective dose window for drugs.

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).

Example 11 Pharmacokinetic evaluation in mice

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

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

Table 3 PK parameters of fusion antibodies

Example 12 Efficacy and toxicity evaluation in MC38-hPSMA mouse tumor model

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 the culture surface, in which MC38-human PSMA cells carry natural mouse PDL1 antigens and heterologously expressed human PSMA antigens on the surface, are cultured in DMEM medium containing 10% FBS, and are taken after trypsin digestion. 6-8 week old C57BL6J mice were taken and 5x10e ⁵ of MMC38-human PSMA cells were inoculated in the armpit. When the tumor size reached a volume of 50-100mm ³ , different doses were administered by tail vein. During this period, the changes in mouse tumor volume were recorded every two days, and when the mouse tumor volume exceeded 1000mm ³ , the mouse was euthanized. The results are shown in Figures 29-30. 17-19 days after inoculation, the tumor volume of the DPBS administration group exceeded 1000mm ³ , and FuAb17-19 showed good tumor inhibitory activity and safety.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: using the symmetrical Y-shaped structure of natural antibodies, with VH-IL-15-CH1-Fc fragment or VH-IL-15Ra-CH1-Fc fragment as the first chain; with VL-IL-15Ra-CL fragment, VL-IL-15-CL-Fc fragment or VL-IL-15-CL fragment, VL-IL-15Ra-CL-Fc fragment as the second chain, the formed fusion antibody has similar stability and half-life as natural antibodies; and significantly reduces the activity of IL15/IL15Ra complex, greatly reduces systemic toxicity, and uses the targeting of antibodies to specifically play a role in the tumor environment, thereby improving the cell targeting of 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.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (19)

A fusion antibody, characterized in that it comprises: 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 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. The fusion antibody according to claim 1, characterized in that the IL-15 is fused directly or through a connecting 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 fused directly or through a connecting 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 fused directly or through a connecting 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 fused directly or through a connecting 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. 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. The fusion antibody according to claim 1 is 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 as shown in SEQ ID NO: 12 or SEQ ID NO: 81. The fusion antibody according to claim 1, characterized in that 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 as shown in SEQ ID NO: 13 or SEQ ID NO: 14. The fusion antibody according to any one of claims 1 to 5, characterized in that the therapeutically relevant cell surface antigen is an immune monitoring point protein or a tumor antigen. The fusion antibody according to claim 6, characterized in that the therapeutically relevant cell surface antigen comprises 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. The fusion antibody according to claim 7, characterized in that the antibody against the therapeutically relevant cell surface antigen comprises an antibody targeting PDL1, which has 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 has a VH sequence as shown in SEQ ID NO: 21, and a VL sequence as shown in SEQ ID NO: 22; or has 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 antibody against the therapeutically relevant cell surface antigen comprises an antibody targeting B7H3, which has 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 has 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 has 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 antibody against the therapeutically relevant cell surface antigen includes an antibody targeting PSMA, which has a heavy chain complementary determining region HCDR1, HCDR2 and HCRD3 sequence as shown in SEQ ID NOs:35-37, and a light chain complementary determining region LCDR1, LCDR2 and LCRD3 sequence as shown in SEQ ID NOs:38-40; or has a VH sequence as shown in SEQ ID NO:41, and a VL sequence as shown in SEQ ID NO:42; or has 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 antibodies against 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, SEQ ID NOs: 126-128 or SEQ ID NOs: 132-134, and light chain complementary determining region 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 have a VH sequence as shown in SEQ ID NO: 138, and a VL sequence as shown in SEQ ID NO: 139; or have 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. 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 fragment of human IgG1, IgG2, IgG3 or IgG4. The fusion antibody according to claim 2, characterized in that the connecting peptide is a cleavable connecting peptide or a non-cleavable connecting peptide; Preferably, each of the connecting peptides 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: 64), GGGGSIPVSLRSGGGGGSG (SEQ ID NO: 65) or GGGGSIPVSLRSGGGSG (SEQ ID NO: 62). The fusion antibody according to any one of claims 1 to 5, characterized in that the fusion antibody comprises a heavy chain and a light chain having any of the following groups 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 I D 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:114; 34) SEQ ID NO:115 and SEQ ID NO:116; 35) SEQ ID NO:115 and SEQ ID NO:117. A DNA molecule, characterized in that the DNA molecule encodes the fusion antibody according to any one of claims 1 to 11. A recombinant plasmid, characterized in that the recombinant plasmid is connected to the DNA molecule according to claim 12. A host cell, characterized in that the recombinant plasmid according to claim 13 is transformed into the host cell. The host cell according to claim 14, characterized in that the host cell comprises a prokaryotic cell or a eukaryotic cell. Use of the fusion antibody according to any one of claims 1 to 11 in the preparation of a medicament for preventing and/or treating cancer. The use according to claim 16 is 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 blood tumor. A drug, characterized in that it comprises the fusion antibody according to any one of claims 1 to 11. The drug according to claim 18, characterized in that the drug further comprises an immune checkpoint inhibitor used in combination with the fusion antibody, and the immune checkpoint inhibitor comprises any one or more of the following: aPDL1, aCTLA4, a41BB or aLAG3.