Therapeutic use of protein heterodimer

Abstract

Use of a protein heterodimer in the preparation of a drug, which drug is used for preventing and / or treating malignant effusion. The protein heterodimer contains a first member and a second member different from the first member, wherein the first member contains a light chain and a heavy chain, the heavy chain contains a first Fc region, and the light chain is complexed with the heavy chain to form a targeting moiety that exhibits binding specificity for a tumor antigen; and the second member contains a polypeptide containing an immunomodulator fused to a second Fc region. The first member and the second member are associated by means of the complexing of the first Fc region with the second Fc region so as to form the heterodimer. In addition, the first Fc region contains a first modification and / or the second Fc region contains a second modification, and these modifications can more effectively promote heterodimerization between the first member and the second member.

Description

Therapeutic uses of protein heterodimers Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to therapeutic uses of protein heterodimers. Background Art

[0002] Malignant pleural effusion (MPE) is one of the most common complications of advanced metastatic cancer. It is caused by the presence of malignant cells in the pleura. Tumor cells infiltrate the pleural cavity through direct invasion, hematogenous metastasis, or lymphatic spread. Pleural effusion may be the result of tumor growth blocking lymphatic drainage.

[0003] Malignant ascites is a clinical manifestation of the late stages of many malignant tumors. Typically, this condition occurs in the late stages of the disease and is often accompanied by distant metastasis of the malignant tumor, at which point drug treatment becomes difficult. Therefore, the formation of ascites indicates that effective treatments are often lacking in the clinic. Ascites formation is particularly prominent in patients with advanced ovarian, pancreatic, and gastrointestinal malignancies. Current clinical treatments mainly include simple ascites extraction, intraperitoneal injection of chemotherapy drugs, and hyperthermic intraperitoneal perfusion therapy. However, these methods are often accompanied by significant toxic effects.

[0004] It can be seen that there is an urgent need to obtain effective methods for treating malignant pleural effusion and malignant ascites.

[0005] Summary of the Invention

[0006] The present application provides a use of a protein heterodimer, a mixture producing the protein heterodimer, a nucleic acid encoding the protein heterodimer, etc., in the treatment of malignant effusions such as malignant pleural effusion and malignant ascites. The use described in the present application has high utilization efficiency of the protein heterodimer and low side effects, and has a very broad application prospect.

[0007] In one aspect, the present application provides a use of a protein heterodimer in the preparation of a medicament for preventing and / or treating malignant effusions, wherein the protein heterodimer may comprise a first member and a second member different from the first member, the first member may comprise a light chain and a heavy chain comprising a first Fc region, the light chain being complexed with the heavy chain to form a targeting moiety exhibiting binding specificity for a tumor antigen; the second member may comprise a polypeptide comprising an immunomodulator fused to a second Fc region; the first member may associate with the second member via complexing of the first Fc region with the second Fc region to form a heterodimer; and the first Fc region may comprise a first modification and / or the second Fc region may comprise a second modification, wherein the first modification and / or the second modification may more effectively promote heterodimerization between the first member and the second member than a knob-hole modification comprising a knob modification and a hole modification. For a knob-hole modification comprising a knob modification and a hole modification, the first Fc region may comprise the knob modification and the second Fc region may comprise the hole modification. Alternatively, the first Fc region may comprise the hole modification and the second Fc region may comprise the knob modification.

[0008] In some embodiments, the first modification is different from the knob modification or the pore modification, and / or the second modification is different from the knob modification or the pore modification.

[0009] In some embodiments, when expressed in mammalian cells, the yield of the protein heterodimer is at least 10% greater than the yield of a control protein, and the control protein differs from the protein heterodimer in that the control protein: i) comprises a knob modification in the first Fc region, ii) comprises a hole modification in the second Fc region, and iii) does not simultaneously comprise the first and second modifications of the protein heterodimer of the present application. The mammalian cell can be selected from HEK293 cells, CHO cells, COS-1 cells, and NS0 cells.

[0010] In some embodiments, the first Fc region comprises a first modification, the second Fc region comprises a second modification, and both the first modification and the second modification are different from a knob modification or a pore modification.

[0011] In some embodiments, the polypeptide contained in the second member is a fusion protein, and the C-terminus of the immunomodulator is fused directly or indirectly to the N-terminus of the second Fc region to form the fusion protein.

[0012] In some embodiments, the tumor antigen is selected from EGFR, EGFR mutant, HER2 / neu, GPC3, FAP, Mucl, MUC5AC, and mesothelin.

[0013] In some embodiments, the light chain of the targeting moiety comprises a CDR comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence contained in a corresponding CDR of a light chain of an antibody specific for a tumor antigen.

[0014] In some embodiments, the light chain of the targeting moiety comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence contained in a corresponding variable region of a light chain of an antibody specific for a tumor antigen.

[0015] In some embodiments, the light chain of the targeting moiety comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the light chain of an antibody specific for a tumor antigen.

[0016] In some embodiments, the heavy chain of the targeting moiety comprises a CDR comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence contained in a corresponding CDR of the heavy chain of an antibody specific for a tumor antigen.

[0017] In some embodiments, the heavy chain of the targeting moiety comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence contained in a corresponding variable region of a heavy chain of an antibody specific for a tumor antigen.

[0018] In some embodiments, the heavy chain of the targeting moiety comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the heavy chain of an antibody specific for a tumor antigen.

[0019] In some embodiments, the light chain of the targeting moiety comprises CDRs comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding CDRs of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting moiety comprises CDRs comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding CDRs of the heavy chain of an antibody specific for a tumor antigen.

[0020] In some embodiments, the light chain of the targeting moiety comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding variable region of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting moiety comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding variable region of the heavy chain of an antibody specific for a tumor antigen.

[0021] In some embodiments, the light chain of the targeting moiety comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting moiety contains an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the heavy chain of an antibody specific for a tumor antigen.

[0022] In some embodiments, the antibody specific for a tumor antigen is selected from anti-EGFR, anti-EGFR mutant, anti-HER2 / neu, anti-GPC3, anti-FAP, anti-Muc1, anti-MUC5AC, and anti-mesothelin.

[0023] In some embodiments, the immunomodulatory agent enhances the immune response. In some embodiments, the immunomodulatory agent decreases the immune response.

[0024] In some embodiments, the immunomodulator is a cytokine. For example, the immunomodulator can be a cytokine selected from interferons, interleukins, chemokines, lymphokines, and tumor necrosis factors.

[0025] In some embodiments, the immunomodulator is an interferon selected from interferon alpha, interferon lambda, and interferon beta.

[0026] In some embodiments, the immunomodulator is an interleukin, and the interleukin comprises interleukin 10, interleukin 2, and / or super interleukin 2.

[0027] In some embodiments, the first Fc region and the second Fc region are from the Fc region of an immunoglobulin.

[0028] For example, the immunoglobulin may be selected from IgG1, IgG2, IgG3, and IgG4.

[0029] In some embodiments, the first Fc region and the second Fc region are from the Fc region of an immunoglobulin, and the immunoglobulin is human IgG1.

[0030] In some embodiments, the second Fc region is fused in-frame to the immunomodulatory agent.

[0031] In some embodiments, the second Fc region is fused in-frame to the immunomodulatory agent via a linker.

[0032] In some embodiments, the polypeptide contained in the second member comprises two or more immunomodulators, the two or more immunomodulators are fused in frame to each other and to the second Fc region, and wherein the two or more immunomodulators are located at the N-terminus of the second Fc region. For example, the two or more immunomodulators can be the same.

[0033] In some embodiments, the first modification comprises an amino acid substitution at position T366, and an amino acid substitution at one or more positions selected from the group consisting of: Y349, F405, K409, D399, K360, Q347, K392, and S354, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0034] In some embodiments, the first modification comprises an amino acid substitution selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D, and T366W.

[0035] In some embodiments, the first modification comprises 2 to 5 amino acid substitutions.

[0036] In some embodiments, the first modification comprises an amino acid substitution at any one of the following positions: 1) Y349 and T366; 2) Y349, T366, and F405; 3) Y349, T366, and K409; 4) Y349, T366, F405, K360, and Q347; 5) Y349, T366, F405, and Q347; 6) Y349, T366, K409, K360 and Q347; 7) Y349, T366, K409 and Q347; 8) T366, K409 and K392; 9) T366 and K409; 10) T366, K409, Y349 and S354; 11) T366 and F405; 12) T366, F405 and D399; 13) T366, F405, Y349 and S354.

[0037] In some embodiments, the first modification comprises an amino acid substitution at any one of the following positions: 1) Y349C and T366W; 2) Y349C, T366W, and F405K; 3) Y349C, T366W, and K409E; 4) Y349C, T366W, and K409A; 5) Y349C, T366W, F405K, K360E, and Q347E; 6) Y349C, T366W, F405K, and Q347R; 7) Y349C, T366W, K409A, K360E, and Q347E. 7E; 8) Y349C, T366W, K409A and Q347R; 9) T366W, K409A and K392D; 10) T366W and K409A; 11) T366W, K409A and Y349D; 12) T366W, K409A, Y349D and S354D; 13) T366W and F405K; 14) T366W, F405K and D399S; 15) T366W, F405K and Y349D; and 16) T366W, F405K, Y349D and S354D.

[0038] In some embodiments, the second modification comprises an amino acid substitution at positions T366, L368, and Y407, and an amino acid substitution at one or more positions selected from D356, D399, E357, F405, K360, K392, K409, and Q347, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0039] In some embodiments, the second modification comprises an amino acid substitution selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A, and Y407V.

[0040] In some embodiments, the second modification comprises an amino acid substitution at position 4 to 6.

[0041] In some embodiments, the second modification comprises an amino acid substitution at any one of the following positions: 1) D356, T366, L368, Y407, and F405; 2) D356, T366, L368, and Y407; 3) D356, T366, L368, Y407, and Q347; 4) D356, T366, L368, Y407, K360, and Q347; 5) D356, T366, L368, Y407, F405, and Q347; 6) D356, T366, L368, Y407, F405, and Q347. 366, L368, Y407, F405, K360 and Q347; 7) T366, L368, Y407, D399 and F405; 8) T366, L368, Y407 and F405; 9) T366, L368, Y407, F405 and E357; 10) T366, L368, Y407 and K409; 11) T366, L368, Y407, K409 and K392; 12) T366, L368, Y407, K409 and E357.

[0042] In some embodiments, the second modification comprises an amino acid substitution at any one of the following positions: 1) D356C, T366S, L368A, Y407V, and F405K; 2) D356C, T366S, L368A, and Y407V; 3) D356C, T366S, L368A, Y407V, and Q347R; 4) D356C, T366S, L368A, Y407V, K360E, and Q347E; 5) D356C, T366S, L368A, Y407V, F405K 405K and Q347R; 6) D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 7) T366S, L368A, Y407V, D399S and F405K; 8) T366S, L368G, Y407A and F405K; 9) T366S, L368A, Y407V, F405K and E357A; 10) T366S, L368A, Y407V and K409A; 11) T366S, L368A, F409K Y407V, K409A and K392D; 12) T366S, L368G, Y407A and K409A; 13) T366S, L368A, Y407V, K409A and E357A.

[0043] In some embodiments, the first Fc region comprises a first modification, and the second Fc region comprises a second modification. The first modification comprises an amino acid substitution at position T366 and an amino acid substitution at one or more positions selected from the group consisting of: Y349, F405, K409, D399, K360, Q347, K392, and S354, wherein the position of the amino acids is determined according to the EU index for KABAT numbering; the second modification comprises an amino acid substitution at positions T366, L368, and Y407, and an amino acid substitution at one or more positions selected from the group consisting of: D356, D399, E357, F405, K360, K392, K409, and Q347, wherein the position of the amino acids is determined according to the EU index for KABAT numbering. The first modification and the second modification may be as defined herein.

[0044] In some embodiments, the first Fc region comprises a first modification, the second Fc region comprises a second modification, and the first modification and the second modification are amino acid substitutions at positions selected from any one of the following groups: 1) first modification: Y349 and T366; second modification: D356, T366, L368, Y407, and F405; 2) first modification: Y349, T366, and F405; second modification: D356, T366, L368, Y407, and F405. 368 and Y407; 3) First modification: Y349, T366 and K409; Second modification: D356, T366, L368, Y407 and F405; 4) First modification: Y349, T366, F405, K360 and Q347; Second modification: D356, T366, L368, Y407 and Q347; 5) First modification: Y349, T366, F405 and Q347; Second modification: D356, T366, L368, Y407, K360, and Q347; 6) First modification: Y349, T366, K409, K360, and Q347; Second modification: D356, T366, L368, Y407, F405, and Q347; 7) First modification: Y349, T366, K409, and Q347; Second modification: D356, T366, L368, Y407, F405, and Q347 7, F405, K360 and Q347; 8) First modification: T366, K409 and K392; Second modification: T366, L368, Y407, D399 and F405; 9) First modification: T366 and K409; Second modification: T366, L368, Y407 and F405; 10) First modification: T366, K409 and Y349; Second modification: T366, L368, Y407 and F405 7, F405 and E357; 11) First modification: T366, K409, Y349 and S354; Second modification: T366, L368, Y407, F405 and E357; 12) First modification: T366 and F405; and second modification: T366, L368, Y407 and K409; 13) First modification: T366, F405 and D399; Second modification: T366, L368 , Y407, K409 and K392; 14) first modification: T366, F405 and Y349; second modification: T366, L368, Y407, K409 and E357; 15) first modification: T366, F405, Y349 and S354; second modification: T366, L368, Y407, K409 and E357; wherein the positions of amino acids are determined according to the EU index of KABAT numbering.

[0045] In some embodiments, the first Fc region comprises a first modification and the second Fc region comprises a second modification, wherein the first modification and the second modification comprise amino acid substitutions at positions selected from any one of the following groups: 1) first modification: Y349C and T366W; second modification: D356C, T366S, L368A, Y407V, and F405K; 2) first modification: Y349C, T366W, and F405K; second modification: D356C, T366S, L368A, and Y407V; 3) first modification: Y349C, T366W, and K409E; second modification: D356C, T366S, L368A, Y407V, and F405K; 4) first modification: Y349C, T366W, and K409E; second modification: D356C, T366S, L368A, Y407V, and F405K. 366W and K409A; second modification: D356C, T366S, L368A, Y407V and F405K; 5) first modification: Y349C, T366W, F405K, K360E and Q347E; second modification: D356C, T366S, L368A, Y407V and Q347R; 6) first modification: Y349C, T366W, F405K and Q347R; second modification: D356C, T366S, L368A, Y407V, K360E and Q347E; 7) first modification: Y349C, T366W, K409A, K360E and Q347E; second modification: D356C, T366S, L368 A, Y407V, F405K and Q347R; 8) First modification: Y349C, T366W, K409A and Q347R; Second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) First modification: T366W, K409A and K392D; Second modification: T366S, L368A, Y407V, D399S and F405K; 10) First modification: T366W and K409A; Second modification: T366S, L368G, Y407A and F405K; 11) First modification: T366W, K409A and Y349D; Second modification: T366S, L3 68A, Y407V, F405K and E357A; 12) First modification: T366W, K409A, Y349D and S354D; Second modification: T366S, L368A, Y407V, F405K and E357A; 13) First modification: T366W and F405K; Second modification: T366S, L368A, Y407V and K409A; 14) First modification: T366W, F405K and D399S; Second modification: T366S, L368A, Y407V, K409A and K392D; 15) First modification: T366W and F405K; Second modification: T366S, L368G, Y407A and K409A;16) First modification: T366W, F405K, and Y349D; second modification: T366S, L368A, Y407V, K409A, and E357A; 17) First modification: T366W, F405K, Y349D, and S354D; second modification: T366S, L368A, Y407V, K409A, and E357A; wherein the amino acid positions are determined according to the EU index with KABAT numbering.

[0046] In some embodiments, the first Fc region comprises a first modification and the second Fc region comprises a second modification, the first modification comprises amino acid substitutions T366W and K409A, and the second modification comprises amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0047] In some embodiments, the targeting moiety specifically binds to EGFR, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 101, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 102, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 103.

[0048] In some embodiments, the targeting moiety specifically binds to EGFR, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:104.

[0049] In some embodiments, the targeting moiety specifically binds EGFR and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:37.

[0050] In some embodiments, the targeting moiety specifically binds to EGFR, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 105, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 106, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 107.

[0051] In some embodiments, the targeting moiety specifically binds to EGFR, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:108.

[0052] In some embodiments, the targeting moiety specifically binds EGFR and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:39.

[0053] In some embodiments, the targeting portion specifically binds to an EGFR mutant, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 109, CDR2 is set forth in SEQ ID NO: 110, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 111.

[0054] In some embodiments, the targeting moiety specifically binds to the EGFR mutant, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:112.

[0055] In some embodiments, the targeting moiety specifically binds to an EGFR mutant, and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:53.

[0056] In some embodiments, the targeting moiety specifically binds to the EGFR mutant, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 113, CDR2 is set forth in SEQ ID NO: 114, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 115.

[0057] In some embodiments, the targeting moiety specifically binds to an EGFR mutant, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:116.

[0058] In some embodiments, the targeting moiety specifically binds to an EGFR mutant, and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:55.

[0059] In some embodiments, the targeting moiety specifically binds to HER2 / neu, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 117 and SEQ ID NO: 125, the amino acid sequence of CDR2 is selected from SEQ ID NO: 118 and SEQ ID NO: 126, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 119 and SEQ ID NO: 127.

[0060] In some embodiments, the targeting moiety specifically binds to HER2 / neu, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 120 and SEQ ID NO: 128.

[0061] In some embodiments, the targeting moiety specifically binds HER2 / neu and the amino acid sequence of the light chain of the first member is selected from the group consisting of SEQ ID NO:45 and SEQ ID NO:49.

[0062] In some embodiments, the targeting moiety specifically binds to HER2 / neu, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 121 and SEQ ID NO: 129, the amino acid sequence of CDR2 is selected from SEQ ID NO: 122 and SEQ ID NO: 130, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 123 and SEQ ID NO: 131.

[0063] In some embodiments, the targeting moiety specifically binds to HER2 / neu, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 124 and SEQ ID NO: 132.

[0064] In some embodiments, the targeting moiety specifically binds HER2 / neu and the amino acid sequence of the heavy chain of the first member is selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:51.

[0065] In some embodiments, the targeting moiety specifically binds GPC3, the antibody light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 133, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 134, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 135.

[0066] In some embodiments, the targeting moiety specifically binds GPC3, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:136.

[0067] In some embodiments, the targeting moiety specifically binds GPC3 and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:57.

[0068] In some embodiments, the targeting moiety specifically binds to GPC3, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 137, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 138, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 139.

[0069] In some embodiments, the targeting moiety specifically binds to GPC3, the antibody heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:140.

[0070] In some embodiments, the targeting moiety specifically binds GPC3 and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:59.

[0071] In some embodiments, the targeting moiety specifically binds FAP, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 141, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 142, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 143.

[0072] In some embodiments, the targeting moiety specifically binds FAP, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:144.

[0073] In some embodiments, the targeting moiety specifically binds FAP and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:61.

[0074] In some embodiments, the targeting moiety specifically binds FAP, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 145, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 146, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 147.

[0075] In some embodiments, the targeting moiety specifically binds to FAP, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:148.

[0076] In some embodiments, the targeting moiety specifically binds FAP and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:63.

[0077] In some embodiments, the targeting moiety specifically binds to Muc1, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 149, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 150, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 151.

[0078] In some embodiments, the targeting moiety specifically binds to Mucl, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:152.

[0079] In some embodiments, the targeting moiety specifically binds to Mucl and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:65.

[0080] In some embodiments, the targeting moiety specifically binds to Muc1, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 153, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 154, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 155.

[0081] In some embodiments, the targeting moiety specifically binds to Mucl, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:156.

[0082] In some embodiments, the targeting moiety specifically binds to Mucl and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:67.

[0083] In some embodiments, the targeting moiety specifically binds mesothelin, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 165, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 166, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 167.

[0084] In some embodiments, the targeting moiety specifically binds mesothelin, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:168.

[0085] In some embodiments, the targeting moiety specifically binds mesothelin and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:73.

[0086] In some embodiments, the targeting portion specifically binds to mesothelin, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 169, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 170, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 171.

[0087] In some embodiments, the targeting moiety specifically binds mesothelin, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:172.

[0088] In some embodiments, the targeting moiety specifically binds mesothelin and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:75.

[0089] In some embodiments, the targeting moiety specifically binds to MUC5AC, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 157, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 158, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 159.

[0090] In some embodiments, the targeting moiety specifically binds to MUC5AC, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:160.

[0091] In some embodiments, the targeting moiety specifically binds MUC5AC and the amino acid sequence of the light chain of the first member is as set forth in SEQ ID NO:69.

[0092] In some embodiments, the targeting moiety specifically binds to MUC5AC, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 161, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 162, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 163.

[0093] In some embodiments, the targeting moiety specifically binds to MUC5AC, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:164.

[0094] In some embodiments, the targeting moiety specifically binds to MUC5AC and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:71.

[0095] In some embodiments, in the heavy chain of the first member, the amino acid sequence of the first Fc region is selected from SEQ ID NO: 1, 4, 5, 6, 7, 9, 11, 13, 15, 17, 19, 21, 22, 24, 26, 27 and 29.

[0096] In some embodiments, the amino acid sequence of the immunomodulator contained in the second member is selected from SEQ ID NOs: 173-180.

[0097] In some embodiments, the amino acid sequence of the second Fc region comprised in the second member is selected from SEQ ID NOs: 2, 3, 8, 10, 12, 14, 16, 18, 20, 23, 25, and 28.

[0098] In some embodiments, the amino acid sequence of the polypeptide contained in the second member is selected from SEQ ID NOs: 77, 80, 82, 84, 86, 89, 91, and 97.

[0099] In some embodiments, the amino acid sequence of the light chain contained in the first member is selected from SEQ ID NO: 37, 45, 49, 53, 57, 61, 65, 69 and 73, the amino acid sequence of the heavy chain contained in the first member is selected from SEQ ID NO: 39, 47, 51, 55, 59, 63, 67, 71 and 75, and the amino acid sequence of the polypeptide contained in the second member is selected from SEQ ID NO: 77, 80, 82, 84, 86, 89, 91 and 97.

[0100] In some embodiments, the knob and pore modifications comprise a knob modification and a pore modification, wherein the knob modification comprises amino acid substitutions Y349C and T366W, and the pore modification comprises amino acid substitutions D356C, T366S, L368A, and Y407V, wherein the positions of the amino acids are determined according to the EU index with KABAT numbering. In some cases, the knob modification is contained in a first Fc region and the pore modification is contained in a second Fc region. In some cases, the knob modification is contained in a second Fc region and the pore modification is contained in a first Fc region.

[0101] In some embodiments, the medicament includes a pharmaceutical composition comprising the protein heterodimer described herein and a pharmaceutically acceptable carrier.

[0102] In some embodiments, the medicament comprises a nucleic acid molecule encoding a protein heterodimer described herein.

[0103] In some embodiments, the medicament comprises a vector comprising the nucleic acid molecule.

[0104] In some embodiments, the medicament comprises a cell comprising the nucleic acid molecule or the vector.

[0105] In some embodiments, the drug includes a protein mixture comprising: 1) a protein heterodimer described herein; and 2) a first homodimer formed by two of the first members of the protein heterodimer; 3) a second homodimer formed by two of the second members of the protein heterodimer; wherein the percentage of the protein heterodimer in the protein mixture is at least 50%.

[0106] In some embodiments, the patient suffering from the malignant effusion comprises a tumor patient.

[0107] In some embodiments, the patient has symptoms including fatigue, vomiting, dorsal edema, loss of appetite, dyspnea, cough, chest pain, and / or abdominal pain.

[0108] In some embodiments, the tumor comprises a solid tumor and / or a non-solid tumor.

[0109] In some embodiments, the tumor comprises lung, lymphoid, liver, stomach, intestinal, breast, pancreatic, ovarian, and / or mesothelial carcinoma.

[0110] In some embodiments, the malignant effusion comprises malignant cells.

[0111] In some embodiments, the malignant effusion comprises blood and / or exudate.

[0112] In some embodiments, the malignant effusion is associated with metastasis of the tumor to the pleura.

[0113] In some embodiments, the malignant effusion is associated with metastasis of the tumor to the peritoneum.

[0114] In some embodiments, the malignant effusion arises from a tumor comprising a primary tumor of the lung, lymphatic, liver, stomach, colon, breast, ovary, pancreas, bile duct, esophagus, brain, nose, and / or prostate.

[0115] In some embodiments, the malignant effusion is caused by metastasis to the thoracic cavity of a primary cancer including lung, lymphatic, liver, stomach, intestine, breast, ovarian, and / or mesothelioma.

[0116] In some embodiments, the malignant effusion is caused by metastasis to the peritoneal cavity of a tumor including: a primary cancer of the ovary, pancreas, bile duct, esophagus, sarcoma, and / or liver.

[0117] In some embodiments, the malignant effusion comprises malignant pleural effusion (MPE) and / or malignant ascites.

[0118] In some embodiments, the malignant pleural effusion is associated with non-small cell lung cancer.

[0119] In some embodiments, the drug is formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous depot.

[0120] In some embodiments, the medicament is configured to include a drainage device.

[0121] In some embodiments, the drug is capable of inhibiting tumor or tumor cell growth.

[0122] On the other hand, the present application also provides a method for constructing an animal model with malignant effusion, the method comprising injecting tumor cells into the thoracic cavity and / or abdominal cavity of the animal.

[0123] In some embodiments, the injection dose is about 1×10 5 to about 2×10 6 cells.

[0124] In some embodiments, the animal comprises a mouse.

[0125] In some embodiments, the injection comprises intraperitoneal injection, intraperitoneal injection and / or intrathoracic injection.

[0126] In some embodiments, the tumor cells are derived from solid tumors and / or non-solid tumors.

[0127] In some embodiments, the tumor comprises lung, lymphoid, liver, stomach, intestine, breast, pancreas, sarcoma, ovarian and / or mesothelial carcinoma.

[0128] In some embodiments, the tumor cells include H22-Luc cells, B16-EGFR cells, MC38-EGFR cells, Pan02 mouse pancreatic cancer cells, S180 mouse sarcoma cells and / or Lewis-luc cells.

[0129] In some embodiments, the tumor cells are capable of causing a tumor in the animal.

[0130] In some embodiments, the tumor metastasizes to the pleura and / or peritoneum of the animal and produces the malignant effusion.

[0131] In some embodiments, the malignant effusion comprises malignant cells.

[0132] In some embodiments, the malignant effusion comprises blood and / or exudate.

[0133] In some embodiments, the malignant effusion comprises malignant pleural effusion (MPE) and / or malignant ascites.

[0134] In some embodiments, the malignant pleural effusion is associated with non-small cell lung cancer.

[0135] The present application also provides tissues or organs or cultures thereof in an animal model with malignant effusion prepared by the above method.

[0136] The present application also provides an animal model with malignant effusion prepared by the above method or its offspring.

[0137] The present application also provides the use of the above-mentioned tissues or organs or their cultures, the animal models with malignant effusions or their offspring in the screening, verification, evaluation and / or research of anti-tumor drugs, or as model systems for pharmacological, immunological, microbiological and medical research.

[0138] Other aspects and advantages of the present application will become apparent to those skilled in the art from the detailed description below, wherein only illustrative embodiments of the present application are shown and described. It will be appreciated that the present application is capable of other and different embodiments, and that its several details are capable of modification in various obvious respects, all without departing from the present application. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0139] Incorporated by Reference

[0140] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] The novel features of the present application are particularly set forth in the appended claims. The features and advantages of the present application may be better understood by referring to the following detailed description taken in conjunction with the accompanying drawings, which set forth illustrative embodiments in which the principles of the present application are utilized:

[0142] Figure 1 shows an example of a protein heterodimer of the present application;

[0143] Figures 2A to 2C show the results of SDS-PAGE analysis of the formation of protein heterodimers of the present application;

[0144] FIG3 shows the survival rate of the H22-Luc mouse ascites model of liver cancer malignant ascites treated with the protein heterodimer of the present application;

[0145] Figures 4A and 4B show malignant abdominal effusion and in vivo imaging data of the H22-Luc mouse ascites model of liver cancer after inoculation in this application;

[0146] Figures 5A and 5B show the protein heterodimers of the present application for treating malignant abdominal effusion of liver cancer in the H22-Luc mouse ascites model and in vivo imaging data;

[0147] Figures 6A and 6B show the effects of the protein heterodimer of the present application on the treatment of malignant pleural effusion and body weight in Lewis mouse lung cancer model;

[0148] FIG7 shows the results of the protein heterodimer of the present application in treating malignant pleural effusion and the number of metastatic lesions in the Lewis mouse lung cancer model. DETAILED DESCRIPTION

[0149] Before describing the embodiments of the present application, it should be understood that such embodiments are provided by way of example only, and that various alternatives of the embodiments of the present application described herein may be adopted when practicing the present application. Many modifications, changes, and substitutions will occur to those skilled in the art without departing from the present application.

[0150] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which this application belongs. Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of this application, suitable methods and materials are described below. In the event of conflict, the patent specification and its definition shall prevail. In addition, materials, methods and embodiments are merely illustrative and are not intended to be limiting. Without departing from the present application, those skilled in the art will appreciate that various modifications, changes and substitutions will be appreciated.

[0151] As used herein, the singular forms "a," "an," and "the" generally include the plural forms as well, unless the context clearly indicates otherwise.

[0152] As used herein, the term "proteinaceous" generally refers to a substance or molecule that is related to, similar to, or is a polypeptide or protein. For example, the protein heterodimer of the present application can be a heterodimeric protein, or a heterodimer comprising two or more polypeptides.

[0153] As used herein, the term "heterodimer" generally refers to a molecule (e.g., a protein molecule) composed of two different members. The two members of a heterodimer may differ in structure, function, activity, and / or composition. For example, the two different members may comprise polypeptides that differ in the order, number, or type of amino acid residues that form these polypeptides. Each of the two different members of a heterodimer may independently comprise one, two, or more units, polypeptide chains, or parts.

[0154] As used herein, the term "targeting moiety" generally refers to a molecule, complex or aggregate that specifically, selectively or preferentially binds to a target molecule, cell, particle, tissue or aggregate. For example, a targeting moiety can be an antibody, an antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecule or compound. Other examples of targeting moieties may include, but are not limited to, aptamers, high-affinity polymers (avimers), receptor binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, etc. The terms "targeting moiety" and "binding moiety" are used interchangeably herein.

[0155] As used herein, the term "tumor antigen" generally refers to an antigenic substance produced in or by a tumor cell that may have the ability to trigger an immune response in a host. For example, a tumor antigen may be a protein, polypeptide, peptide, or fragment thereof that constitutes a part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. Tumor antigen peptides may be peptides produced due to degradation of tumor antigens in tumor cells, and may induce or activate tumor-specific cytotoxic T lymphocytes after expression on the cell surface by binding to HLA molecules. In some embodiments, the term "tumor antigen" may also refer to a biomolecule (such as a protein, carbohydrate, glycoprotein, etc.) that is specifically or preferentially or differentially expressed and / or found to be associated with a cancer cell on a cancer cell, thereby providing a preferential or specific target for cancer. For example, preferential expression may be preferential expression compared to any other cell in an organism, or may be preferentially expressed in a specific region of an organism (such as in a specific organ or tissue).

[0156] The terms "tumor antigen epitope" and "tumor antigen determinant" are used interchangeably herein and generally refer to sites of amino acid sequences present in tumor antigens that can induce tumor-specific cytotoxic T lymphocytes.

[0157] The terms "immunoregulator" and "immunomodulator" are used interchangeably herein and generally refer to substances that affect immune system function. Immunoregulators can enhance or reduce immune responses. For example, immunoregulators can be the active agents of immunotherapy, including but not limited to, for example, cytokines from bacteria, chemokines, interleukins, cytosine phosphate-guanosine (CpG) oligodeoxynucleotides and dextran, granulocyte colony stimulating factor (G-CSF), interferon, imiquimod, cell membrane fractions, recombinant, synthetic and / or natural preparations. In some instances, the immunoregulator is a cytokine. In some cases, the immunoregulator is not an antibody or an antigen binding fragment thereof. In some cases, the immunoregulator is not an immunoglobulin molecule or a fragment thereof (such as an antigen binding fragment).

[0158] In some embodiments, the immunomodulator is selected from interferons, interleukins, chemokines, lymphokines, and tumor necrosis factors. For example, the immunomodulator can be selected from interferon alpha, interferon lambda, interferon beta, interleukin 10, interleukin 2, and super interleukin 2.

[0159] The term "expression yield" as used herein in the context of protein heterodimers generally refers to the amount of protein heterodimer produced in a functional form upon expression, eg, when expressed by a host cell.

[0160] As used herein, the term "dimerization sequence" generally refers to an amino acid sequence that can form a dimer or undergo dimerization. In some embodiments, a dimer is a heterodimer formed by two different members. In some cases, the two different members of a heterodimer can comprise different dimerization sequences.

[0161] As used herein, the term "heterodimerization" generally refers to the process of forming a heterodimer between two different members (e.g., two different polypeptides), such as by complexation, association, or aggregation, with or without the formation of a covalent bond between the two or more members.

[0162] As used herein, the term "covalent bond" generally refers to a chemical bond formed between atoms by the sharing of electrons. For example, a covalent bond can be polar or non-polar. In some embodiments, the covalent bond is a disulfide bond.

[0163] As used herein, the term "non-covalent pair-wise affinity" generally refers to dimerization sequences or heterodimerization sequences that are capable of binding to each other through non-covalent interactions, such as via ion pairs, hydrogen bonds, dipole-dipole interactions, charge transfer interactions, π-π interactions, cation-π electron interactions, van der Waals interactions and dispersion interactions, hydrophobic (lipophilic) interactions, complex formation (e.g., complex formation with transition metal cations), or a combination of these interactions.

[0164] As used herein, the term "linker" generally refers to a link or a connection between two polypeptide sequences, such as a synthetic amino acid sequence connecting two polypeptide domains. A linker can connect two amino acid sequences via a peptide bond. In some embodiments, the linker of the present application connects a biologically active portion to a second portion in a linear sequence.

[0165] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation with a labeling component). The term can apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term can also include variants on the traditional peptide bond that connects the amino acids that make up the polypeptide. For example, "peptide," "polypeptide," and "protein" can be chains of amino acids whose alpha carbons are linked by peptide bonds. Thus, the terminal amino acid at one end of the chain (the amino terminus) can have a free amino group, while the terminal amino acid at the other end of the chain (the carboxyl terminus) can have a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) generally refers to the free alpha-amino group on the amino acid at the amino terminus of a peptide or the alpha-amino group (imino group when involved in the alpha terminus) of an amino acid at any other position within the peptide. Similarly, the term "carboxyl terminus" generally refers to the free carboxyl group at the carboxyl terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides may also comprise essentially any polyamino acid, including but not limited to peptide mimetics, such as amino acids linked by ether rather than amide bonds.

[0166] As used herein, the term "amino acid" generally refers to natural and / or unnatural or synthetic amino acids, including but not limited to D or L optical isomers or both, amino acid analogs and peptidomimetics. Standard single-letter or three-letter codes are used to designate amino acids.

[0167] As used herein, the term "natural L-amino acids" generally refers to glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine ​​(C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).

[0168] As used herein, the term "non-naturally occurring" generally refers to a polypeptide or polynucleotide sequence that does not correspond to, is not complementary to, or does not have a high degree of homology to a wild-type or naturally occurring sequence (e.g., a sequence found in a subject). For example, when compared to a native sequence, a non-naturally occurring polypeptide or fragment can share less than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity when properly aligned. Alternatively, when compared to a native sequence, a non-naturally occurring polypeptide or fragment can share more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even more amino acid sequence identity when properly aligned.

[0169] As used herein, the terms "hydrophilic" and "hydrophobic" generally refer to the affinity of a substance for water. Hydrophilic substances have a strong affinity for water and tend to dissolve, mix, or be wetted by water, while hydrophobic substances essentially lack affinity for water, tend to repel and not absorb water, and tend to be insoluble in water or mix with or be wetted by water. Amino acids can be characterized based on their hydrophobicity. Many scales have been developed. One example is a scale developed by Levitt, M ​​et al., J Mol Biol (1976) 104:59, which is listed in Hopp, TP et al., Proc Natl Acad Sci USA (1981) 78:3824. Examples of "hydrophilic amino acids" are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartic acid, glutamic acid, and serine, as well as glycine. Examples of "hydrophobic amino acids" are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine and valine.

[0170] When used in the context of proteinaceous molecules (eg, polypeptides or proteins), the term "fragment" generally refers to a truncated form of a native biologically active protein that may or may not retain a portion of the therapeutic and / or biological activity.

[0171] When used in the context of a protein molecule (e.g., a polypeptide or protein), the term "variant" generally refers to a protein molecule having sequence homology to a native biologically active protein that retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, the variant protein may share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared to a control biologically active protein. In some embodiments, a "variant" may include a deliberately modified protein, such as one modified by site-directed mutagenesis, synthesis of an encoding gene, insertion, or accidental mutation.

[0172] The terms "conjugated," "linked," "fused," and "fusion" are used interchangeably herein and generally refer to the joining together of two or more chemical elements, sequences, or components, for example, by means including chemical conjugation or recombination. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are continuous and in the reading segment or frame. "In-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a continuous, longer ORF in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting "fusion polypeptide" is a single protein comprising two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not typically so joined in nature). A "fusion site" refers to the sequence where two or more segments are joined together. In some cases, a fusion site can be a sequence that is identical to a sequence in the two or more segments to be joined. In some cases, the fusion site can further comprise a gap segment that is not identical in sequence to either of the two or more segments being joined.

[0173] In the context of polypeptides, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide in the amino to carboxyl terminus direction, wherein the residues adjacent to each other in the sequence are contiguous in the primary structure of the polypeptide. A "partial sequence" is a linear sequence that forms a portion of a polypeptide, which linear sequence is known to contain additional residues in one or both directions.

[0174] The terms "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (or locus) defined according to linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation to a labeling component.

[0175] The terms "gene" and "gene fragment" are used interchangeably herein and generally refer to a polynucleotide comprising at least one open reading frame that is capable of encoding a specific protein after transcription and translation. The gene or gene fragment can be genomic or cDNA, and as long as the polynucleotide comprises at least one open reading frame, it can cover the entire coding region or a fragment thereof. A "fusion gene" is a gene composed of at least two heterologous polynucleotides linked together.

[0176] As used herein, the term "antibody" generally refers to a protein comprising one or more polypeptides that are substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Immunoglobulin genes may include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. As used herein, light chains can be classified as κ or λ. Heavy chains can be classified as γ, μ, α, δ, or ε, which define immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Antibodies as used in this application can have a structural unit comprising a tetramer. Each tetramer can be composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25KD) and a "heavy" chain (about 50-70KD). The N-terminus of each chain can define a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. As used herein, the terms "light chain variable region" (VL) and "heavy chain variable region" (VH) generally refer to these regions of the light chain and heavy chain, respectively. Antibodies can exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases or expressed de novo. Thus, for example, pepsin can digest an antibody below the disulfide bonds in the hinge region to produce F(ab)'2 (a dimer of Fab, which itself is a light chain connected to VH-CH1 by a disulfide bond). F(ab)'2 can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab with a partial hinge region (see Fundamental Immunology, ed. W.E. Paul, Raven Press, New York (1993) for a more detailed description of other antibody fragments). Although various antibody fragments are defined based on digestion of intact antibodies, those of ordinary skill in the art will appreciate that such Fab' fragments can be synthesized de novo chemically or using recombinant DNA methods. Therefore, the term antibody as used herein may also include antibody fragments synthesized de novo by modifying whole antibodies or using recombinant DNA methods, including but not limited to Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nanobodies, monobodies and diabodies. In some embodiments, antibodies include but are not limited to Fab'2, IgG, IgM, IgA, IgE and single-chain antibodies, for example, single-chain Fv (scFv) antibodies in which the variable heavy chain and the variable light chain are linked together (directly or through a peptide linker) to form a continuous polypeptide.

[0177] As used herein, the term "antigen binding site" or "binding portion" generally refers to a part of an antibody that participates in antigen binding. The antigen binding site can be formed by amino acid residues of the N-terminal variable ("V") region of the heavy ("H") chain and / or light ("L") chain. The three highly divergent stretches within the V region of the heavy and light chains are called "hypervariable regions," which are located between more conserved flanking segments, called "framework regions" or "FRs." Therefore, the term "FR" as used herein generally refers to the amino acid sequence between and near the hypervariable regions naturally present in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form a "surface" for antigen binding. This surface can mediate the recognition and binding of the target antigen. The three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs" and are characterized, for example, in Kabat et al., Sequences of proteins of immunological interest, 4th ed., U.S. Department of Health and Human Services, Public Health Service, Bethesda, MD (1987).

[0178] In some embodiments, the antibody used herein and its fragment can be bispecific. Bispecific antibodies or its fragment can have various configurations. For example, bispecific antibodies can be similar to single antibodies (or antibody fragments), but have two different antigen binding sites (variable regions). In various embodiments, bispecific antibodies can be by chemical technology (Kranz et al. (1981) Proc. Natl. Acad. Sci, U.S., 78: 5807), by " polyoma (polyoma) " technology (see, for example, U.S. Patent number 4,474,893), or by recombinant DNA technology. In some embodiments, bispecific antibodies used herein can have binding specificity for at least two different epitopes, at least one of which is a tumor antigen. In some embodiments, antibodies and their fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies or antibody binding fragments (such as Fab) linked together, and each antibody or fragment has different specificities.

[0179] As used herein, the terms "homology," "homologous" or "sequence identity" generally refer to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When determining sequence identity, similarity or homology between two different amino acid sequences using a program (e.g., Emboss Needle or BestFit), the default settings can be used, or an appropriate scoring matrix (such as blosum45 or blosum80) can be selected to optimize identity, similarity or homology scores. In some embodiments, homologous polynucleotides are those that hybridize under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or even 100% sequence identity compared to these sequences. When sequences having a considerable length are optimally aligned, homologous polypeptides have at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% sequence identity.

[0180] The terms "percent identity" and "% identity" used in the context of polynucleotide sequences generally refer to the percentage of residue matches between at least two polynucleotide sequences compared using a standardized algorithm. Such an algorithm can insert spaces in the compared sequences in a standardized and reproducible manner to optimize the comparison between the two sequences, thereby achieving a more meaningful comparison of the two sequences. Percent identity can be measured over the length of the entire limited polynucleotide sequence, or can be measured over a shorter length, for example, over the length of a fragment obtained from a larger limited polynucleotide sequence. It should be understood that any fragment length supported by the sequences shown in this paper, table, accompanying drawings, or sequence listing can be used to describe the length over which percent identity can be measured.

[0181] The term "percentage (%) of sequence identity" as used in the context of the polypeptide sequences identified herein generally refers to the percentage of amino acid residues in a query sequence that are identical to the amino acid residues in a second reference polypeptide sequence, after aligning the sequences and introducing gaps, partial sequences or portions thereof may be used, if desired, to achieve maximum percentage sequence identity, and any conservative substitutions are not considered to be part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. Percentage identity can be measured over the length of the entire determined polypeptide sequence, or can be measured over a shorter length, for example, over the length of a fragment obtained from a larger determined polypeptide sequence. It should be understood that any fragment length supported by the sequences shown in this article, table, accompanying drawings or sequence listing can be used to describe the length over which percentage identity can be measured.

[0182] As used herein, the term "host cell" generally includes individual cells, cell lines or cell cultures, which can be or have been the recipient of the application's plasmid or vector, polynucleotides comprising the application, protein heterodimers (such as heterodimeric proteins) expressed herein. Host cells can include the offspring of a single host cell. Due to natural, accidental or deliberate mutations, offspring are not necessarily identical (morphologically or on the genome of the total DNA complement) with the original parent cell. Host cells can include cells transfected with the application's vector in vitro. Host cells can be bacterial cells (such as Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or myeloma cells. In some embodiments, host cells are mammalian cells. In some embodiments, mammalian cells are HEK293 cells.

[0183] As used herein, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The term can include a vector primarily used for the function of inserting DNA or RNA into cells, a vector primarily used for the replication of the function of DNA or RNA replication, and an expression vector for the transcription and / or translation of the DNA or RNA. Also included are vectors that provide one of the above functions." expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. "Expression system" generally refers to a suitable host cell comprising an expression vector that can function to produce a desired expression product.

[0184] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition (e.g., a protein heterodimer as described herein) sufficient to achieve the intended application, including but not limited to disease treatment. The therapeutically effective amount can vary depending on the intended application (e.g., in vitro or in vivo) or the subject and the disease condition being treated (e.g., the subject's weight and age, the severity of the disease condition, the treatment regimen). One of ordinary skill in the art can readily determine the mode of administration, etc. The term can also apply to doses that will induce a specific response in the target cell, such as targeted gene induction, proliferation and / or apoptosis. The specific dosage will vary depending on the specific compound selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the time of administration, the tissue to be administered, and the physical delivery system in which it is administered.

[0185] The terms "treatment" or "treating" or "palliating" or "ameliorating" are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. As used herein, therapeutic benefit generally refers to the eradication or reduction in severity of the underlying disease being treated. Likewise, therapeutic benefit is obtained by eliminating, reducing the severity of, or reducing the incidence of one or more physiological symptoms associated with the underlying disease, such that an improvement is observed in the subject, even though the subject may still be suffering from the underlying condition. For prophylactic benefit, the composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

[0186] As used herein, the term "therapeutic effect" generally includes therapeutic benefits and / or prophylactic benefits as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof.

[0187] As used herein, the terms "co-administered," "administered in conjunction with," and their grammatical equivalents generally include administering two or more agents to an animal such that the agents and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0188] The terms "antagonist" and "inhibitor" are used interchangeably herein and generally refer to compounds that inhibit the biological function of a target protein by inhibiting the activity or expression of the target protein. Thus, the terms "antagonist" and "inhibitor" are defined in the context of the biological effects of the target protein. Although preferred antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit the biological activity of the target protein by interacting with other members of the signal transduction pathway of which the target protein is a member are also explicitly included in the definition. The preferred biological activity inhibited by the antagonist is related to the occurrence, growth, or spread of a tumor.

[0189] As used herein, the term "agonist" generally refers to a compound that has the ability to initiate or enhance the biological function of a target protein by inhibiting or enhancing the activity or expression of the target protein. Thus, the term "agonist" is defined in the context of the biological effect of the target polypeptide. Although preferred agonists herein specifically interact with (e.g., bind to) the target, compounds that initiate or enhance the biological activity of the target polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are also specifically included in this definition.

[0190] As used herein, the term "agent" or "bioactive agent" generally refers to a biological, pharmaceutical or chemical compound or other moiety. Non-limiting examples include simple or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, vitamin derivatives, carbohydrates, toxins or chemotherapeutic compounds. Various compounds can be synthesized, such as small molecules and oligomers (e.g., oligopeptides and oligonucleotides), as well as synthetic organic compounds based on various core structures. In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, etc.

[0191] As used herein, the term "anticancer agent," "antitumor agent," or "chemotherapeutic agent" generally refers to any agent useful in treating a neoplastic disease. One class of anticancer agents includes chemotherapeutic agents.

[0192] As used herein, the term "chemotherapy" generally refers to the administration of one or more chemotherapeutic drugs and / or other agents to a cancer patient by various methods including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal, or inhalation or suppository form.

[0193] As used herein, the term "cell proliferation" generally refers to a phenomenon in which the number of cells changes due to division. For example, cell proliferation can result in an increase in the number of cells. The term also includes cell growth, by which the cell morphology has changed (e.g., increased in size) in accordance with a proliferation signal.

[0194] As used herein, the term "in vivo" generally refers to events that occur within the body of a subject.

[0195] As used herein, the term "in vitro" generally refers to an event that occurs outside the body of a subject. For example, an in vitro assay encompasses any assay performed outside of a subject. In vitro assays include cell-based assays using dead or live cells. In vitro assays also include cell-free assays, in which intact cells are not used.

[0196] As used herein, the term "interferon" (IFN) generally refers to a signaling protein produced and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. There are three main types of interferons, namely type I, type II, and type III, wherein type I interferons may include IFN-α and IFN-β, and IFN-α may further include IFN-α subtypes, such as IFN-α2, IFN-α4, etc. Type I interferons can inhibit viral replication, have antiparasitic activity, inhibit cell proliferation, stimulate the cytotoxic activity of immune cells, participate in immunomodulation, and show anti-tumor effects. Type II and type III interferons may include IFN-γ, IFN-λ1 (IL-29), IFN-λ2 (IL-28a), and IFN-λ3 (IL-28b). As used herein, the term "interferon" may include full-length interferon or a fragment thereof (e.g., a truncated form) or a variant thereof, substantially maintaining the biological activity of the corresponding wild-type interferon (e.g., at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even at least 100% of the biological activity of the corresponding wild-type interferon). As used herein, interferon can be from any mammal. In some embodiments, interferon is selected from humans, horses, cows, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, and non-human primates.

[0197] As used herein, the term "interleukin" generally refers to a secretory protein or signaling molecule that can promote the development and differentiation of T and / or B lymphocytes and / or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes as well as monocytes, macrophages and endothelial cells. As used herein, interleukin (IL) may include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL- 18. IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and / or IL-36. As used herein, the term "interleukin" can include full-length interleukins, or fragments (e.g., truncated forms) or variants thereof that substantially maintain the biological activity of the corresponding wild-type interleukin (e.g., having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or even at least 100% of the biological activity of the corresponding wild-type interleukin. The interleukins used herein can be from any mammal. In some embodiments, the interleukin is selected from humans, horses, cows, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, and non-human primates. In some embodiments, the interleukin can be a mutant form, for example, with increased or decreased affinity for its receptor. In a specific embodiment, the interleukin can be super IL-2 (also known as sIL2, see Nature 484, 529-533, April 26, 2012), which can be obtained by modifying IL-2 to increase its binding affinity to IL-2Rβ. Mutations in sIL-2 are primarily located in the cytokine core. Molecular dynamics simulations indicate that these evolved mutations stabilize IL-2, reducing the flexibility of the IL-2Rβ binding site helix, leading to a conformation similar to the receptor's optimal conformation when bound to CD25. Compared to IL-2, sIL-2 induces superior expansion of cytotoxic T cells, leading to enhanced anti-tumor responses in vivo, proportionally reducing the expansion of T regulatory cells, and reducing pulmonary edema.

[0198] As used herein, the term "anti-HER2 / neu antibody" generally refers to an antibody that specifically or preferentially binds to the HER2 / neu receptor. For example, the anti-HER2 / neu antibody or anti-HER2 antibody can be trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0199] As used herein, the term "anti-EGFR antibody" generally refers to an antibody that specifically or preferentially binds to EGFR. In some cases, the anti-EGFR antibody can bind to a mutant form of EGFR (e.g., EGFR variant III (also known as EGFRvIII), which is the most common extracellular domain mutation of EGFR, which results in the deletion of exons 2–7 of the EGFR gene and is characterized by a truncated extracellular domain with ligand-independent constitutive activity. For example, the anti-EGFR antibody can be cetuximab, Mab806, or an antigen-binding fragment thereof.

[0200] As used herein, the term "subject" generally refers to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, goat, rabbit, mouse, rat, or monkey.

[0201] As used herein, the term "anti-EGFR family antibody" generally refers to an antibody that specifically binds to a member of the epidermal growth factor receptor family. For example, it can be an antibody that specifically binds to ErbB-1 (also known as epidermal growth factor receptor (EGFR)), ErbB-2 (also known as HER2 in humans and neu in rodents), ErbB-3 (also known as (e.g., HER3) and / or ErbB-4 (also known as HER4). Examples of anti-EGFR family antibodies include, but are not limited to, one or more of the following antibodies: C6.5, C6mL3-9, C6MH3-B1, C6-B1D2, F5, HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12, EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8, HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8 and HER4.C7, etc., see also, for example, U.S. Patent Publications US2006 / 0099205A1 and US2004 / 0071696A1, which are incorporated herein by reference.

[0202] As used herein, the term "single-chain Fv" ("sFv" or "scFv") polypeptide generally refers to a covalently linked VH (heavy chain variable region): VL (light chain variable region) heterodimer, which can be expressed as a nucleic acid comprising the VH and VL coding sequences linked directly or through a peptide-encoded linker. (See Huston et al., Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988)).

[0203] When used in conjunction with cancer cells, the term "inhibit growth and / or proliferation" generally refers to a decrease in the growth rate and / or proliferation rate of cancer cells. For example, this can include the death of cancer cells (e.g., by apoptosis). In some embodiments, the term can also refer to inhibiting the growth and / or proliferation of solid tumors and / or inducing a decrease in tumor size or elimination.

[0204] As used herein, the term "cancer cell surface marker" or "cancer cell-associated marker" generally refers to a biological molecule, such as a protein, carbohydrate, glycoprotein, etc., that is exclusively, preferentially, or differentially expressed on cancer cells and / or is found to be associated with cancer cells and thus provides a preferential or specific target for cancer. In some embodiments, preferential expression can be preferential expression in a particular region of an organism (e.g., within a particular organ or tissue) compared to any other cell in the organism.

[0205] As used herein, the term "member" generally refers to a polypeptide, subunit, or portion that is a component of a protein heterodimer.

[0206] As used herein, the term "Fc region" generally refers to the carboxyl terminal portion of the constant region of an immunoglobulin heavy chain or an analog thereof or a portion capable of binding to an Fc receptor. As is well known, each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named in the following order: CH1-hinge-CH2-CH3 (-CH4). CH4 is present in IgM without a hinge region. The immunoglobulin heavy chain constant region that can be used in the present application may comprise an immunoglobulin hinge region and may further comprise a CH3 domain. For example, an immunoglobulin heavy chain constant region may comprise an immunoglobulin hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the Fc region according to the present application consists of a hinge-CH2-CH3 domain.

[0207] As used herein, the term "complexed with" generally refers to the association (e.g., binding) of one member / subunit with another member / subunit of a molecule (e.g., an antibody). For example, a light chain can complex with a heavy chain to form a targeting moiety.

[0208] As used herein, the term "binding specificity" generally refers to the ability to specifically bind to (e.g., immunoreact with) a given target (while not binding or substantially binding to non-targets). The targeting moiety of the present application can be monospecific and contain one or more binding sites that specifically bind to a target, or can be multispecific (e.g., bispecific or trispecific) and contain two or more binding sites that specifically bind to the same or different targets.

[0209] As used herein, the term "associates with" or "associated with" generally refers to the physical association or contact of one entity with another entity. For example, a first member of a protein heterodimer can be "associated" with a second member, either covalently or non-covalently. In some embodiments, the first member of a protein heterodimer is associated with the second member via an interface, and the interface is formed by amino acid residues from the first member and the second member, respectively (i.e., interface residues).

[0210] As used herein, the term "modification" generally refers to any manipulation of the polypeptide backbone (e.g., amino acid sequence) or any post-translational modification of the polypeptide (e.g., glycosylation). For example, a modification is compared to the sequence of a corresponding wild-type polypeptide. A modification can be a substitution, addition, and / or deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

[0211] As used herein, the term "knob-and-hole modification" generally refers to the introduction of a modification at the interface of a polypeptide to form a protrusion (knob modification), and the introduction of a modification at the corresponding position of another polypeptide to form a cavity (hole modification), and the size of the protrusion is the same or similar to the size of the cavity. For example, the knob-and-hole modification enables the formation of heterodimers while inhibiting the formation of homodimers. See U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Therefore, the term "knob-and-hole modification" as used herein generally refers to a modification at the polypeptide interface to replace an amino acid with a smaller side chain (e.g., tyrosine or tryptophan) with an amino acid with a larger side chain (e.g., alanine or threonine) to form a protrusion. As used herein, the term "pore modification" generally refers to a modification made at a corresponding position in another polypeptide to replace an amino acid with a larger side chain (e.g., alanine or threonine) with an amino acid with a smaller side chain (e.g., tyrosine or tryptophan) to form a cavity. Knob modifications and pore modifications can be performed by altering the nucleic acid encoding the polypeptide, for example, by altering the nucleic acid. By site-specific mutagenesis, or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitutions Y349C and T366W in one of the two subunits of the Fc region, and the pore modification comprises the amino acid substitutions D356C, T366S, L368A, and Y407V in the other of the two subunits of the Fc region.

[0212] As used herein, the term "HEK293 cells" generally refers to a clonal isolate derived from transformed human embryonic kidney (HEK) cells. The HEK293 strain is a variant of the 293 cell line that exhibits better adherence in monolayer culture and is readily used for plaque assays and other anchorage-dependent applications. They have been adapted for suspension culture in serum-free media, such as 293SFMII.

[0213] As used herein, the term "CHO ​​cells" generally refers to Chinese hamster ovary cells, which are non-secreting, immortalized fibroblasts. CHO cells rarely secrete CHO endogenous proteins, thus facilitating the isolation and purification of target proteins.

[0214] As used herein, the term "COS-1 cells" generally refers to a fibroblast-like cell line derived from monkey kidney tissue. COS cells are derived by immortalizing CV-1 cells with a form of the SV40 virus, which produces a large T antigen but is defective in genome replication. One commonly used form of the COS cell line is COS-1.

[0215] As used herein, the term "NS0 cells" generally refers to a model cell line derived from a non-secretory murine myeloma. This cell line is a cholesterol-dependent cell line derived from a subline of NSI / 1.

[0216] As used herein, the term "fusion protein" generally refers to a polypeptide comprising or consisting of the amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to the amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to the previous polypeptide or domains thereof).

[0217] As used herein, the term "C-terminus" generally refers to the carboxyl terminus of a polypeptide.

[0218] As used herein, the term "N-terminus" generally refers to the amino terminus of a polypeptide.

[0219] As used herein, the term "EGFR" generally refers to epidermal growth factor receptor. See, for example, Carpenter et al., Ann. Rev. Biochem. 56:881-914 (1987), including naturally occurring mutant forms thereof.

[0220] As used herein, the term "EGFR mutant" generally refers to a mutant form of EGFR (e.g., EGFR variant III (also known as EGFRvIII), which is the most common extracellular domain mutation of EGFR that results in the deletion of exons 2-7 of the EGFR gene and is characterized by a truncated extracellular domain with ligand-independent constitutive activity.

[0221] As used herein, the term "HER2 / neu" generally refers to the human HER2 protein as described, for example, in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al., Nature 319:230-234 (1986) (GenBank Accession No. X03363).

[0222] As used herein, the term "GPC3" generally refers to a protein encoded by the gene glypican 3 (NCBI database gene ID: 2719), which is an early marker for liver cancer. GPC3 is highly expressed in hepatocellular carcinoma and has been detected in tissues of early-stage hepatocellular carcinoma patients.

[0223] As used herein, the term "anti-GPC3 antibody" generally refers to an antibody that specifically or preferentially binds to GPC3. For example, the anti-GPC3 antibody can be codrituzumab or an antigen-binding fragment thereof.

[0224] As used herein, the term "FAP" generally refers to fibroblast activation protein (FAP). FAP is present in tumor stroma fibroblasts and acts on the cell surface. FAP is a membrane serine peptidase, which is a member of the type II serine protease family and has dipeptidyl peptidase and collagenase activities.

[0225] As used herein, the term "anti-FAP antibody" generally refers to an antibody that specifically or preferentially binds to FAP. For example, the anti-FAP antibody can be antibody 28H1 or an antigen-binding fragment thereof.

[0226] As used herein, the term "Muc1" generally refers to a glycoprotein encoded by the muc1 gene. Muc1 is primarily found in epithelial tissues and organs such as the breast, pancreas, and ovary. It is highly expressed on the surface of cancer epithelial cells and, therefore, becomes a target of immune response.

[0227] As used herein, the term "anti-Muc1 antibody" generally refers to an antibody that specifically or preferentially binds to Muc1. For example, the anti-Muc1 antibody can be antibody 5E5, a humanized form of antibody 5E5, or an antigen-binding fragment thereof.

[0228] As used herein, the term "MUC5AC" generally refers to the mucin MUC5AC. MUC5AC is highly expressed in colorectal cancer, gastric signet ring cell carcinoma, colon cancer, rectal cancer, and pancreatic cancer.

[0229] As used herein, the term "anti-MUC5AC antibody" generally refers to an antibody that specifically or preferentially binds to MUC5AC. For example, the anti-MUC5AC antibody can be the antibody ensituximab or an antigen-binding fragment thereof.

[0230] As used herein, the term "mesothelin" generally refers to a cell surface glycoprotein with a molecular weight of 40 KD. Mesothelin is highly expressed in various tumor tissues (such as early pancreatic tumors) and can be expressed in normal pleural, pericardial and peritoneal mesothelial cells.

[0231] As used herein, the term "anti-mesothelin antibody" generally refers to an antibody that specifically or preferentially binds to mesothelin. For example, the anti-mesothelin antibody can be the antibody amatuximab, a humanized form of the antibody amatuximab, or an antigen-binding fragment thereof.

[0232] As used herein, the term "chemokine" generally refers to a number of low molecular weight (mostly 8-10 kD) proteins that can attract white blood cells to sites of infection. For example, common structural features of chemokine proteins can include a small molecular weight and four cysteine ​​residues in conserved positions that ensure tertiary structure. Some chemokines are involved in promoting inflammatory responses, while some chemokines are involved in controlling cell migration during normal repair or development.

[0233] As used herein, the term "lymphokine" generally refers to a hormone-like polypeptide produced by activated lymphocytes, which can act on corresponding target cells, thereby causing changes in the characteristics or functions of the target cells. Lymphocytes act on adjacent or distant target cells through lymphokines to achieve immunomodulation and immune effects. Common lymphokines include, but are not limited to, monocyte-macrophage migration inhibitory factor (MIF), leukocyte motility inhibitory factor (LIF), natural killer cell cytotoxin (NKCF) and lymphotoxin (LB).

[0234] As used herein, the term "tumor necrosis factor" generally refers to tumor necrosis factor produced by activated macrophages, NK cells, and T lymphocytes. Among them, TNF produced by macrophages is called TNF-α, and T lymphocytes produced by lymphotoxin (LT) are called TNF-β.

[0235] As used herein, the term "immunoglobulin" generally refers to a protein consisting of one or more polypeptides that are essentially encoded by immunoglobulin genes. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as countless immunoglobulin variable region genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer, consisting of two pairs of identical immunoglobulin chains, each pair having a light chain and a heavy chain. In each pair, the light chain and heavy chain variable regions are jointly responsible for binding to the antigen, and the constant region is responsible for the function of the antibody effector. In addition to antibodies, immunoglobulins can exist in a variety of other forms, including, for example, Fv, Fab, Fab', and (Fab')2.

[0236] As used herein, the term "in-frame fusion" generally refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF in a manner that maintains the correct reading frame of the original ORFs.

[0237] As used herein, the term "linker" generally refers to a synthetic amino acid sequence that connects or joins two polypeptide sequences, such as two polypeptide domains. A linker can connect two amino acid sequences via a peptide bond. In some embodiments, the linker of the present application connects the immunomodulator to the second Fc region in a linear sequence.

[0238] As used herein, the term "located at its N-terminus" generally refers to being located at the N-terminal position of another molecule (eg, another polypeptide). For example, according to the present application, two or more immunomodulators can be located at the N-terminus of the second Fc region.

[0239] As used herein, the term "amino acid substitution" generally refers to the substitution of one amino acid at a specific position in a polypeptide with another amino acid.

[0240] As used herein, the term "EU index of KABAT number" generally refers to the sequence corresponding to the sequence according to Kabat et al., Ann. NY Acad, Sci. 190:382-391 and Kabat, EA et al. (1991) Sequences of proteins of immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242.

[0241] As used herein, the term "isolated polynucleotide" generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs, separated from their natural environment or artificially synthesized.

[0242] As used herein, the term "protein mixture" generally refers to a mixture of two or more proteins.

[0243] As used herein, the term "homodimer" generally refers to a molecule formed by two identical monomers (e.g., two identical members or subunits). Two monomers can aggregate, complex, or associate with each other through covalent and / or non-covalent interactions. For example, two monomers of a protein homodimer can associate with each other through interactions between interfacial amino acid residues from each of the two monomers.

[0244] As used herein, the term "malignant effusion" generally refers to an effusion caused by a malignant tumor.

[0245] As used herein, the term "malignant cell" generally refers to a cell that is undergoing malignant proliferation. The malignant cell may include a tumor cell. The malignant cell may migrate from the primary tissue to other parts of the body through the circulatory system or the lymphatic system.

[0246] As used herein, the term "malignant pleural effusion (MPE)" generally refers to fluid accumulation within the pleural cavity. For example, MPE may include pleural metastasis caused by direct invasion or hemorrhagic spread of tumor cells, resulting in effusion. MPE may also include inflammatory pleural effusions caused by tumors combined with heart failure, tuberculosis, etc. Malignant pleural effusions are commonly seen in lung cancer, breast cancer, lymphoma, leukemia, etc., but are also seen in ovarian cancer, gastrointestinal tumors, pleural mesothelioma, etc. Malignant pleural effusions can cause severe discomfort symptoms such as dyspnea, dysphagia, chest pain, cough, and fever, and in severe cases, can lead to respiratory and circulatory dysfunction and death. Malignant pleural effusions can be considered a special tumor microenvironment that, in addition to tumor cells, also contains a large number of lymphocytes, macrophages, and fibroblasts. Symptomatic MPE can generally be treated with drainage, and a non-fixed pleural catheter (Pleur-X™) has been shown to be a viable alternative to standard chest tube thoracostomy for this purpose.

[0247] As used herein, the term "malignant ascites" generally refers to the abdominal cavity and peritoneal metastasis of a malignant tumor; and / or symptoms caused by portal hypertension (for example). The malignant ascites may include malignant ascites caused by a malignant tumor. For example, the malignant ascites may include ascites caused by a malignant tumor due to obstruction of lymphatic vessels and vascular thrombosis by tumor cells, resulting in obstruction of lymphatic blood flow; ascites caused by increased vascular permeability due to peritoneal metastasis of tumor cells, damage to vascular endothelial cells or even malignant changes, resulting in excessive entry of water and other substances into the abdominal cavity; and / or ascites caused by decreased plasma colloid osmotic pressure due to nutritional depletion and cachexia in patients. The malignant ascites may be treated with diuretics, chemotherapy (including systemic chemotherapy and intraperitoneal chemotherapy), targeted drug therapy, and / or surgery combined with hyperthermic peritoneal perfusion (HIPEC).

[0248] As used herein, the term "substantially free" generally means that a composition (e.g., a mixture) contains little or no substance. For example, the substance is present at a percentage of, for example, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%.

[0249] As used herein, the term "pharmaceutically acceptable excipient" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0250] Protein heterodimers, protein mixtures, isolated polynucleotides, vectors and host cells

[0251] The protein heterodimers described herein may comprise a first member and a second member different from the first member. The first member may comprise a light chain and a heavy chain comprising a first Fc region, and the light chain may complex with the heavy chain to form a targeting moiety exhibiting binding specificity for a tumor antigen. The second member may comprise a polypeptide, and the polypeptide may comprise an immunomodulatory agent fused to a second Fc region. The first member may associate with the second member through complexation of the first Fc region with the second Fc region to form a heterodimer.

[0252] In some cases, the protein heterodimer of the present application can be a protein complex. The complex can include at least three polypeptide chains, such as a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain. In some embodiments, the complex is composed of or essentially consists of three polypeptide chains. For example, the first polypeptide chain can include the heavy chain of an antibody specific for a tumor antigen, and the second polypeptide chain can include the light chain of an antibody specific for a tumor antigen. The heavy chain can include a first Fc region. The heavy chain (i.e., the heavy chain of the first polypeptide chain) and the light chain (i.e., the light chain of the second polypeptide chain) can be compounded to form the first member of a protein heterodimer. The third polypeptide chain can include (e.g., from N-terminus to C-terminus) one or more immunomodulators optionally fused to the second Fc region via one or more linkers. The third polypeptide chain can be the second member of a protein heterodimer. The first member can associate with the second member through a compound of the first Fc region and the second Fc region to form a heterodimer.

[0253] For example, the protein heterodimer of the present application can be a protein complex, which can contain (1) heavy chains and light chains of an antibody specific for a tumor antigen; and (2) a fusion protein comprising one or more immunomodulators, optionally fused to the Fc region of the antibody via one or more linkers, from the N-terminus to the C-terminus.

[0254] The first Fc region may comprise a first modification, and / or the second Fc region may comprise a second modification, wherein the first modification and / or the second modification may promote heterodimerization between the first member and the second member more effectively than a knob-hole modification comprising both a knob modification and a hole modification. For example, the first modification may be in the CH3 domain of the first Fc region, and the second modification may be in the CH3 domain of the second Fc region. For example, the first modification and / or the second modification are each compared to the sequence of their corresponding wild-type Fc region.

[0255] In some embodiments, the first Fc region comprises a first modification and the second Fc region does not comprise any modifications, and the first modification promotes heterodimerization between the first and second members more effectively than a knob-hole modification comprising both a knob modification and a hole modification.

[0256] In some embodiments, the second Fc region comprises a second modification and the first Fc region does not comprise any modification, and the second modification promotes heterodimerization between the first and second members more effectively than a knob-hole modification comprising both a knob modification and a hole modification.

[0257] In some embodiments, the first Fc region comprises a first modification and the second Fc region comprises a second modification, wherein the first modification and the second modification promote heterodimerization between the first member and the second member more effectively than a knob-hole modification comprising a knob modification and a hole modification.

[0258] In some embodiments, the first modification is different from the knob modification or the pore modification, and / or the second modification is different from the knob modification or the pore modification. For example, the first modification can be different from the knob modification or the pore modification, while the second modification is the same as the pore modification. In some cases, the first modification is the same as the knob modification, while the second modification is different from the knob modification or the pore modification. In some embodiments, the first Fc region comprises the first modification and the second Fc region comprises the second modification, and both the first modification and the second modification are the same as the knob modification or the pore modification.

[0259] In some embodiments, the first modification comprises an amino acid substitution at position T366, and an amino acid substitution at one or more positions selected from the group consisting of: Y349, F405, K409, D399, K360, Q347, K392, and S354, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0260] In some embodiments, the first modification comprises an amino acid substitution selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D, and T366W, wherein the position of the amino acid is determined according to the EU index with KABAT numbering.

[0261] In some embodiments, the first modification comprises 2-5 amino acid substitutions.

[0262] In some embodiments, the first modification comprises an amino acid substitution at any one of the following positions: 1) Y349 and T366; 2) Y349, T366, and F405; 3) Y349, T366, and K409; 4) Y349, T366, F405, K360, and Q347; 5) Y349, T366, F405, and Q347; 6) Y349, T366, K409, K360, and Q347. 47; 7) Y349, T366, K409 and Q347; 8) T366, K409 and K392; 9) T366 and K409; 10) T366, K409, Y349 and S354; 11) T366 and F405; 12) T366, F405 and D399; 13) T366, F405, Y349 and S354, where the positions of amino acids are determined according to the EU index using KABAT numbering.

[0263] In some embodiments, the first modification comprises any one of the following amino acid substitutions: 1) Y349C and T366W; 2) Y349C, T366W and F405K; 3) Y349C, T366W and K409E; 4) Y349C, T366W and K409A; 5) Y349C, T366W, F405K, K360E and Q347E; 6) Y349C, T366W, F405K and Q347R; 7) Y349C, T366W, K409A, K360E and Q347E; 8) Y349C, T366W, F405K and Q347R. 6W, K409A and Q347R; 9) T366W, K409A and K392D; 10) T366W and K409A; 11) T366W, K409A and Y349D; 12) T366W, K409A, Y349D and S354D; 13) T366W and F405K; 14) T366W, F405K and D399S; 15) T366W, F405K and Y349D; 16) T366W, F405K, Y349D and S354D, where the positions of amino acids are determined according to the EU index using KABAT numbering.

[0264] In some embodiments, the second modification comprises an amino acid substitution at position T366, L368, and Y407, and an amino acid substitution at one or more positions selected from D356, D399, E357, F405, K360, K392, K409, and Q347, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0265] In some embodiments, the second modification comprises an amino acid substitution selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A, and Y407V, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0266] In some embodiments, the second modification comprises an amino acid substitution at position 4-6.

[0267] In some embodiments, the second modification comprises an amino acid substitution at any one of the following positions: 1) D356, T366, L368, Y407, and F405; 2) D356, T366, L368, and Y407; 3) D356, T366, L368, Y407, and Q347; 4) D356, T366, L368, Y407, K360, and Q347; 5) D356, T366, L368, Y407, F405, and Q347; 6) D356, T366, L368, Y407, and Q347. 7) F405, K360 and Q347; 7) T366, L368, Y407, D399 and F405; 8) T366, L368, Y407 and F405; 9) T366, L368, Y407, F405 and E357; 10) T366, L368, Y407 and K409; 11) T366, L368, Y407, K409 and K392; 12) T366, L368, Y407, K409 and E357, where the positions of amino acids are determined according to the EU index based on KABAT values.

[0268] In some embodiments, the second modification comprises an amino acid substitution selected from any one of the following groups: 1) D356C, T366S, L368A, Y407V, and F405K; 2) D356C, T366S, L368A, and Y407V; 3) D356C, T366S, L368A, Y407V, and Q347R; 4) D356C, T366S, L368A, Y407V, K360E, and Q347E; 5) D356C, T366S, L368A, Y407V, F405K, and Q347R; 6) D356C, T366S, L368A, Y407V, F405K, K360E, and Q347E; 7) T366S, L368A, Y407V, D399S, and F405K; 8) T366S, L368G, Y407A, and F405K; 9) T366S, L368A, Y407V, F405K and E357A; 10) T366S, L368A, Y407V and K409A; 11) T366S, L368A, Y407V, K409A and K392D; 12) T366S, L368G, Y407A and K409A; 13) T366S, L368A, Y407V, K409A and E357A, where the positions of amino acids are determined according to the EU index according to the KABAT value.

[0269] In some embodiments, the first Fc region comprises a first modification, the second Fc region comprises a second modification, and the first modification and the second modification comprise amino acid substitutions at positions selected from any one of the following groups: 1) first modification: Y349 and T366; 2) second modification: D356, T366, L368, Y407, and F405; 2) first modification: Y349, T366, and F405; 3) first modification: Y349, T366, and K409; 4) first modification: Y349, T366, L368, Y407, and F405; 6) F405, K360 and Q347; second modification: D356, T366, L368, Y407 and Q347; 5) First modification: Y349, T366, F405 and Q347; second modification: D356, T366, L368, Y407, K360 and Q347; 6) First modification: Y349, T366, K409, K360 and Q347; second modification: D356, T366, L368, Y407, F405 and Q347; 7) First modification: Y349, T366, K409 and Q347; second modification: D356, T366, L368, Y407 , F405, K360 and Q347; 8) First modification: T366, K409 and K392; Second modification: T366, L368, Y407, D399 and F405; 9) First modification: T366 and K409; Second modification: T366, L368, Y407 and F405; 10) First modification: T366, K409 and Y349; Second modification: T366, L368, Y407, F405 and E357; 11) First modification: T366, K409, Y349 and S354; Second modification: T366, L368, Y407, F405 and E357; 12) First modification: T366, K409, Y349 and S354; Second modification: T366, L368, Y407, F405 and E357 modification: T366 and F405; second modification: T366, L368, Y407 and K409; 13) first modification: T366, F405 and D399; second modification: T366, L368, Y407, K409 and K392; 14) first modification: T366, F405 and Y349; second modification: T366, L368, Y407, K409 and E357; 15) first modification: T366, F405, Y349 and S354; second modification: T366, L368, Y407, K409 and E357; wherein the positions of amino acids are determined according to the EU index of KABAT numbering.

[0270] In some embodiments, the first Fc region comprises a first modification and the second Fc region comprises a second modification, wherein the first modification and the second modification comprise amino acid substitutions selected from any one of the following groups: 1) first modification: Y349C and T366W; second modification: D356C, T366S, L368A, Y407V, and F405K; 2) first modification: Y349C, T366W, and F405K; second modification: D356C, T366S, L368A, and Y407V; 3) first modification: Y349C, T366W, and K409E; second modification: D356C, T366S, L368A , Y407V and F405K; 4) first modification: Y349C, T366W and K409A; second modification: D356C, T366S, L368A, Y407V and F405K; 5) first modification: Y349C, T366W, F405K, K360E and Q347E; second modification: D356C, T366S, L368A, Y407V and Q347R; 6) first modification: Y349C, T366W, F405K and Q347R; second modification: D356C, T366S, L368A, Y407V, K360E and Q347E; 7) First modification: Y349C, T366W, K409A, K360E and Q347E; Second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) First modification: Y349C, T366W, K409A and Q347R; Second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) First modification: T366W, K409A and K392D; Second modification: T366S, L368A, Y407V, D399S and F405K; 10) First modification: T366W and K409A; Second modification: T366S, L368G, Y407A and F405K; 11) First modification: T36 6W, K409A and Y349D; second modification: T366S, L368A, Y407V, F405K and E357A; 12) first modification: T366W, K409A, Y349D and S354D; second modification: T366S, L368A, Y407V, F405K and E357A; 13) first modification: T366W and F405K; Second modification: T366S, L368A, Y407V and K409A; 14) First modification: T366W, F405K and D399S; Second modification: T366S, L368A, Y407V, K409A and K392D; 15) First modification: T366W and F405K; Second modification: T366S, L368G, Y407A and K409A;16) First modification: T366W, F405K, and Y349D; second modification: T366S, L368A, Y407V, K409A, and E357A; 17) First modification: T366W, F405K, Y349D, and S354D; second modification: T366S, L368A, Y407V, K409A, and E357A; wherein the amino acid positions are determined according to the EU index with KABAT numbering.

[0271] In some embodiments, the first Fc region comprises a first modification and the second Fc region comprises a second modification, the first modification comprises amino acid substitutions T366W and K409A, and the second modification comprises amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0272] In some embodiments, the knob and pore modification comprises a knob modification and a pore modification, wherein the knob modification comprises amino acid substitutions Y349C and T366W, and the pore modification comprises amino acid substitutions D356C, T366S, L368A, and Y407V, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0273] In some embodiments, when expressed in mammalian cells, the yield of the protein heterodimer of the present application is at least 10% higher (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more) than that of a control protein. The control protein differs from the protein heterodimer in that the control protein: i) comprises a knob modification in the first Fc region, ii) comprises a hole modification in the second Fc region, and iii) does not comprise both the first modification and the second modification. The mammalian cell can be selected from HEK293 cells, CHO cells, COS-1 cells, and NS0 cells. In some embodiments, the knob modification comprises amino acid substitutions Y349C and T366W, and the hole modification comprises amino acid substitutions D356C, T366S, L368A, and Y407V, wherein the positions of the amino acids are determined according to the EU index of KABAT numbering.

[0274] In some embodiments, the polypeptide included in the second member is a fusion protein, and the C-terminus of the immunomodulator is fused directly or indirectly to the N-terminus of the second Fc region to form a fusion protein. In some embodiments, the C-terminus of the immunomodulator is indirectly fused to the N-terminus of the second Fc region. For example, the second Fc region can be fused to the immunomodulator in frame through a joint. The joint can be, for example, a synthetic amino acid sequence that connects or connects two polypeptide sequences through a peptide bond. In some embodiments, the joint is a peptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. For example, the linker can comprise 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids), 1-15 amino acids (e.g., 1-11, 12, 13, 14, 15 amino acids), 1-20 amino acids, 1-30 amino acids or more. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 79 or 88. In some embodiments, the linker is resistant to proteolysis or substantially resistant to proteolysis.

[0275] In some embodiments, the tumor antigen is selected from EGFR, EGFR mutant, HER2 / neu, GPC3, FAP, Mucl, MUC5AC, and mesothelin.

[0276] The light chain of the targeting moiety may contain CDRs comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding CDR of the light chain of an antibody specific for a tumor antigen. In some embodiments, the light chain of the targeting moiety comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding variable region of the light chain of an antibody specific for a tumor antigen. In some embodiments, the light chain of the targeting moiety comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence comprised by the corresponding amino acid sequence of the light chain of an antibody specific for a tumor antigen.

[0277] The heavy chain of the targeting moiety may contain CDRs comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding CDR of the heavy chain of an antibody specific for a tumor antigen. In some embodiments, the heavy chain of the targeting moiety comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding variable region of the heavy chain of an antibody specific for a tumor antigen. In some embodiments, the heavy chain of the targeting moiety contains an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence contained in the corresponding amino acid sequence of the heavy chain of an antibody specific for a tumor antigen.

[0278] In some embodiments, the light chain of the targeting moiety contains CDRs that comprise an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding CDRs of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting moiety contains CDRs that comprise an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding CDRs of the heavy chain of an antibody specific for a tumor antigen.

[0279] In some embodiments, the light chain of the targeting moiety contains a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding variable region of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting moiety contains a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding variable region of the heavy chain of an antibody specific for a tumor antigen.

[0280] In some embodiments, the light chain of the targeting moiety comprises an amino acid sequence that comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting moiety comprises an amino acid sequence that comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the heavy chain of an antibody specific for a tumor antigen.

[0281] Antibodies specific for tumor antigens may be selected from anti-EGFR, anti-EGFR mutants, anti-HER2 / neu, anti-GPC3, anti-FAP, anti-Muc1, anti-MUC5AC, and anti-mesothelin. In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, the anti-EGFR mutant antibody is an anti-EGFR variant III antibody, such as Mab806. In some embodiments, the anti-HER2 / neu antibody is trastuzumab or pertuzumab. In some embodiments, the anti-GPC3 antibody is trastuzumab. In some embodiments, the anti-FAP antibody is 28H1. In some embodiments, the anti-Muc1 antibody is 5E5 or a humanized form of 5E5. In some embodiments, the anti-MUC5AC antibody is ensituximab. In some embodiments, the anti-mesothelin antibody is amatuximab or a humanized form of amatuximab.

[0282] In some embodiments, the targeting portion specifically binds to EGFR, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 101, the amino acid sequence of CDR2 is shown in SEQ ID NO: 102, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 103.

[0283] In some embodiments, the targeting moiety specifically binds to EGFR, and the light chain of the first member comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:104.

[0284] In some embodiments, the targeting moiety specifically binds to EGFR and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:37.

[0285] In some embodiments, the targeting moiety specifically binds to EGFR, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 105, the amino acid sequence of CDR2 is shown in SEQ ID NO: 106, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 107.

[0286] In some embodiments, the targeting moiety specifically binds to EGFR, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 108.

[0287] In some embodiments, the targeting moiety specifically binds to EGFR and the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:39.

[0288] In some embodiments, the targeting portion specifically binds to EGFR, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO: 101, the light chain CDR2 is shown in SEQ ID NO: 102, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO: 103; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 105, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO: 106, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO: 107.

[0289] In some embodiments, the targeting portion specifically binds to EGFR, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 104; the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 108.

[0290] In some embodiments, the targeting moiety specifically binds to EGFR, and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:37; the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:39.

[0291] In some embodiments, the targeting portion specifically binds to an EGFR mutant, and the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 109, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 110, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 111.

[0292] In some embodiments, the targeting moiety specifically binds to an EGFR mutant, and the light chain of the first member comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:112.

[0293] In some embodiments, the targeting moiety specifically binds to an EGFR mutant, and the amino acid sequence of the light chain of the first member is as shown in SEQ ID NO:53.

[0294] In some embodiments, the targeting portion specifically binds to an EGFR mutant, and the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 113, the amino acid sequence of CDR2 is shown in SEQ ID NO: 114, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 115.

[0295] In some embodiments, the targeting moiety specifically binds to an EGFR mutant, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:116.

[0296] In some embodiments, the targeting moiety specifically binds to an EGFR mutant and the amino acid sequence of the heavy chain of the first member is as shown in SEQ ID NO:55.

[0297] In some embodiments, the targeting portion specifically binds to an EGFR mutant, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO: 109, the amino acid sequence of amino light chain CDR2 is shown in SEQ ID NO: 110, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO: 111; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 113, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO: 114, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO: 115.

[0298] In some embodiments, the targeting portion specifically binds to the EGFR mutant, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 112; the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 116.

[0299] In some embodiments, the targeting moiety specifically binds to an EGFR mutant, and the amino acid sequence of the light chain of the first member is shown in SEQ ID NO: 53; the amino acid sequence of the heavy chain of the first member is shown in SEQ ID NO: 55.

[0300] In some embodiments, the targeting moiety specifically binds to HER2 / neu, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NOs: 117 and 125, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 118 and 126, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 119 and 127.

[0301] In some embodiments, the targeting moiety specifically binds HER2 / neu, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is selected from SEQ ID NOs: 120 and 128.

[0302] In some embodiments, the targeting moiety specifically binds HER2 / neu and the amino acid sequence of the light chain of the first member is selected from the group consisting of SEQ ID NOs: 45 and 49.

[0303] In some embodiments, the targeting portion specifically binds to HER2 / neu, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NOs: 121 and 129, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 122 and 130, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 123 and 131.

[0304] In some embodiments, the targeting moiety specifically binds to HER2 / neu, the antibody heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is selected from SEQ ID NOs: 124 and 132.

[0305] In some embodiments, the targeting moiety specifically binds HER2 / neu and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NOs: 47 and 51.

[0306] In some embodiments, the targeting portion specifically binds to HER2 / neu, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is selected from SEQ ID NOs: 117 and 125, the amino acid sequence of amino light chain CDR2 is selected from SEQ ID NOs: 118 and 126, and the amino acid sequence of light chain CDR3 is selected from SEQ ID NOs: 119 and 127; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is selected from SEQ ID NOs: 121 and 129, the amino acid sequence of heavy chain CDR2 is selected from SEQ ID NOs: 122 and 130, and the amino acid sequence of heavy chain CDR3 is selected from SEQ ID NOs: 123 and 131.

[0307] In some embodiments, the targeting portion specifically binds to HER2 / neu, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is selected from SEQ ID NOs: 120 and 128; the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is selected from SEQ ID NOs: 124 and 132.

[0308] In some embodiments, the targeting moiety specifically binds to HER2 / neu, the amino acid sequence of the light chain of the first member is selected from SEQ ID NOs: 45 and 49; the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NOs: 47 and 51.

[0309] In some embodiments, the targeting moiety specifically binds GPC3, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 133, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 134, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 135.

[0310] In some embodiments, the targeting moiety specifically binds GPC3, and the light chain of the first member comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:136.

[0311] In some embodiments, the targeting moiety specifically binds GPC3 and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:57.

[0312] In some embodiments, the targeting moiety specifically binds to GPC3, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 137, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 138, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 139.

[0313] In some embodiments, the targeting moiety specifically binds to GPC3, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:140.

[0314] In some embodiments, the targeting moiety specifically binds GPC3 and the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:59.

[0315] In some embodiments, the targeting portion specifically binds to GPC3, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 133, the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 134, and the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 135; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is set forth in SEQ ID NO: 137, the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 138, and the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 139.

[0316] In some embodiments, the targeting moiety specifically binds to GPC3, the light chain of the first member comprises a light chain variable region, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 136; and the heavy chain of the first member comprises a heavy chain variable region, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 140.

[0317] In some embodiments, the targeting moiety specifically binds to GPC3, and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:57; the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:59.

[0318] In some embodiments, the targeting portion specifically binds to FAP, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 141, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 142, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 143.

[0319] In some embodiments, the targeting moiety specifically binds to FAP, and the light chain of the first member comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:144.

[0320] In some embodiments, the targeting moiety specifically binds FAP and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:61.

[0321] In some embodiments, the targeting moiety specifically binds to FAP, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 145, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 146, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 147.

[0322] In some embodiments, the targeting moiety specifically binds to FAP, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:148.

[0323] In some embodiments, the targeting moiety specifically binds FAP and the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:63.

[0324] In some embodiments, the targeting portion specifically binds to FAP, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 141, the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 142, and the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 143; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is set forth in SEQ ID NO: 145, the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 146, and the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 147.

[0325] In some embodiments, the targeting portion specifically binds to FAP, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 148; the chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 152.

[0326] In some embodiments, the targeting moiety specifically binds to FAP, and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:61; the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:63.

[0327] In some embodiments, the targeting portion specifically binds to Muc1, the antibody light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 149, the amino acid sequence of CDR2 is shown in SEQ ID NO: 150, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 151.

[0328] In some embodiments, the targeting moiety specifically binds to Muc1, and the antibody light chain of the first member comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:152.

[0329] In some embodiments, the targeting moiety specifically binds to Mucl and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:65.

[0330] In some embodiments, the targeting portion specifically binds to Muc1, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 153, the amino acid sequence of CDR2 is shown in SEQ ID NO: 154, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 155.

[0331] In some embodiments, the targeting moiety specifically binds to Muc1, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:156.

[0332] In some embodiments, the targeting moiety specifically binds to Mucl and the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:67.

[0333] In some embodiments, the targeting portion specifically binds to Muc1, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO: 149, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO: 150, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO: 151; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 153, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO: 154, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO: 155.

[0334] In some embodiments, the targeting portion specifically binds to Muc1, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 152; the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 156.

[0335] In some embodiments, the targeting moiety specifically binds to Muc1, and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:65; the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:67.

[0336] In some embodiments, the targeting portion specifically binds to mesothelin, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 165, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 166, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 167.

[0337] In some embodiments, the targeting moiety specifically binds mesothelin, and the light chain of the first member comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:168.

[0338] In some embodiments, the targeting moiety specifically binds mesothelin and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:73.

[0339] In some embodiments, the targeting portion specifically binds to mesothelin, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 169, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 170, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 171.

[0340] In some embodiments, the targeting moiety specifically binds mesothelin, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:172.

[0341] In some embodiments, the targeting moiety specifically binds mesothelin and the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:75.

[0342] In some embodiments, the targeting portion specifically binds to mesothelin, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO: 165, the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO: 166, and the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO: 167; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is set forth in SEQ ID NO: 169, the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO: 170, and the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO: 171.

[0343] In some embodiments, the targeting portion specifically binds to mesothelin, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 168; the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 172.

[0344] In some embodiments, the targeting moiety specifically binds to mesothelin, and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:73; the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:75.

[0345] In some embodiments, the targeting moiety specifically binds to MUC5AC, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 157, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 158, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 159.

[0346] In some embodiments, the targeting moiety specifically binds to MUC5AC, and the light chain of the first member comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:160.

[0347] In some embodiments, the targeting moiety specifically binds to MUC5AC and the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:69.

[0348] In some embodiments, the targeting moiety specifically binds to MUC5AC, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is set forth in SEQ ID NO: 161, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 162, and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 163.

[0349] In some embodiments, the targeting moiety specifically binds to MUC5AC, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:164.

[0350] In some embodiments, the targeting moiety specifically binds to MUC5AC and the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:71.

[0351] In some embodiments, the targeting moiety specifically binds to MUC5AC, the light chain of the first member comprises light chain CDR1-3, the amino acid sequence of light chain CDR1 is set forth in SEQ ID NO:157, the amino acid sequence of light chain CDR2 is set forth in SEQ ID NO:158, and the amino acid sequence of light chain CDR3 is set forth in SEQ ID NO:159; the heavy chain of the first member comprises heavy chain CDR1-3, the amino acid sequence of heavy chain CDR1 is set forth in SEQ ID NO:161, the amino acid sequence of heavy chain CDR2 is set forth in SEQ ID NO:162, and the amino acid sequence of heavy chain CDR3 is set forth in SEQ ID NO:163.

[0352] In some embodiments, the targeting moiety specifically binds to MUC5AC, the light chain of the first member comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 160, and the heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 164.

[0353] In some embodiments, the targeting moiety specifically binds to MUC5AC, the amino acid sequence of the light chain of the first member is set forth in SEQ ID NO:69; and the amino acid sequence of the heavy chain of the first member is set forth in SEQ ID NO:71.

[0354] In some embodiments, the immunomodulator enhances the immune response. Examples of immunomodulators that can enhance the immune response include, but are not limited to, IL-2, IFNα, IFNβ, IFNγ, IFNλ, tumor necrosis factor (TNF)α, IL-12, and IL-10.

[0355] In some embodiments, the immunomodulator reduces an immune response. Non-limiting examples of immunomodulators capable of reducing an immune response include IL-10 and transforming growth factor (TGF)-β.

[0356] In some embodiments, the immunomodulator is a cytokine. For example, the immunomodulator can be a cytokine selected from interferons, interleukins, chemokines, lymphokines, and tumor necrosis factors.

[0357] In some embodiments, the immunomodulator is an interferon selected from interferon alpha, interferon lambda, and interferon beta.

[0358] In some embodiments, the immunomodulator is an interleukin, and the interleukin comprises interleukin 10, interleukin 2, and / or super interleukin 2.

[0359] In some embodiments, the first Fc region and the second Fc region are from the Fc region of an immunoglobulin. For example, the immunoglobulin can be selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the first Fc region and the second Fc region are from the Fc region of an immunoglobulin, and the immunoglobulin is human IgG1.

[0360] In some embodiments, the first modification and / or the second modification is compared to the wild-type amino acid sequence of the Fc region of human IgG1.

[0361] In some embodiments, the second Fc region is fused in-frame to the immunomodulatory agent.

[0362] In some embodiments, the polypeptide included in the second member includes two or more immunomodulators, and the two or more immunomodulators are fused to each other in frame and fused to the second Fc region, and wherein the two or more immunomodulators are located at the N-terminus of the second Fc region. In some embodiments, the two or more immunomodulators can be fused to each other in frame and / or fused to the second Fc region through a joint. The joint can be, for example, a synthetic amino acid sequence that connects or links two polypeptide sequences through a peptide bond. In some embodiments, the joint is comprised of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. The two or more immunomodulators can be of the same type or can be of different types. For example, the two or more immunomodulators can be identical. In some embodiments, the two or more immunomodulators are interleukin 10.

[0363] In some embodiments, in the heavy chain of the first member, the amino acid sequence of the first Fc region is selected from SEQ ID NO: 1, 4, 5, 6, 7, 9, 11, 13, 15, 17, 19, 21, 22, 24, 26, 27 and 29.

[0364] In some embodiments, the amino acid sequence of the immunomodulator contained in the second member is selected from SEQ ID NOs: 173-180.

[0365] In some embodiments, the amino acid sequence of the second Fc region comprised in the second member is selected from SEQ ID NOs: 2, 3, 8, 10, 12, 14, 16, 18, 20, 23, 25, and 28.

[0366] In some embodiments, the amino acid sequence of the polypeptide contained in the second member is selected from SEQ ID NOs: 77, 80, 82, 84, 86, 89, 91, and 97.

[0367] In some embodiments, the amino acid sequence of the light chain contained in the first member is selected from the group consisting of SEQ ID NOs: 37, 45, 49, 53, 57, 61, 65, 69, and 73. The amino acid sequence of the heavy chain contained in the first member is selected from the group consisting of SEQ ID NOs: 39, 47, 51, 55, 59, 63, 67, 71, and 75, and the amino acid sequence of the polypeptide contained in the second member is selected from the group consisting of SEQ ID NOs: 77, 80, 82, 84, 86, 89, 91, and 97.

[0368] On the other hand, the application provides a kind of protein mixture, it includes: 1) according to the protein heterodimer of the present application; And 2) by two first members of protein heterodimer, form first homodimer; 3) by two second homodimers formed in the second member of protein heterodimer.In the protein mixture, the percentage of protein heterodimer can be at least 50% (for example at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or higher).

[0369] In some embodiments, in the protein mixture, the second homodimer percentage is less than the first homodimer percentage. For example, the first homodimer percentage can be at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times at least 2.9 times, at least 3.0 times, at least 3.1 times, at least 3.2 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 7.0 times, at least 8.0 times or more higher than the second homodimer percentage.

[0370] In some embodiments, the percentage of the second homodimer in the protein mixture is at most 10% (e.g., at most 0.0%, at most 0.01%, at most 0.1%, at most 0.5%, at most 1%, at most 1.5%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%). In some embodiments, the protein mixture does not substantially comprise the second homodimer.

[0371] In another aspect, the present application provides an isolated polynucleotide encoding a protein heterodimer according to the present application. In some embodiments, the isolated polynucleotide encodes a subunit (eg, member) or fragment of a protein heterodimer according to the present application.

[0372] Polynucleotides can be synthesized using recombinant techniques well known in the art. For example, polynucleotides can be synthesized using an automated DNA synthesizer.

[0373] Standard recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis), and T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishers Associates and Wiley-Interscience (1987). Briefly, the nucleic acids of the present application can be prepared from genomic DNA fragments, cDNA, and RNA, all of which can be extracted directly from cells or produced recombinantly by various amplification methods, including, but not limited to, PCR and RT-PCR.

[0374] Direct chemical synthesis of nucleic acids generally involves the sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5' hydroxyl group of a growing nucleotide polymer chain, wherein each addition is effected by nucleophilic attack by the terminal 5' hydroxyl group of the growing chain at the 3' position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, etc. See, for example, Matteuci et al., Tet. Lett. 521:719 (1980); U.S. Pat. No. 4,500,707 to Caruthers et al.; and U.S. Pat. Nos. 5,436,327 and 5,700,637 to Southern et al.

[0375] In another aspect, the present application provides a vector comprising the isolated polynucleotide of the present application.

[0376] The vector can be any linear nucleic acid, plasmid, phagemid, cosmid, RNA vector, viral vector, etc. Non-limiting examples of viral vectors can include retroviruses, adenoviruses, and adeno-associated viruses. In some embodiments, the vector is an expression vector, such as a phage display vector.

[0377] An expression vector may be suitable for use with certain types of host cells but not with other types. For example, an expression vector may be introduced into a host organism and then monitored for viability and expression of any gene / polynucleotide contained in the vector.

[0378] The expression vector may also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits that can be used to select or otherwise identify host cells carrying the expression vector. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

[0379] The subject vector can be introduced stably or transiently into the host cell by a variety of established techniques. For example, one method involves calcium chloride treatment, wherein the expression vector is introduced via a calcium precipitate. Other salts, such as calcium phosphate, can also be used following similar procedures. In addition, electroporation (i.e., application of an electric current to increase the permeability of the cell to the nucleic acid) can be used. Other examples of transformation methods include microinjection, DEAE dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipoplexes, liposomes, and dendrimers can also be used to transfect host cells.

[0380] After the heterologous sequence has been introduced into the host cells, a variety of methods can be practiced to identify host cells into which the subject vector has been introduced. One exemplary selection method involves subculturing individual cells to form individual colonies, which are then tested for expression of the desired protein product. Another method entails selecting host cells containing the heterologous sequence based on the phenotypic trait conferred by expression of a selectable marker gene contained in the expression vector.

[0381] For example, various heterologous sequences of the present application can be confirmed to be introduced into host cells by methods such as PCR, Southern blotting or Northern blot hybridization. For example, nucleic acid can be prepared from the gained host cells, and primers specific to the target sequence can be used to amplify the target specific sequence by PCR. The amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, and then stained with ethidium bromide, SYBR Green solution or the like, or the DNA is detected by UV detection. Alternatively, a nucleic acid probe specific to the target sequence can be used in a hybridization reaction. The expression of a specific gene sequence can be determined by immunoassay to detect the corresponding mRNA by reverse transcription, Northern blot hybridization or using a reactive antibody with the encoded gene product combined with PCR. Exemplary immunoassays include but are not limited to ELISA, radioimmunoassay and sandwich immunoassay.

[0382] In addition, the enzymatic activity of the enzyme (e.g., enzyme labeling) encoded by the heterologous sequence can be used to confirm that the various heterologous sequences of the present application are introduced into the host cell. The enzyme can be measured by various methods known in the art. Usually, the enzymatic activity can be determined by the formation of the product of the enzyme reaction studied or the conversion of the substrate. The reaction can be carried out in vitro or in vivo.

[0383] On the other hand, the present application provides an isolated host cell comprising the isolated polynucleotide or vector of the present application, and / or capable of expressing protein heterodimers, and / or an isolated polynucleotide encoding protein heterodimers, and / or the protein mixture of the present application.

[0384] In some embodiments, cell expression is the heterodimeric protein of the application, and the polynucleotide of separation encodes the heterodimeric protein and / or protein mixture of the application.The cell can be a eukaryotic cell or a prokaryotic cell.Can be transformed or transfected with the polynucleotide of the application or suitable cell, and use it for the expression and / or secretion of heterodimeric protein and / or protein mixture.For example, cell can be Escherichia coli cells, other bacterial host cells, yeast cells or various higher eukaryotic cells (such as immortal hybridoma cells, NSO myeloma cells, HEK293 cells, Chinese hamster ovary cells, HeLa cells, COS cells etc.).In some embodiments, the polynucleotide encoding protein heterodimer (such as heterodimeric protein) can be operably connected to the expression control sequence suitable for expressing in specific host cells.

[0385] drug

[0386] The present application provides the use of the protein heterodimer described in the present application in the preparation of a medicament, wherein the medicament is used to prevent and / or treat malignant effusion.

[0387] In the present application, the drug may be a pharmaceutical composition, which may further comprise a pharmaceutically acceptable excipient.

[0388] Examples of pharmaceutically acceptable excipients include, but are not limited to, inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers, and adjuvants.

[0389] In some embodiments, the drug may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous depot.

[0390] This medication can be used to prevent and / or treat malignant effusions.

[0391] In certain instances, the drug can inhibit or delay the development or progression of a disease, can reduce the size of a tumor (or even substantially eliminate a tumor), and can alleviate and / or stabilize the disease state.

[0392] Described below are non-limiting exemplary pharmaceuticals and methods for their preparation.

[0393] The medicine can be, for example, a form suitable for oral administration, such as a tablet, capsule, pill, powder, sustained-release preparation, solution, suspension, a sterile solution for parenteral injection, a suspension or an emulsion, for topical application, as an ointment or cream, or as a suppository for rectal administration. The medicine can be a unit dosage form suitable for single administration of an exact dose. The medicine can further comprise a protein heterodimer (such as a heterodimer protein) or a protein mixture according to the application as an active ingredient, and can comprise conventional pharmaceutical carriers or excipients. In addition, it can include other drugs or medicaments, carriers, adjuvants, etc.

[0394] Exemplary parenteral administration forms include, but are not limited to, solutions or suspensions of active protein heterodimers (e.g., heterodimeric proteins) in sterile aqueous solutions such as propylene glycol aqueous solutions or dextrose solutions. If desired, these dosage forms can be suitably buffered with salts such as histidine and / or phosphate.

[0395] In some embodiments, the present application provides a drug for injection, which comprises the protein heterodimer (e.g., heterodimeric protein) or protein mixture of the present application and a pharmaceutical excipient suitable for injection. The ingredients and amounts of the reagents in the composition are as described in the present application.

[0396] Forms in which the drugs of the present application may be incorporated for administration by injection include aqueous or oily suspensions or emulsions, using sesame oil, corn oil, cottonseed oil or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions and similar pharmaceutical vehicles.

[0397] Aqueous solutions in saline can also be used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, etc. (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, and by using a surfactant to maintain the desired particle size when dispersed. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0398] Can be by by the protein heterodimer (such as heterodimer protein) of the present application of an appropriate amount or the protein mixture of the present application with various other compositions as listed above in suitable solvent, as needed, then filter sterilization prepares sterile injection solution.Usually, dispersion is prepared by the active component of various sterilizations being mixed in sterile carrier, and this sterile carrier contains basic dispersion medium and other compositions listed above as needed or desired.In the case of the sterile powder for the preparation of sterile injection solution, some desired preparation method is vacuum drying and freeze drying technology, and it produces the powder of active component and any other desired composition from previous sterile filtered solution.

[0399] In some embodiments, the present application provides a medicament for oral administration, which comprises the protein heterodimer (eg, heterodimeric protein) or protein mixture of the present application, and a pharmaceutical excipient suitable for oral administration.

[0400] In some embodiments, the present application provides a solid drug for oral administration, which comprises: (i) a certain amount of a protein heterodimer (e.g., a heterodimeric protein) or a protein mixture of the present application; optionally (ii) a certain amount of a second agent; (iii) a pharmaceutical excipient suitable for oral administration. In some embodiments, the composition further comprises: (iv) a certain amount of a third agent. In some embodiments, the amount of the protein heterodimer or protein mixture, the second agent, and the optional third agent is an amount that is effective, alone or in combination, to treat the subject's condition.

[0401] In some embodiments, the pharmaceutical composition can be a liquid medication suitable for oral administration. The drugs of the present application suitable for oral administration can exist in discrete dosage forms, such as capsules, cachets or tablets, or powders or granules containing a predetermined amount of active ingredient, solutions, or suspensions in aqueous or non-aqueous liquids, oil-in-water emulsions or water-in-oil liquid emulsions. Such dosage forms can be prepared by any pharmaceutical method, but all methods generally include the step of combining the active ingredient with a carrier that constitutes one or more other ingredients. Typically, the composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired appearance.

[0402] Since water can promote the degradation of certain polypeptides, the present application also covers anhydrous drugs and dosage forms comprising active ingredients (e.g., protein heterodimers or heterodimeric proteins of the present application). For example, in the pharmaceutical field, water (e.g., 5%) can be added as a means of simulating long-term storage to determine characteristics such as shelf life or the stability of the formulation over time. Anhydrous pharmaceutical compositions can be prepared and stored to maintain their anhydrous properties. Therefore, anhydrous compositions can be packaged using known materials that prevent exposure to water so that they can be included in suitable formula kits. Examples of suitable packaging include, but are not limited to, airtight foils, plastics, etc., unit dose containers, blister packs, and strip packs.

[0403] According to conventional drug mixing technology, the protein heterodimer of the present application (such as heterodimer protein or complex) or protein mixture can be closely mixed with a pharmaceutical carrier. The carrier can take various forms, depending on the desired formulation form of administration. When preparing a composition for oral dosage form, any conventional pharmaceutical medium can be used as a carrier, in the case of oral liquid preparations (such as suspensions, solutions and elixirs) or aerosols, such as, for example, water, ethylene glycol, oil, alcohol, flavorings, preservatives, coloring agents, etc.; or, in some embodiments, lactose is not used, in the case of oral solid preparations, carriers or disintegrants such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants can be used. For example, suitable carriers include powders, capsules and tablets and solid oral preparations. If necessary, tablets can be coated by standard aqueous or non-aqueous technology.

[0404] When aqueous suspensions and / or elixirs are required for oral administration, the active ingredient therein may be mixed with various sweetening or flavoring agents, coloring agents or dyes and emulsifying and / or suspending agents, if desired, and with diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof.

[0405] The tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

[0406] Surfactants that can be used to form the pharmaceutical compositions and dosage forms of the present application include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. A mixture of hydrophilic surfactants can be used, a mixture of lipophilic surfactants can be used, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant can be used.

[0407] In some embodiments, the medicine can include a solubilizing agent to ensure good dissolution and / or dispersion of the protein heterodimer or protein mixture of the present application and to minimize the precipitation of the protein heterodimer or protein mixture of the present application. This is especially important for compositions for non-oral use, such as compositions for injection. A solubilizing agent can also be added to increase the solubility of hydrophilic drugs and / or other components such as surfactants, or to maintain the composition as a stable or uniform solution or dispersion.

[0408] The composition may further comprise one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, but are not limited to, detackifiers, defoamers, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, growth enhancers, flavorings, colorants, flavoring agents, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[0409] Additionally, acids or bases may be incorporated into the composition to facilitate processing, enhance stability, or for other reasons.

[0410] The pharmaceutical composition of the present application may include a therapeutically effective amount of an active agent (e.g., a protein heterodimer or protein mixture of the present application). A therapeutically effective amount is a pharmaceutical composition of the present application that is capable of (at least partially) preventing and / or curing a condition or disorder (e.g., cancer) and / or any complications thereof in a subject suffering from or having a risk of developing the condition or disorder. The disease or disorder. The specific amount / concentration of the active agent included can vary according to the method of administration and the needs of the patient, and can be determined based on, for example, the patient's volume, viscosity, and / or body weight. For example, a suitable dose can be about 0.1 mg or 1 mg / kg / day to about 50 mg / kg / day; sometimes, the dose can be even higher. In some embodiments, the dose administered can be about 3 mg / kg / day to about 3.5 mg / kg / day, 3.5 mg / kg / day to about 7.2 mg / kg / day, about 7.2 mg / kg / day to about 11.0 mg / kg / day, about 11.0 mg / kg / day to about 15.0 mg / kg / day. In some embodiments, the dosage administered is from about 10 mg / kg / day to about 50 mg / kg / day, for example, from about 20 mg to about 50 mg per day, administered twice a day. It should be understood that those skilled in the art (e.g., a doctor or pharmacist) can conveniently adjust these specific dosages based on the conditions of a particular patient, formulation, and / or disease.

[0411] The protein heterodimer or pharmaceutical composition of the present application may further comprise one or more additional therapeutically active components. Such additional therapeutically active components may be present in the composition alone, or may be attached, conjugated, or associated with the protein heterodimer of the present application.

[0412] Medical uses and treatments

[0413] In another aspect, the present application provides use of a protein heterodimer or a protein mixture according to the present application in preparing a medicament and / or kit for preventing and / or treating malignant effusion.

[0414] In the present application, the drug may be further used in combination with chemotherapeutic agents, surgical procedures, catheter devices for achieving pleural drainage, and / or radiation.

[0415] In some embodiments, the medicament and / or kit is used to specifically and / or preferentially inhibit the growth or differentiation of or kill target cells (eg, cancer cells).

[0416] In another aspect, the present application provides a method for inhibiting the growth of a tumor or tumor cell. The method may include contacting the tumor or tumor cell with an effective amount of a protein heterodimer according to the present application or a protein mixture according to the present application. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo.

[0417] In some embodiments, the contacting comprises administering the protein heterodimer (e.g., heterodimeric protein), protein mixture, pharmaceutical composition, or drug of the present application to a subject (e.g., a mammal) systemically or topically. In some embodiments, the contacting comprises administering the protein heterodimer (e.g., heterodimeric protein), protein mixture, pharmaceutical composition, or drug of the present application directly to the tumor site. In some embodiments, administration is by oral administration, intravenous administration, intramuscular administration, administration in situ at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir.

[0418] In some embodiments, the tumor (e.g., cancer) or tumor cell (e.g., cancer cell) is or is from a solid tumor. For example, the cancer can be selected from B-cell lymphoma, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, melanoma, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, liposarcoma, testicular cancer, and malignant fibrous histiocytoma.

[0419] In some embodiments, the cancer cell or cancer cells are located in a subject, such as a human or a non-human animal (eg, a mammal).

[0420] In some embodiments, the mammal is a human. In some embodiments, the mammal is a mouse, rat, cat, dog, rabbit, pig, sheep, horse, cattle, goat, gerbil, hamster, guinea pig, monkey or any other mammal. Many such mammals can be subjects of preclinical models of certain diseases or conditions (including solid tumors and / or other cancers) known in the art (e.g., Talmadge et al., 2007 Am. J. Pathol. 170: 793; Kerbel, Canc. Biol. Therap. 2 (4 Suppl 1): S134; Man et al., 2007 Canc. Met. Rev. 26: 737; Cespedes et al., 2006 Clin. Trans L Oncol. 8: 318).

[0421] Method for preparing protein heterodimer or protein mixture

[0422] On the other hand, the present application provides a method for producing protein heterodimers or a protein mixture containing protein heterodimers, which method comprises (i) culturing the host cell of the present application under conditions that affect the expression of protein heterodimers, and (ii) harvesting the expressed protein heterodimers or a protein mixture containing the expressed protein heterodimers (such as the protein mixture of the present application).

[0423] In some embodiments, the method of producing protein heterodimers comprises the following steps:

[0424] (1) providing a first member of a heterodimer, wherein the first member comprises a light chain and a heavy chain, the heavy chain comprising a first Fc region, wherein the light chain is complexed with the heavy chain to form a targeting moiety that exhibits binding specificity for a tumor antigen;

[0425] (2) providing a second member of the heterodimer, the second member being different from the first member, wherein the second member comprises a polypeptide comprising an immunomodulator fused to a second Fc region; the first member associates with the second member to form a heterodimer through complexation of the first Fc region with the second Fc region; and the first Fc region comprises a first modification and / or the second Fc region comprises a second modification, wherein the first modification and / or the second modification promotes heterodimerization between the first member and the second member more effectively than a knob-hole comprising a knob modification and a hole modification; and

[0426] (3) Obtain protein heterodimers.

[0427] In some embodiments, the method further comprises the step of isolating and / or purifying the protein heterodimer or protein mixture.

[0428] In some embodiments, the method further comprises the step of transfecting / transforming the host cell with a polynucleotide / vector encoding / expressing the heterodimer of the present application, one or more members thereof, or a fragment thereof.

[0429] In some embodiments, the protein heterodimer or protein mixture of the present application is produced by expressing a vector in a cell under conditions suitable for protein expression. In some embodiments, the protein heterodimer or protein mixture of the present application is produced in a single cell clone.

[0430] Factors that may vary between suitable conditions for protein expression include factors such as incubation time, temperature, and culture medium, and may depend on the cell type and can be readily determined by one of ordinary skill in the art.

[0431] In some embodiments, in the process of producing the protein heterodimers or protein mixtures of the present application, host cells are grown in culture and in any device (including fermentors) that can be used to grow cultures. The cells can be grown in a monolayer or attached to a surface. Alternatively, the host cells can be grown in suspension. The cells can be grown in a serum-free culture medium. The culture medium can be a commercially available culture medium, such as, but not limited to, Opti-CHO (Invitrogen, catalog number 12681) supplemented with glutamine, such as 8mM L-glutamine; and RPMI 1640 culture medium supplemented with 10% calf serum, 10.5ng / ml mIL-1 -3 and L-glutamine; or 5% FCS medium.

[0432] Animal models and their offspring

[0433] On the other hand, the present application also provides a method for constructing an animal model with malignant effusion, which may include injecting tumor cells into the thoracic cavity and / or abdominal cavity of the animal.

[0434] In the present application, the injection dose may be about 1E5 to about 2E6 cells per animal. (wherein 1E5 is 1×10 5 , 2E6 is 2×10 6 )

[0435] In the present application, the injected dose can be about 1E5 to about 1.5E5 cells, about 1.5E5 to about 2E5 cells, about 2E5 to about 2.5E5 cells, about 2.5E5 to about 3E5 cells, about 3E5 to about 3.5E5 cells, about 3.5E5 to about 4E5 cells, about 4E5 to about 4.5E5 cells, about 4.5E5 to about 5E5 cells, about 5E5 to about 5.5E5 cells, about 5.5E5 to about 6E5 cells, about 6E5 to about 6.5E5 cells, about 6.5E5 to about 7E5 cells, about 7E5 to about 7.5E5 cells, about 7.5E5 to about 8E5 cells, or about 8.5E5 to about 9E5 cells. cells, about 8E5 to about 8.5E5 cells, about 8.5E5 to about 9E5 cells, about 9E5 to about 9.5E5 cells, about 9.5E5 to about 1E6 cells, about 1E6 to about 1.1E6 cells, about 1.1E6 to about 1.2E6 cells, about 1.2E6 to about 1.3E6 cells, about 1.3E6 to about 1.4E6 cells, about 1.4E6 to about 1.5E6 cells, about 1.5E6 to about 1.6E6 cells, about 1.6E6 to about 1.7E6 cells, about 1.7E6 to about 1.8E6 cells, about 1.8E6 to about 1.9E6 cells, or about 1.9E6 to about 2E6 cells.

[0436] In the present application, the animal may include a mammal. The animal may include a rodent. The animal may include a mouse. The mouse may be an inbred mouse or an outbred mouse. The mouse may be a normal mouse or an immunodeficient mouse. The mouse may include, but is not limited to, one or more of the following groups: C57BL / 6 (including C57BL / 6N and C57BL / 6J), Balb / c, 615 mice, China No. 1 (C-1) mice, Kunming mice (KM mice), NIH mice, LACA mice, ICR mice, nude mice, CBA / N mice, Beige mice, severe combined immunodeficiency mice (SCID mice), non-obese diabetic / severe combined immunodeficiency mice (NOD / SCID mice), NPG mice, NRG mice, BRG mice, and NOD mice.

[0437] In the present application, the injection may include intraperitoneal injection, intraperitoneal injection and / or intrathoracic injection.

[0438] For example, the injection may comprise intraperitoneal injection, intraperitoneal injection or intrathoracic injection into the animal.

[0439] For example, the injection may comprise intraperitoneal injection into the animal. In some embodiments, the injection may comprise intrathoracic injection into the animal.

[0440] In the present application, the tumor cell can be derived from a solid tumor and / or a non-solid tumor. For example, the tumor can include but is not limited to Hodgkin's lymphoma, lung cancer, breast cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, prostate cancer, sarcoma (including osteosarcoma and chondrosarcoma, etc.), lymphoma, leukemia, glioma, melanoma, lipoma and / or thyroid cancer. For example, the tumor can include lung, lymph, liver, stomach, intestine, breast, pancreas, sarcoma, ovary and / or mesothelioma.

[0441] For example, the tumor cells may include H22-Luc cells, B16-EGFR cells, MC38-EGFR cells, Pan02 mouse pancreatic cancer cells, S180 mouse sarcoma cells and / or Lewis-luc cells.

[0442] In the present application, the tumor cells can cause the animal to suffer from a tumor.

[0443] The tumor cells can cause C57BL / 6 (including C57BL / 6N and C57BL / 6J), Balb / c, 615 mice, China No. 1 (C-1) mice, Kunming mice (KM mice), NIH mice, LACA mice, ICR mice, nude mice, CBA / N mice, Beige mice, severe combined immunodeficiency mice (SCID mice), non-obese diabetic / severe combined immunodeficiency mice (NOD / SCID mice), NPG mice, NRG mice, BRG mice and / or NOD mice to have tumors.

[0444] In the present application, the tumor cells can cause animals to develop corresponding tumors. For example, BALB / c mice injected with H22-Luc cells can develop liver cancer, BALB / c mice injected with B16-EGFR cells can develop colon cancer, BALB / c mice injected with Pan02 mouse pancreatic cancer cells can develop pancreatic cancer, and C57BL / 6 mice injected with Lewis-luc cells can develop lung cancer.

[0445] In the present application, the tumor may metastasize to the pleura and / or peritoneum of the animal and produce the malignant effusion. The malignant effusion may contain malignant cells, blood and / or exudative fluid. The malignant effusion includes malignant pleural effusion (MPE) and / or malignant ascites.

[0446] In the present application, when the metastatic tumor is a thoracic tumor, the pleura of the animal can produce the malignant effusion. For example, when the metastatic tumor is lung cancer, the pleura of the animal can produce the malignant effusion, such as malignant pleural effusion (MPE). For example, the malignant pleural effusion is associated with non-small cell lung cancer.

[0447] In the present application, when the metastatic tumor is an abdominal tumor, the peritoneum of the animal can produce the malignant effusion. For example, when the metastatic tumor is colon cancer, pancreatic cancer and / or liver cancer, the peritoneum of the animal can produce the malignant effusion, such as malignant ascites.

[0448] In the present application, the malignant cells may include tumor cells. The malignant cells may be transferred from the primary tissue to other parts of the body through the circulatory system or the lymphatic system.

[0449] The present application also provides tissues or organs or cultures thereof in an animal model with malignant effusion prepared by the above method.

[0450] The animal model with malignant effusion prepared according to the above method or its offspring can specifically include: C57BL / 6 (including C57BL / 6N and C57BL / 6J) with malignant effusion, Balb / c, 615 mice, China No. 1 (C-1) mice, Kunming mice (KM mice), NIH mice, LACA mice, ICR mice, nude mice, CBA / N mice, Beige mice, severe combined immunodeficiency mice (SCID mice), non-obese diabetic / severe combined immunodeficiency mice (NOD / SCID mice), NPG mice, NRG mice, BRG mice and tissues or organs of NOD mice or their cultures.

[0451] In the present application, the animal model with malignant effusion or its offspring prepared according to the above method can specifically include: tissues or organs or cultures thereof of BALB / c mice with liver cancer and malignant effusion, BALB / c mice with colon cancer and malignant effusion, BALB / c mice with pancreatic cancer and C57BL / 6 mice with malignant effusion.

[0452] For example, tissues, organs, or cultures thereof from BALB / c mice with liver cancer and malignant effusions can be prepared using the above method. For example, tissues, organs, or cultures thereof from BALB / c mice with colon cancer and malignant effusions can be prepared using the above method. For example, tissues, organs, or cultures thereof from BALB / c mice with pancreatic cancer and malignant effusions can be prepared using the above method. For example, tissues, organs, or cultures thereof from C57BL / 6 mice with lung cancer and malignant effusions can be prepared using the above method.

[0453] The present application also provides an animal model with malignant effusion prepared by the above method or its offspring.

[0454] The animal models with malignant effusions prepared according to the above method or their offspring can specifically include: C57BL / 6 (including C57BL / 6N and C57BL / 6J) with malignant effusions, Balb / c, 615 mice, China No. 1 (C-1) mice, Kunming mice (KM mice), NIH mice, LACA mice, ICR mice, nude mice, CBA / N mice, Beige mice, severe combined immunodeficiency mice (SCID mice), non-obese diabetic / severe combined immunodeficiency mice (NOD / SCID mice), NPG mice, NRG mice, BRG mice, and NOD mice or their offspring. For example, the animal models with malignant effusions prepared according to the above method or their offspring can specifically include: BALB / c mice with liver cancer, BALB / c mice with colon cancer, BALB / c mice with pancreatic cancer, and C57BL / 6 mice with lung cancer or their offspring. For example, the above method can be used to prepare a BALB / c mouse model of liver cancer with malignant effusion or its offspring. For example, the above method can be used to prepare a BALB / c mouse model of colon cancer with malignant effusion or its offspring. For example, the above method can be used to prepare a BALB / c mouse model of pancreatic cancer with malignant effusion or its offspring. For example, the above method can be used to prepare a C57BL / 6 mouse model of lung cancer with malignant effusion or its offspring.

[0455] The present application also provides the use of the above-mentioned tissues or organs or their cultures, the animal models with malignant effusions or their offspring in the screening, verification, evaluation and / or research of anti-tumor drugs, or as model systems for pharmacological, immunological, microbiological and medical research.

[0456] This application includes the following implementation plans:

[0457] 1. A use of a protein heterodimer in preparing a drug, wherein the drug is used to prevent and / or treat malignant effusion, wherein the protein heterodimer comprises a first member and a second member, and the second member is different from the first member, wherein: the first member comprises a light chain and a heavy chain, the heavy chain comprises a first Fc region, and the light chain is complexed with the heavy chain to form a targeting portion that exhibits binding specificity to a tumor antigen; the second member comprises a polypeptide, and the polypeptide comprises an immunomodulator fused to the second Fc region; the first member and the second member associate through the complex of the first Fc region and the second Fc region to form the heterodimer; the first Fc region comprises a first modification and / or the second Fc region comprises a second modification, wherein the first modification and / or the second modification promotes heterodimerization between the first member and the second member more effectively than a knob-hole modification comprising a knob modification and a hole modification.

[0458] 2. Use according to embodiment 1, wherein the first modification is different from the knob modification or the pore modification, and / or the second modification is different from the knob modification or the pore modification.

[0459] 3. The use according to any of the preceding embodiments, wherein the yield of the protein heterodimer is at least 10% higher than the yield of a control protein when expressed in a mammalian cell, and the control protein is different from the protein heterodimer in that the control protein: i) comprises a knob modification in the first Fc region, ii) comprises a hole modification in the second Fc region, and iii) does not comprise both the first modification and the second modification.

[0460] 4. The use according to embodiment 3, wherein the mammalian cells are selected from HEK293 cells, CHO cells, COS-1 cells and NSO cells.

[0461] 5. The use according to any one of the preceding embodiments, wherein the first Fc region comprises a first modification, the second Fc region comprises a second modification, and both the first modification and the second modification are different from a knob modification or a hole modification.

[0462] 6. The use according to any one of the preceding embodiments, wherein the polypeptide comprised in the second member is a fusion protein, and the C-terminus of the immunomodulator is fused directly or indirectly to the N-terminus of the second Fc region to form the fusion protein.

[0463] 7. The use according to any one of the preceding embodiments, wherein the tumor antigen is selected from EGFR, EGFR mutant, HER2 / neu, GPC3, FAP, Mucl, MUC5AC and mesothelin.

[0464] 8. The use according to any of the preceding embodiments, wherein the light chain of the targeting portion contains a CDR comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acids contained in the corresponding CDR of the light chain of an antibody specific for a tumor antigen.

[0465] 9. The use according to any of the preceding embodiments, wherein the light chain of the targeting portion contains a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding variable region of the light chain of an antibody specific for a tumor antigen.

[0466] 10. The use according to any of the preceding embodiments, wherein the light chain of the targeting portion comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the light chain of an antibody specific for a tumor antigen.

[0467] 11. The use according to any of the preceding embodiments, wherein the heavy chain of the targeting portion contains a CDR comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding CDR of the heavy chain of an antibody specific for a tumor antigen.

[0468] 12. The use according to any of the preceding embodiments, wherein the heavy chain of the targeting portion contains a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding variable region of the heavy chain of an antibody specific for a tumor antigen.

[0469] 13. The use according to any of the preceding embodiments, wherein the heavy chain of the targeting portion comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the heavy chain of an antibody specific for a tumor antigen.

[0470] 14. The use according to any of the preceding embodiments, wherein the light chain of the targeting portion comprises CDRs comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acids contained in the corresponding CDRs of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting portion comprises CDRs comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding CDRs of the heavy chain of an antibody specific for a tumor antigen.

[0471] 15. The use according to any of the preceding embodiments, wherein the light chain of the targeting portion comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding variable region of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting portion comprises a variable region comprising an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding variable region of the heavy chain of an antibody specific for a tumor antigen.

[0472] 16. The use according to any of the preceding embodiments, wherein the light chain of the targeting moiety comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the light chain of an antibody specific for a tumor antigen; and the heavy chain of the targeting moiety comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence contained in the corresponding amino acid sequence of the heavy chain of an antibody specific for a tumor antigen.

[0473] 17. The use according to any one of embodiments 8 to 16, wherein the antibody specific for a tumor antigen is selected from anti-EGFR, anti-EGFR mutant, anti-HER2 / neu, anti-GPC3, anti-FAP, anti-Muc1, anti-MUC5AC and anti-mesothelin.

[0474] 18. The use according to any one of the preceding embodiments, wherein the immunomodulator enhances the immune response.

[0475] 19. The use according to any one of embodiments 1-17, wherein the immunomodulator reduces the immune response.

[0476] 20. The use according to any one of the preceding embodiments, wherein the immunomodulator is a cytokine.

[0477] 21. The use according to embodiment 20, wherein the immunomodulator is a cytokine selected from interferons, interleukins, chemokines, lymphokines and tumor necrosis factors.

[0478] 22. The use according to embodiment 21, wherein the immunomodulator is an interferon selected from interferon alpha, interferon lambda and interferon beta.

[0479] 23. The use according to embodiment 21, wherein the immunomodulator is an interleukin comprising interleukin 10, interleukin 2 and / or super interleukin 2.

[0480] 24. The use according to any one of the preceding embodiments, wherein the first Fc region and the second Fc region are from the Fc region of an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0481] 25. The use according to embodiment 24, wherein the first Fc region and the second Fc region are from the Fc region of an immunoglobulin, and the immunoglobulin is human IgG1.

[0482] 26. The use according to any one of the preceding embodiments, wherein the second Fc region is fused in-frame to an immunomodulator.

[0483] 27. The use according to any one of the preceding embodiments, wherein the second Fc region is fused in-frame to the immunomodulator via a linker.

[0484] 28. The use according to any of the preceding embodiments, wherein the polypeptide comprised in the second member comprises two or more immunomodulators, said two or more immunomodulators being fused in-frame to each other and to a second Fc region, wherein said two or more immunomodulators are located at the N-terminus of the second Fc region.

[0485] 29. The use according to embodiment 28, wherein the two or more immunomodulatory agents are the same.

[0486] 30. The use according to any one of the preceding embodiments, wherein the first modification comprises an amino acid substitution at position T366, and an amino acid substitution at one or more positions selected from the group consisting of: Y349, F405, K409, D399, K360, Q347, K392, and S354, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0487] 31. The use according to embodiment 30, wherein the first modification comprises an amino acid substitution selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D and T366W.

[0488] 32. Use according to embodiment 30 or 31, wherein the first modification comprises 2 to 5 amino acid substitutions.

[0489] 33. The use according to any one of embodiments 30-32, wherein the first modification comprises an amino acid substitution at any one of the following positions: 1) Y349 and T366; 2) Y349, T366 and F405; 3) Y349, T366 and K409; 4) Y349, T366, F405, K360 and Q347; 5) Y349, T366, F405 and Q347; 6) Y3 49, T366, K409, K360, and Q347; 7) Y349, T366, K409, and Q347; 8) T366, K409, and K392; 9) T366 and K409; 10) T366, K409, Y349, and S354; 11) T366 and F405; 12) T366, F405, and D399; 13) T366, F405, Y349, and S354.

[0490] 34. The use according to any one of embodiments 30-33, wherein the first modification comprises an amino acid substitution at any one of the following positions: 1) Y349C and T366W; 2) Y349C, T366W and F405K; 3) Y349C, T366W and K409E; 4) Y349C, T366W and K409A; 5) Y349C, T366W, F405K, K360E and Q347E; 6) Y349C, T366W, F405K and Q347R; 7) Y349C, T366W, K409A, K347E 60E and Q347E; 8) Y349C, T366W, K409A and Q347R; 9) T366W, K409A and K392D; 10) T366W and K409A; 11) T366W, K409A and Y349D; 12) T366W, K409A, Y349D and S354D; 13) T366W and F405K; 14) T366W, F405K and D399S; 15) T366W, F405K and Y349D; and 16) T366W, F405K, Y349D and S354D.

[0491] 35. The use of any one of embodiments 30-34, wherein the second modification comprises an amino acid substitution at positions T366, L368, and Y407, and an amino acid substitution at one or more positions selected from D356, D399, E357, F405, K360, K392, K409, and Q347, wherein the positions of the amino acids are determined according to the EU index of KABAT numbering.

[0492] 36. The use according to embodiment 35, wherein the second modification comprises an amino acid substitution selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A and Y407V.

[0493] 37. Use according to embodiment 35 or 36, wherein the second modification comprises 4 to 6 amino acid substitutions.

[0494] 38. The use according to any one of embodiments 35-37, wherein the second modification comprises an amino acid substitution at any one of the following positions: 1) D356, T366, L368, Y407 and F405; 2) D356, T366, L368 and Y407; 3) D356, T366, L368, Y407 and Q347; 4) D356, T366, L368, Y407, K360 and Q347; 5) D356, T366, L368, Y407, F405 and Q347; 6) D356, T366, L368, Y407, F405, K360 and Q347; 7) T366, L368, Y407, D399 and F405; 8) T366, L368, Y407 and F405; 9) T366, L368, Y407, F405 and E357; 10) T366, L368, Y407 and K409; 11) T366, L368, Y407, K409 and K392; 12) T366, L368, Y407, K409 and E357.

[0495] 39. The use according to any one of embodiments 35-38, wherein the second modification comprises an amino acid substitution at any one of the following positions: 1) D356C, T366S, L368A, Y407V and F405K; 2) D356C, T366S, L368A and Y407V; 3) D356C, T366S, L368A, Y407V and Q347R; 4) D356C, T366S, L368A, Y407V, 360E and Q347E; 5) D356C, T366S, L368A, Y407V, F405K and Q347R; 6) D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 7) T366S, L368A, Y407V, D399S and F405K; 8) T366S, L368G, Y407A and F405K; 9) T366S, L368A, Y407V, F405K and E357A; 10) T366S, L368A, Y407V and K409A; 11) T366S, L368A, Y407V, K409A and K392D; 12) T366S, L368G, Y407A and K409A; 13) T366S, L368A, Y407V, K409A and E357A.

[0496] 40. The use according to any one of the preceding embodiments, wherein the first Fc region comprises a first modification, the second Fc region comprises a second modification, and the first modification and the second modification comprise amino acid substitutions at any one of the following positions: 1) first modification: Y349 and T366; second modification: D356, T366, L368, Y407, and F405; 2) first modification: Y349, T366, and F405; second modification: D356, T366, L368, and Y407; 3) first modification: Y349, T366, and K409; second modification: D356, T366, L368, Y407, and F405; 4) first modification: modification: Y349, T366, F405, K360 and Q347; second modification: D356, T366, L368, Y407 and Q347; 5) first modification: Y349, T366, F405 and Q347; second modification: D356, T366, L368, Y407, K360 and Q347; 6) first modification: Y349, T366, K409, K360 and Q347; second modification: D356, T366, L368, Y407, F405 and Q347; 7) first modification: Y349, T366, K409 and Q347; second modification: D356, T366, L3 68, Y407, F405, K360 and Q347; 8) First modification: T366, K409 and K392; Second modification: T366, L368, Y407, D399 and F405; 9) First modification: T366 and K409; Second modification: T366, L368, Y407 and F405; 10) First modification: T366, K409 and Y349; Second modification: T366, L368, Y407, F405 and E357; 11) First modification: T366, K409, Y349 and S354; Second modification: T366, L368, Y407, F405 and E357; 12 ) first modification: T366 and F405; second modification: T366, L368, Y407 and K409; 13) first modification: T366, F405 and D399; second modification: T366, L368, Y407, K409 and K392; 14) first modification: T366, F405 and Y349; second modification: T366, L368, Y407, K409 and E357; 15) first modification: T366, F405, Y349 and S354; second modification: T366, L368, Y407, K409 and E357; wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0497] 41. The use according to any of the preceding embodiments, wherein the first Fc region comprises a first modification and the second Fc region comprises a second modification, wherein the first modification and the second modification comprise amino acid substitutions at any one of the following positions: 1) first modification: Y349C and T366W; second modification: D356C, T366S, L368A, Y407V, and F405K; 2) first modification: Y349C, T366W, and F405K; second modification: D356C, T366S, L368A, and Y407V; 3) first modification: Y349C, T366W, and K409E; second modification: D356C, T366S, L368A, Y407V, and F405K; 4) First modification: Y349C, T366W and K409A; Second modification: D356C, T366S, L368A, Y407V and F405K; 5) First modification: Y349C, T366W, F405K, K360E and Q347E; Second modification: D356C, T366S, L368A, Y407V and Q347R; 6) First modification: Y349C, T366W, F405K and Q347R; Second modification: D356C, T366S, L368A, Y407V, K360E and Q347E; 7) First modification: Y349C, T366W, K409A, K360E and Q347E; second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) first modification: Y349C, T366W, K409A and Q347R; second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) first modification: T366W, K409A and K392D; second modification: T366S, L368A, Y407V, D399S and F405K; 10) first modification: T366W and K409A; second modification: T366S, L368G, Y407A and F405K; 11) first modification: T36 6W, K409A and Y349D; second modification: T366S, L368A, Y407V, F405K and E357A; 12) first modification: T366W, K409A, Y349D and S354D; second modification: T366S, L368A, Y407V, F405K and E357A; 13) first modification: T366W and F405K; second modification: T366S, L368A, Y407V and K409A; 14) first modification: T366W, F405K and D399S; second modification: T366S, L368A, Y407V, K409A and K392D; 15) first modification: T366W and F405K;Second modification: T366S, L368G, Y407A, and K409A; 16) First modification: T366W, F405K, and Y349D; Second modification: T366S, L368A, Y407V, K409A, and E357A; 17) First modification: T366W, F405K, Y349D, and S354D; Second modification: T366S, L368A, Y407V, K409A, and E357A; wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0498] 42. The use according to embodiment 41, wherein the first Fc region comprises a first modification and the second Fc region comprises a second modification, the first modification comprises amino acid substitutions T366W and K409A, and the second modification comprises amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the positions of amino acids are determined according to the EU index with KABAT numbering.

[0499] 43. The use according to any one of the preceding embodiments, wherein the targeting portion specifically binds to EGFR, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 101, the amino acid sequence of CDR2 is selected from SEQ ID NO: 102, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 103.

[0500] 44. The use according to any one of the preceding embodiments, wherein the targeting moiety specifically binds to EGFR, the light chain of the first member comprises a light chain variable region, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 104.

[0501] 45. The use according to any one of the preceding embodiments, wherein the targeting moiety specifically binds to EGFR and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 37.

[0502] 46. ​​The use according to any one of embodiments 43-45, wherein the targeting portion specifically binds to EGFR, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 105, the amino acid sequence of CDR2 is selected from SEQ ID NO: 106, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 107.

[0503] 47. The use according to any one of embodiments 43-45, wherein the targeting moiety specifically binds to EGFR, the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 108.

[0504] 48. The use according to any one of embodiments 43-45, wherein the targeting moiety specifically binds to EGFR and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NO: 39.

[0505] 49. The use according to any one of embodiments 1-42, wherein the targeting portion specifically binds to an EGFR mutant, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 1 NO: 109, the amino acid sequence of CDR2 is selected from SEQ ID NO: 110, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 111.

[0506] 50. The use according to any one of embodiments 1-42, wherein the targeting portion specifically binds to the EGFR mutant, and the light chain of the first member comprises a light chain variable region, and the amino acid sequence region of the light chain variable region is selected from SEQ ID NO: 112.

[0507] 51. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to an EGFR mutant and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 53.

[0508] 52. The use according to any one of embodiments 49-51, wherein the targeting portion specifically binds to an EGFR mutant, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 1 NO: 113, the amino acid sequence of CDR2 is selected from SEQ ID NO: 114, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 115.

[0509] 53. The use according to any one of embodiments 49-51, wherein the targeting portion specifically binds to the EGFR mutant, and the heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence region of the heavy chain variable region is selected from SEQ ID NO: 116.

[0510] 54. The use according to any one of embodiments 49-51, wherein the targeting moiety specifically binds to an EGFR mutant and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NO: 55.

[0511] 55. The use according to any one of embodiments 1-42, wherein the targeting portion specifically binds to HER2 / neu, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NOs: 117 and 125, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 118 and 126, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 119 and 127.

[0512] 56. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to HER2 / neu, and the light chain of the first member comprises a light chain variable region, and the amino acid sequence region of the light chain variable region is selected from SEQ ID NO: 120 and 128.

[0513] 57. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to HER2 / neu and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 45 and 49.

[0514] 58. The use according to any one of embodiments 55-57, wherein the targeting portion specifically binds to HER2 / neu, the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 1 NO: 121 and 129, the amino acid sequence of CDR2 is selected from SEQ ID NO: 122 and 130, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 123 and 131.

[0515] 59. The use according to any one of embodiments 55-57, wherein the targeting moiety specifically binds to HER2 / neu, and the antibody heavy chain of the first member comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 124 and 132.

[0516] 60. The use according to any one of embodiments 55-57, wherein the targeting moiety specifically binds to HER2 / neu and the amino acid sequence of the heavy chain of the first member is selected from the group consisting of SEQ ID NOs: 47 and 51.

[0517] 61. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to GPC3, the antibody light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 133, the amino acid sequence of CDR2 is selected from SEQ ID NO: 134, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 135.

[0518] 62. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to GPC3, and the antibody light chain of the first member comprises a light chain variable region, the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 136.

[0519] 63. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to GPC3 and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 57.

[0520] 64. The use according to any one of embodiments 61-63, wherein the targeting moiety specifically binds to GPC3, the antibody heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 137, the amino acid sequence of CDR2 is selected from SEQ ID NO: 138, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 139.

[0521] 65. The use according to any one of embodiments 61-63, wherein the targeting moiety specifically binds to GPC3, and the antibody heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 140.

[0522] 66. The use according to any one of embodiments 61-63, wherein the targeting moiety specifically binds to GPC3 and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NO: 59.

[0523] 67. The use according to any one of embodiments 1-42, wherein the targeting portion specifically binds to FAP, the antibody light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 141, the amino acid sequence of CDR2 is selected from SEQ ID NO: 142, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 143.

[0524] 68. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to FAP, and the antibody light chain of the first member comprises a light chain variable region, the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 144.

[0525] 69. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to FAP and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 61.

[0526] 70. The use according to any one of embodiments 67-69, wherein the targeting moiety specifically binds to FAP, the antibody heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 145, the amino acid sequence of CDR2 is selected from SEQ ID NO: 146, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 147.

[0527] 71. The use according to any one of embodiments 67-69, wherein the targeting moiety specifically binds to FAP, and the antibody heavy chain of the first member comprises a heavy chain variable region, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 148.

[0528] 72. The use according to any one of embodiments 67-69, wherein the targeting moiety specifically binds to FAP and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NO: 63.

[0529] 73. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to Muc1, the antibody light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 149, the amino acid sequence of CDR2 is selected from SEQ ID NO: 150, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 151.

[0530] 74. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to Mucl, and the antibody light chain of the first member comprises a light chain variable region, the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 152.

[0531] 75. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to Mucl and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 65.

[0532] 76. The use according to any one of embodiments 73-75, wherein the targeting moiety specifically binds to Muc1, the antibody heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 153, the amino acid sequence of CDR2 is selected from SEQ ID NO: 154, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 155.

[0533] 77. The use according to any one of embodiments 73-75, wherein the targeting moiety specifically binds to Muc1, and the antibody heavy chain of the first member comprises a heavy chain variable region, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 156.

[0534] 78. The use according to any one of embodiments 73-75, wherein the targeting moiety specifically binds to Muc1 and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NO: 67.

[0535] 79. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to mesothelin, the antibody light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 165, the amino acid sequence of CDR2 is selected from SEQ ID NO: 166, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 167.

[0536] 80. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to mesothelin, and the antibody light chain of the first member comprises a light chain variable region, the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 168.

[0537] 81. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to mesothelin and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 73.

[0538] 82. The use according to any one of embodiments 79-81, wherein the targeting portion specifically binds to mesothelin, the antibody heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 169, the amino acid sequence of CDR2 is selected from SEQ ID NO: 170, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 171.

[0539] 83. The use according to any one of embodiments 79-81, wherein the targeting moiety specifically binds to mesothelin, and the antibody heavy chain of the first member comprises a heavy chain variable region, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 172.

[0540] 84. The use according to any one of embodiments 79-81, wherein the targeting moiety specifically binds to mesothelin and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NO: 75.

[0541] 85. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to MUC5AC, the antibody light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 157, the amino acid sequence of CDR2 is selected from SEQ ID NO: 158, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 159.

[0542] 86. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to MUC5AC, and the antibody light chain of the first member comprises a light chain variable region, the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 160.

[0543] 87. The use according to any one of embodiments 1-42, wherein the targeting moiety specifically binds to MUC5AC and the amino acid sequence of the light chain of the first member is selected from SEQ ID NO: 69.

[0544] 88. The use according to any one of embodiments 85-87, wherein the targeting moiety specifically binds to MUC5AC, the antibody heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NO: 161, the amino acid sequence of CDR2 is selected from SEQ ID NO: 162, and the amino acid sequence of CDR3 is selected from SEQ ID NO: 163.

[0545] 89. The use according to any one of embodiments 85-87, wherein the targeting moiety specifically binds to MUC5AC, and the antibody heavy chain of the first member comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 164.

[0546] 90. The use according to any one of embodiments 85-87, wherein the targeting moiety specifically binds to MUC5AC and the amino acid sequence of the heavy chain of the first member is selected from SEQ ID NO: 71.

[0547] 91. The use according to any one of the preceding embodiments, wherein in the heavy chain of the first member, the amino acid sequence of the first Fc region is selected from SEQ ID NO: 1, 4, 5, 6, 7. 9, 11, 13, 15, 17, 19, 21, 22, 24, 26, 27 and 29.

[0548] 92. The use according to any one of the preceding embodiments, wherein the amino acid sequence of the immunomodulator comprised in the second member is selected from the group consisting of SEQ ID NOs: 173-180.

[0549] 93. The use according to any one of the preceding embodiments, wherein the amino acid sequence of the second Fc region comprised in the second member is selected from the group consisting of SEQ ID NO: 2, 3, 8, 10, 12, 14, 16 18, 20, 23, 25 and 28.

[0550] 94. The use according to any one of the preceding embodiments, wherein the amino acid sequence of the polypeptide comprised in the second member is selected from the group consisting of SEQ ID NOs: 77, 80, 82, 84, 86, 89, 91 and 97.

[0551] 95. The use according to any one of the preceding embodiments, wherein the amino acid sequence of the light chain contained in the first member is SEQ ID NO: 37, 45, 49, 53, 57, 61, 65, 69 and 73, the amino acid sequence of the heavy chain contained in the first member is SEQ ID NO: 39, 47, 51, 55, 59, 63, 67, 71 and 75, and the amino acid sequence of the polypeptide contained in the second member is SEQ ID NO: 77, 80, 82, 84, 86, 89, 91 and 97.

[0552] 96. The use according to any one of the preceding embodiments, wherein the knob and hole modification comprises a knob modification and a hole modification, wherein the knob modification comprises amino acid substitutions Y349C and T366W, and the hole modification comprises amino acid substitutions D356C, T366S, L368A, and Y407V, wherein the positions of the amino acids are determined according to the EU index with KABAT numbering.

[0553] 97. The use according to any one of the preceding embodiments, wherein the medicament comprises a pharmaceutical composition comprising the protein heterodimer according to any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0554] 98. The use according to any one of the preceding embodiments, wherein the drug comprises a nucleic acid molecule encoding the protein heterodimer of any one of the preceding embodiments.

[0555] 99. The use according to embodiment 98, wherein the medicament comprises a vector comprising the nucleic acid molecule.

[0556] 100. The use according to any one of the preceding embodiments 98-99, wherein the medicament comprises a cell comprising the nucleic acid molecule or the vector.

[0557] 101. The use according to any one of the aforementioned embodiments 98-100, wherein the drug comprises a protein mixture, the protein mixture comprising: 1) a protein heterodimer according to any one of embodiments 1-96; and 2) a first homodimer formed by two of the first members of the protein heterodimer; 3) a second homodimer formed by two of the second members of the protein heterodimer; wherein the percentage of the protein heterodimer in the protein mixture is at least 50%.

[0558] 102. The use according to any one of the preceding embodiments, wherein the patient suffering from the malignant effusion comprises a tumor patient.

[0559] 103. The use according to the preceding embodiment 102, wherein the patient has symptoms including fatigue, vomiting, edema of the dorsal part of the foot, loss of appetite, dyspnea, cough, chest pain and / or abdominal pain.

[0560] 104. The use according to any one of the preceding embodiments 102-103, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

[0561] 105. The use according to any one of the preceding embodiments 102-104, wherein the tumor comprises lung, lymphoid, liver, stomach, intestinal, breast, pancreatic, ovarian and / or mesothelial cancer.

[0562] 106. The use according to any one of the preceding embodiments, wherein the malignant effusion comprises malignant cells.

[0563] 107. The use according to any one of the preceding embodiments, wherein the malignant effusion comprises blood and / or exudate.

[0564] 108. The use according to any one of the preceding embodiments 102-107, wherein the malignant effusion is associated with metastasis of the tumor to the pleura.

[0565] 109. The use according to any one of the preceding embodiments 102-108, wherein the malignant effusion is associated with metastasis of the tumor to the peritoneum.

[0566] 110. The use according to any one of the preceding embodiments, wherein the malignant effusion is caused by a tumor comprising a primary tumor of the lung, lymphatic, liver, stomach, colon, breast, ovarian, pancreatic, bile duct, esophagus, brain, nose and / or prostate.

[0567] 111. The use according to any one of the preceding embodiments, wherein the malignant effusion is caused by metastasis to the thoracic cavity of a tumor comprising: a primary cancer of the lung, lymphatic, liver, stomach, intestine, breast, ovarian cancer and / or mesothelioma.

[0568] 112. The use according to any one of the preceding embodiments, wherein the malignant effusion is caused by metastasis to the abdominal cavity of a tumor comprising: ovarian cancer, pancreatic cancer, bile duct, esophagus, sarcoma and / or primary cancer of the liver.

[0569] 113. The use according to any one of the preceding embodiments, wherein the malignant effusion comprises malignant pleural effusion (MPE) and / or malignant ascites.

[0570] 114. The use according to any one of the preceding embodiments, wherein the malignant pleural effusion is associated with non-small cell lung cancer.

[0571] 115. The use according to any one of the preceding embodiments, wherein the medicament is formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous depot.

[0572] 116. The use according to any one of the preceding embodiments, wherein the medicament is configured to include a drainage device.

[0573] 117. The use according to any one of the preceding embodiments, wherein the medicament is capable of inhibiting tumor or tumor cell growth.

[0574] 118. A method for constructing an animal model with malignant effusion, the method comprising injecting tumor cells into the thoracic cavity and / or abdominal cavity of the animal.

[0575] 119. The method of embodiment 118, wherein the injected dose is about 1×10 5 to about 2×10 6 cells.

[0576] 120. The method of any of embodiments 118-119, wherein the animal comprises a mouse.

[0577] 121. The method of any one of embodiments 118-120, wherein the injection comprises intraperitoneal injection, intraperitoneal injection, and / or intrathoracic injection.

[0578] 122. The method of any one of embodiments 118-121, wherein the tumor cells are derived from solid tumors and / or non-solid tumors.

[0579] 123. The method of embodiment 122, wherein the tumor comprises lung, lymphatic, liver, stomach, intestine, breast, pancreas, sarcoma, ovarian and / or mesothelial carcinoma.

[0580] 124. A method according to any one of embodiments 118-123, wherein the tumor cells include H22-Luc cells, B16-EGFR cells, MC38-EGFR cells, Pan02 mouse pancreatic cancer cells, S180 mouse sarcoma cells and / or Lewis-luc cells.

[0581] 125. The method of any one of embodiments 118-124, wherein the tumor cells are capable of causing the animal to have a tumor.

[0582] 126. The method of embodiment 125, wherein the tumor metastasizes to the pleura and / or peritoneum of the animal and produces the malignant effusion.

[0583] 127. The method of any one of embodiments 118-126, wherein the malignant effusion comprises malignant cells.

[0584] 128. The method of any one of embodiments 118-127, wherein the malignant effusion comprises blood and / or exudate.

[0585] 129. The method of any one of embodiments 118-128, wherein the malignant effusion comprises malignant pleural effusion (MPE) and / or malignant ascites.

[0586] 130. The method of any one of embodiments 118-129, wherein the malignant pleural effusion is associated with non-small cell lung cancer.

[0587] 131. The tissue or organ or culture thereof in an animal model with malignant effusion prepared by the method of any one of embodiments 118-120.

[0588] 132. An animal model with malignant effusion or its offspring prepared by the method described in any one of embodiments 118-130.

[0589] 133. Use of the tissue or organ or culture thereof, the animal model with malignant effusion or its progeny described in any one of embodiments 131-132 in the screening, verification, evaluation and / or research of anti-tumor drugs, or as a model system for pharmacological, immunological, microbiological and medical research.

[0590] Although the preferred embodiments of the present application have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided only by way of example. Without departing from the present application, those skilled in the art will now appreciate that many modifications, changes, and substitutions are possible. It should be understood that the various alternatives of the embodiments of the present application as described herein can be adopted in practicing the present application. It is intended that the following claims limit the scope of the application, and thus encompass methods and structures and equivalents thereof within the scope of these claims.

[0591] Example

[0592] The examples and preparations provided below further illustrate and exemplify the protein heterodimers of the present application and their use and preparation methods. It should be understood that the scope of the present application is not limited in any way by the following examples and preparations.

[0593] Example 1: Modification and preparation of polypeptides

[0594] 1.1 Determination of amino acid modifications in the Fc region

[0595] Determination of amino acid residues at the CH3-CH3 domain interface of human IgG1. Wild-type human IgG1 comprises a first heavy chain (chain A) and a second heavy chain (chain B). The amino acid interfaces of chains A and B are shown in Table 1 below. The positions of the amino acids are determined according to the EU index using KABAT numbering:

[0596] Table 1. CH3-CH3 interface residues in the wild-type human IgG1 antibody Fc region (PDB code 1DN2)

[0597] Then, amino acid modifications (e.g., amino acid substitutions) are performed on the interface residues to obtain the following modification groups (as shown in Table 2 below, with reference to KH knuckle hole modifications). In this application, chain A is also referred to as Fc9 or the first Fc region, and chain B is also referred to as Fc6 or the second Fc region:

[0598] Table 2. Amino acid modification groups

[0599] Subsequently, the formation of heterodimeric proteins comprising the modification groups listed in Table 2 above was examined using the ScFv-Fc / Fc system, as explained in detail below.

[0600] First, the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region was obtained from the Uniprot database (P01857) to obtain the wild-type human IgG1-Fc region amino acid sequence (SEQ ID NO: 30). A polynucleotide fragment encoding wild-type human IgG1-Fc was obtained from human PBMC total RNA (SEQ ID NO: 31, referred to as the Fc gene fragment) by RT-PCR. A polynucleotide fragment encoding the mouse kappa III signal peptide (SEQ ID NO: 32) was added to the 5' end of the Fc gene by overlapping PCR and then subcloned into the vector pcDNA4 (Invitrogen, Catalog No. V86220) for recombinant expression. This vector was used to express human IgG1-Fc in mammalian cells.

[0601] In some cases, a polypeptide encoding the variable region (VhH) of a camelid single-domain antibody is fused to the N-terminus of an Fc gene fragment to obtain a fusion gene fragment (as shown in SEQ ID NO: 33) encoding a fusion protein VhH-Fc (as shown in SEQ ID NO: 34). This fragment is then subcloned into the vector pcDNA4 (Invitrogen, Catalog No. V86220) to obtain a recombinant expression vector for expressing the fusion protein VhH-Fc in mammalian cells.

[0602] A nucleic acid molecule encoding a ScFv-Fc fusion protein (SEQ ID NO: 35) was synthesized, where ScFv refers to an anti-Her2 single-chain antibody, and the amino acid sequence of the ScFv-Fc fusion protein is shown in SEQ ID NO: 36. The ScFv-Fc gene fragment was then subcloned into the pcDNA4 vector (Invitrogen, Catalog No. V86220) to generate a recombinant expression vector for expressing the ScFv-Fc fusion protein in mammalian cells.

[0603] Then, the amino acid modifications listed in Table 2 above were introduced into ScFv-Fc (KH and 1-17 groups), VhH-Fc (9-12, 14, 15, and 17 groups), and Fc gene fragments (KH and 1-8 groups) by overlapping PCR, where chain A refers to the Fc region in ScFv-Fc, and chain B refers to an independent Fc region or the Fc region in VhH-Fc. The gene fragments with amino acid modifications were subcloned into the vector pcDNA4 (Invitrogen, Catalog No. V86220) to obtain recombinant expression vectors for expressing modified ScFv-Fc fusion proteins, modified Fc proteins, and modified VhH-Fc fusion proteins in mammalian cells.

[0604] Then, HEK293 cells (ATCC CRL-1573™) in suspension culture were transfected with the constructed expression vector containing PEI. For each group, an expression vector expressing the A chain (ScFv-Fc fusion protein) and an expression vector expressing the B chain (Fc protein or VhH-Fc fusion protein) were co-transfected at a 1:1 ratio. After 5-6 days of culture, the supernatant of the transient expression product was collected, and the expression product containing the corresponding protein heterodimer was initially purified using Protein A affinity chromatography. Each of the initially purified expression products contained the homodimeric protein ScFv-Fc / ScFv-Fc, the homodimeric protein Fc / Fc (or the homodimeric protein VhH-Fc / VhH-Fc), and the heterodimeric protein ScFv-Fc / Fc (or the heterodimeric protein ScFv-Fc / VhH-Fc) at varying percentages. Since the molecular weights of these proteins (i.e., homodimers and heterodimers) are different, their respective percentages can be determined based on the intensity of the corresponding bands reflected on the non-reducing SDS-PAGE gel. The intensity was quantified and the results are summarized in Tables 3-6 below.

[0605] Table 3 Percentages of homodimeric and heterodimeric proteins in the expression products

[0606] Table 4 Percentages of homodimeric and heterodimeric proteins in the expression products

[0607] Table 5 Percentages of homodimeric and heterodimeric proteins in the expression products

[0608] Table 6 Percentage of homodimeric proteins and heterodimeric proteins in the expression products

[0609] As can be seen from Tables 3-6 above, all modification groups promote heterodimer formation more effectively than the reference modification knothole. For illustrative purposes, modifications in Group 10 (modifications in chain A: T366W+K409A; modifications in chain B: T366S+L368G+Y407A+F405K) are used in the following examples to produce protein heterodimers or protein mixtures of the present application.

[0610] 1.2 Preparation of anti-EGFR (cetuximab)

[0611] The full-length amino acid sequences of the heavy and light chains of cetuximab (also known as Erbitux or Erb, an antibody against the epidermal growth factor receptor, EGFR) were obtained, and the corresponding DNA sequences encoding these amino acid sequences were obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the cetuximab light chain (Erb-LC) was then synthesized. The amino acid sequence of Erb-LC is shown in SEQ ID NO:37, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO:38. Point mutations (T366W and K409A) were then introduced into the polynucleotide sequence encoding the Fc region of the cetuximab heavy chain gene to synthesize a nucleic acid molecule encoding the modified cetuximab heavy chain (referred to herein as erb-Fc9). The corresponding polypeptide encoding it is referred to as Erb-Fc9. The amino acid sequence of Erb-Fc9 is shown in SEQ ID NO:39, and the polynucleotide sequence encoding it is shown in SEQ ID NO:40.

[0612] In another experiment, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the polynucleotide sequence encoding the Fc region of the cetuximab heavy chain gene. A nucleic acid molecule encoding the modified cetuximab heavy chain (referred to herein as erb-Fc6) was synthesized, and the corresponding polypeptide encoded thereby was designated Erb-Fc6. The amino acid sequence of Erb-Fc6 is set forth in SEQ ID NO:41, and the polynucleotide sequence encoding it is set forth in SEQ ID NO:42.

[0613] In another experiment, to prepare a reference protein, point mutations (Y349C and T366W) were introduced into the polynucleotide sequence encoding the Fc region of the cetuximab heavy chain gene. A nucleic acid molecule encoding the modified cetuximab heavy chain (referred to herein as erb-knob) was synthesized, and the corresponding polypeptide encoding it was named Erb-Knob. The amino acid sequence of Erb-Knob is set forth in SEQ ID NO:43, and the polynucleotide sequence encoding it is set forth in SEQ ID NO:44.

[0614] 1.3 Preparation of anti-HER2 (trastuzumab)

[0615] The full-length amino acid sequences of the heavy and light chains of trastuzumab were obtained according to U.S. Patent No. 7,879,325B2 (incorporated herein by reference). The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the light chain of trastuzumab (T-LC) was then synthesized. The amino acid sequence of T-LC is shown in SEQ ID NO: 45, and the corresponding polynucleotide sequence encoding this T-LC is shown in SEQ ID NO: 46. Point mutations (T366W and K409A) were then introduced into T-LC. A polynucleotide sequence encoding the Fc region of the trastuzumab heavy chain gene and a nucleic acid molecule encoding the modified trastuzumab heavy chain (referred to herein as t-Fc9) were synthesized, and the corresponding polypeptide encoding it was named T-Fc9. The amino acid sequence of T-Fc9 is shown in SEQ ID NO: 47, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 48.

[0616] 1.4 Preparation of anti-HER2 (Pertuzumab)

[0617] The full-length amino acid sequences of the heavy and light chains of Pertuzumab were obtained according to US Pat. No. 7,879,325 B2 (incorporated herein by reference). The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the light chain of Pertuzumab (P-LC) was then synthesized. The amino acid sequence of P-LC is shown in SEQ ID NO:49, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO:50. Point mutations (T366W and K409A) were then introduced into P-LC. A polynucleotide sequence encoding the Fc region of the Pertuzumab heavy chain gene and a nucleic acid molecule encoding the modified Pertuzumab heavy chain (referred to herein as p-Fc9) were synthesized, and the corresponding polypeptide encoding it is referred to as P-Fc9. The amino acid sequence of P-Fc9 is shown in SEQ ID NO:51, and the polynucleotide sequence encoding it is shown in SEQ ID NO:52.

[0618] 1.5 Preparation of anti-EGFR mutant (Mab806)

[0619] The full-length amino acid sequences of the heavy and light chains of Mab806 were obtained according to US Pat. No. 7,589,180B2 (incorporated herein by reference). The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the light chain of Mab806 (Mab806-LC) was then synthesized. The amino acid sequence of Mab806-LC is shown in SEQ ID NO: 53, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO: 54. Point mutations (T366W and K409A) were then introduced into the polynucleotide sequence encoding the Fc region of the Mab806 heavy chain gene, and a nucleic acid molecule encoding the modified Mab806 heavy chain (referred to herein as mab806-Fc9) was synthesized. The corresponding polypeptide encoded thereby is referred to as Mab806-Fc9. The amino acid sequence of Mab806-Fc9 is shown in SEQ ID NO: 55, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 56.

[0620] 1.6 Preparation of anti-GPC3 (codrituzumab)

[0621] The full-length amino acid sequences of the heavy and light chains of codrituzumab were obtained according to US Pat. No. 7,919,086 B2 (incorporated herein by reference). The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the light chain of codrituzumab (C-mab-LC) was then synthesized. The amino acid sequence of C-mab-LC is shown in SEQ ID NO: 57, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO: 58. Point mutations (T366W and K409A) were then introduced into the polynucleotide sequence encoding the Fc region of the codrituzumab heavy chain gene, and a nucleic acid molecule encoding the modified codrituzumab heavy chain (referred to herein as C-mab-Fc9) was synthesized. The corresponding polypeptide encoding it is referred to as C-mab-Fc9. The amino acid sequence of C-mab-Fc9 is shown in SEQ ID NO: 59, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 60.

[0622] 1.7 Preparation of anti-FAP (28H1)

[0623] The full-length amino acid sequences of the heavy and light chains of 28H1 were obtained according to US20120128591A1 (incorporated herein by reference). The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the light chain of 28H1 (28H1-LC) was then synthesized. The amino acid sequence of 28H1-LC is shown in SEQ ID NO: 61, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO: 62. Point mutations (T366W and K409A) were then introduced into the polynucleotide sequence encoding the Fc region of the 28H1 heavy chain gene, and a nucleic acid molecule encoding the modified 28H1 heavy chain (referred to herein as 28H1-Fc9) was synthesized, and the corresponding polypeptide encoding it was named 28H1-Fc9. The amino acid sequence of 28H1-Fc9 is shown in SEQ ID NO: 63, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 64.

[0624] 1.8 Preparation of anti-Muc1 (5E5)

[0625] The full-length amino acid sequences of the heavy and light chains of 5E5 were obtained according to US Pat. No. 8,440,798 B2 (incorporated herein by reference). The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the 5E5 light chain (5E5-LC) was then synthesized. The amino acid sequence of 5E5-LC is shown in SEQ ID NO: 65, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO: 66. Point mutations (T366W and K409A) were then introduced into the polynucleotide sequence encoding the Fc region of the 5E5 heavy chain gene, and a nucleic acid molecule encoding the modified 5E5 heavy chain (referred to herein as 5E5-Fc9) was synthesized. The corresponding polypeptide encoding it is referred to as 5E5-Fc9. The amino acid sequence of 5E5-Fc9 is shown in SEQ ID NO: 67, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 68.

[0626] 1.9 Preparation of Anti-MUC5AC (Enstuximab)

[0627] The full-length amino acid sequences of the heavy and light chains of Enstuximab were obtained according to WO2006113546A2 (incorporated herein by reference). The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the light chain of Enstuximab (E-mab-LC) was then synthesized. The amino acid sequence of E-mab-LC is shown in SEQ ID NO: 69, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO: 70. Point mutations (T366W and K409A) were then introduced into the polynucleotide sequence encoding the Fc region of the Enstuximab heavy chain gene, and a nucleic acid molecule encoding the modified Enstuximab heavy chain (referred to herein as E-mab-Fc9) was synthesized, and the corresponding polypeptide encoding it was referred to as E-mab-FC9. The amino acid sequence of E-mab-Fc9 is shown in SEQ ID NO: 71, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 72.

[0628] 1.10 Preparation of anti-mesothelin (amatuximab)

[0629] The full-length amino acid sequences of the heavy and light chains of amatuximab were obtained from http: / / www.imgt.org / mAb-DB / index. The corresponding DNA sequences encoding these amino acid sequences were then obtained using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A nucleic acid molecule encoding the light chain of amatuximab (A-mab-LC) was then synthesized. The amino acid sequence of A-mab-LC is shown in SEQ ID NO:73, and the corresponding polynucleotide sequence encoding it is shown in SEQ ID NO:74. Point mutations (T366W and K409A) were then introduced into the polynucleotide sequence encoding the Fc region of the amatuximab heavy chain gene, and a nucleic acid molecule encoding the modified xxx heavy chain (referred to herein as A-mab-Fc9) was synthesized. The corresponding polypeptide encoded by it is referred to as A-mab-FC9. The amino acid sequence of A-mab-Fc9 is shown in SEQ ID NO:75, and the polynucleotide sequence encoding it is shown in SEQ ID NO:76.

[0630] 1.11 Preparation of muIFNa4-Fc6

[0631] First, the sequence information of mouse interferon α4 (IFNa4) (NM_010504.2) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding the sequence was obtained. Then, based on the amino acid sequence of the human immunoglobulin γ1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. The linker sequence "GSGGG" (SEQ ID NO: 79) was then added to the N-terminus of Fc6 to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding mouse IFNa4 was added to the 5' end of the polynucleotide sequence encoding linker-Fc6 to obtain and synthesize the polynucleotide sequence encoding the fusion protein muIFNa4-Fc6. The amino acid sequence of muIFNa4-Fc6 is shown in SEQ ID NO: 77, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 78.

[0632] 1.12 Preparation of huIFNa2-Fc6

[0633] First, the sequence information of human interferon α2 (IFNa2) (NM_000605.3) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin γ1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the polypeptide thus obtained was called Fc6. Then, the linker sequence "GSGGG" (SEQ ID NO: 79) was added to the N-terminus of Fc6 to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding human IFNa2 was added to the 5' end of the polynucleotide sequence encoding linker-Fc6 to obtain and synthesize the polynucleotide sequence encoding the fusion protein huIFNa2-Fc6. The amino acid sequence of huIFNa2-Fc6 is shown in SEQ ID NO: 80, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 81.

[0634] 1.13 Preparation of muIFNb-Fc6

[0635] First, the sequence information of mouse interferon β (IFNβ) (NM_005018.2) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin γ1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. The linker sequence "GSGGG" (SEQ ID NO: 79) was then added to the N-terminus of Fc6 to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding mouse IFNβ was added to the 5' end of the polynucleotide sequence encoding linker-Fc6 to obtain and synthesize the polynucleotide sequence encoding the fusion protein muIFNb-Fc6. The amino acid sequence of muIFNb-Fc6 is shown in SEQ ID NO: 82, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 83.

[0636] 1.14 Preparation of huIFNb-Fc6

[0637] First, the sequence information of interferon beta (IFNβ) (EF064725.1) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 and 105 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. The linker sequence "GSGGG" (SEQ ID NO: 79) was then added to the N-terminus of Fc6 to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding human IFNβ was added to the 5' end of the polynucleotide sequence encoding linker-Fc6 to obtain and synthesize the polynucleotide sequence encoding the fusion protein huIFNb-Fc6. The amino acid sequence of huIFNb-Fc6 is shown in SEQ ID NO: 84, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 85.

[0638] Preparation of 1.15huIFNL-Fc6

[0639] First, the sequence information of human interferon λ (IFNL) (BC117482.1) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin γ1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 and 105 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. The linker sequence "GSGGG" (SEQ ID NO: 79) was then added to the N-terminus of Fc6 to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding human IFNL was added to the 5' end of the polynucleotide sequence encoding linker-Fc6 to obtain and synthesize the polynucleotide sequence encoding the fusion protein huIFNL-Fc6. The amino acid sequence of huIFNL-Fc6 is shown in SEQ ID NO: 86, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 87.

[0640] 1.16 Preparation of huIL10-Fc6

[0641] First, the sequence information of human interleukin 10 (huIL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 and 105 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. The linker sequence "(GGGGS)3" (SEQ ID NO: 88) was then added to the N-terminus of Fc6 to obtain linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding huIL10 was added to the 5' end of the polynucleotide sequence encoding linker-Fc6 to obtain and synthesize the polynucleotide sequence encoding the fusion protein huIL10-Fc6. The amino acid sequence of huIL10-Fc6 is shown in SEQ ID NO: 89, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 90.

[0642] 1. Preparation of 17(huIL10)2-Fc6

[0643] First, the sequence information of human interleukin 10 (huIL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 and 105 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. The linker sequence "(GGGGS)3" (SEQ ID NO: 88) was then added to the N-terminus of Fc6 to obtain Linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The linker sequence "(GGGGS)3" (SEQ ID NO: 88) was then added between two copies of huIL10 to obtain (huIL10)2. The polynucleotide sequence encoding (huIL10)2 was then added to the 5' end of the polynucleotide sequence encoding Linker-Fc6, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein (huIL10)2-Fc6. The amino acid sequence of (huIL10)2-Fc6 is shown in SEQ ID NO:91, and the polynucleotide sequence encoding it is shown in SEQ ID NO:92.

[0644] 1. Preparation of 18(huIL10)2-Fc9

[0645] First, the sequence information of human interleukin 10 (huIL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 and 105 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366W and K409A) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc9. The linker sequence "(GGGGS)3" (SEQ ID NO: 88) was then added to the N-terminus of Fc9 to obtain Linker-Fc9. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The linker sequence "(GGGGS)3" (SEQ ID NO: 88) was then added between two copies of huIL10 to obtain (huIL10)2. The polynucleotide sequence encoding (huIL10)2 was then added to the 5' end of the polynucleotide sequence encoding Linker-Fc9, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein (huIL10)2-Fc9. The amino acid sequence of (huIL10)2-Fc9 is shown in SEQ ID NO: 93, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 94.

[0646] 1.19(huIL10)2-Fc-pore preparation

[0647] First, the sequence information of human interleukin 10 (huIL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Then, based on the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 and 105 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated the Fc pore. The linker sequence "(GGGGS)3" (SEQ ID NO: 88) was then added to the N-terminus of the Fc-pore to obtain the linker-Fc-pore. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The linker sequence "(GGGGS)3" (SEQ ID NO: 88) was then added between two copies of huIL10 to obtain (huIL10)2. The polynucleotide sequence encoding (huIL10)2 was then added to the 5' end of the polynucleotide sequence encoding the linker-Fc-pore, thereby obtaining and synthesizing a polynucleotide sequence encoding the fusion protein (huIL10)2-Fc-pore. The amino acid sequence of (huIL10)2-Fc-pore is shown in SEQ ID NO:95, and the polynucleotide sequence encoding it is shown in SEQ ID NO:96.

[0648] 1.20 Preparation of husIL2-Fc6

[0649] First, the sequence information of human super interleukin 2 (husIL2) was obtained according to Nature 484, 529-533 (April 26, 2012) (incorporated herein by reference), and the full-length polynucleotide sequence encoding the sequence was obtained. Then, based on the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. The linker sequence "GGGGS" (SEQ ID NO: 79) was then added to the N-terminus of Fc6 to obtain Linker-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding husIL2 was added to the 5' end of the polynucleotide sequence encoding Linker-Fc6 to obtain and synthesize the polynucleotide sequence encoding the fusion protein husIL2-Fc6. The amino acid sequence of husIL2-Fc6 is shown in SEQ ID NO: 97, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 98.

[0650] 1.21 Preparation of husIL2 wells

[0651] First, the sequence information of human super interleukin 2 (husIL2) was obtained according to Nature 484, 529-533 (April 26, 2012) (incorporated herein by reference), and the full-length polynucleotide sequence encoding the sequence was obtained. Then, based on the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857), the amino acid sequence of human IgG1-Fc (i.e., residues 104 of P01857) was obtained from the protein database Uniprot. Residue 330). Subsequently, point mutations (D356C, T366S, L368A, and Y407V) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated the Fc pore. The linker sequence "GGGGS" (SEQ ID NO: 79) was then added to the N-terminus of the Fc-pore to obtain the linker-Fc-pore. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding husIL2 was added to the 5' end of the polynucleotide sequence encoding the linker-Fc-pore to obtain and synthesize the polynucleotide sequence encoding the fusion protein husIL2-Fc-pore. The amino acid sequence of husIL2-Fc-pore is shown in SEQ ID NO: 99, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 100.

[0652] Example 2: Construction of recombinant plasmid

[0653] Nucleic acid molecules (encoding Erb-Fc9, Erb-Fc6, Erb-Knob, T-Fc9, P-Fc9, Mab806-Fc9, C-mab-Fc9, 28H1-Fc9, 5E5-Fc9, E-mab-Fc9, A-mab-Fc9, T-LC (trastuzumab light chain), P-LC (pertuzumab light chain), Erb-LC (cetuximab light chain), Mab806-LC (Mab806 light chain), C-mab-LC (codrituzumab light chain)), 28H1-LC (28H1 light chain), 5E5-LC (5E5 light chain), E-mab-LC (ensutuximab light chain) The products obtained according to Example 1 were digested with HindIII and EcoRI (Takara), and then subcloned into the vector pcDNA4 / myc-HisA (Invitrogen, V863-20), respectively.The obtained plasmids were verified by sequencing, and the correct recombinant plasmids were named as follows: pcDNA4-Erb-Fc9, pcDNA4-Erb-Fc6, pcDNA4-Erb-Knob, pcDNA4-T-Fc9, pcDNA4-P-Fc9, pcDNA4-Mab806-Fc9, pcDNA4-C-mab-Fc9, pcDNA4-28H1-Fc9, pcDNA4-5E5-Fc9, pcDNA4-E-mab-Fc9, pcDNA4-A-mab-Fc9, pcDNA4-T-LC, pcDNA4-P-LC, pcDNA4-Erb-LC, pcDNA4-Mab806-LC, pcDNA4-C-mab-LC, pcDNA4-28H1-LC, pcDNA4-5E5-LC, pcDNA4-E-mab-LC, pcDNA4-A-mab-LC, pcDNA4-muIFNa4-Fc6, pcDNA4-huIFNa2-Fc6, pcDNA4-muIFNb-Fc6, pcDNA4-huIFNb-Fc6 , pcDNA4-huIFNL-Fc6, pcDNA4-huIL10-Fc6, pcDNA4-(huIL10)2-Fc6, pcDNA4-(huIL10)2-Fc9, pcDNA4-(huIL10)2-Fc-pore, pcDNA4-husIL2-Fc6 and pcDNA4-husIL2-pore.

[0654] Example 3 Expression and purification of protein heterodimers

[0655] Two days before transfection, prepare 12 × 600 mL suspension-acclimated HEK293 (ATCC, CRL-1573 TM ) cells were used for transient transfection and 0.8×10 6 Two days later, three aliquots of the cell suspension were centrifuged and resuspended in 600 mL of Freestyle 293 medium.

[0656] The recombinant expression vectors obtained in Example 2 are divided into the following groups:

[0657] Group 1: pcDNA4-Erb-Knob(200μg)+pcDNA4-Erb-LC(200μg)+pcDNA4-(huIL10)2-Fc-hole(200μg)

[0658] Group 2: pcDNA4-Erb-Fc6(200μg)+pcDNA4-Erb-LC(200μg)+pcDNA4-(huIL10)2-Fc9(200μg)

[0659] Group 3: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0660] Group 4: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0661] Group 5: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0662] Group 6: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0663] Group 7: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0664] Group 8: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-huIFNL-Fc6 (200 μg)

[0665] Group 9: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0666] Group 10: pcDNA4-Erb-Knob (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-husIL2-hole (200 μg)

[0667] Group 11: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0668] Group 12: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0669] Group 13: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0670] Group 14: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0671] Group 15: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0672] Group 16: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-huIFNL- Fc6 (200 μg)

[0673] Group 17: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0674] Group 18: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0675] Group 19: pcDNA4-Mab806-Fc9 (200 μg) + pcDNA4-Mab806-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0676] Group 20: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0677] Group 21: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0678] Group 22: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0679] Group 23: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0680] Group 24: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-huIFNL-Fc6 (200 μg)

[0681] Group 25: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0682] Group 26: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0683] Group 27: pcDNA4-T-Fc9 (200 μg) + pcDNA4-T-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0684] Group 28: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0685] Group 29: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0686] Group 30: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0687] Group 31: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0688] Group 32: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-huIFNL-Fc6 (200 μg)

[0689] Group 33: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0690] Group 34: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0691] Group 35: pcDNA4-P-Fc9 (200 μg) + pcDNA4-P-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0692] Group 36: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0693] Group 37: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0694] Group 38: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0695] Group 39: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0696] Group 40: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-huIFNL-Fc6 (二百微克)

[0697] Group 41: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0698] Group 42: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0699] Group 43: pcDNA4-C-mab-Fc9 (200 μg) + pcDNA4-C-mab-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0700] Group 44: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0701] Group 45: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0702] Group 46: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0703] Group 47: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0704] Group 48: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-huIFNL-Fc6 (200 μg)

[0705] Group 49: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0706] Group 50: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0707] Group 51: pcDNA4-28H1-Fc9 (200 μg) + pcDNA4-28H1-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0708] Group 52: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0709] Group 53: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0710] Group 54: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0711] Group 55: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0712] Group 56: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-huIFNL-Fc6 (200 μg)

[0713] Group 57: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0714] Group 58: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0715] Group 59: pcDNA4-5E5-Fc9 (200 μg) + pcDNA4-5E5-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0716] Group 60: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0717] Group 61: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0718] Group 62: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0719] Group 63: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0720] Group 64: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-huIFNL-Fc6 (200 μg)

[0721] Group 65: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0722] Group 66: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0723] Group 67: pcDNA4-E-mab-Fc9 (200 μg) + pcDNA4-E-mab-LC (200 μg) + pcDNA4-husIL2-Fc6 (200 μg)

[0724] Group 68: pcDNA4-A-mab-Fc9 (200 μg) + pcDNA4-A-mab-LC (200 μg) + pcDNA4-muIFNa4-Fc6 (200 μg)

[0725] Group 69: pcDNA4-A-mab-Fc9 (200 μg) + pcDNA4-A-mab-LC (200 μg) + pcDNA4-huIFNa2-Fc6 (200 μg)

[0726] Group 70: pcDNA4-A-mab-Fc9 (200 μg) + pcDNA4-A-mab-LC (200 μg) + pcDNA4-muIFNb-Fc6 (200 μg)

[0727] Group 71: pcDNA4-A-mab-Fc9 (200 μg) + pcDNA4-A-mab-LC (200 μg) + pcDNA4-huIFNb-Fc6 (200 μg)

[0728] Group 72: pcDNA4-A-mab-Fc9 (200 μg) + pcDNA4-A-mab-LC (200 μg) + pcDNA4-huIFNL-Fc6 (200 μg)

[0729] Group 73: pcDNA4-A-mab-Fc9 (200 μg) + pcDNA4-A-mab-LC (200 μg) + pcDNA4-huIL10-Fc6 (200 μg)

[0730] Group 74: pcDNA4-A-mab-Fc9 (200 μg) + pcDNA4-A-mab-LC (200 μg) + pcDNA4-(huIL10)2-Fc6 (200 μg)

[0731] Group 75: pcDNA4-A-mab-Fc9 (200μg) + pcDNA4-A-mab-LC (200μg) + pcDNA4-husIL2-Fc6 (200μg)

[0732] Each plasmid mixture was diluted with 6 mL of Freestyle 293 medium and PEI (polyethyleneimine) solution was added for transfection. Each plasmid / PEI mixture was added to 600 mL of cell suspension, and then cultured at 37 ° C, 10% CO2, and 90 rpm, and 50 μg / L IGF-1 (insulin-like growth factor I) was added to the culture medium. After 4 hours, 600 mL of EX293 medium, 2 mM glutamine and 50 μg / L IGF-1 were supplemented in the culture and cultured at 135 rpm. After 24 hours, 3.8 mM VPA was added. After 5-6 days, the supernatant of 5 × 1200 mL of cells was collected, and the crude protein heterodimer sample was purified by protein A affinity chromatography. The obtained sample was first detected by SDS-PAGE, and the target band was clearly seen, as shown in Figure 2.

[0733] Figure 2A shows the SDS-PAGE measurement results of Tmab-(huIL10)2-6-9, lane 2 is loaded with protein marker (MW marker 26619), lane 1 is loaded with reduced Tmab-(huIL10)2-6-9 heterodimeric protein, lane 3 is loaded with unreduced Tmab-(huIL10)2-6-9 heterodimeric protein, the MW of T-LC is approximately 25KD, the MW of T-Fc9 is approximately 50KD, the MW of (huIL10)2-Fc6 is approximately 65KD, and the MW of Tmab-(huIL10)2-6-9 heterodimeric protein is approximately 140KD.

[0734] Figure 2B shows the SDS-PAGE measurement results of Mab806-(huIL10)2-6-9, lane 2 is loaded with a protein marker (MW marker 26619), lane 1 is loaded with reduced Mab806-(huIL10)2-6-9 heterodimeric protein, and lane 3 is loaded with unreduced Mab806-(huIL10)2-6-9 heterodimeric protein. The MW of Mab806-LC is approximately 25KD, the MW of Mab806-Fc9 is approximately 50KD, the MW of (huIL10)2-Fc6 is approximately 65KD, and the MW of Mab806-(huIL10)2-6-9 heterodimeric protein is approximately 140KD.

[0735] Figure 2C shows the SDS-PAGE measurement results of Erb-muIFNa4-6-9, lane 2 is loaded with protein marker (MW marker 26619), lane 1 is loaded with reduced Erb-muIFNa4-6-9 heterodimeric protein, and lane 3 is loaded with unreduced Erb-muIFNa4-6-9 heterodimeric protein. The MW of Erb-LC is approximately 25KD, the MW of Erb-Fc9 is approximately 50KD, the MW of muIFNa4-Fc6 is approximately 45KD, and the MW of Erb-muIFNa4-6-9 heterodimeric protein is approximately 120KD.

[0736] Figure 2D shows the SDS-PAGE measurement results of Erb-husIL2-6-9, lane 2 is loaded with a protein marker (MW marker 26619), lane 1 is loaded with reduced Erb-husIL2-6-9 heterodimeric protein, and lane 3 is loaded with unreduced Erb-husIL2-6-9 heterodimeric protein. The molecular weight of Erb-LC is approximately 25KD, the molecular weight of Erb-Fc9 is approximately 50KD, the molecular weight of husIL2-Fc6 is approximately 41KD, and the molecular weight of Erb-husIL2-6-9 heterodimeric protein is approximately 120KD.

[0737] Figure 2E shows the SDS-PAGE measurement results of Erb-(huIL10)2-6-9, lane 2 is loaded with a protein marker (MW marker 26619), lane 1 is loaded with reduced Erb-(huIL10)2-6-9 heterodimeric protein, and lane 3 is loaded with unreduced Erb-(huIL10)2-6-9 heterodimeric protein. The MW of Erb-LC is approximately 25KD, the MW of Erb-Fc9 is approximately 50KD, the MW of (huIL10)2-Fc6 is approximately 65KD, and the MW of Erb-(huIL10)2-6-9 heterodimeric protein is approximately 140KD.

[0738] Figure 2F shows the SDS-PAGE measurement results of Tmab-husIL2-6-9, lane 2 is loaded with protein marker (MW marker 26619), lane 1 is loaded with reduced Tmab-husIL2-6-9 heterodimeric protein, and lane 3 is loaded with unreduced Tmab-husIL2-6-9 heterodimeric protein. The molecular weight of T-LC is approximately 25KD, the molecular weight of T-Fc9 is approximately 50KD, the molecular weight of husIL2-Fc6 is approximately 41KD, and the molecular weight of Tmab-husIL2-6-9 heterodimeric protein is approximately 116KD.

[0739] Figure 2G shows the SDS-PAGE measurement results of Erb-huIFNa2-6-9, where lane 2 is loaded with a protein marker (MW marker 26619), lane 1 is loaded with reduced Erb-huIFNa2-6-9 heterodimeric protein, and lane 3 is loaded with unreduced Erb-huIFNa2-6-9 heterodimeric protein. The molecular weight of Erb-LC is approximately 25KD, the molecular weight of Erb-Fc9 is approximately 50KD, the molecular weight of huIFNa2-Fc6 is approximately 45KD, and the MW of the Erb-huIFNa2-6-9 heterodimeric protein is approximately 120KD.

[0740] Similarly, the expression and purification results of other protein heterodimers of the present application were verified and confirmed by SDS-PAGE.

[0741] The protein heterodimers thus obtained were named (from group 1 to group 75): Erb-(huIL10)2-KH, Erb-(huIL10)2-6-9, Erb-(huIL10)2-9-6, Erb-muIFNa4-6-9, Erb-huIFNa2-6-9, Erb-muIFNb- 6-9, Erb-huIFNb-6-9, Erb-huIFNL-6-9, Erb-huIL10-6-9, Erb-husIL2-6-9, Erb-husIL2-KH, Mab806-muIFNa4-6-9, Mab806-huIFN a2-6-9, Mab806-muIFNb-6-9, Mab806-huIFNb-6-9, Mab806-huIFNL-6-9, Mab806-huIL10-6-9, Mab806-husIL2-6-9, Mab806-(huIL 10)2-6-9, Tmab-muIFNa4-6-9, Tmab-huIFNa2-6-9, Tmab-muIFNb-6-9, Tmab-huIFNb-6-9, Tmab-huIFNL-6-9, Tmab-huIL10-6-9, Tm ab-husIL2-6-9, Tmab-(huIL10)2-6-9, Pmab-muIFNa4-6-9, Pmab-huIFNa2-6-9, Pmab-muIFNb-6-9, Pmab-huIFNb-6-9, Pmab-huIFNL -6-9, Pmab-huIL10-6-9, Pmab-husIL2-6-9, Pmab-(huIL10)2-6-9, C-mab-muIFNa4-6-9, C-mab-huIFNa2-6-9, C-mab-muIFNb-6-9, C-mab-huIFNb-6-9, C-mab-huIFNL-6-9, C-mab-huIL10-6-9, C-mab-husIL2-6-9, C-mab-(huIL10)2-6-9, 28H1-muIFNa4-6-9, 28H1 -huIFNa2-6-9, 28H1-muIFNb-6-9, 28H1-huIFNb-6-9, 28H1-huIFNL-6-9, 28H1-huIL10-6-9, 28H1-husIL2-6-9, 28H1-(huIL10)2-6 -9, 5E5-muIFNa4-6-9, 5E5-huIFNa2-6-9, 5E5-muIFNb-6-9, 5E5-huIFNb-6-9, 5E5-huIFNL-6-9, 5E5-huIL10-6-9, 5E5-husIL2-6-9,5E5-(huIL10)2-6-9, E-mab-muIFNa4-6-9, E-mab-huIFNa2-6-9, E-mab-muIFNb-6-9, E-ma b-huIFNb-6-9, E-mab-huIFNL-6-9, E-mab-huIL10-6-9, E-mab-husIL2-6-9, E-mab-(huIL 10)2-6-9, A-mab-muIFNa4-6-9, A-mab-huIFNa2-6-9, A-mab-muIFNb-6-9, A-mab-huIFNb- 6-9, A-mab-huIFNL-6-9, A-mab-huIL10-6-9, A-mab-husIL2-6-9 and A-mab-(huIL10)2-6-9. 、

[0742] Example 4: Activity of protein heterodimers in treating malignant effusions

[0743] 4.1 Activity of the Erb-interleukin heterodimer of the present application in treating malignant peritoneal effusions of liver cancer

[0744] The BALB / c mouse model was used to test the in vivo activity of Erb-(huIL10)2-6-9 in treating malignant effusions of hepatocellular carcinoma. BALB / c mice were given 1×10 5 Mouse H22-Luc cells were injected intraperitoneally with a dose of 100 cells (mouse H22-Luc cells were purchased from Shanghai South Model Organisms Science Co., Ltd.). Two days after inoculation, mice were imaged in vivo, grouped according to the imaging data, and drug administration was started (see Table 7 for details of the administration), which was counted as day 0 of administration. Erb-(huIL10)2-6-9 heterodimers were injected intraperitoneally (ip) twice a week into mice. Specifically, mice were treated with Erb-(huIL10)2-6-9 heterodimers at two different doses (0.5 mg / kg and 2 mg / kg, respectively) on days 7, 10, and 14. After inoculation, mouse body weight, abdominal circumference, and abdominal imaging fluorescence data were measured twice a week.

[0745] The experimental results are shown in Figures 3-5. The data from in vivo imaging of mice on the 10th and 17th days after administration indicate that the ascites production in mice in Groups 2 and 3 administered with Erb-(huIL10)2-6-9 was significantly reduced (Figures 4-5), demonstrating that the Erb-(huIL10)2-6-9 heterodimer has a good effect in treating malignant abdominal effusions in liver cancer.

[0746] Drug withdrawal was performed on day 35 after dosing. Experimental data showed that the survival of mice in Groups 2 and 3 treated with the drug was significantly prolonged, and the survival rate of mice within each group was significantly improved (Figure 3), demonstrating the effectiveness of Erb-(huIL10)2-6-9 heterodimer in treating malignant abdominal effusions in patients with liver cancer. Survival rates increased with increasing doses, demonstrating that Erb-(huIL10)2-6-9 has a dose-dependent effect on prolonging mouse survival.

[0747] Table 7 Administration of Erb-(huIL10)2-6-9 heterodimer in the treatment of malignant abdominal effusion in patients with liver cancer

[0748] 4.2 Activity of the Erb-interleukin heterodimer of the present application in treating malignant peritoneal effusions of colon cancer

[0749] The BALB / c mouse model was used to test the in vivo activity of Erb-(huIL10)2-6-9 in treating malignant effusions of colon cancer. BALB / c mice were given 0.5×10 5 to about 10×10 5 Mouse B16-EGFR cells or MC38-EGFR cells were injected intraperitoneally with a dose of 100 cells. Two days after inoculation, mice were imaged in vivo, grouped according to the imaging data, and drug administration was started, which was counted as day 0 of administration. Erb-(huIL10)2-6-9 heterodimers were injected intraperitoneally twice a week in mice. Specifically, mice were treated with Erb-(huIL10)2-6-9 heterodimers at two different doses (0.5 mg / kg and 2 mg / kg, respectively) on days 7, 10, and 14. After inoculation, mouse body weight, abdominal circumference, and abdominal imaging fluorescence data were measured twice a week.

[0750] The experimental results showed that the data from in vivo mouse imaging indicated that the ascites production of mice in Group 2 and Group 3 administered with Erb-(huIL10)2-6-9 was significantly reduced, proving that Erb-(huIL10)2-6-9 heterodimers have a good effect in treating malignant abdominal effusions in colon cancer.

[0751] The experimental results showed that the survival of mice in Groups 2 and 3 treated with the drug was significantly prolonged, and the survival rate of mice within each group was significantly improved, demonstrating that Erb-(huIL10)2-6-9 heterodimers are effective in treating malignant abdominal effusions caused by colon cancer. The survival rate of mice also increased with increasing dosage, demonstrating that Erb-(huIL10)2-6-9's effect on extending mouse survival is dose-dependent.

[0752] 4.3 Activity of the Erb-interleukin heterodimer of the present application in treating malignant peritoneal effusion in pancreatic cancer

[0753] The BALB / c mouse model was used to test the in vivo activity of Erb-(huIL10)2-6-9 in treating pancreatic cancer malignant effusions. BALB / c mice were given 0.5×10 5 to about 10×10 5 Pan02 mouse pancreatic cancer cells were injected intraperitoneally at a dose of 100 cells. Two days after inoculation, mice were imaged in vivo, grouped according to the imaging data, and dosing began, which was counted as day 0 of dosing. Erb-(huIL10)2-6-9 heterodimers were injected intraperitoneally (IP) twice a week into the mice. Specifically, mice were treated with Erb-(huIL10)2-6-9 heterodimers at two different doses (0.5 mg / kg and 2 mg / kg, respectively) on days 7, 10, and 14. After inoculation, mouse body weight, abdominal circumference, and abdominal imaging fluorescence data were measured twice a week.

[0754] The experimental results showed that the data from in vivo mouse imaging indicated that the ascites production of mice in Group 2 and Group 3 administered with Erb-(huIL10)2-6-9 was significantly reduced, proving that the Erb-(huIL10)2-6-9 heterodimer has a good effect in treating malignant abdominal effusions in pancreatic cancer.

[0755] The experimental results showed that the survival of mice in Groups 2 and 3 treated with the drug was significantly prolonged, and the survival rate of mice within each group was significantly improved, demonstrating that Erb-(huIL10)2-6-9 heterodimers are effective in treating malignant abdominal effusions caused by pancreatic cancer. The survival rate of mice increased with increasing dosage, indicating that Erb-(huIL10)2-6-9 has a dose-dependent effect on extending mouse survival.

[0756] 4.4 Activity of the Erb-interleukin heterodimer of the present application in treating malignant peritoneal effusions of ascites cancer

[0757] The activity of Erb-(huIL10)2-6-9 in treating malignant effusions of ascites carcinoma was tested in BALB / c mice. 5 to about 10×10 5S180 mouse sarcoma cells were injected intraperitoneally at a dose of 100 cells. Two days after inoculation, mice were imaged in vivo, grouped according to the imaging data, and drug administration began, which was counted as day 0 of administration. Erb-(huIL10)2-6-9 heterodimers were injected intraperitoneally (ip) twice a week into the mice. Specifically, mice were treated with Erb-(huIL10)2-6-9 heterodimers at two different doses (0.5 mg / kg and 2 mg / kg, respectively) on days 7, 10, and 14. After inoculation, mouse body weight, abdominal circumference, and abdominal imaging fluorescence data were measured twice a week.

[0758] The experimental results showed that the data from in vivo mouse imaging indicated that the ascites production in mice in Group 2 and Group 3 administered with Erb-(huIL10)2-6-9 was significantly reduced, proving that the Erb-(huIL10)2-6-9 heterodimer has a good effect in treating malignant abdominal effusions caused by ascites cancer.

[0759] The experimental results showed that the survival of mice in Groups 2 and 3 treated with the drug was significantly prolonged, and the survival rate of mice within each group was significantly improved, demonstrating that the Erb-(huIL10)2-6-9 heterodimer is effective in treating malignant abdominal effusions caused by ascites cancer. The survival rate of mice also increased with increasing dosage, indicating that Erb-(huIL10)2-6-9 has a dose-dependent effect on extending mouse survival.

[0760] 4.5 Activity of the Erb-interleukin heterodimer of the present application in treating malignant pleural effusion of lung cancer

[0761] After anesthetizing C57BL / 6 mice, the right chest skin of the mice was disinfected and 5×10 5 Lewis-luc cells (Lewis-luc cells are purchased from Shanghai South Model Organisms Technology Co., Ltd.) were injected at a dose of 100 cells. Mice were observed daily after inoculation. On the second day after inoculation, the mice were randomly divided into groups according to the imaging results and then given the drug (see Table 8 for specific drug administration), with 8 mice in each group. Live imaging was performed twice a week. After two weeks, 4 mice in each group were dissected to observe the production of pleural effusion. Pleural effusion was extracted with a 1 mL syringe with the needle removed, and the volume of pleural effusion was calculated. Lung tissue was taken to take a picture and calculate the number of lung surface metastases. The number of chest wall metastases was also counted.

[0762] Table 8 Administration of Erb-(huIL10)2-6-9 heterodimer in the treatment of malignant thoracic effusion

[0763] The experimental results are detailed in Figures 6 and 7. Figure 6 shows that by day 12 after dosing, the weight of mice in Group 3 treated with Erb-(huIL10)2-6-9 had stabilized, and in vivo imaging indicated less pleural effusion. By day 15 after dosing, both mice in Control 1 and Positive Control 2 had died, but mice in Group 3 had maintained their weight and remained intact. By the end of the experiment, both mice in Control 1 and Positive Control 2 had died, but two mice in Group 3 treated with Erb-(huIL10)2-6-9 survived.

[0764] The results in Figure 7 show the amount of pleural effusion, the number of lung surface metastases, and the number of chest metastases in mice euthanized on day 14 from administration in each of Groups 1 to Group 3. The results in Figure 7 show that, compared with the mice in Control Group 1 and Positive Control Group 2, the mice in Group 3 treated with Erb-(huIL10)2-6-9 developed less pleural effusion and significantly reduced numbers of lung surface and chest metastases after administration.

Claims

1. Use of a protein heterodimer in the preparation of a medicament for preventing and / or treating malignant effusion, wherein the protein heterodimer comprises a first member and a second member different from the first member, wherein: i. the first member comprises a light chain and a heavy chain, the heavy chain comprising a first Fc region, the light chain complexing with the heavy chain to form a targeting portion exhibiting binding specificity to a tumor antigen; ii. the second member comprises a polypeptide comprising an immunomodulator fused to a second Fc region; iii. the first member and the second member associate through the complex of the first Fc region and the second Fc region to form the heterodimer; and iv. the first Fc region comprises a first modification and / or the second Fc region comprises a second modification, wherein the first modification and / or the second modification more effectively promotes heterodimerization between the first member and the second member compared to a knob-hole modification comprising a knob modification and a hole modification.

2. The use according to claim 1, wherein the polypeptide contained in the second member is a fusion protein, and the C-terminus of the immunomodulator is fused to the N-terminus of the second Fc region to form the fusion protein, optionally via a linker.

3. Use according to any one of the preceding claims, wherein the tumor antigen is selected from EGFR, EGFR mutants, HER2 / neu, GPC3, FAP, Mucl, MUC5AC and mesothelin.

4. Use according to any one of the preceding claims, wherein the immunomodulator is a cytokine selected from interferons, interleukins, chemokines, lymphokines and tumor necrosis factor.

5. Use according to any one of the preceding claims, wherein the immunomodulator is an interferon selected from interferon alpha, interferon lambda and interferon beta.

6. The use according to any one of the preceding claims, wherein the immunomodulator is an interleukin comprising interleukin 10, interleukin 2 and / or super interleukin 2.

7. Use according to any one of the preceding claims, wherein the first Fc region and the second Fc region are from the Fc region of an immunoglobulin, the immunoglobulin being human IgGl.

8. The use according to any one of the preceding claims, wherein the polypeptide contained in the second member comprises two or more immunomodulators, and the two or more immunomodulators are fused in-frame to each other and to the second Fc region, wherein the two or more immunomodulators are located at the N-terminus of the second Fc region.

9. The use according to any one of the preceding claims, wherein the first modification comprises an amino acid substitution at position T366, and an amino acid substitution at one or more positions selected from Y349, F405, K409, D399, K360, Q347, K392 and S354, wherein the positions of the amino acids are determined according to the EU index of KABAT numbering.

10. The use according to claim 9, wherein the first modification comprises an amino acid substitution selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D and T366W.

11. The method according to any one of claims 9 to 10, wherein the first modification comprises an amino acid substitution at any position selected from the following groups: 1) Y349 and T366; 2) Y349, T366 and F405; 3) Y349, T366 and K409; 4) Y349, T366, F405, K360 and Q347; 5) Y349, T366, F405 and Q347; 6) Y349, T366, K409, K360 and Q347; 7) Y349, T366, K409 and Q347; 8) T366, K409 and K392; 9) T366 and K409; 10) T366, K409, Y349 and S354; 11) T366 and F405; 12) T366, F405 and D399; and 13) T366, F405, Y349 and S354.

12. The method according to any one of claims 9 to 11, wherein the first modification comprises an amino acid substitution selected from any one of the following groups: 1) Y349C and T366W; 2) Y349C, T366W and F405K; 3) Y349C, T366W and K409E; 4) Y349C, T366W and K409A; 5) Y349C, T366W, F405K, K360E and Q347E; 6) Y349C, T366W, F405K and Q347R; 7) Y349C, T366W, K409A, K360E and Q347E; 8) Y349C, T366W, K409A and Q347R; 9) T366W, K409A and K392D; 10) T366W and K409A; 11) T366W, K409A and Y349D; 12) T366W, K409A, Y349D and S354D; 13) T366W and F405K; 14) T366W, F405K and D399S; 15) T366W, F405K and Y349D; and 16)T366W, F405K, Y349D and S354D.

13. The method of claim 9 , wherein the second modification comprises amino acid substitutions at positions T366, L368 and Y407, and amino acid substitutions at one or more positions selected from the group consisting of D356, D399, E357, F405, K360, K392, K409 and Q347, wherein the positions of the amino acids are determined according to the EU index of KABAT numbering.

14. The use according to claim 13, wherein the amino acid substitution comprised by the second modification is selected from D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A and Y407V.

15. The method according to any one of claims 13 to 14, wherein the second modification comprises an amino acid substitution at any position selected from the following groups: 1) D356, T366, L368, Y407 and F405; 2) D356, T366, L368 and Y407; 3) D356, T366, L368, Y407 and Q347; 4) D356, T366, L368, Y407, K360 and Q347; 5) D356, T366, L368, Y407, F405 and Q347; 6) D356, T366, L368, Y407, F405, K360 and Q347; 7) T366, L368, Y407, D399 and F405; 8) T366, L368, Y407 and F405; 9) T366, L368, Y407, F405 and E357; 10) T366, L368, Y407 and K409; 11) T366, L368, Y407, K409 and K392; and 12) T366, L368, Y407, K409 and E357.

16. The method according to any one of claims 13 to 15, wherein the second modification comprises any one of the following amino acid substitutions: 1) D356C, T366S, L368A, Y407V and F405K; 2) D356C, T366S, L368A and Y407V; 3) D356C, T366S, L368A, Y407V and Q347R; 4) D356C, T366S, L368A, Y407V, K360E and Q347E; 5) D356C, T366S, L368A, Y407V, F405K and Q347R; 6) D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 7) T366S, L368A, Y407V, D399S and F405K; 8) T366S, L368G, Y407A and F405K; 9) T366S, L368A, Y407V, F405K and E357A; 10) T366S, L368A, Y407V and K409A; 11) T366S, L368A, Y407V, K409A and K392D; 12) T366S, L368G, Y407A and K409A; 13) T366S, L368A, Y407V, K409A and E357A.

17. The use according to any one of the preceding claims, wherein the first Fc region comprises the first modification, the second Fc region comprises the second modification, and the first modification and the second modification comprise an amino acid substitution at any one of the positions selected from the following group: 1) First modification: Y349 and T366; Second modification: D356, T366, L368, Y407 and F405; 2) First modification: Y349, T366 and F405; Second modification: D356, T366, L368 and Y407; 3) First modification: Y349, T366 and K409; Second modification: D356, T366, L368, Y407 and F405; 4) First modification: Y349, T366, F405, K360 and Q347; Second modification: D356, T366, L368, Y407 and Q347; 5) First modification: Y349, T366, F405 and Q347; Second modification: D356, T366, L368, Y407, K360 and Q347; 6) First modification: Y349, T366, K409, K360 and Q347; Second modification: D356, T366, L368, Y407, F405 and Q347; 7) First modification: Y349, T366, K409 and Q347; Second modification: D356, T366, L368, Y407, F405, K360 and Q347; 8) First modification: T366, K409 and K392; Second modification: T366, L368, Y407, D399 and F405; 9) First modification: T366 and K409; Second modification: T366, L368, Y407 and F405; 10) First modification: T366, K409 and Y349; Second modification: T366, L368, Y407, F405 and E357; 11) First modification: T366, K409, Y349 and S354; Second modification: T366, L368, Y407, F405 and E357; 12) First modification: T366 and F405; Second modification: T366, L368, Y407 and K409; 13) First modification: T366, F405 and D399; Second modification: T366, L368, Y407, K409 and K392; 14) First modification: T366, F405 and Y349; Second modification: T366, L368, Y407, K409 and E357; 15) First modification: T366, F405, Y349 and S354; Second modification: T366, L368, Y407, K409 and E357; The positions of the amino acids are determined according to the EU index using KABAT numbering.

18. The use according to any one of the preceding claims, wherein the first Fc region comprises the first modification, and the second Fc region comprises the second modification, wherein the first modification and the second modification comprise any one group of amino acid substitutions selected from the following groups: 1) First modification: Y349C and T366W; Second modification: D356C, T366S, L368A, Y407V and F405K; 2) First modification: Y349C, T366W and F405K; Second modification: D356C, T366S, L368A and Y407V; 3) First modification: Y349C, T366W and K409E; Second modification: D356C, T366S, L368A, Y407V and F405K; 4) First modification: Y349C, T366W and K409A; Second modification: D356C, T366S, L368A, Y407V and F405K; 5) First modification: Y349C, T366W, F405K, K360E and Q347E; Second modification: D356C, T366S, L368A, Y407V and Q347R; 6) First modification: Y349C, T366W, F405K and Q347R; Second modification: D356C, T366S, L368A, Y407V, K360E and Q347E; 7) First modification: Y349C, T366W, K409A, K360E and Q347E; Second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) First modification: Y349C, T366W, K409A and Q347R; Second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) First modification: T366W, K409A and K392D; Second modification: T366S, L368A, Y407V, D399S and F405K; 10) First modification: T366W and K409A; Second modification: T366S, L368G, Y407A and F405K; 11) First modification: T366W, K409A and Y349D; Second modification: T366S, L368A, Y407V, F405K and E357A; 12) First modification: T366W, K409A, Y349D and S354D; Second modification: T366S, L368A, Y407V, F405K and E357A; 13) First modification: T366W and F405K; Second modification: T366S, L368A, Y407V and K409A; 14) First modification: T366W, F405K and D399S; Second modification: T366S, L368A, Y407V, K409A and K392D; 15) First modification: T366W and F405K; Second modification: T366S, L368G, Y407A and K409A; 16) First modification: T366W, F405K and Y349D; Second modification: T366S, L368A, Y407V, K409A and E357A; 17) First modification: T366W, F405K, Y349D and S354D; Second modification: T366S, L368A, Y407V, K409A and E357A; The positions of the amino acids are determined according to the EU index using KABAT numbering.

19. The use according to any one of the preceding claims, wherein the first Fc region comprises the first modification, and the second Fc region comprises the second modification, wherein the first modification comprises amino acid substitutions T366W and K409A, and the second modification comprises amino acid substitutions T366S, L368G, Y407A and F405K, wherein the positions of the amino acids are determined according to the EU index of KABAT numbers.

20. The use according to any one of the preceding claims, wherein the targeting portion specifically binds to EGFR, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 101, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 102, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 103; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 105, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 106, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:

107.

21. The use according to any one of claims 1-19, wherein the targeting portion specifically binds to an EGFR mutant, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 109, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 110, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 111; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 113, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 114, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:

115.

22. The use according to any one of claims 1-19, wherein the targeting portion specifically binds to HER2 / neu, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NOs: 117 and 125, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 118 and 126, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 119 and 127; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is selected from SEQ ID NOs: 121 and 129, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 122 and 130, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 123 and 131.

23. The use according to any one of claims 1-19, wherein the targeting portion specifically binds to GPC3, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 133, the amino acid sequence of CDR2 is shown in SEQ ID NO: 134, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 135; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 137, the amino acid sequence of CDR2 is shown in SEQ ID NO: 138, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

139.

24. The use according to any one of claims 1-19, wherein the targeting portion specifically binds to FAP, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 141, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 142, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 143; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 145, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 146, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:

147.

25. The use according to any one of claims 1-19, wherein the targeting portion specifically binds to Muc1, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 149, the amino acid sequence of CDR2 is shown in SEQ ID NO: 150, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 151; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 153, the amino acid sequence of CDR2 is shown in SEQ ID NO: 154, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

155.

26. The use according to any one of claims 1-19, wherein the targeting portion specifically binds to mesothelin, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 165, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 166, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 167; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 169, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 170, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:

171.

27. The use according to any one of claims 1-19, wherein the targeting portion specifically binds to MUC5AC, the light chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 157, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 158, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 159; the heavy chain of the first member comprises CDR1-3, the amino acid sequence of CDR1 is as shown in SEQ ID NO: 161, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 162, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:

163.

28. The use according to any one of the preceding claims, wherein in the heavy chain of the first member, the amino acid sequence of the first Fc region is selected from SEQ ID NO: 1, 4, 5, 6, 7, 9, 11, 13, 15, 17, 19, 21, 22, 24, 26, 27 and 29.

29. The use according to any one of the preceding claims, wherein the amino acid sequence of the immunomodulator contained in the second member is selected from SEQ ID NOs: 173-180.

30. The use according to any one of the preceding claims, wherein the amino acid sequence of the second Fc region comprised in the second member is selected from SEQ ID NO: 2, 3, 8, 10, 12, 14, 16, 18, 20, 23, 25 and 28.

31. The use according to any one of the preceding claims, wherein the amino acid sequence of the polypeptide comprised in the second member is selected from SEQ ID NO: 77, 80, 82, 84, 86, 89, 91 and 97.

32. The use according to any one of the preceding claims, wherein the amino acid sequence of the light chain contained in the first member is selected from SEQ ID NO: 37, 45, 49, 53, 57, 61, 65, 69 and 73, the amino acid sequence of the heavy chain contained in the first member is selected from SEQ ID NO: 39, 47, 51, 55, 59, 63, 67, 71 and 75, and the amino acid sequence of the polypeptide contained in the second member is selected from SEQ ID NO: 77, 80, 82, 84, 86, 89, 91 and 97.

33. The use according to any one of the preceding claims, wherein the medicament comprises a pharmaceutical composition comprising the protein heterodimer according to any one of the preceding claims and a pharmaceutically acceptable carrier.

34. The use according to any one of the preceding claims, wherein the medicament comprises a nucleic acid molecule encoding the protein heterodimer according to any one of the preceding claims.

35. Use according to the preceding claim 34, wherein the medicament comprises a vector comprising the nucleic acid molecule.

36. Use according to any one of the preceding claims 34-35, wherein the medicament comprises a cell comprising the nucleic acid molecule or the vector.

37. The use according to any one of the preceding claims, wherein the drug comprises a protein mixture, the protein mixture comprising: 1) a protein heterodimer according to any one of claims 1-32; and 2) a first homodimer formed by two of the first members of the protein heterodimer; 3) a second homodimer formed by two of the second members of the protein heterodimer; wherein the percentage of the protein heterodimer in the protein mixture is at least 50%.

38. Use according to any one of the preceding claims, wherein the patient suffering from the malignant effusion comprises a tumor patient.

39. The use according to claim 38, wherein the patient has symptoms including fatigue, vomiting, edema of the dorsal part of the foot, loss of appetite, dyspnea, cough, chest pain and / or abdominal pain.

40. The use according to any one of the preceding claims 38-39, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

41. Use according to any one of the preceding claims 38-40, wherein the tumor comprises lung, lymphatic, liver, stomach, intestine, breast, pancreatic cancer, ovarian and / or mesothelioma.

42. Use according to any one of the preceding claims, wherein the malignant effusion comprises malignant cells.

43. Use according to any one of the preceding claims, wherein the malignant effusion comprises blood and / or exudative fluid.

44. Use according to any one of the preceding claims 38-43, wherein the malignant effusion is associated with metastasis of the tumor to the pleura.

45. The use according to any one of the preceding claims 38-44, wherein the malignant effusion is associated with metastasis of the tumor to the peritoneum.

46. ​​Use according to any one of the preceding claims, wherein the malignant effusion is caused by a tumor comprising a primary tumor of the lung, lymph, liver, stomach, colon, breast, ovarian cancer, pancreatic cancer, bile duct, esophagus, brain, nose and / or prostate.

47. Use according to any one of the preceding claims, wherein the malignant effusion is caused by metastasis to the thoracic cavity of a tumour including: a primary cancer of the lung, lymph, liver, stomach, intestine, breast, ovarian cancer and / or mesothelioma.

48. Use according to any one of the preceding claims, wherein the malignant effusion is caused by metastasis to the abdominal cavity of a tumor including: ovarian cancer, pancreatic cancer, bile duct, esophagus, sarcoma, and / or a primary cancer of the liver.

49. Use according to any one of the preceding claims, wherein the malignant effusion comprises malignant pleural effusion (MPE) and / or malignant ascites.

50. Use according to any one of the preceding claims, wherein the malignant pleural effusion is associated with non-small cell lung cancer.

51. The use according to any one of the preceding claims, wherein the medicament is formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or administration via a subcutaneous depot.

52. Use according to any one of the preceding claims, wherein the medicament is configured to include a drainage device.

53. Use according to any one of the preceding claims, wherein the medicament is capable of inhibiting tumour or tumour cell growth.

54. A method for constructing an animal model with malignant effusion, the method comprising injecting tumor cells into the thoracic cavity and / or abdominal cavity of the animal.

55. The method of claim 54, wherein the injection dose is about 1×10 5 About 2×10 6 cells.

56. The method of any one of claims 54-55, wherein the animal comprises a mouse.

57. The method of any one of claims 54-56, wherein the injection comprises intraperitoneal injection, intraperitoneal injection and / or intrathoracic injection.

58. The method according to any one of claims 54-57, wherein the tumor cells are derived from solid tumors and / or non-solid tumors.

59. The method of claim 58, wherein the tumor comprises lung, lymphatic, liver, stomach, intestine, breast, pancreatic, sarcoma, ovarian and / or mesothelioma.

60. The method of any one of claims 54-59, wherein the tumor cells comprise H22-Luc cells, B16-EGFR cells, MC38-EGFR cells, Pan02 mouse pancreatic cancer cells, S180 mouse sarcoma cells and / or Lewis-luc cells.

61. The method of any one of claims 54-60, wherein the tumor cells are capable of causing the animal to suffer from a tumor.

62. The method of claim 61, wherein the tumor metastasizes to the pleura and / or peritoneum of the animal and produces the malignant effusion.

63. The method of any one of claims 54-62, wherein the malignant effusion comprises malignant cells.

64. The method of any one of claims 54-63, wherein the malignant effusion comprises blood and / or exudative fluid.

65. The method of any one of claims 54-64, wherein the malignant effusion comprises malignant pleural effusion (MPE) and / or malignant ascites.

66. The method of any one of claims 54-65, wherein the malignant pleural effusion is associated with non-small cell lung cancer.

67. A tissue or organ or a culture thereof in an animal model with malignant effusion prepared by the method of any one of claims 54 to 66.

68. An animal model with malignant effusion prepared by the method of any one of claims 54-66 or its offspring.

69. The use of the tissue or organ or culture thereof described in any one of claims 67-68, the animal model with malignant effusion or its progeny in the screening, verification, evaluation and / or research of anti-tumor drugs, or as a model system for pharmacological, immunological, microbiological and medical research.

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