Antibody fusion protein as well as preparation therefor and use thereof

Abstract

Provided is an antibody fusion protein having a masking peptide. The fusion protein comprises: an antibody that specifically binds to a target, a masking peptide, a cytokine, and a linker. Further provided are a preparation method for the antibody fusion protein and the use of same in the treatment and / or prevention of tumors or autoimmune diseases.

Description

Antibody fusion protein and preparation and application thereof

[0001] This application claims priority to the Chinese application with the application date of April 16, 2024, the application number of 202410458955.5, and the invention title of "Antibody fusion protein and preparation and application thereof". The entire contents of the priority application are incorporated into this application as part of this application. The entire contents of all cited references in this application are incorporated into this application as part of this application. TECHNICAL FIELD

[0002] The present application relates to an antibody fusion protein targeting tumor-specific antigens and having cytokines. The present application also relates to the preparation and application of the antibody fusion protein. BACKGROUND

[0003] Antibody-cytokine fusion proteins (Immunokine) are a class of very promising tumor immunotherapy products, which can reduce the peripheral immunotoxicity of cytokines through antibody targeting, and can prolong the half-life of cytokines and enhance the immunoregulatory effects of cytokines and antibodies through fusion with antibodies (Xue, Hsu, Fu, & Peng, 2021).

[0004] Cytokines are a class of small molecular proteins with broad biological activities synthesized and secreted by immune cells (such as mononuclear cells, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. They generally regulate cell growth, differentiation, maturation, function maintenance, immune response, participate in body inflammatory response, wound healing, and tumor growth and regression, etc. through binding to corresponding receptors.

[0005] Cytokine Interleukin-2 (IL-2) is essential for the survival and expansion of T cells, especially natural killer CD8+ T cells and NK cells. High-dose recombinant human IL-2 aldesleukin (Proleukin) was approved by the U.S. Food and Drug Administration (FDA) in 1992 and 1998 for the treatment of metastatic renal cell carcinoma and metastatic melanoma, respectively, but its short half-life and high toxicity (capillary leakage and multi-organ failure) have greatly limited its clinical application. Currently, several tumor-targeting antibody fusion IL-2 cytokines are in clinical development, including L19-IL-2 (Darleukin) in phase II clinical trials, GD2-IL-2, CD20-IL-2, and EpCAM-IL-2 (Pires, Hammond, & Irvine, 2021). Although antibody conjugation can reduce the peripheral immunotoxicity of IL-2 to some extent, natural or IL-2Rβγ-biased IL-2 molecules can still activate peripheral lymphocytes, potentially leading to peripheral immunotoxicity.

[0006] Therefore, reducing peripheral immunotoxicity and stably exerting therapeutic effect are still problems to be solved for antibody conjugated fusion proteins. SUMMARY

[0007] The present application provides antibody cytokine fusion proteins with a shielding peptide and a preparation method thereof. The fusion protein of the present application blocks the binding site of the cytokine and the receptor through the shielding peptide, which can reduce peripheral toxicity while maintaining the activity of the antibody and the cytokine, greatly improving the effectiveness and safety of the fusion protein.

[0008] The present application also provides nucleic acid molecules encoding the shielding peptide antibody fusion protein, vectors or host cells comprising the nucleic acid molecules, and their medical uses. Specifically, the technical solutions adopted by the present application are as follows:

[0009] The present application provides an antibody fusion protein with a shielding peptide, which comprises an antibody specifically binding to a target, a shielding peptide and a cytokine, wherein the shielding peptide is fused to the antibody through a linker peptide L1, and the cytokine is fused to the shielding peptide through a linker peptide L2.

[0010] The antibody fusion protein with a shielding peptide combines the cytokine with the antibody, which on the one hand increases the targeting of the cytokine and on the other hand prolongs the half-life of the cytokine; at the same time, the shielding peptide inhibits the activity of the cytokine by being fused to the linker and the cytokine, which on the one hand reduces the activity of the cytokine that is detrimental to normal tissues and reduces peripheral toxicity, and on the other hand, when reaching the tumor, it also reduces the transient over-activation of cells while exerting a therapeutic effect, so that the antibody fusion protein can exert its effect more stably and safely.

[0011] In the present application, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art, unless otherwise specified.

[0012] In the present application, the term "about" or "approximately" means within 10% of a given value or range. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a structural schematic diagram of the antibody fusion protein of the present application.

[0014] Figure 2 is an SDS-PAGE diagram of specific antibody fusion proteins provided by the present application, wherein:

[0015] A) is an SDS-PAGE diagram of antibody fusion proteins such as AF94;

[0016] B) is an SDS-PAGE diagram of antibody fusion proteins such as AF72;

[0017] C) is an SDS-PAGE diagram of antibody fusion proteins such as AF1000.

[0018] Figure 3 is a SEC-HPLC diagram of specific antibody fusion proteins provided by the present application, wherein:

[0019] A) is a SEC-HPLC diagram of antibody fusion proteins such as AF94;

[0020] B) is a SEC-HPLC diagram of antibody fusion proteins such as AF71;

[0021] C) is a SEC-HPLC diagram of antibody fusion proteins such as AF75;

[0022] D) is a SEC-HPLC diagram of antibody fusion proteins such as AF1000.

[0023] Figure 4 shows the effect of specific antibody fusion proteins provided by the present application on the proliferation of NK-92 cells.

[0024] Figure 5 shows the effect of specific antibody fusion proteins provided by the present application on the proliferation of CTLL-2 cells.

[0025] Figure 6 shows the effect of specific antibody fusion proteins provided by the present application on the STAT5 phosphorylation signal of cells, wherein:

[0026] A) shows the effect of specific antibody fusion proteins provided by the present application on the STAT5 phosphorylation signal of T cells;

[0027] B) shows the effect of specific antibody fusion proteins provided by the present application on the STAT5 phosphorylation signal of CD8+ T cells;

[0028] C) indicates the influence of the specific antibody fusion protein provided by the present application on the STAT5 phosphorylation signal of CD4+ T cells;

[0029] D) indicates the influence of the specific antibody fusion protein provided by the present application on the STAT5 phosphorylation signal of NK cells;

[0030] E) indicates the influence of the specific antibody fusion protein provided by the present application on the STAT5 phosphorylation signal of Treg cells.

[0031] Figure 7 shows the influence of the specific antibody fusion protein provided by the present application on the proliferation of mouse T mother cells.

[0032] Figure 8 shows the inhibitory activity of the specific antibody fusion protein provided by the present application on MC-38-hCLDN18.2-A11 mouse colon cancer cell transplanted tumors, wherein

[0033] A) shows the tumor volume change of mice in each administration group;

[0034] B) shows the body weight change curve of mice in each administration group;

[0035] C-F) respectively show the tumor volume change of mice in each administration group.

[0036] Figure 9 shows the immune memory of each administration group in Figure 8 when re-implanted after tumor regression.

[0037] Figure 10 shows the pulmonary edema caused by the administration of AF49 and the like.

[0038] A) shows the pulmonary edema of mice in each administration group 48 hours after administration;

[0039] B) shows the pulmonary edema of mice in each administration group 96 hours after administration.

[0040] Figure 11 shows the inhibitory activity of antibody fusion protein AF1003 on MC-38-hCLDN6 mouse colon cancer cell transplanted tumors.

[0041] Figure 12 shows the inhibitory activity of antibody fusion protein AF1000 on MC-38-hCD228 mouse colon cancer cell transplanted tumors.

[0042] Figure 13 shows the charge distribution of antibody fusion proteins AF48 and the like, wherein:

[0043] A) shows the charge distribution of antibody fusion protein AF48 without enzyme cutting;

[0044] B) shows the charge distribution of antibody fusion protein AF49 without enzyme cutting;

[0045] C) shows the charge distribution of antibody fusion protein AF94 without enzyme cleavage;

[0046] D) shows the charge distribution of antibody fusion protein AF48 with sialic acid enzyme cleavage;

[0047] E) shows the charge distribution of antibody fusion protein AF49 with sialic acid enzyme cleavage;

[0048] F) shows the charge distribution of antibody fusion protein AF94 with sialic acid enzyme cleavage.

[0049] Figure 14 shows the protein purity analysis of antibody fusion proteins AF48 and the like based on capillary electrophoresis:

[0050] A) shows the purity of the intact protein of antibody fusion protein AF48 under non-reducing conditions;

[0051] B) shows the purity of the intact protein of antibody fusion protein AF49 under non-reducing conditions;

[0052] C) shows the purity of the intact protein of antibody fusion protein AF94 under non-reducing conditions;

[0053] D) shows the purity of the light chain and heavy chain proteins of antibody fusion protein AF48 under reducing conditions;

[0054] E) shows the purity of the light chain and heavy chain proteins of antibody fusion protein AF49 under reducing conditions;

[0055] F) shows the purity of the light chain and heavy chain proteins of antibody fusion protein AF94 under reducing conditions. DETAILED DESCRIPTION

[0056] In some embodiments, the antibody fusion protein of the present application comprises, in order from N-terminus to C-terminus: an antibody that specifically binds to a target, a linker peptide LI, a masking peptide, a linker peptide L2, and a cytokine, as shown in Figure 1.

[0057] In some embodiments, the target is a tumor-specific antigen, wherein the tumor-specific antigen is selected from one or more of the following: CLDN18.2 (Claudin 18.2), CLDN6 (Claudin 6), CA125, AFP, CEA, EGFR, HER2, B7H3, B7H6, MUC1, MUC16, GPC3, CD20, CD228, CD24. Preferably, the tumor-specific antigen is CLDN18.2, CLDN6, CD20, or CD228. More preferably, the tumor-specific antigen is CLDN18.2.

[0058] In some embodiments, the amino acid sequence of the heavy chain Fd of the antibody of the present application is as set forth in SEQ ID No. 51, 52, 53, or 54.

[0059] In some embodiments, the amino acid sequence of the Fc of the antibody of the present application is as set forth in SEQ ID No. 61, 62, 63, or 64.

[0060] In some embodiments, the cytokine is selected from one or more of the following: interleukin-2 (IL-2), interferon alpha (IFNa), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNy), interleukin-7 (IL-7), interleukin-12 (IL-12), and interleukin-21 (IL-21). Preferably, the cytokine is IL-2.

[0061] In some embodiments, the cytokine is wild-type IL-2, or a mutant thereof, or a truncation of the wild-type or mutant. IL-2 truncation refers to a form of IL-2 obtained by truncating one or more amino acids from the C- and / or N-terminus of the wild-type or mutant IL-2.

[0062] In some embodiments, the amino acid sequence of the cytokine is as set forth in SEQ ID No. 65, 66, 67, or 68.

[0063] In some embodiments, the masking peptide is a receptor or binding fragment thereof of the cytokine, or an antibody or binding fragment thereof that specifically binds to the cytokine, which can inhibit the activity of the cytokine by binding to the cytokine.

[0064] In some embodiments, the masking peptide inhibits the activity of the IL-2 cytokine by inhibiting the binding of the IL-2 cytokine to IL-2RaPy and / or IL-2RPy and / or IL-2Ra in normal tissues.

[0065] In some embodiments, the masking peptide is selected from the following: wild-type IL-2Ra, IL-2Rb, IL-2Ry, or a mutant thereof, or a truncation of the wild-type or mutant. Preferably, the masking peptide is IL-2Ra. Truncation of the masking peptide refers to a form of the masking peptide obtained by truncating one or more amino acids from the C- and / or N-terminus of the wild-type or mutant masking peptide.

[0066] In some embodiments, the amino acid sequence of the masking peptide is as set forth in SEQ ID No. 69, 70, 71, 72, 73, 74, 75, 76, or 77.

[0067] In some embodiments, the linker peptides L1, L2 are selected from flexible linker peptides comprising glycine (G) and serine (S) residues, preferably having 1-50 amino acids. Preferably, the linker peptides L1, L2 comprise (GGGGS) n n is an integer from 1 to 10, more preferably the amino acid sequence of the linker peptide L1, L2 is as set forth in SEQ ID No. 55, 56, 57, 58, 59, or 60.

[0068] In some embodiments, the antibody fusion protein heavy chain comprises, in order from N-terminus to C-terminus: an Fd as set forth in any one of SEQ ID Nos. 51, 52, 53, or 54, an Fc as set forth in any one of SEQ ID Nos. 61, 62, 63, or 64, a linker peptide L1 as set forth in any one of SEQ ID Nos. 55, 56, 57, 58, 59, or 60, a masking peptide as set forth in any one of SEQ ID Nos. 69, 70, 71, 72, 73, 74, 75, 76, or 77, a linker peptide L2 as set forth in any one of SEQ ID Nos. 55, 56, 57, 58, 59, or 60, and a cytokine as set forth in any one of SEQ ID Nos. 65, 66, 67, or 68.

[0069] In some embodiments, the fusion protein of the present application comprises a heavy chain and a light chain, the light chain having an amino acid sequence as set forth in SEQ ID No. 1, 20, 22, or 24; and / or the heavy chain having an amino acid sequence as set forth in SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 78, 79, 21, 23, or 25.

[0070] In some embodiments, the light chain amino acid sequence of the fusion protein of the present invention is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.2; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.3; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.4; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.5; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.6; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.7; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.8; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.9; or the light chain amino acid sequence is as shown in SEQ ID No.1, and the heavy chain amino acid sequence is as shown in SEQ ID No.10; or the light chain amino acid sequence is as shown in SEQ ID or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.11; or the light chain amino acid sequence is shown in SEQ ID No.1; the heavy chain amino acid sequence is shown in SEQ ID No.12; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.13; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.14; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.15; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.16; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.17; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.18; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No.78; or the light chain amino acid sequence is shown in SEQ ID No.1, and the heavy chain amino acid sequence is shown in SEQ ID No. No.79; or the light chain amino acid sequence is shown in SEQ ID No.20, and the heavy chain amino acid sequence is shown in SEQ ID No.21; or the light chain amino acid sequence is shown in SEQ ID No.22, and the heavy chain amino acid sequence is shown in SEQ ID No.23; or the light chain amino acid sequence is shown in SEQ ID No.24, the heavy chain amino acid sequence is shown as SEQ ID No. 25.

[0071] In some embodiments, the antibody fusion protein of the application is selected from the antibody fusion proteins shown in Table 1.

[0072] Table 1 : Antibody fusion protein constructs

[0073] In some embodiments, the sequences of the parts of the fusion protein constructs of the application are shown in Table 2.

[0074] Table 2: Sequences of the parts of the antibody fusion protein constructs

[0075] In specific embodiments, the antibody fusion protein of the application is the fusion protein shown in Table 3.

[0076] Table 3: Sequences of the antibody fusion proteins

[0077] In a second aspect, the application provides an isolated nucleic acid molecule comprising a polynucleotide encoding an antibody fusion protein of the application. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from the group consisting of SEQ ID No. 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 80, 81.

[0078] In a third aspect, the application provides a vector comprising a nucleic acid molecule of the application as described above.

[0079] In a fourth aspect, the application provides a host cell comprising a nucleic acid molecule or a vector of the application as described above.

[0080] In some embodiments, the host cell is selected from the group consisting of CHO cells, COS cells, HeLa cells, HEK cells, and the like, for example HEK 293 cells.

[0081] In a fifth aspect, the application provides a method for producing an antibody fusion protein of the application, comprising culturing a host cell according to the application to express the fusion protein, and isolating the expressed fusion protein.

[0082] Further, the present application provides a pharmaceutical composition comprising an effective amount of the antibody fusion protein and a pharmaceutically acceptable excipient. The present application also provides use of the antibody fusion protein, nucleic acid molecule, vector or host cell of the present application in the preparation of a medicament or reagent for the diagnosis, treatment or prevention of a tumor or an autoimmune disease. In some embodiments, the tumor is a CLDN18.2-related tumor, a CLDN6-related tumor, a CD20-related tumor or a CD228-related tumor. In further embodiments, the tumor is a solid tumor such as gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, breast cancer; a hematological cancer such as lymphoma (e.g. non-Hodgkin lymphoma, follicular non-Hodgkin lymphoma, diffuse large B-cell non-Hodgkin lymphoma, follicular lymphoma, etc.), leukemia (e.g. chronic lymphocytic leukemia, etc.). In some embodiments, the autoimmune disease is rheumatoid arthritis, autoimmune hemolytic anemia, pure red cell anemia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura, Evans syndrome, vasculitis (e.g. granulomatosis with polyangiitis, etc.), or bullous skin disease (e.g. pemphigus, pemphigoid, etc.).

[0083] The antibody fusion protein of the present application targets a tumor-specific antigen, thereby carrying the cytokine to the target tumor site, achieving effective targeting, while the coupling of the Fc of the antibody and the cytokine prolongs the half-life of the cytokine. The shielding peptide inhibits the activity of the cytokine by fusion with the linker and the cytokine, on the one hand, reducing the activity of the cytokine against normal tissues, reducing the peripheral toxicity; on the other hand, when reaching the tumor, it also reduces the transient over-activation of the cells while exerting a therapeutic effect, so that the antibody fusion protein exerts a more stable and safer effect.

[0084] In the following, the examples listed are for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can appreciate that the concept of the present application is also applicable to various tumor-specific antigens and cytokines, without being limited by their specific sequences.

[0085] The amino acid sequences and nucleotide sequences described in the present application are as follows:

[0086] SEQ ID No. 1: AF94 light chain amino acid sequence

[0087] SEQ ID No. 2: AF94 heavy chain amino acid sequence

[0088] SEQ ID No. 3: AF81 heavy chain amino acid sequence

[0089] SEQ ID No. 4: AF82 heavy chain amino acid sequence

[0090] SEQ ID No. 5: AF48 heavy chain amino acid sequence

[0091] SEQ ID No. 6: AF49 heavy chain amino acid sequence

[0092] SEQ ID No. 7: AF56 heavy chain amino acid sequence

[0093] SEQ ID No. 8: AF71 heavy chain amino acid sequence

[0094] SEQ ID No. 9: AF72 heavy chain amino acid sequence

[0095] SEQ ID No. 10: AF73 heavy chain amino acid sequence

[0096] SEQ ID No. 11: AF74 heavy chain amino acid sequence

[0097] SEQ ID No. 12: AF75 heavy chain amino acid sequence

[0098] SEQ ID No. 13: AF76 heavy chain amino acid sequence

[0099] SEQ ID No. 14: AF77 heavy chain amino acid sequence

[0100] SEQ ID No. 15: AF78 heavy chain amino acid sequence

[0101] SEQ ID No. 16: AF79 heavy chain amino acid sequence

[0102] SEQ ID No. 17: AF80 heavy chain amino acid sequence

[0103] SEQ ID No. 18: AF1006 heavy chain amino acid sequence

[0104] SEQ ID No. 19: AF50 heavy chain amino acid sequence

[0105] SEQ ID No. 20: AF1003 light chain amino acid sequence

[0106] SEQ ID No. 21: AF1003 heavy chain amino acid sequence

[0107] SEQ ID No. 22: AF1005 light chain amino acid sequence

[0108] SEQ ID No. 23: AF1005 heavy chain amino acid sequence

[0109] SEQ ID No. 24: AF1000 light chain amino acid sequence

[0110] SEQ ID No. 25: AF1000 heavy chain amino acid sequence

[0111] SEQ ID No. 26: AF94 light chain nucleotide sequence

[0112] SEQ ID No. 27: AF94 heavy chain nucleotide sequence

[0113] SEQ ID No. 28: AF81 heavy chain nucleotide sequence

[0114] SEQ ID No. 29: AF82 heavy chain nucleotide sequence

[0115] SEQ ID No. 30: AF48 heavy chain nucleotide sequence

[0116] SEQ ID No. 31: AF49 heavy chain nucleotide sequence

[0117] SEQ ID No. 32: AF56 heavy chain nucleotide sequence

[0118] SEQ ID No. 33: AF71 heavy chain nucleotide sequence

[0119] SEQ ID No. 34: AF72 heavy chain nucleotide sequence

[0120] SEQ ID No. 35: AF73 heavy chain nucleotide sequence

[0121] SEQ ID No. 36: AF74 heavy chain nucleotide sequence

[0122] SEQ ID No. 37: AF75 heavy chain nucleotide sequence

[0123] SEQ ID No. 38: AF76 heavy chain nucleotide sequence

[0124] SEQ ID No. 39: AF77 heavy chain nucleotide sequence

[0125] SEQ ID No. 40: AF78 heavy chain nucleotide sequence

[0126] SEQ ID No. 41: AF79 heavy chain nucleotide sequence

[0127] SEQ ID No. 42: AF80 heavy chain nucleotide sequence

[0128] SEQ ID No. 43: AF1006 heavy chain nucleotide sequence

[0129] SEQ ID No. 44: AF50 heavy chain nucleotide sequence

[0130] SEQ ID No. 45: AF1003 light chain nucleotide sequence

[0131] SEQ ID No. 46: AF1003 heavy chain nucleotide sequence

[0132] SEQ ID No. 47: AF1005 light chain nucleotide sequence

[0133] SEQ ID No. 48: AF1005 heavy chain nucleotide sequence

[0134] SEQ ID No. 49: AF1000 light chain nucleotide sequence

[0135] SEQ ID No. 50: AF1000 heavy chain nucleotide sequence

[0136] SEQ ID No. 51 : Fd-1

[0137] SEQ ID No. 52: Fd-2

[0138] SEQ ID No. 53: Fd-3

[0139] SEQ ID No. 54: Fd-4

[0140] SEQ ID No. 55: Linker 1

[0141] SEQ ID No. 56: Linker 2

[0142] SEQ ID No. 57: Linker 3

[0143] SEQ ID No. 58: Linker 4

[0144] SEQ ID No. 59: Linker 5

[0145] SEQ ID No. 60: Linker 6

[0146] SEQ ID No. 61 : FC-1

[0147] SEQ ID No. 62: FC-2

[0148] SEQ ID No. 63: FC-3

[0149] SEQ ID No. 64: FC-4

[0150] SEQ ID No. 65: IL-2-1

[0151] SEQ ID No. 66: IL-2-2

[0152] SEQ ID No. 67: IL-2-3

[0153] SEQ ID No. 68: IL-2-4

[0154] SEQ ID No. 69: IL-2Ra-1

[0155] SEQ ID No. 70: IL-2Ra-2

[0156] SEQ ID No. 71 : IL-2Ra-3

[0157] SEQ ID No. 72: IL-2Ra-4

[0158] SEQ ID No. 73: IL-2Ra-5

[0159] SEQ ID No. 74: IL-2Ra-6

[0160] SEQ ID No. 75: IL-2Ra-7

[0161] SEQ ID No. 76: IL-2Ra-8

[0162] SEQ ID No. 77: IL-2Ra-9

[0163] SEQ ID No 78: AF1007 heavy chain amino acid sequence

[0164] SEQ ID No 79: AF1008 heavy chain amino acid sequence

[0165] SEQ ID No 80: AF1007 heavy chain nucleotide sequence

[0166] SEQ ID No 81: AF1008 heavy chain nucleotide sequence

[0167] Example

[0168] The application will now be demonstrated with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for the purposes of illustration only and do not limit the scope of the application in any way.

[0169] Example 1: Design of IL-2 antibody fusion proteins

[0170] The specific antibody fusion protein provided in the embodiments of the present application is that the human CLDN18.2, CDLN6, CD20 or CD228 antibody heavy chain Fd (amino acid sequence such as SEQ ID No. 51-SEQ ID No. 54) is sequentially connected with the heavy chain Fc (amino acid sequence such as SEQ ID No. 61-SEQ ID No. 64), linker 1 (amino acid sequence such as SEQ ID No. 55-SEQ ID No. 60), interleukin-2 receptor subunit alpha (amino acid sequence such as SEQ ID No. 69-SEQ ID No. 77), linker 2 (amino acid sequence such as SEQ ID No. 55-SEQ ID No. 60), interleukin-2 (amino acid sequence such as SEQ ID No. 65-SEQ ID No. 68) to obtain the heavy chain of the antibody fusion protein, and combine with the light chain (amino acid sequence such as SEQ ID No. 1, 20, 22, 24) to obtain the antibody fusion proteins AF94, AF81, AF82, AF48, AF49, AF56, AF71, AF72, AF73, AF74, AF75, AF76, AF77, AF78, AF79, AF80, AF1006, AF1007, AF1008, AF1003, AF1005 and AF1000, the amino acid sequences are shown in Table 3 above.

[0171] In which AF50 does not include a shielding part, as a control.

[0172] The connection of each part of the antibody fusion protein is realized by DNA homologous recombination, and the heavy chain nucleotide sequences obtained after connection are SEQ ID No. 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 41, 43, 80, 81, 46, 48, 50; and the light chain nucleotide sequences are SEQ ID No. 26, 45, 47, 49.

[0173] Example 2: Preparation of antibody fusion protein

[0174] The gene synthesized after DNA homologous recombination is cloned into the vector pCDNA TM 3.4TOPO TM TA (Thermo fisher, item number A14697), and the vector construction is completed according to the company's operation manual pcDNA3.4-TOPO TA Cloning Kit User Guide. The CHO-S cells are cultured at 37℃, 8% CO2, 100 rpm to a cell density of 6x10 61 mg / mL, and the concentration of liposome was referred to ExpiCHO TM Expression System kit, and incubated at 32℃, 5% CO2, 100 rpm for 12-14 days. The cells were fed once at 18-22h after transfection and between the 5th day. The culture product was centrifuged, 0.22 μm filter membrane was used to filter and collect the supernatant, and the antibody fusion protein was purified by Protein A ion column.

[0175] The specific operation steps of purification are as follows: the supernatant is taken after high-speed centrifugation of the cell culture solution, and affinity chromatography is performed by using the Protein A chromatography column of Cytiva. The equilibrium buffer used in the chromatography is 1x PBS (pH 7.4), and after the cell supernatant is loaded, PBS is used for washing until the ultraviolet light returns to the baseline, then the elution buffer 0.1M glycine (pH 3.0) is used to elute the target protein, and the pH is adjusted to neutral by using Tris for storage. Then, the eluate obtained after purification is ultrafiltrated into the buffer. The protein purity and content are detected by SDS-PAGE gel electrophoresis, as shown in Figures 2A-2C.

[0176] Further SEC-HPLC is used to determine the purity of the fusion protein, and the results show that after one-step purification, most of the target fusion proteins have a purity of more than 90%, and some proteins can reach more than 90% after two-step purification, and the purity is high, as shown in Figures 3A-3D and Table 4.

[0177] Table 4: Purity results of one-step purification of each antibody fusion protein

[0178] Example 3: Detection of NK-92 and CTLL-2 cell proliferation activity of CLDN18.2-IL-2 antibody fusion protein

[0179] The human NK cell line NK-92 (CRL-2407, ATCC) and the mouse T lymphocyte cell line CTLL-2 (purchased from the Institute of Biophysics, Chinese Academy of Sciences) are IL-2 dependent cell lines, and the NK-92 and CTLL-2 cell lines cultured in vitro can be used to evaluate the cell proliferation activity of the antibody fusion protein after starvation culture.

[0180] In 96-well plates, 3000 cells / well were added, then the antibody fusion protein was added, the initial concentration of the antibody fusion protein was 26 nM, 5-fold gradient dilution to obtain 9 concentration points, and PBS negative control was set. After incubation, a flat-bottom 96-well plate was taken out and equilibrated to room temperature, 30 μL of CTG-Glo reagent (Promega, Madison, WI) was added, shaken well and fully lysed for 10 minutes, and after 10 minutes of room temperature standing, 50 μL of supernatant was transferred to a flat-bottom 384-well plate, and the fluorescence signal was detected on an Envision multifunctional microplate detector (Perkin Elmer, Waltham, MA). The data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was fitted by nonlinear S curve regression, and the EC 50 values were calculated, and the results are shown in Figures 4 and 5. The EC 50 ± SEM of NK-92 (n = 3) and CTLL2 (n = 3) cells are summarized in Tables 5 and 6. Since the above-mentioned cells are IL-2 dependent, the PBS group showed apoptosis due to the lack of cytokines.

[0181] Table 5: Effect of antibody fusion protein on cell proliferation activity of NK-92

[0182] Table 6: Effect of antibody fusion protein on cell proliferation activity of CTLL-2

[0183] From the above results, in the proliferation experiment of NK-92 cells, the activation ability of AF94, AF81 and AF82 was reduced by about 33-62 times compared with IL-2, but compared with the PBS control group, there was still a significant activation effect; in the proliferation experiment of CTLL-2 cells, the activation ability of AF94, AF81 and AF82 was reduced by about 2.2-12.7 times compared with IL-2, but compared with the PBS control group, there was still an activation effect. These results show that AF94, AF81 and AF82 are weak IL-2 activation molecules.

[0184] Example 4: Detection of CLDN18.2-IL-2 antibody fusion protein-mediated STAT5 phosphorylation in different T cells and NK cells

[0185] IL-2 activates the downstream JAK1 / JAK3-STAT5 signaling pathway by binding to IL-2Rβγ dimers on the surface of NK cells or CD8+ T cells, or IL-2Rαβγ trimers on the surface of Treg cells, promoting STAT5 phosphorylation (p-STAT5). Therefore, by testing the ability of antibody fusion proteins to promote STAT5 phosphorylation in cells such as NK cells, CD8+ T cells, or CD4+ T cells, their function can be further determined.

[0186] After thawing frozen human PBMCs at 37°C and 5% CO2 for 1 hour, 2.22×10 5 / mL cells were resuspended with MEM basal medium and added to a 96-well cell culture plate, 90 μL per well. The starting concentration of the antibody fusion protein was 3 nM, and it was serially diluted 3 times with MEM basal medium to obtain 10 concentration points. 10 μL of the antibody fusion protein solution was added to the 96-well plate and incubated in a 37°C incubator for 15 minutes. After stimulation, the cells were treated with Foxp3 Fix / Perm buffer set (eBioscience, catalog number 00-5523-00) according to the instructions. Then, eFluor TM Cells were stained with 450 anti-human FOXP3 (236A / E7, eBioscience, Cat. No. 48-4777-42), FITC anti-human CD8a (RPA-T8, Biolegend, Cat. No. 301060), PE mouse anti-human CD3 (UCHT1, BD, Cat. No. 555333), and PE / Cyanine7 anti-human CD56 (NCAM, Biolegend, Cat. No. 318318). Following staining, cells were fixed with 2% paraformaldehyde at 4°C for 30 minutes and then treated with 90% pre-cooled methanol at 4°C for 30 minutes. Following treatment, cells were stained with Alexa Fluor 647 mouse anti-Stat5 (pY694) (47 / STAT5, BD, Cat. No. 562076).

[0187] After staining, flow cytometry (Novocyte Quanteon, Agilent) was used to detect the proportion of pSTAT-5+ cells in CD3+CD8α- (CD4+ T cells), CD3+CD8α+ (CD8+ T cells), CD3+CD8-Foxp3+ (Treg), and CD3-CD56+ (NK cells). The experimental results were analyzed by 4-parameter nonlinear fitting using GraphPad Prism to obtain the EC of each molecule. 50The experimental results are shown in Figures 6A-6E and Tables 7-11. Wherein n.a. represents that the EC 50 value is too large, and the EC 50 value cannot be fitted by the model.

[0188] Table 7: Effect of antibody fusion protein on T cell STAT5 phosphorylation

[0189] Table 8: Effect of antibody fusion protein on CD8+T cell STAT5 phosphorylation

[0190] Table 9: Effect of antibody fusion protein on CD4+T cell STAT5 phosphorylation

[0191] Table 10: Effect of antibody fusion protein on NK cell STAT5 phosphorylation

[0192] Table 11: Effect of antibody fusion protein on Treg cell STAT5 phosphorylation

[0193] The results show that AF94, AF48, AF49, AF81 and AF82 down-regulate the pSTAT5 phosphorylation levels of T cells, CD8+T cells, CD4+T cells, Treg cells and NK cells, etc. compared with IL-2 with full activation ability. The over-activation of CD8+T cells and NK cells, especially in the periphery, often leads to capillary leakage, cytokine storm and other toxicities, and the antibody fusion proteins in the application significantly reduce the transient over-activation of these cells.

[0194] Example 5: Detection of in vitro proliferation activity of antibody fusion protein on mouse T blast cells

[0195] T blast cells (Tblast) are a type of cells that highly express IL-2 receptors (CD25 / CD122 / CD132), and IL-2 stimulation will cause them to proliferate. After obtaining the TbIast cells by stimulating the cryopreserved mouse PBMC cells with 2 μg / mL of concanavalin A (Con A, Sigma-Aldrich, item number C2575) for 72 hours at 37°C, 5% CO2, they were inoculated into a U-shaped 96-well plate. Then the antibody fusion protein was added at a starting maximum final concentration of 36 nM, 3-fold dilution, to obtain 10 concentration points. After incubation at 37°C, 5% CO2 for 72 hours, the proliferation of TbIast cells was detected according to the instructions using the Cell-Titer glow kit (Promega, item number G7570).

[0196] The experimental results were analyzed by 4-parameter nonlinear fitting using GraphPad Prism to obtain the EC of each fusion protein molecule. 50 The results are shown in Figure 7 and Table 12, indicating that the antibody fusion protein reduced the proliferation of T cells that highly expressed IL-2 receptor (CD25 / CD122 / CD132) to a certain extent compared to IL-2.

[0197] Table 12: Effects of antibody fusion proteins on the proliferation activity of mouse Tblast cells

[0198] Example 6: Efficacy of CLDN18.2-IL-2 Antibody Fusion Protein on MC-38-hCLDN18.2 Mouse Colon Cancer Cell Transplants

[0199] The PBS group was used as the control group to detect the efficacy of three antibody fusion proteins AF94, AF48 and AF49 on MC-38-hCLDN18.2-A11 mouse colon cancer cell transplanted tumors.

[0200] MC-38-hCLDN18.2-A11 mouse colon cancer cells (purchased from Nanjing Bowang) in the logarithmic growth phase were inoculated into 6-8 week old female C57BL / 6 mice (Weitong Lihua) at an inoculation volume of 5×10 5 / mouse, the inoculation volume was 0.1mL. When the tumor volume reached 60-80mm 3 At 14:00, intraperitoneal administration was started, with each mouse receiving 1.2 mg / kg, twice a week for a total of 5 doses. After tumor inoculation, routine monitoring included tumor growth and the effects of treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. Clinical symptoms observed during the experiment were recorded in the original data. The average tumor volume of the control group exceeded 2000 mm 3 The experimental endpoint was set as the experimental end point, at which spleen, draining lymph nodes, and tumor tissues were collected for immune cell infiltration analysis.

[0201] Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter).

[0202] The relative tumor inhibition rate (TGI) was calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the changes in tumor volume (TW) at a specific time point relative to the initial tumor volume before drug administration in the treatment group and the PBS control group, respectively.)

[0203] The tumor growth curve is shown in Figure 8A.

[0204] In the MC-38-hCLDN18.2-A11 model, on day 21, the tumor inhibition rate TGI of AF94 was 102.02%, the tumor inhibition rate TGI of AF48 was 98.58%, and the tumor inhibition rate TGI of AF49 was 99.59% (p < 0.01, One-way ANOVA), which had a significant difference compared with the PBS control group (Figure 8C).

[0205] The tumor growth curves of individual mice are shown in Figures 8C-8F, in which 7 / 10 (70%) mice in the AF94 administration group achieved complete response (CR, complete response) (Figure 8D), 5 / 10 (50%) mice in the AF48 administration group achieved complete response (CR, complete response) (Figure 8E), and 7 / 10 (70%) mice in the AF49 administration group achieved complete response (CR, complete response) (Figure 8F), showing that AF94, AF48 and AF49 monotherapy have excellent anti-tumor ability in the MC-38hCLDN18.2 mouse colon cancer model. At the same time, compared with the PBS control group, AF94, AF48 and AF49 did not show obvious weight loss and other abnormal behaviors (as shown in Figure 8B).

[0206] Example 7: CLDN18.2-IL-2 antibody fusion protein induces immune memory formation in MC-38-hCLDN18.2 mouse colon cancer tumor model

[0207] The tumor inhibition ability and the ability to promote immune memory formation of antibody fusion proteins AF94, AF48 and AF49 in the MC-38-hCLDN18.2-A11 (purchased from Nanjing Bowang) mouse colon cancer tumor model were compared.

[0208] Mice with complete tumor regression (from the AF94, AF48 and AF49 groups) were continued to be routinely fed and monitored for 6 weeks, after which 5x10 5 MC-38-hCLDN18.2-A11 (purchased from Nanjing Bowang) cells / mouse were inoculated on the contralateral side, and 8 wild-type mice (Vivinent) were inoculated at the same time as tumor occurrence controls. The tumor growth was measured every week, and the average tumor volume of the control group of mice was about 2000 mm 3 at the end of the observation. The results are shown in Figure 9, and the AF94, AF48 and AF49 administration groups can form immune memory, and the tumor does not grow after re-inoculation.

[0209] Example 8: Toxicity study of CLDN18.2-IL-2 antibody fusion protein in MC-38-hCLDN18.2 mouse colon cancer tumor model

[0210] The main toxicity of IL-2 is to cause capillary leakage and induce multi-organ failure. We used MC-38-hCLDN18.2 tumor-bearing mice to study the toxicity of CLDN18.2-IL-2.

[0211] The mouse tumor cell inoculation and tumor measurement were as described in Example 6. When the tumor occurred and reached 500-800 mm 3 , the PBS, equimolar Fc and IL2 fusion protein IL-2-Fc and AF49 were administered respectively, twice intraperitoneally, the dose of each administration was 13 mg / kg, and each experimental group contained 5 mice. At 48 hours and 96 hours after administration, the mouse lungs were collected and weighed, and after drying at 37°C for 48 hours, they were weighed again, and the difference was the net weight.

[0212] The results are shown in Figures 10A and 10B. Compared with the control group, IL-2-Fc had a significant increase in lung net weight at 48 hours after administration, indicating that it caused capillary leakage; at 96 hours after administration, four of the five mice died, and the remaining one was in a state of debilitation. The experimental group AF49 had no significant difference in lung net weight compared with the control group at 48 hours after administration, and had no obvious abnormalities at 96 hours after administration, with a slight increase in lung net weight, which was not significant.

[0213] As can be seen from Example 6, the antibody fusion protein can achieve complete tumor inhibition at a dose of 1.2 mg / kg, and has no obvious toxicity at a dose of 13 mg / kg, with a treatment window of about 11 times, which is expected to greatly improve the clinical effectiveness and safety of IL-2 drugs.

[0214] Example 9: Efficacy study of antibody fusion proteins targeting different antibodies in a mouse colon cancer cell model overexpressing antigens

[0215] The PBS group was used as the control group, and the antibody fusion protein AF1003 was used as the test group. The MC-38-hCLDN6 (purchased from Kangyuan Bochuang) mouse colon cancer cells in the logarithmic growth phase were inoculated into 6-8 week old female C57BL / 6 mice (Vitron Lihua) at a dose of 1 x 10 6 cells per mouse and a volume of 0.1 mL. The administration and tumor volume detection scheme were carried out according to Example 6.

[0216] The change in tumor volume is shown in Figure 11.

[0217] In the MC-38-hCLDN6 model, on day 23, the tumor inhibition rate TGI of AF1003 was 60.87% (p<0.01, One way ANOVA), which had a significant difference compared with the PBS control group. The AF1003 administration group had 2 / 8 (25%) mice that achieved complete response (CR).

[0218] A method of lentivirus transfection was used to construct a MC38 cell line overexpressing human CD228 protein.

[0219] The cDNA of human CD228 was cloned into the lentivirus vector pCDH-CMV-MCS-EF1-Puro to obtain the lentivirus vector plenti-hCD228 loaded with the CD228 cDNA fragment. 2-3 x 10 6 HEK-293T cells (Promocell, Cat. No. CL-0005) were seeded in 100 mm dishes (Coming, Cat. No. CLS430167) and cultured at 37°C, 5% CO2 to a cell density of about 60% using DMEM (Gibico, Cat. No. 11965092) + 10% FBS (ExCell, Cat. No. FSP500) medium. The plenti-CD228 was mixed with the packaging plasmids psPAX2 and pMD2.G in a mass ratio of 4:3:1 (total mass 10 μg) with 1 mg / mL PEI 25K (Polyscience, Cat. No. 23966-100) to prepare a solution with a final volume of 1 mL of OPTI-MEM (Gibico, Cat. No. 31985070). The solution was added dropwise to the 100 mm dishes seeded with HEK-293T cells, and after 48 hours of culture at 37°C, 5% CO2, the supernatant was collected and stored at -80°C after 0.45 μm filtration for later use.

[0220] 0.1 x 10 6 MC38 cells (Nanjing Kebai, Cat. No. CBP60825) were seeded in 6-well plates (Coming, Cat. No. 3615), and after the cells adhered, the medium was changed. The changed medium was DMEM (Gibico, Cat. No. 11965092) plus 10% FBS (ExCell, Cat. No. FSP500) mixed 1:1 with the aforementioned stored supernatant. After 72 hours of culture at 37°C, 5% CO2, the medium was changed to DMEM (Gibico, Cat. No. 11965092) plus 10% FBS (ExCell, Cat. No. FSP500) + Puromycin (2 μg / mL, Invivogen, Cat. No. ant-pr-1). After 7 days of maintenance under the selection pressure of puromycin, single clone cells were selected. After obtaining the single clone, the expression of human CD228 was detected using flow cytometry, and finally the MC38 monoclonal cell line overexpressing CD228 was obtained.

[0221] PBS group as control group, antibody fusion protein AF1000 as test group, MC-38-hCD228 mouse colon cancer cells in logarithmic growth phase were inoculated into 6-8 week old female C57BL / 6 mice (Vitron), the inoculation amount was 3 x 105 The cell number was 5 x 106cells / rat, and the inoculation volume was 0.1 mL. The administration and tumor volume detection scheme was performed according to Example 6.

[0222] The tumor growth curve is shown in Figure 12.

[0223] In the MC-38-hCD228 model, on day 27, the tumor inhibition rate TGI of AF1000 was 53.34% (p < 0.01, One-way ANOVA), which had a significant difference compared with the PBS control group.

[0224] Example 10: Charge analysis of CLDN18.2-IL-2 antibody fusion protein

[0225] Capillary isoelectric focusing (cIEF) is a technique that can quantitatively analyze the isoelectric focus point (pI) and charge isomer of a protein. Under the action of an electric field within a pH gradient, an amphoteric substance (such as a protein) is separated and finally focused into a very narrow section at a place where the pH value in the pH gradient is equal to the isoelectric point (pI) value of the amphoteric substance. cIEF has high resolution and good repeatability, and is a powerful tool for characterizing the charge homogeneity of a protein. When the protein isoelectric point band is wide, it indicates that the protein has poor homogeneity, strong heterogeneity, and a more difficult production process.

[0226] A 10 M urea solution and a 200 mM iminodiacetic acid solution were prepared and stored at room temperature. PI 3.38 and PI 8.4 standards were prepared and stored at -20°C. Antibody fusion protein samples were prepared at a concentration range of 0.15 mg / mL to 2 mg / mL, and if the salt ion concentration was higher than 15 mM, desalting treatment was required. 4 μL of the amphoteric electrolyte solution, 35 μL of methyl cellulose, 1 μL of PI 3.38, and 1 μL of PI 8.4 standard were mixed, 100 μg of the sample was added, and deionized water was added to a volume of 100 μL. 80 μL was transferred to a sample tube for machine analysis. The machine conditions were 1500 V focusing for 1 minute, 3000 V focusing for 9 minutes, 55 seconds of sample loading, and data analysis. The sialidase digestion conditions were 100 μg of the sample added to 2 mM sialidase, and the reaction was performed at 37°C for 2 hours. The results are shown in Figures 13A-13F and Table 13.

[0227] Table 13: Antibody fusion protein sample pI isoelectric point results

[0228] As shown in FIGS. 13A-13C, the main peaks of AF48 and AF49 are relatively obvious, and the main peak pI is 8.88 and 8.80, respectively, and the pI band ranges are 8.12-9.03 and 8.20-8.97, respectively; in contrast, the pI band range of AF94 is larger, from 7.48 to 9.03, and the main peak is not obvious, estimated to be 8.57. AF48 and AF49 each have 9 acidic peaks with a relatively high proportion before the main peak, to determine the contribution of sialic acid modification to the charge heterogeneity, the test sample was further treated with sialidase, and the results are shown in FIGS. 13D-13F. The acidic peaks of AF48 and AF49 changed from 9 to 3, while AF94 still had about 9 or more acidic peaks after sialidase treatment, and the main peak proportion was low. It can be inferred that AF94 has complex post-translational modification and strong heterogeneity; while AF48 and AF49 have relatively uniform post-translational modification and stronger developability.

[0229] Example 11: Purity analysis of CLDN18.2-IL-2 antibody fusion protein

[0230] Large molecules such as antibodies have large relative molecular mass and complex structure, and are easily affected by amino acid sequence, formulation prescription, and post-translational modification during protein expression, purification, and storage, resulting in the production of aggregates and fragments. The breakage of the hinge region of a monoclonal antibody can affect the function and half-life of the crystallizable fragment of the monoclonal antibody, and aggregates not only reduce the in vivo efficacy of the monoclonal antibody drug, but also have stronger immunogenicity than monomers. Therefore, the detection of fragments and aggregates of a monoclonal antibody product, i.e., size heterogeneity, is crucial for evaluating the stability, safety, and production process stability of an antibody fusion protein. CE-SDS, i.e., capillary electrophoresis with sodium dodecyl sulfate, has the characteristics of small sample size, high resolution, and more objective ultraviolet absorption quantitative analysis, and is a main analysis method for fusion proteins.

[0231] The preparation of non-reduced samples is as follows: take 50 μL of buffer solution (40 mM phosphate solution, 1% SDS, PH 6.5) of PH 6.5, add 1M IAM 1.5 μL, add 100 μg of sample, add deionized water to 100 μL; the preparation of reduced sample is as follows: take 50 μL of buffer solution (40 mM phosphate solution, 1% SDS, PH 6.5) of PH 6.5, add 5 μL of 2-mercaptoethanol, add 100 μg of sample, add deionized water to 100 μL. After the sample is prepared, mix well, centrifuge at 12000 rpm for 2 min, heat in 65℃ metal bath for 5 min, cool to room temperature, centrifuge at 12000 rpm for 2 min again, remove 80 μL in the inner cannula with a pipette gun and detect on the machine. The instrument uses PA800plus, the on-machine conditions are: capillary inner diameter 50 μm, effective length 20 cm, hole plug 100 μm*200 μm, temperature 25℃; sample chamber temperature 10℃; detector PDA, detection wavelength 220 nm; sample voltage 5kV, sample time 20 seconds, separation voltage 15kV, separation time 40 minutes. The results are shown in Figures 14A-14F and Table 14.

[0232] Table 14: Antibody fusion protein sample CE-SDS purity results

[0233] Under non-reducing conditions, the purity of AF48 and AF49 is 97% and 96.4% respectively; while the purity of AF94 is 87.3%, and its peak width is wider, and its aggregate content is 8.3%, indicating that AF94 has poor purity and strong heterogeneity. The results of CE-SDS under reducing conditions show that the light chain of AF48 and AF49 accounts for 26.3% and 27.2% respectively, and the heavy chain accounts for 63.7% and 63.9% respectively, and the light and heavy chains account for 90% and 91.1% respectively, while the heavy chain of AF94 is split into two peaks, further proving its complexity. The results of CE-SDS show that AF48 and AF49 have higher purity and developability.

Claims

1. An antibody fusion protein, having a structure of Ab-L1-B-L2-D, wherein: Ab is an antibody capable of specifically binding to a target; B is a masking peptide; L1 and L2 are respectively a linker peptide 1 and a linker peptide 2; D is a cytokine. 2.The antibody fusion protein of claim 1, wherein: the cytokine is selected from one or more of interleukin-2 (IL-2), interferon alpha (IFNα), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin-7 (IL-7), interleukin-12 (IL-12), and interleukin-21 (IL-21) ; and / or the masking peptide is a receptor or a binding fragment thereof of the cytokine or an antibody or a binding fragment thereof specifically binding to the cytokine, which can inhibit the activity of the cytokine by binding to the cytokine. 3.The antibody fusion protein of claim 1 or 2, wherein: the masking peptide B is selected from IL-2Rα, IL-2Rβ, IL-2Rγ or a mutant or a truncate thereof; the linker peptides L1 and L2 are respectively a polypeptide of 1-50 amino acids; each is independently selected from a flexible linker peptide comprising glycine (G) and serine (S) residues, preferably a linker peptide comprising (GGGGS) n repeats, wherein n is an integer selected from 1-10; the cytokine D is IL-2.

4. The antibody fusion protein of any one of claims 1-3, wherein, the heavy chain Fd amino acid sequence of the antibody is selected from any one of SEQ ID No. 51-SEQ ID No. 54; the Fc portion of the antibody has an amino acid sequence selected from any one of SEQ ID No. 61-SEQ ID No. 64; the masking peptide B is wild-type IL-2Rα or a mutant or a truncate thereof; preferably an amino acid sequence selected from any one of SEQ ID No. 69-SEQ ID No. 77; L1 and L2 are each independently selected from any one of SEQ ID No. 55-SEQ ID No. 60; the cytokine D is wild-type IL-2 or a mutant or a truncate thereof; preferably an amino acid sequence selected from any one of SEQ ID No. 65-SEQ ID No.

68. 5.The antibody fusion protein of any one of claims 1-4, having a light chain with an amino acid sequence selected from the sequence set forth in SEQ ID No. 1, 20, 22 or 24, and a heavy chain with an amino acid sequence selected from the sequence set forth in SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 78, 79, 21, 23 or 25. 6.The antibody fusion protein of any one of claims 1-5, having a light chain with an amino acid sequence as set forth in SEQ ID No. 1 and a heavy chain with an amino acid sequence as set forth in SEQ ID No.

2. ​ or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 3; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 4; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 5; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 6; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 7; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 8; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 9; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 10; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 11; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 12; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 13; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 14; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 15; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 16; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 17; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 18; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 78; or the amino acid sequence of the light chain is shown as SEQ ID No. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 79; or the amino acid sequence of the light chain is shown as SEQ ID No. 20, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 21; or the amino acid sequence of the light chain is as set forth in SEQ ID No. 22, and the amino acid sequence of the heavy chain is as set forth in SEQ ID No. 23; or the amino acid sequence of the light chain is as set forth in SEQ ID No. 24, and the amino acid sequence of the heavy chain is as set forth in SEQ ID No.

25.

7. A pharmaceutical composition comprising an effective amount of the antibody fusion protein of any one of claims 1-6 and a pharmaceutically acceptable excipient.

8. A nucleic acid molecule comprising a polynucleotide encoding the antibody fusion protein of any one of claims 1-6.

9. The nucleic acid molecule of claim 8, wherein the nucleotide sequence of the light chain is selected from the group consisting of SEQ ID No. 26, 45, 47 or 49, and the nucleotide sequence of the heavy chain is selected from the group consisting of the sequences set forth in SEQ ID No. 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 80, 81, 46, 48 or 50.

10. A vector comprising the nucleic acid molecule of claim 8 or 9.

11. A host cell comprising the nucleic acid molecule of claim 8 or 9, or the vector of claim 10.

12. A method of producing the antibody fusion protein of any one of claims 1-6, comprising culturing the host cell of claim 11 to express the antibody fusion protein, and isolating the expressed antibody fusion protein.

13. A method for diagnosing, treating or preventing a tumor or an autoimmune disease, the method comprising administering to a subject in need thereof an effective amount of the antibody fusion protein of any one of claims 1-6.

14. Use of the antibody fusion protein of any one of claims 1-6, the nucleic acid molecule of claim 8 or 9, the vector of claim 10, or the host cell of claim 11 in the manufacture of a medicament or reagent for diagnosing, treating or preventing a tumor or an autoimmune disease.

15. Use according to claim 14, wherein, the tumor is a CLDN18.2-related tumor, a CLDN6-related tumor, a CD20-related tumor or a CD228-related tumor; and the autoimmune disease is selected from the group consisting of rheumatoid arthritis, autoimmune hemolytic anemia, pure red cell anemia, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, Evans syndrome, vasculitis, and bullous skin disease.

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