Methods and compositions for cancer treatment

Abstract

Provided are compositions for treating cancer, the compositions comprising immunoconjugates for use in combination with chemotherapeutic drugs. Also provided are methods for treating cancer, the methods comprising use of the compositions.

Description

Background Technology

[0001] Although immune responses to tumor antigens can be detected (Disis et al. (1997), *Journal of Clinical Oncology*, 15:3363-3367), the malignant cells causing disease often fail to elicit an immune response leading to rejection. Studies have shown that it is possible to enhance the immunogenicity of tumor cells by introducing immunomodulatory molecules such as cytokines and costimulatory molecules; however, eradicating residual cancer cells may require targeting widely dispersed micrometastatic tumor deposits that cannot guide gene transfer. Furthermore, the expression and stability of the introduced immunomodulatory molecules are often far from satisfactory. Immunomodulators such as cytokines produced by cells of the immune system can directly or indirectly activate cells that initiate adaptive immune responses and can play an important role in triggering protective anti-tumor immunity. The innate immune system can be triggered by bacterial products or “danger” signals that lead to the release of pro-inflammatory cytokines such as interleukins.

[0002] Multiple studies have shown that immunomodulators can exert antitumor effects in animal models and cancer patients. However, the short half-life and systemic toxicity associated with the use of immunomodulators largely limit their application.

[0003] Besides immunotherapy, surgery, chemotherapy, hormone therapy, and radiation therapy have also been used to treat cancer (see, for example, Stockdale, 1998, Principles of Cancer Patient Management, in Scientific American: Medicine, Vol. 3, eds. Rubenstein and Federman, Chapter 12, Section IV). However, surgery may be impossible or unacceptable due to the patient's health condition or advanced stage of the disease, and often fails to completely remove the cancer cells after surgery. Hormone therapy is rarely given as a single agent and, although potentially effective, is often used to prevent or delay cancer recurrence after other treatments have removed most of the cancer cells. Furthermore, patients may quickly develop resistance to chemotherapy agents.

[0004] Therefore, there is still a great expectation for novel and effective therapies for cancer treatment. Summary of the Invention

[0005] This disclosure provides compositions and methods for treating cancer, the compositions and methods including the combined use of an immunoconjugate with a chemotherapeutic agent, and the invention of this disclosure has shown a significant synergistic effect in cancer treatment.

[0006] In one aspect, this disclosure provides a composition comprising an immunoconjugate and a chemotherapeutic agent, wherein: the immunoconjugate comprises 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, the first Fc subunit associating with the second Fc subunit to form a dimer; the one or more interleukins are fused to the Fc domain; and wherein the chemotherapeutic agent comprises fluorouracil and / or oxaliplatin.

[0007] In some embodiments, at least one of the one or more interleukins is fused to the amino-terminal amino acid of the Fc domain.

[0008] In some implementations, the immunoconjugate comprises two or more interleukins.

[0009] In some embodiments, at least two of the two or more interleukins are fused with the amino-terminal amino acid of the Fc domain.

[0010] In some embodiments, one or more of the interleukins are fused to the Fc domain via peptide linkers and / or immunoglobulin hinge regions.

[0011] In some embodiments, at least two of the two or more interleukins are fused together via peptide linkers to form an interleukin dimer.

[0012] In some embodiments, at least one of the interleukin dimers is fused to the N-terminal amino acid of the Fc domain. For example, at least one of the interleukin dimers can be fused to the N-terminal amino acid of the Fc domain via a peptide linker or an immunoglobulin hinge region.

[0013] In some implementations, the two or more interleukins are two or more copies of the same interleukin.

[0014] In some implementations, the two or more interleukins are two or more copies of IL10.

[0015] In some implementations, the one or more interleukins include one or more IL10.

[0016] In some embodiments, the immunoconjugate further includes a targeting portion fused to the Fc domain, wherein the targeting portion exhibits binding specificity for tumor antigens.

[0017] In some embodiments, the targeting portion is fused with the amino-terminal amino acid of the Fc domain.

[0018] In some implementations, the targeting portion is fused to the Fc domain via a peptide linker or an immunoglobulin hinge region.

[0019] In some implementations, the targeting portion includes the antigen-binding domain of the antibody.

[0020] In some implementations, the antigen-binding domain of the antibody is the Fab portion.

[0021] In some implementations, the tumor antigen is EGFR.

[0022] In some implementations, the targeting portion includes the antigen-binding domain of an anti-EGFR antibody.

[0023] In some implementations, the anti-EGFR antibody is cetuximab.

[0024] In some embodiments, the targeting portion includes the heavy chains CDR1-3 of cetuximab, where HCDR1 includes the amino acid sequence shown in SEQ ID NO:52, HCDR2 includes the amino acid sequence shown in SEQ ID NO:53, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:54.

[0025] In some embodiments, the targeting portion comprises the light chains CDR1-3 of cetuximab, LCDR1 comprising the amino acid sequence shown in SEQ ID NO:48, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:49, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:50.

[0026] In some embodiments, the targeting portion includes a heavy chain variable region of cetuximab, and the heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO:55.

[0027] In some embodiments, the targeting portion includes a light chain variable region of cetuximab, and the light chain variable region includes an amino acid sequence as shown in SEQ ID NO:51.

[0028] In some implementations, the Fc domain is an IgG Fc domain.

[0029] In some implementations, the IgG is IgG1.

[0030] In some implementations, the IgG is human IgG1.

[0031] In some embodiments, the immunoconjugate is an asymmetric immunoconjugate comprising a first member and a second member different from the first member, wherein the first member comprises a first Fc subunit, and the second member comprises one or more interleukins fused to the second Fc subunit, and the first Fc subunit associates with the second Fc subunit to form the dimer of the Fc domain.

[0032] In some embodiments, in the second member, at least one of the one or more interleukins is fused to the N-terminal amino acid of the second Fc subunit.

[0033] In some embodiments, in the second member, at least two of the one or more interleukins are fused together to form an interleukin dimer, and the interleukin dimer is further fused with the amino-terminal amino acid of the second Fc subunit.

[0034] In some implementations, the first member further includes the target portion fused with the first Fc subunit.

[0035] In some implementations, in the first member, the targeting portion is fused with the amino-terminal amino acid of the first Fc subunit.

[0036] In some implementations, the first Fc subunit is different from the second Fc subunit, and the Fc domain includes modifications that promote heterodimerization between the first Fc subunit and the second Fc subunit.

[0037] In some implementations, the first Fc subunit includes a first modification, and the second Fc subunit includes a second modification.

[0038] In some embodiments, the first modification includes 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 said amino acid is determined according to the EU index of the KABAT number.

[0039] In some embodiments, the amino acid substitutions included in the first modification are selected from the group consisting of:

[0040] 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 of the KABAT number.

[0041] In some implementations, the first modification includes substitution of 2-5 amino acids.

[0042] In some embodiments, the first modification includes amino acid substitutions at a set of positions selected from any of the following: 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) 8) Y349, T366, K409 and Q347; 9) T366, K409 and K392; 10) T366 and K409; 11) T366, K409, Y349 and S354; 12) T366 and F405; 13) T366, F405 and D399; and 14) T366, F405, Y349 and S354; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0043] In some embodiments, the first modification comprises a group of amino acid substitutions selected from the group consisting of: 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, and K409A; 9) Y349C, T366W, and K409E; 10) Y349C, T366W, and K409A; 11) Y349C, T366W, and K409E; 12) Y349C, T366W, and K409A; 13) Y349C, T366W, and K409E; 14) Y349C, T366W, and K409E; 15) Y349C, T366W, and K409A; 16) Y349C, T366W, and K409E; 17) Y349C, T366W, and K409E; 18) Y349C, T366W, and K409E; 19 ... 409A 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0044] In some embodiments, the second modification includes 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 the KABAT number.

[0045] In some embodiments, the amino acid substitutions included in the second modification are selected from the group consisting of:

[0046] D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A, and Y407V, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0047] In some embodiments, the second modification includes the substitution of 4-6 amino acids.

[0048] In some embodiments, the second modification includes amino acid substitutions at a set of positions selected from the group consisting of: 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 F405. 5) 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0049] In some embodiments, the second modification comprises a group of amino acid substitutions selected from the group consisting of: 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0050] In some embodiments, the first Fc subunit includes the first modification, the second Fc subunit includes the second modification, and the first and second modifications include amino acid substitutions at a set of positions selected from any of the following: 1) the first modification: Y349 and T366; and the second modification: D356, T366, L368, Y407, and F405; 2) the first modification: Y349, T366, and F405; and the second modification: D356, T366, L368, and Y407; 3) the first modification: Y349, T366, and K409; and the second modification: D356, T366, L368, Y407, and F405. 4) The first modification: Y349, T366, F405, K360 and Q347; and the second modification: D356, T366, L368, Y407 and Q347; 5) The first modification: Y349, T366, F405 and Q347; and the second modification: D356, T366, L368, Y407, K360 and Q347; 6) The first modification: Y349, T366, K409, K360 and Q347; and the second modification: D356, T366, L368, Y407, F405 and Q347; 7) The first modification: Y349, T366, K409 and Q347; and the second modification Modifications: D356, T366, L368, Y407, F405, K360 and Q347; 8) First modification: T366, K409 and K392; and second modification: T366, L368, Y407, D399 and F405; 9) First modification: T366 and K409; and second modification: T366, L368, Y407 and F405; 10) First modification: T366, K409 and Y349; and second modification: T366, L368, Y407, F405 and E357; 11) First modification: T366, K409, Y349 and S354; and second modification: T366, L368, Y407, F405 and E357; 68, 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; and second modification: T366, L368, Y407, K409 and K392; 14) First modification: T366, F405 and Y349; and second modification: T366, L368, Y407, K409 and E357; 15) First modification: T366, F405, Y349 and S354; and second modification: T366, L368, Y407, K409 and E357;The position of the amino acid is determined according to the EU index of the KABAT number.

[0051] In some embodiments, the first Fc subunit includes the first modification, and the second Fc subunit includes the second modification, wherein the first modification and the second modification include a group of amino acid substitutions selected from any of the following: 1) the first modification: Y349C and T366W; and the second modification: D356C, T366S, L368A, Y407V, and F405K; 2) the first modification: Y349C, T366W, and F405K; and the second modification: D356C, T366S, L368A, and Y407V; 3) the first modification: Y349C, T366W, and K409E; and the second modification: D356C and T366S. 4) The first modification: Y349C, T366W, and K409A; and the second modification: D356C, T366S, L368A, Y407V, and F405K; 5) The first modification: Y349C, T366W, F405K, K360E, and Q347E; and the second modification: D356C, T366S, L368A, Y407V, and Q347R; 6) The first modification: Y349C, T366W, F405K, and Q347R; and the second modification: D356C, T366S, L368A, Y407V, K360E, and Q347E; 7) The first modification: Y 349C, T366W, K409A, K360E and Q347E; and the second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) the first modification: Y349C, T366W, K409A and Q347R; and the second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) the first modification: T366W, K409A and K392D; and the second modification: T366S, L368A, Y407V, D399S and F405K; 10) the first modification: T366W and K409A; and the second modification: D356C, T366S, L368A, Y407V, D399S and F405K; 11) First modification: T366W, K409A, and Y349D; and second modification: T366S, L368A, Y407V, F405K, and E357A; 12) First modification: T366W, K409A, Y349D, and S354D; and second modification: T366S, L368A, Y407V, F405K, and E357A; 13) First modification: T366W and F405K; and second modification: T366S, L368A, Y407V, and K409A; 14) First modification: T366W, F405K, and D399S;The second modification: T366S, L368A, Y407V, K409A, and K392D; 15) the first modification: T366W and F405K; and the second modification: T366S, L368G, Y407A, and K409A; 16) the first modification: T366W, F405K, and Y349D; and the second modification: T366S, L368A, Y407V, K409A, and E357A; 17) the first modification: T366W, F405K, Y349D, and S354D; and the second modification: T366S, L368A, Y407V, K409A, and E357A; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0052] In some embodiments, the first Fc subunit includes the first modification, the second Fc subunit includes the second modification, the first modification includes the amino acid substitutions T366W and K409A, and the second modification includes the amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0053] In some implementations, the first member does not include any interleukins.

[0054] In some embodiments, the first Fc subunit includes the amino acid sequence shown in any of SEQ ID NO:17.

[0055] In some embodiments, the second Fc subunit includes the amino acid sequence shown in any of SEQ ID NO:18.

[0056] In some implementations, the interleukin is a human interleukin.

[0057] In some embodiments, the interleukin comprises the amino acid sequence shown in any of SEQ ID NO:56.

[0058] In some embodiments, the immunoconjugate includes a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO:37, the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO:39, and the third polypeptide chain includes the amino acid sequence shown in SEQ ID NO:42.

[0059] In some embodiments, the first member includes a first polypeptide chain and a second polypeptide chain, the second member includes a third polypeptide chain, the first polypeptide chain includes an amino acid sequence as shown in SEQ ID NO:37, the second polypeptide chain includes an amino acid sequence as shown in SEQ ID NO:39, and the third polypeptide chain includes an amino acid sequence as shown in SEQ ID NO:42.

[0060] In some embodiments, the first member comprises a first polypeptide chain, and the second member comprises a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:17, and the second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:42.

[0061] In some embodiments, the fluorouracil comprises 5-Fu.

[0062] In some implementations, the chemotherapy drug further includes leucovorin.

[0063] In some embodiments, the chemotherapy drug includes tetrahydrofolate and / or calcium tetrahydrofolate.

[0064] In some implementations, the chemotherapy drugs include the FOLFOX regimen.

[0065] In some embodiments, the fluorouracil and the oxaliplatin are not mixed with each other in the composition.

[0066] In other respects, this disclosure provides immunoconjugates for use in combination with chemotherapy drugs for the treatment of cancer, wherein the immunoconjugate is as defined in this disclosure and the chemotherapy drug is as defined in this disclosure.

[0067] In some implementations, the cancer is selected from pancreatic cancer and colorectal cancer.

[0068] In some implementations, the pancreatic cancer is metastatic pancreatic cancer.

[0069] In some implementations, the colorectal cancer is metastatic colorectal cancer.

[0070] In some implementations, the cancer or its cells have increased EGFR expression.

[0071] In other respects, this disclosure provides the use of an immunoconjugate in combination with a chemotherapeutic agent for the preparation of an agent for the treatment of cancer in a subject of need, wherein the immunoconjugate is as defined in this disclosure and the chemotherapeutic agent is as defined in this disclosure.

[0072] In some implementations, the cancer is selected from pancreatic cancer and colorectal cancer.

[0073] In some implementations, the pancreatic cancer is metastatic pancreatic cancer.

[0074] In some implementations, the colorectal cancer is metastatic colorectal cancer.

[0075] In some implementations, the cancer or its cells have increased EGFR expression.

[0076] In other respects, this disclosure provides a method for treating cancer in a subject in need, comprising administering to the subject (a) an effective amount of an immunoconjugate as defined in this disclosure and (b) an effective amount of a chemotherapeutic agent as defined in this disclosure.

[0077] In some implementations, the immunoconjugate is administered to the subject after the chemotherapy drug is administered.

[0078] In some implementations, the immunoconjugate is administered to the subject no more than 10 days after the administration of the chemotherapy drug.

[0079] In some implementations, the immunoconjugate is administered to the subject no more than 3 days after the administration of the chemotherapy drug.

[0080] In some implementations, the immunoconjugate is administered to the subject two or more times.

[0081] In some implementations, the cancer is selected from pancreatic cancer, melanoma, and colorectal cancer.

[0082] In some implementations, the pancreatic cancer is metastatic pancreatic cancer.

[0083] In some implementations, the colorectal cancer is metastatic colorectal cancer.

[0084] In some implementations, the cancer is melanoma.

[0085] In some implementations, the cancer or its cells have increased EGFR expression.

[0086] Additional aspects and advantages of this disclosure will become apparent to those skilled in the art from the following embodiments, wherein only illustrative embodiments of this disclosure are shown and described. As will be appreciated, this disclosure is capable of other and different embodiments, and certain details thereof can be modified in various obvious ways, all of which do not depart from this disclosure. Therefore, the drawings and embodiments should be considered illustrative in nature and not restrictive.

[0087] By incorporating references

[0088] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually indicated as incorporated by reference. Attached Figure Description

[0089] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention can be obtained by referring to the following embodiments and accompanying drawings (also referred to herein as “figures”), which illustrate illustrative implementations in which the principles of the invention are employed, as shown in the drawings:

[0090] Figure 1A-1E The purification results of the immunoconjugates disclosed herein are illustrated, as shown by SDS-PAGE and SEC-HPLC analyses.

[0091] Figures 2A-2D The effects of various chemotherapy therapies combined with the immunoconjugates disclosed herein are illustrated.

[0092] Figure 3 A-3C illustrates embodiments of the immunoconjugates disclosed herein.

[0093] Figures 4A-4B The diagram illustrates that Erb-(IL10)2, FOLFOX, and the combination of Erb-(IL10)2 and FOLFOX inhibit tumor growth.

[0094] Figure 4C The study demonstrated that Erb-(IL10)2, FOLFOX, and the combination of Erb-(IL10)2 and FOLFOX prolonged mouse survival.

[0095] Figures 5A-5B The diagram illustrates that Erb-(IL10)2, FOLFOX, and the combination of Erb-(IL10)2 and FOLFOX inhibit tumor growth. Detailed Implementation

[0096] Before describing embodiments of this disclosure, it should be understood that the embodiments described are provided by way of example only, and various alternatives to the embodiments of this disclosure described herein may be adopted in the practice of this disclosure. Many changes, modifications, and substitutions will appear to those skilled in the art without departing from this disclosure.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although similar or equivalent methods and materials to those described herein may be used in practice or testing with respect to this disclosure, suitable methods and materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and embodiments are illustrative only and not intended to be restrictive. Many variations, modifications, and substitutions will appear to those skilled in the art without departing from this disclosure.

[0098] Unless the context clearly specifies otherwise, the singular forms “a / an” and “the” used herein generally include plural references.

[0099] As used herein, the term "immunoconjugate" generally refers to a protein molecule formed by conjugating one or more antibodies or fragments thereof to one or more second molecules. The second molecule may be the same or different and may contain, for example, an effector protein.

[0100] As used herein, the term "protein" generally refers to materials or molecules that are, relate to, are similar to, or are polypeptides or proteins. For example, the immunoconjugates disclosed herein may be heterodimeric proteins or heterodimers comprising two or more polypeptides.

[0101] As used herein, the term "heterodimer" generally refers to a molecule (e.g., a protein molecule) composed of two distinct members. The two members of a heterodimer may differ in structure, function, activity, and / or composition. For example, the two distinct members may include polypeptides that differ in the order, number, or type of amino acid residues that form them. Each of the two distinct members of a heterodimer may independently comprise one, two, or more units, polypeptide chains, or portions.

[0102] 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 molecules or compounds. Other embodiments of a targeting moiety may include, but are not limited to, aptamers, high-affinity multimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides, or proteins. The terms "targeting moiety" and "binding moiety" are used interchangeably herein.

[0103] As used herein, the term "tumor antigen" generally refers to an antigenic substance produced or generated by tumor cells that can trigger an immune response in the host. For example, a tumor antigen can be a protein, polypeptide, peptide, or fragment thereof that constitutes part of a tumor cell and can induce tumor-specific cytotoxic T lymphocytes. A tumor antigen peptide can be a peptide produced due to the degradation of a tumor antigen in a tumor cell and, when expressed on the cell surface by binding to HLA molecules, can induce or activate tumor-specific cytotoxic T lymphocytes. In some embodiments, the term "tumor antigen" can also refer to a biomolecule (e.g., a protein, carbohydrate, glycoprotein, etc.) that is exclusively, preferentially, or differentially expressed on cancer cells and / or found to be associated with cancer cells and thus provides a cancer-preferred or cancer-specific target. For example, preferential expression can be preferential expression relative to any other cell in the organism or preferential expression in a specific region of the organism (e.g., within a specific organ or tissue).

[0104] As used in this article, the term "heterodimerization" generally refers to the formation of a heterodimer between two distinct members (e.g., two non-identical polypeptides), such as through complexation, association, or aggregation, and the process of forming or not forming covalent bonds between two distinct members.

[0105] As used herein, the term "covalent bond" generally refers to a chemical bond formed between atoms by sharing electrons. For example, a covalent bond can be polar or nonpolar. In some embodiments, the covalent bond is a disulfide bond.

[0106] As used herein, the term “non-covalent pairing affinity” generally refers to the ability of dimerized or heterodimerized sequences to bind together through non-covalent interactions, such as 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 of transition metal cations), or combinations of these interactions.

[0107] As used herein, the term "linker" generally refers to a synthetic amino acid sequence that links or connects two polypeptide sequences, such as linking two polypeptide domains. A linker can link two amino acid sequences via peptide bonds. In some embodiments, the linkers of this disclosure link a biologically active portion to a second portion within a linear sequence.

[0108] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of any length of amino acids. These polymers may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acids. The terms also cover modified amino acid polymers, for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation such as coupling with a labeled component. The terms may apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, or to naturally occurring amino acid polymers. The terms may also include variations of the conventional peptide bond linking the amino acids constituting a polypeptide. For example, “peptide,” “polypeptide,” and “protein” are used interchangeably. "Peptide" and "protein" can be chains of amino acids linked by peptide bonds at their α-carbons. Therefore, the terminal amino acid at one end of the chain (amino terminus) can have a free amino group, while the terminal amino acid at the other end of the chain (carboxyl terminus) can have a free carboxyl group. As used herein, the term "amino terminus" (abbreviated as N-terminus) generally refers to the free α-amino group on the amino acid at the amino terminus of a peptide, or the α-amino group (the amino group involved in the peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxyl terminus" generally refers to the free carboxyl group on the carboxyl terminus of a peptide, or the carboxyl group of an amino acid at any other position within the peptide. Peptides can also contain essentially any polyamino acid, including, but not limited to, peptide mimics, such as amino acids linked by ether bonds rather than amide bonds.

[0109] As used herein, the term "amino acid" generally refers to natural and / or non-natural or synthetic amino acids, including but not limited to D or L optical isomers or both, amino acid analogs, and peptide mimics. Standard single-letter or three-letter codes are used to designate amino acids.

[0110] The term "variant," when used in the context of protein molecules (e.g., polypeptides or proteins), generally refers to a protein molecule that has sequence homology with a native bioactive protein, retaining at least a portion of the therapeutic and / or biological activity of said bioactive protein. For example, a variant protein may share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a reference bioactive protein. In some embodiments, a "variant" may comprise a protein that has been intentionally modified, for example, through site-directed mutagenesis, coding gene synthesis, insertion, or accidental modification by mutation.

[0111] The terms “coupled,” “linked,” “fused,” and “fusion” are used interchangeably herein and generally refer to the joining of two or more chemical elements, sequences, or components together, for example, by means of chemical coupling or recombination. For example, if a promoter or enhancer affects the transcription of a coding sequence, then it is operatively linked to said sequence. Generally, “operatively linked” means that the linked DNA sequences are joined together and are within a reading frame or frame. “In-frame fusion” refers to the joining of two or more ORFs in a manner that maintains the correct reading frame of the original open reading frame (ORF) to form a joined, longer ORF. Thus, the resulting “fusion polypeptide” is a single protein containing two or more segments (segments that are not normally joined in nature) corresponding to the polypeptide encoded by the original ORF. A “fusion site” refers to the sequence where two or more segments are joined together. In some cases, the fusion site may be the same sequence as the sequences in the two or more joined segments. In some cases, the fusion site may further contain a gap segment that is not identical to any of the sequences in the two or more joined segments.

[0112] In the case of polypeptides, a "linear sequence" or "sequence" refers to a series of amino acids in the polypeptide running from the amino terminus to the carboxyl terminus, where adjacent residues in the sequence are connected in the primary structure of the polypeptide. A "partial sequence" is a linear sequence that forms part of a polypeptide and is known to include additional residues in one or both directions.

[0113] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, which is a deoxynucleotide or ribonucleotide, or an analogue thereof. Polynucleotides 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 genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure may be imposed before or after polymer assembly, if present. The sequence of the nucleotide may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by coupling with labeled components.

[0114] The terms "gene" and "gene segment" are used interchangeably herein and generally refer to a polynucleotide containing at least one open reading frame that encodes a specific protein after transcription and translation. A gene or gene segment can be genomic or cDNA, provided the polynucleotide contains at least one open reading frame that covers the entire coding region or a segment thereof. A "fusion gene" is a gene consisting of at least two linked heteropolynucleotides.

[0115] As used herein, the term "antibody" generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or segments of immunoglobulin genes. Immunoglobulin genes may contain κ, λ, α, γ, δ, ε, and μ constant regions and myriad immunoglobulin variable regions. The light chain, as used herein, may be classified as κ or λ. The heavy chain may be classified as γ, μ, α, δ, or ε, which respectively define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies used in this disclosure may have structural units comprising tetramers. Each tetramer may consist of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The N-terminus of each chain may define a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "light chain variable region" (VL) and "heavy chain variable region" (VH), as used herein, generally refer to these regions containing light and heavy chains, respectively. Antibodies can exist either as intact immunoglobulins or as a number of well-characterized fragments produced or re-expressed by digestion with various peptidases. Thus, for example, pepsin can digest antibodies below the disulfide bonds in the hinge region to produce F(ab)'2 (a Fab dimer, itself a light chain linked to VH-CH1 by disulfide bonds). 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 Fab' monomers. Fab' monomers are essentially Fab with a portion of the hinge region (see Fundamental Immunology, ed. W.E. Paul, Raven Press, NY (1993) for a more detailed description of other antibody fragments). While the various antibody fragments are defined in relation to the digestion of intact antibodies, those skilled in the art will understand that said Fab' fragments can be resynthesized chemically or by methods utilizing recombinant DNA. Therefore, the term antibody as used herein can also encompass antibody fragments generated by modifying the whole antibody or resynthesized using recombinant DNA methods, including but not limited to Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nanobodies, monofunctional antibodies, and bifunctional antibodies. In some embodiments, antibodies comprise, but are not limited to, Fab'2, IgG, IgM, IgA, IgE, and single-chain antibodies such as single-chain Fv (scFv) antibodies, wherein variable heavy chains and variable light chains are linked together (directly or via peptide linkers) to form a continuous polypeptide.

[0116] As used herein, the terms "antigen binding site" or "binding moiety" generally refer to the portion of an antibody involved in antigen binding. An antigen binding site can be formed by amino acid residues in the N-terminal variable ("V") region of the heavy ("H") chain and / or light ("L") chain. Three highly variable segments within the V region of the heavy and light chains are called "hypervariable regions," which are inserted between more conserved flanking segments called "frame regions" or "FRs." Therefore, as used herein, the term "FR" generally refers to the amino acid sequence between and adjacent to 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 opposite each other in three-dimensional space to form an antigen-binding "surface." This surface mediates the recognition and binding of the target antigen. The three hypervariable regions of each heavy and light chain are called “complementarity-determining regions” or “CDRs”, and they are characterized, for example, by Kabat et al., “Sequences of proteins of immunological interest”, 4th edition, U.S. Department of Health and Human Services, Public Health Services, Bethesda, Md. (1987).

[0117] As used herein, the terms “homology,” “homology,” or “sequence identity” generally refer to the sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program (such as Emboss Needle or BestFit) to determine the sequence identity, similarity, or homology between two different amino acid sequences, default settings can be used, or an appropriate scoring matrix such as blosum45 or blosum80 can be selected to optimize the identity, similarity, or homology score. In some embodiments, homologous polynucleotides are polynucleotides that are heterozygous 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%, and even 100% sequence identity compared to those sequences. When optimally aligned to sequences of a comparable length, homologous polypeptides have at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity, or at least 99% sequence identity.

[0118] The term "effective amount" or "therapeutic effective amount" refers to an amount of composition sufficient to achieve the intended application, including but not limited to the treatment of a disease. Therapeutic effective amounts can vary depending on the intended application (e.g., in vitro or in vivo) that can be readily determined by a person skilled in the art, or on the subject being treated and the disease condition, such as the subject's weight and age, the severity of the disease condition, the method of administration, etc. The term can also be applied to doses that induce specific responses in target cells, such as target gene induction, proliferation, and / or apoptosis. Specific doses will vary depending on the specific compound selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the time of administration, the tissue to which it is administered, and the physical delivery system used to deliver it.

[0119] The terms “treatment / treating,” “relief,” or “improvement” are used interchangeably herein and refer to methods for achieving beneficial or desired outcomes (including, but not limited to, therapeutic and / or preventative benefits). Therapeutic benefits, as used herein, generally refer to the eradication of the underlying condition being treated or a reduction in its severity. Furthermore, therapeutic benefits are achieved through the eradication, reduction in severity, or decrease in incidence of one or more physical symptoms associated with the underlying condition, resulting in an observed improvement in the subject, although the subject may still be suffering from the underlying condition. To obtain preventative benefits, the composition may be administered to subjects at risk of developing a specific disease, or to subjects who report one or more physical symptoms of a disease, even if such a disease may not yet have been diagnosed.

[0120] The term "therapeutic effect" as used herein generally encompasses the therapeutic benefits and / or preventive benefits described above. Preventive effects include delaying the onset of a disease or condition or eliminating said disease or condition, delaying the onset of symptoms of a disease or condition or eliminating said symptoms, slowing, stopping, or reversing the development of a disease or condition, or any combination thereof.

[0121] As used herein, the terms “co-administration,” “combined administration,” “conjunctive use,” and their grammatical equivalents generally cover the administration of two or more agents or therapies to a subject such that both agents and / or their metabolites or both therapies are present in the subject and / or act in the subject. Co-administration includes simultaneous administration in separate compositions or forms, administration in separate compositions or forms at different times, or administration in a composition in which two agents are present.

[0122] As used herein, the term "pharmaceutical" generally refers to a biological, pharmaceutical, or chemical compound or other component. Non-limiting embodiments include simple or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, vitamin derivatives, carbohydrates, toxins, chemotherapeutic compounds, or pharmaceutical agents capable of generating / emitting radiation.

[0123] As used herein, the term "interleukin" generally refers to a secretory protein or signaling molecule capable of promoting the development and differentiation of T lymphocytes and / or B lymphocytes and / or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes as well as by monocytes, macrophages, and endothelial cells. Interleukin (IL) as used herein may comprise IL10. The term "interleukin" as used herein may comprise a full-length interleukin or a fragment (e.g., a truncated form) or variant thereof that substantially maintains 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). Interleukins as used herein may be derived from any mammalian species. In some embodiments, interleukins are derived from species selected from the group consisting of humans, horses, cattle, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, and non-human primates. In some implementations, interleukins may be in mutant forms, for example, with increased or decreased affinity for their receptors.

[0124] The term “subject” as used in this article generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, goats, rabbits, mice, rats, or monkeys.

[0125] As used in this article, the term "EGFR family member" generally refers to a member of the epidermal growth factor receptor family. For example, it could be ErbB-1 (also known as epidermal growth factor receptor (EGFR)).

[0126] The term "member" as used in this article generally refers to a polypeptide, subunit, or portion that is a component of an immunoconjugate.

[0127] As used herein, the term "Fc domain" generally refers to the Fc portion or Fc fragment of an antibody heavy chain. For example, it can refer to the carboxyl-terminal portion of the immunoglobulin heavy chain constant region or an analogue or portion thereof capable of binding to an Fc receptor. It is well known that each immunoglobulin heavy chain constant region comprises four or five domains. These domains are named sequentially as follows: CH1-hinge-CH2-CH3(-CH4). CH4 is present in IgM that does not have a hinge region. Immunoglobulin heavy chain constant regions used in this disclosure may include an immunoglobulin hinge region and may also include a CH3 domain. For example, an immunoglobulin heavy chain constant region may include an immunoglobulin hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the Fc domain of this disclosure consists of a hinge-CH2-CH3 domain.

[0128] The term "Fc subunit" as used in this paper generally refers to a component of an Fc domain. For example, an Fc domain can be formed by two or more members, and each member can be considered an Fc subunit.

[0129] As used herein, the term "composite with" generally refers to the association (e.g., binding) of one member / subunit of a molecule (e.g., an antibody) with another member / subunit. For example, a light chain can composite with a heavy chain to form a targeting moiety.

[0130] As used herein, the term "binding specificity" generally refers to the ability to specifically bind to a given target (e.g., to which an immune response occurs) while not binding to or substantially not binding to non-targets. The targeting portion of this disclosure may be monospecific and contain one or more binding sites that specifically bind to targets, or may be multispecific (e.g., bispecific or trispecific) and contain two or more binding sites that specifically bind to the same or different targets.

[0131] As used herein, the term "associates with" generally refers to the physical association or contact between one entity and another entity. For example, the first member of an immunoconjugate may covalently or non-covalently "associate" with the second member. In some embodiments, the first member of the immunoconjugate associates with the second member via an interface, and said interface is formed by amino acid residues of the first and second members (i.e., interface residues).

[0132] 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 contrasts with the sequence of the corresponding wild-type polypeptide. A modification can be the 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).

[0133] As used herein, the term "fusion protein" generally refers to a polypeptide comprising or composed of an amino acid sequence of a polypeptide, the amino acid sequence of which is fused directly or indirectly (e.g., through a linker) to an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide independent of the preceding polypeptide or its domains).

[0134] The term "C-terminus" used in this article generally refers to the carboxyl terminus of a polypeptide.

[0135] The term "N-terminus" as used in this article generally refers to the amino terminus of a polypeptide.

[0136] As used herein, the term "immunoglobulin" generally refers to a protein composed of one or more polypeptides largely encoded by immunoglobulin genes. Recognized immunoglobulin genes include the κ, λ, α, γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ constant region genes, as well as the Melyad immunoglobulin variable region genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two pairs of identical immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains work together to bind to the antigen, while the constant regions are responsible for antibody effector functions. Besides antibodies, immunoglobulins can also exist in a variety of other forms, including, for example, Fv, Fab, Fab′, and (Fab′)2.

[0137] The term "intra-frame fusion" as used in this article generally refers to connecting two or more ORFs in a manner that maintains the correct reading frame of the original open reading frame (ORF) to form a longer, contiguous ORF.

[0138] The term "amino acid substitution" as used in this article generally refers to the replacement of one amino acid at a specific position in a polypeptide with another amino acid.

[0139] The term “EU index of KABAT number” as used in this article generally refers to the index corresponding to the EU number of the amino acid sequence, according to Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, E.A. et al. (1991) Sequences of Immunologically Significant Proteins, 5th Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services.

[0140] The terms “isolated polynucleotide” and “isolated nucleic acid” are used interchangeably here and generally refer to a polymer of any length of nucleotide, which is a deoxyribonucleotide or ribonucleotide, or an analogue thereof, isolated from its natural environment or synthesized artificially.

[0141] As used in this article, the term "pharmaceuticalally acceptable excipient" generally refers to any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0142] The terms “immunogenic cell death” and “immunogenic apoptosis” are used interchangeably in this document and generally refer to a form of cell death that induces an effective antitumor immune response by activating, for example, dendritic cells (DCs) and subsequently specific T cell responses. Immunogenic cell death can be characterized by secretory damage-associated molecular patterns (DAMPs). DAMPs may include calreticulin (CRT), heat shock proteins (HSPs), secreted neural axonal growth factor (HMGB1), ATP, etc. CRTs are typically located within the lumen of the endoplasmic reticulum (ER) and can translocate to the surface of dying cells after inducing immunogenic apoptosis, where they act as an “eat me” signal for specialized phagocytes. HSPs include HSP70 and HSP90, which can also translocate to the plasma membrane under stress conditions. HMGB1 is considered a marker of late apoptosis, and its release into the extracellular space appears to be essential for the optimal release and presentation of tumor antigens to dendritic cells. ATP, upon secretion, can act as a “find me” signal for monocytes and induce their attraction to apoptotic sites.

[0143] As used in this article, the term "FOLFOX regimen" generally refers to a chemotherapy regimen containing leucovorin calcium (leucovorin, leucovorin, leucovorin), fluorouracil (5-FU), and oxaliplatin, which can be used to treat advanced and metastatic colorectal cancer. FOLFOX regimens differ in drug dosage and administration schedule, and include FOLFOX 4, FOLFOX 6, modified FOLFOX 6 (mFOLFOX 6), and FOLFOX 7.

[0144] The terms “fluorouracil,” “5-fluorouracil,” or “5-FU” as used in this article generally refer to fluorouracil analogs, an antimetabolites of nucleoside pyrimidines used as anticancer chemotherapy drugs.

[0145] The terms “oxaliplatin” or “eloxatin” as used in this article generally refer to antitumor drugs that inhibit cell growth and can be used in the therapeutic treatment of various types of susceptible cancers and tumors.

[0146] The terms “leucovorin,” “tetrahydrofolate,” “leucovorin calcium / calcium leucovorin” used in this article generally refer to chemopreventive agents used in combination with other chemotherapy drugs.

[0147] The term "increased expression" as used in this article generally refers to higher production expression under certain conditions than under normal conditions. For example, EGFR is increased in metastatic pancreatic cancer (e.g., pancreatic ductal adenocarcinoma PDAC) and metastatic colorectal cancer cells, including wild-type or normal cells.

[0148] Compositions including immunoconjugates and chemotherapy drugs

[0149] In one aspect, this disclosure provides a composition comprising an immunoconjugate and a chemotherapeutic agent. The immunoconjugate may comprise: 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer. One or more interleukins may be fused to the Fc domain. The chemotherapeutic agent may comprise fluorouracil and oxaliplatin.

[0150] For the immunoconjugates of this disclosure, at least one of one or more interleukins (e.g., IL10) may be fused to an N-terminal amino acid of the Fc domain (e.g., within a frame). The immunoconjugate may include two or more interleukins. In some embodiments, at least two of the two or more interleukins are fused to an N-terminal amino acid of the Fc domain. In some embodiments, one or more of the interleukins are fused to the Fc domain via a peptide linker and / or an immunoglobulin hinge region (e.g., within a frame). In some embodiments, at least two of the two or more interleukins are fused to each other via a peptide linker (e.g., within a frame) to form an interleukin dimer. At least one of the interleukin dimers may be fused to an N-terminal amino acid of the Fc domain. In some embodiments, the two or more interleukins are two or more copies of the same interleukin. For example, the two or more interleukins are two or more copies of IL10. Therefore, in some embodiments, two IL10s are fused in-frame with each other (e.g., via a peptide linker) to form an IL10 dimer, and subsequently, the carboxyl terminus of the IL10 dimer can be fused with the amino-terminal amino acid of the Fc domain (e.g., in-frame, e.g., via a peptide linker).

[0151] The linker can be 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 amino acids. In some embodiments, the linker comprises an amino acid sequence as shown in SEQ ID NO:41.

[0152] The immunoconjugate may further include a targeting portion fused to an Fc domain, wherein the targeting portion may exhibit binding specificity to the tumor antigen. The tumor antigen may be any immunogenic entity or a portion thereof specifically expressed or present in the tumor environment or on the surface of tumor cells. In some embodiments, the tumor antigen is selected from the group consisting of members of the EGFR family (e.g., EGFR).

[0153] The targeting portion can fuse with the N-terminal amino acid of the Fc domain. In some embodiments, the targeting portion fuses with the Fc domain via a peptide linker or an immunoglobulin hinge region.

[0154] The targeting portion may include the antigen-binding domain of the antibody; for example, the antigen-binding domain of the antibody may be a Fab portion, a domain antibody, or an ScFv portion. In some embodiments, the antigen-binding domain of the antibody is a Fab portion. The antibody may be selected from the group consisting of anti-EGFR antibodies.

[0155] In some embodiments, the antibody is an anti-EGFR antibody. For example, an anti-EGFR antibody may be cetuximab. In some embodiments, the targeting portion includes the heavy chain CDR1-3, the light chain CDR1-3, the heavy chain variable region, the light chain variable region, and / or the light chain of cetuximab. For example, the targeting portion may be the Fab portion, which includes both the heavy chain variable region and the light chain variable region of cetuximab.

[0156] For example, the targeting portion may include a heavy chain CDR having 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 included in the corresponding heavy chain CDRs 1-3 of the cetuximab. Alternatively, the targeting portion may include a light chain CDR having 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 included in the corresponding light chain CDRs 1-3 of the cetuximab. For example, the targeting region may include a heavy chain variable region having 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 included in the heavy chain variable region of the corresponding cetuximab. For example, the targeting region may include a light chain variable region having 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 included in the light chain variable region of the corresponding cetuximab. For example, the targeting portion may include a light chain having 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 included in the light chain of the corresponding cetuximab.

[0157] The heavy chain CDRs 1-3 of cetuximab are shown in SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3), respectively. The light chain CDRs 1-3 of cetuximab are shown in SEQ ID NO:48 (CDR1), SEQ ID NO:49 (CDR2), and SEQ ID NO:50 (CDR3), respectively. The variable region of the heavy chain of cetuximab is shown in SEQ ID NO:55. The variable region of the light chain of cetuximab is shown in SEQ ID NO:51.

[0158] For the immunoconjugates of this disclosure, the Fc domain may be an IgG Fc domain. IgG may be selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG is human IgG1, and the Fc domain is a human IgG1 Fc domain (wild-type or modified).

[0159] In some embodiments, the immunoconjugate is a protein homodimer composed of two identical members. Each of the two identical members may include one or more interleukins (e.g., IL10) fused to a subunit of the Fc domain (e.g., within a frame, e.g., via a peptide linker). For example, the carboxyl terminus of one or more interleukins may be fused to the N-terminal amino acid of the Fc subunit. In some embodiments, the carboxyl terminus of an interleukin (e.g., IL10) is fused within a frame to the N-terminal amino acid of one of the two Fc subunits to form one member of the homodimer, and the two identical members associate with each other through interactions between the two Fc subunits to form the homodimer (e.g., Figure 3 (As illustrated in A). In some embodiments, two interleukins (e.g., two IL10s) are fused in-frame with each other (e.g., via peptide linkers) to form an interleukin dimer, and then the carboxyl terminus of the interleukin dimer is fused in-frame with the amino-terminal amino acid of one of the two Fc subunits to form a member of a homodimer, and two identical members associate with each other through interactions between the two Fc subunits to form a homodimer.

[0160] In some embodiments, the immunoconjugate is a protein heterodimer comprising a first member and a second member different from the first member, wherein the first member comprises a first Fc subunit and the second member comprises one or more interleukins fused to the second Fc subunit, and the first Fc subunit associates with the second Fc subunit to form the heterodimer.

[0161] In some embodiments, in the second member, at least one of the one or more interleukins is fused to the N-terminal amino acid of the second Fc subunit.

[0162] In some embodiments, in the second member, at least two of the one or more interleukins are fused together to form an interleukin dimer, and the interleukin dimer is further fused with the N-terminal amino acid of the second Fc subunit. For example, two IL10s may be fused in-frame with each other (e.g., via a peptide linker to form an IL10 dimer) and then fused in-frame with the second Fc subunit to form a second member of a protein heterodimer. For example, the C-terminus of the IL10 dimer may be fused with the N-terminal amino acid of the second Fc subunit (e.g., ...). Figure 3 (as shown in B and 3C).

[0163] For example, the second member of the immunoconjugate can be a fusion protein, wherein the second Fc subunit can be fused to the interleukin frame. In some embodiments, the C-terminus of (multiple) interleukins is fused directly or indirectly to the N-terminus of the second Fc subunit to form the fusion protein. In some embodiments, the second Fc subunit is fused to the interleukin frame via a peptide linker or an immunoglobulin hinge region.

[0164] The peptide linker disclosed herein can be, for example, a synthetic amino acid sequence that links or connects two polypeptide sequences via peptide bonds. In some embodiments, the linker 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 may 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 amino acids. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the linker is resistant to or substantially resistant to protein hydrolysis. The hinge region may comprise the amino acid sequence shown in SEQ ID NO:60.

[0165] In some embodiments, the first member further includes the targeting portion fused to the first Fc subunit. For example, the targeting portion may be fused to the N-terminal amino acid of the first Fc subunit. For example, the first member may include the Fab portion of an antibody, whose carboxyl terminus (e.g., the carboxyl terminus of a heavy chain portion such as the CH1 domain or hinge region) is fused to the N-terminal amino acid of the first Fc subunit (e.g., Figure 3 (as shown in C).

[0166] In some implementations, the protein heterodimer does not include any targeting portion. For example, the first member may include only the first Fc subunit (e.g., Figure 3 (as shown in B).

[0167] In some implementations, the first Fc subunit is the same as the second Fc subunit (e.g., a subunit of the wild-type human IgG1 Fc domain).

[0168] In some implementations, the first Fc subunit differs from the second Fc subunit, and the Fc domain includes modifications that promote heterodimerization between the first and second Fc subunits. For example, the first Fc subunit may include a first modification, and the second Fc subunit may include a second modification. For example, the first modification may be in the CH3 domain of the first Fc subunit, and the second modification may be in the CH3 domain of the second Fc subunit. For example, the first modification and / or the second modification are respectively sequence-dependent on their corresponding wild-type Fc domains.

[0169] For example, the first modification may include 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 said amino acid is determined according to the EU index of the KABAT number.

[0170] In some embodiments, the first modification includes 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 said amino acid is determined according to the EU index of the KABAT number.

[0171] For example, the amino acid substitutions included in the first modification may be selected from the group consisting of: Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D and T366W.

[0172] In some implementations, the first modification includes substitution of 2-5 amino acids.

[0173] In some embodiments, the first modification includes amino acid substitutions at a set of positions selected from any of the following: 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) 8) Y349, T366, K409 and Q347; 9) T366, K409 and K392; 10) T366 and K409; 11) T366, K409, Y349 and S354; 12) T366 and F405; 13) T366, F405 and D399; and 14) T366, F405, Y349 and S354; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0174] In some embodiments, the first modification comprises a group of amino acid substitutions selected from the group consisting of: 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, and K409A; 9) Y349C, T366W, and K409E; 10) Y349C, T366W, and K409A; 11) Y349C, T366W, and K409E; 12) Y349C, T366W, and K409A; 13) Y349C, T366W, and K409E; 14) Y349C, T366W, and K409E; 15) Y349C, T366W, and K409A; 16) Y349C, T366W, and K409E; 17) Y349C, T366W, and K409E; 18) Y349C, T366W, and K409E; 19 ... 409A 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0175] In some embodiments, the second modification includes 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 the KABAT number.

[0176] In some embodiments, the amino acid substitutions included in the second modification are selected from the group consisting of: D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A, and Y407V.

[0177] In some embodiments, the second modification includes amino acid substitutions at 4-6 positions.

[0178] In some embodiments, the second modification includes amino acid substitutions at a set of positions selected from the group consisting of: 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 F405. 5) 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0179] In some embodiments, the second modification comprises a group of amino acid substitutions selected from the group consisting of: 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0180] In some embodiments, the first Fc subunit includes the first modification, the second Fc subunit includes the second modification, and the first and second modifications include amino acid substitutions at a set of positions selected from any of the following: 1) the first modification: Y349 and T366; and the second modification: D356, T366, L368, Y407, and F405; 2) the first modification: Y349, T366, and F405; and the second modification: D356, T366, L368, and Y407; 3) the first modification: Y349, T366, and K409; and the second modification: D356, T366, L368, Y407, and F405. 5; 4) First modification: Y349, T366, F405, K360 and Q347; and second modification: D356, T366, L368, Y407 and Q347; 5) First modification: Y349, T366, F405 and Q347; and second modification: D356, T366, L368, Y407, K360 and Q347; 6) First modification: Y349, T366, K409, K360 and Q347; and second modification: D356, T366, L368, Y407, F405 and Q347; 7) First modification: Y349, T366, K409 and Q347; and second modification : D356, T366, L368, Y407, F405, K360 and Q347; 8) the first modification: T366, K409 and K392; and the second modification: T366, L368, Y407, D399 and F405; 9) the first modification: T366 and K409; and the second modification: T366, L368, Y407 and F405; 10) the first modification: T366, K409 and Y349; and the second modification: T366, L368, Y407, F405 and E357; 11) the first modification: T366, K409, Y349 and S354; and the second modification: T366, L368, D369, and Q347; 8. 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; and second modification: T366, L368, Y407, K409, and K392; 14) First modification: T366, F405, and Y349; and second modification: T366, L368, Y407, K409, and E357; 15) First modification: T366, F405, Y349, and S354; and second modification: T366, L368, Y407, K409, and E357;The position of the amino acid is determined according to the EU index of the KABAT number.

[0181] In some embodiments, the first Fc subunit includes the first modification, and the second Fc subunit includes the second modification, wherein the first modification and the second modification include a group of amino acid substitutions selected from any of the following: 1) the first modification: Y349C and T366W; and the second modification: D356C, T366S, L368A, Y407V, and F405K; 2) the first modification: Y349C, T366W, and F405K; and the second modification: D356C, T366S, L368A, and Y407V; 3) the first modification: Y349C, T366W, and K409E; and the second modification: D356C, T366S, L368A, and F405K. 368A, Y407V, and F405K; 4) First modification: Y349C, T366W, and K409A; and second modification: D356C, T366S, L368A, Y407V, and F405K; 5) First modification: Y349C, T366W, F405K, K360E, and Q347E; and second modification: D356C, T366S, L368A, Y407V, and Q347R; 6) First modification: Y349C, T366W, F405K, and Q347R; and second modification: D356C, T366S, L368A, Y407V, K360E, and Q347E; 7) First modification: Y3 49C, T366W, K409A, K360E and Q347E; and the second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) the first modification: Y349C, T366W, K409A and Q347R; and the second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) the first modification: T366W, K409A and K392D; and the second modification: T366S, L368A, Y407V, D399S and F405K; 10) the first modification: T366W and K409A; and the second modification: D356C, T366S, L368A, Y407V, D399S and F405K; Modifications: T366S, L368G, Y407A, and F405K; 11) First modification: T366W, K409A, and Y349D; and second modification: T366S, L368A, Y407V, F405K, and E357A; 12) First modification: T366W, K409A, Y349D, and S354D; and second modification: T366S, L368A, Y407V, F405K, and E357A; 13) First modification: T366W and F405K; and second modification: T366S, L368A, Y407V, and K409A; 14) First modification: T366W, F405K, and D399S;The second modification: T366S, L368A, Y407V, K409A, and K392D; 15) the first modification: T366W and F405K; and the second modification: T366S, L368G, Y407A, and K409A; 16) the first modification: T366W, F405K, and Y349D; and the second modification: T366S, L368A, Y407V, K409A, and E357A; 17) the first modification: T366W, F405K, Y349D, and S354D; and the second modification: T366S, L368A, Y407V, K409A, and E357A; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0182] In some embodiments, the first Fc subunit includes the first modification, the second Fc subunit includes the second modification, the first modification includes the amino acid substitutions T366W and K409A, and the second modification includes the amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0183] For example, the amino acid sequence of the first Fc subunit may be selected from SEQ ID NO:17. The amino acid sequence of interleukin may be as shown in SEQ ID NO:56. The amino acid sequence of the second Fc subunit may be selected from SEQ ID NO:18.

[0184] In some embodiments, the immunoconjugate of this disclosure is a protein heterodimer, and the amino acid sequence of the second member may be selected from SEQ ID NO:42.

[0185] In some embodiments, the immunoconjugate may include a first member and a second member. The first member may include a first polypeptide chain and a second polypeptide chain. The second member may include a third polypeptide chain. The first polypeptide chain may include the light chain variable region of cetuximab. The second polypeptide chain may include a first Fc subunit and a heavy chain variable region of cetuximab. The third polypeptide chain may include a second Fc subunit and one or more interleukins. The target moiety may include the heavy chain variable region and the light chain variable region of cetuximab.

[0186] In some embodiments, the immunoconjugate may include a first member comprising a first polypeptide chain, and a second member comprising a second polypeptide chain, wherein the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO:17, and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO:42.

[0187] The different components of the composition may be packaged independently (e.g., not mixed with each other before application) or pre-mixed and packaged in the same packaging unit.

[0188] The compositions disclosed herein may be pharmaceutical compositions and may further include pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include, but are not limited to, inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers and adjuvants.

[0189] In some embodiments, the pharmaceutical composition 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 reservoir.

[0190] The compositions disclosed herein may include therapeutically effective amounts of active agents (e.g., immunoconjugates and chemotherapeutic drugs). A therapeutically effective amount is the amount of the composition of the invention capable of (at least partially) preventing and / or curing a condition or ailment (e.g., cancer) or at risk of developing said condition or ailment and / or any complications thereof in a subject. The specific amount / concentration of the included active agent may vary depending on the method of administration and patient needs, and may be determined based on, for example, the patient's volume, viscosity, and / or weight.

[0191] Methods and uses for treating cancer

[0192] In another aspect, this disclosure provides immunoconjugates for use in combination with chemotherapy drugs to treat cancer. The immunoconjugate may include: 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer. One or more interleukins may be fused to the Fc domain.

[0193] In another aspect, this disclosure provides the use of an immunoconjugate in combination with a chemotherapeutic agent for the preparation of an agent for treating cancer in a subject of need. The immunoconjugate may include: 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit and the second Fc subunit are associated to form a dimer. One or more interleukins may be fused to the Fc domain.

[0194] In another aspect, this disclosure provides a method for treating cancer in a subject of need. The method includes administering to the subject (a) an effective amount of an immunoconjugate from other parts of this disclosure and (b) an effective amount of a chemotherapeutic agent from other parts of this disclosure. The immunoconjugate may include: 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, wherein the first Fc subunit associates with the second Fc subunit to form a dimer. One or more interleukins may be fused to the Fc domain.

[0195] Immunoconjugates are as defined in other parts of this disclosure. For example, an immunoconjugate may be an immunoconjugate included in the compositions of this disclosure.

[0196] Immunoconjugates may be administered to subjects after chemotherapy. In some implementations, subjects are given more than one dose of chemotherapy, and an immunoconjugate is administered after the last (or final) dose of chemotherapy.

[0197] For example, a subject may be given 2-15 doses of chemotherapy (e.g., 2-14 doses, 2-13 doses, 2-12 doses, 2-11 doses, 2-10 doses, 2-9 doses, 2-8 doses, 3-10 doses, 4-10 doses, or 4-12 doses), and an immunoconjugate may be given to the subject after the last (or final) dose of chemotherapy.

[0198] The immunoconjugate may be administered to the subject no more than 10 days after the administration of chemotherapy (e.g., no more than 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or immediately after the administration of chemotherapy). In some embodiments, the immunoconjugate may be administered to the subject 0–7 days after the administration of chemotherapy (e.g., 0–6 days, 0–5 days, 0–4 days, 0–3 days, 0–2 days, or 0–1 days).

[0199] The immunoconjugate may be administered to the subject two or more times (e.g., at least two, at least three, at least four or more times).

[0200] Cancer can include solid tumors. For example, cancer can be selected from the group consisting of colorectal cancer, pancreatic cancer, and melanoma.

[0201] In some embodiments, the cancer is present in the subject, for example, the cancer or cancer cells are present in a human or in a non-human animal (e.g., a mammal). In some embodiments, the mammal is a human. In some embodiments, the mammal is a mouse, rat, cat, dog, rabbit, pig, sheep, horse, cow, goat, gerbil, hamster, guinea pig, monkey, or any other mammal. Many of the said mammals may be subjects who are preclinical models of certain diseases or conditions known in the art, including solid tumors and / or other cancers (e.g., Talmadge et al., 2007 *American Journal of Pathology* 170:793; Kerbel, 2003 *Cancer Biology and Therapy* 2(4 Supplement 1):S134; Man et al., 2007 *Cancer and Metastasis Review* 26:737; Cespedes et al., 2006 *Clinical and Translational Oncology* 8:318).

[0202] This disclosure also includes the following implementation schemes:

[0203] 1. A composition comprising an immunoconjugate and a chemotherapeutic agent, wherein: the immunoconjugate comprises 1) one or more interleukins, and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, the first Fc subunit associating with the second Fc subunit to form a dimer; the one or more interleukins are fused to the Fc domain; and wherein the chemotherapeutic agent comprises fluorouracil and / or oxaliplatin.

[0204] 2. The composition according to embodiment 1, wherein at least one of the one or more interleukins is fused to the amino-terminal amino acid of the Fc domain.

[0205] 3. The composition according to any one of embodiments 1 to 2, wherein the immunoconjugate comprises two or more interleukins.

[0206] 4. The composition according to embodiment 3, wherein at least two of the two or more interleukins are fused to the amino-terminal amino acid of the Fc domain.

[0207] 5. The composition according to any one of embodiments 1 to 4, wherein one or more of the interleukins are fused to the Fc domain via a peptide linker and / or an immunoglobulin hinge region.

[0208] 6. The composition according to any one of embodiments 3 to 5, wherein at least two of the two or more interleukins are fused together via peptide linkers to form an interleukin dimer.

[0209] 7. The composition according to embodiment 6, wherein at least one of the interleukin dimers is fused to the amino-terminal amino acid of the Fc domain.

[0210] 8. The composition according to any one of embodiments 3 to 7, wherein the two or more interleukins are two or more copies of the same interleukin.

[0211] 9. The composition according to embodiment 8, wherein the two or more interleukins are two or more copies of IL10.

[0212] 10. The composition according to embodiments 1 to 9, wherein the one or more interleukins comprise one or more IL10.

[0213] 11. The composition according to any one of embodiments 1 to 10, wherein the immunoconjugate further comprises a targeting portion fused to the Fc domain, wherein the targeting portion exhibits binding specificity for tumor antigens.

[0214] 12. The composition according to embodiment 11, wherein the targeting portion is fused to the amino-terminal amino acid of the Fc domain.

[0215] 13. The composition according to any one of embodiments 11 to 12, wherein the targeting portion is fused to the Fc domain via a peptide linker or an immunoglobulin hinge region.

[0216] 14. The composition according to any one of embodiments 11 to 13, wherein the targeting portion comprises an antigen-binding domain of an antibody.

[0217] 15. The composition according to embodiment 14, wherein the antigen-binding domain of the antibody is the Fab moiety.

[0218] 16. The composition according to any one of embodiments 11 to 15, wherein the tumor antigen is EGFR.

[0219] 17. The composition according to any one of embodiments 11 to 16, wherein the targeting portion comprises an antigen-binding domain of an anti-EGFR antibody.

[0220] 18. The composition according to embodiment 17, wherein the anti-EGFR antibody is cetuximab.

[0221] 19. The composition according to embodiment 18, wherein the targeting portion comprises the heavy chain CDR1-3 of cetuximab, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:53, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:54.

[0222] 20. The composition according to any one of embodiments 18 to 19, wherein the targeting portion comprises the light chain CDR1-3 of cetuximab, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:49, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:50.

[0223] 21. The composition according to any one of embodiments 18 to 20, wherein the targeting portion comprises a heavy chain variable region of cetuximab, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:55.

[0224] 22. The composition according to any one of embodiments 18 to 21, wherein the targeting portion comprises a light chain variable region of cetuximab, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:51.

[0225] 23. The composition according to any one of embodiments 1 to 22, wherein the Fc domain is an IgG Fc domain.

[0226] 24. The composition according to embodiment 23, wherein the IgG is IgG1.

[0227] 25. The composition according to embodiment 24, wherein the IgG is human IgG1.

[0228] 26. The composition according to any one of embodiments 1 to 25, wherein the immunoconjugate is an asymmetric immunoconjugate comprising a first member and a second member different from the first member, wherein the first member comprises a first Fc subunit, and the second member comprises one or more interleukins fused to the second Fc subunit, and the first Fc subunit associates with the second Fc subunit to form the dimer of the Fc domain.

[0229] 27. The composition according to embodiment 26, wherein in the second member, at least one of the one or more interleukins is fused to the amino-terminal amino acid of the second Fc subunit.

[0230] 28. The composition according to embodiments 26 to 27, wherein in the second member, at least two of the one or more interleukins are fused to each other to form an interleukin dimer, and the interleukin dimer is further fused with the amino-terminal amino acid of the second Fc subunit.

[0231] 29. The composition according to any one of embodiments 26 to 28, wherein the first member further comprises the targeting portion fused with the first Fc subunit.

[0232] 30. The composition according to embodiment 29, wherein in the first member, the targeting portion is fused with the amino-terminal amino acid of the first Fc subunit.

[0233] 31. The composition according to any one of embodiments 1 to 30, wherein the first Fc subunit is different from the second Fc subunit, and the Fc domain includes modifications that promote heterodimerization between the first Fc subunit and the second Fc subunit.

[0234] 32. The composition according to embodiment 31, wherein the first Fc subunit includes a first modification and the second Fc subunit includes a second modification.

[0235] 33. The composition according to embodiment 32, 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 position of the amino acid is determined according to the EU index of the KABAT number.

[0236] 34. The composition according to embodiment 33, wherein the amino acid substitutions included in the first modification are 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 of the KABAT number.

[0237] 35. The composition according to any one of embodiments 32 to 34, wherein the first modification comprises 2-5 amino acid substitutions.

[0238] 36. The composition according to any one of embodiments 32 to 35, wherein the first modification comprises amino acid substitutions at a set of positions selected from the group consisting of: 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, and K360. 60 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0239] 37. The composition according to any one of embodiments 32 to 36, wherein the first modification comprises an amino acid substitution selected from the group consisting of: 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, K360E and Q347E; 9) Y349C, T366W, K409A, K360E and Q347E; 10) Y349C, T366W, K409A, K360E and Q347E; 11) Y349C, T366W, K409A, K360E and Q347E; 12) Y349C, T366W, K409A, K360E and Q347E; 13) Y349C, T366W, K409A, K360E and Q347E; 14) Y349C, T366W, K409C, T366W, K409A, K360E and Q347E; 15) Y349C, T366W, K409C, T366W, K409A, K360E and Q347E; 16) Y349C, C) 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0240] 38. The composition according to any one of embodiments 32 to 37, 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 the KABAT number.

[0241] 39. The composition according to embodiment 39, wherein the amino acid substitutions included in the second modification are selected from the group consisting of: D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A, and Y407V, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0242] 40. The composition according to any one of embodiments 32 to 39, wherein the second modification comprises 4-6 amino acid substitutions.

[0243] 41. The composition according to any one of embodiments 32 to 40, wherein the second modification comprises amino acid substitutions at a set of positions selected from the group consisting of: 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, L368, Y407, F405, and Q347. 8) 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0244] 42. The composition according to any one of embodiments 32 to 41, wherein the second modification comprises amino acid substitutions selected from the group consisting of: 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, and K360E. 0E 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; wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0245] 43. The composition according to any one of embodiments 32 to 42, wherein the first Fc subunit comprises the first modification, the second Fc subunit comprises the second modification, and the first modification and the second modification comprise amino acid substitutions at a set of positions selected from any of the following: 1) the first modification: Y349 and T366; and the second modification: D356, T366, L368, Y407, and F405; 2) the first modification: Y349, T366, and F405; and the second modification: D356, T366, L368, and Y407; 3) the first modification: Y349, T366, and K409; and the second modification: D356, T366, L368, and Y407. 6. L368, Y407, and F405; 4) First modification: Y349, T366, F405, K360, and Q347; and second modification: D356, T366, L368, Y407, and Q347; 5) First modification: Y349, T366, F405, and Q347; and second modification: D356, T366, L368, Y407, K360, and Q347; 6) First modification: Y349, T366, K409, K360, and Q347; and second modification: D356, T366, L368, Y407, F405, and Q347; 7) First modification: Y349, T366, K409, and Q3 47; and the second modification: D356, T366, L368, Y407, F405, K360 and Q347; 8) the first modification: T366, K409 and K392; and the second modification: T366, L368, Y407, D399 and F405; 9) the first modification: T366 and K409; and the second modification: T366, L368, Y407 and F405; 10) the first modification: T366, K409 and Y349; and the second modification: T366, L368, Y407, F405 and E357; 11) the first modification: T366, K409, Y349 and S354; and the second modification: T36 6. 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; and second modification: T366, L368, Y407, K409, and K392; 14) First modification: T366, F405, and Y349; and second modification: T366, L368, Y407, K409, and E357; 15) First modification: T366, F405, Y349, and S354; and second modification: T366, L368, Y407, K409, and E357;The position of the amino acid is determined according to the EU index of the KABAT number.

[0246] 44. The composition according to any one of embodiments 32 to 43, wherein the first Fc subunit comprises the first modification, the second Fc subunit comprises the second modification, wherein the first modification and the second modification comprise a group of amino acid substitutions selected from any one of the following: 1) the first modification: Y349C and T366W; and the second modification: D356C, T366S, L368A, Y407V, and F405K; 2) the first modification: Y349C, T366W, and F405K; and the second modification: D356C, T366S, L368A, and Y407V; 3) the first modification: Y349C, T366W, and K409E. ; and the second modification: D356C, T366S, L368A, Y407V and F405K; 4) the first modification: Y349C, T366W and K409A; and the second modification: D356C, T366S, L368A, Y407V and F405K; 5) the first modification: Y349C, T366W, F405K, K360E and Q347E; and the second modification: D356C, T366S, L368A, Y407V and Q347R; 6) the first modification: Y349C, T366W, F405K and Q347R; and the second modification: D356C, T366S, L368A, Y407V 7) First modification: Y349C, T366W, K409A, K360E and Q347E; and second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) First modification: Y349C, T366W, K409A and Q347R; and second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) First modification: T366W, K409A and K392D; and second modification: T366S, L368A, Y407V, D399S and F405K; 1 0) First modification: T366W and K409A; and second modification: T366S, L368G, Y407A and F405K; 11) First modification: T366W, K409A and Y349D; and second modification: T366S, L368A, Y407V, F405K and E357A; 12) First modification: T366W, K409A, Y349D and S354D; and second modification: T366S, L368A, Y407V, F405K and E357A; 13) First modification: T366W and F405K; and second modification: T366S, L368A, Y407V and K409A;14) The first modification: T366W, F405K, and D399S; and the second modification: T366S, L368A, Y407V, K409A, and K392D; 15) The first modification: T366W and F405K; and the second modification: T366S, L368G, Y407A, and K409A; 16) The first modification: T366W, F405K, and Y349D; and the second modification: T366S, L368A, Y407V, K409A, and E357A; 17) The first modification: T366W, F405K, Y349D, and S354D; and the second modification: T366S, L368A, Y407V, K409A, and E357A; wherein the position of the amino acid is determined according to the EU index of the KABAT number. ;

[0247] 45. The composition according to embodiment 44, wherein the first Fc subunit includes the first modification, the second Fc subunit includes the second modification, the first modification includes the amino acid substitutions T366W and K409A, and the second modification includes the amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the position of the amino acid is determined according to the EU index of the KABAT number.

[0248] 46. ​​The composition according to any one of embodiments 26 to 45, wherein the first member does not include any interleukin.

[0249] 47. The composition according to any one of embodiments 1 to 46, wherein the first Fc subunit comprises an amino acid sequence as shown in SEQ ID NO:17.

[0250] 48. The composition according to any one of embodiments 1 to 47, wherein the second Fc subunit comprises an amino acid sequence as shown in any one of SEQ ID NO:18.

[0251] 49. The composition according to any one of embodiments 1 to 48, wherein the interleukin is a human interleukin.

[0252] 50. The composition according to any one of embodiments 1 to 49, wherein the interleukin comprises an amino acid sequence as shown in any one of SEQ ID NO: 56.

[0253] 51. The composition according to any one of embodiments 1 to 50, wherein the immunoconjugate comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, the first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:37, the second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:39, and the third polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:42.

[0254] 52. The composition according to any one of embodiments 26 to 50, wherein the first member comprises a first polypeptide chain and a second polypeptide chain, the second member comprises a third polypeptide chain, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO:37, the second polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO:39, and the third polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO:42.

[0255] 53. The composition according to any one of claims 26 to 50, wherein the first member comprises a first polypeptide chain and the second member comprises a second polypeptide chain, the first polypeptide chain comprising an amino acid sequence as shown in SEQ ID NO:17 and the second polypeptide chain comprising an amino acid sequence as shown in SEQ ID NO:42.

[0256] 54. The composition according to any one of embodiments 1 to 53, wherein the fluorouracil comprises 5-Fu.

[0257] 55. The composition according to any one of embodiments 1 to 54, wherein the chemotherapeutic agent further comprises leucovorin.

[0258] 56. The composition according to any one of embodiments 1 to 55, wherein the chemotherapeutic agent comprises tetrahydrofolate and / or calcium formyltetrahydrofolate.

[0259] 57. The composition according to any one of embodiments 1 to 56, wherein the chemotherapy drug comprises the FOLFOX regimen.

[0260] 58. The composition according to any one of embodiments 1 to 57, wherein the fluorouracil and the oxaliplatin are not mixed with each other in the composition.

[0261] 59. An immunoconjugate, said immunoconjugate in combination with a chemotherapy drug for the treatment of cancer, wherein said immunoconjugate is as defined in any one of embodiments 1 to 57, and said chemotherapy drug is as defined in any one of embodiments 1 to 58.

[0262] 60. The immune conjugate according to embodiment 59, wherein the cancer is selected from pancreatic cancer and colorectal cancer.

[0263] 61. The immunoconjugate according to embodiment 59, wherein the pancreatic cancer is metastatic pancreatic cancer.

[0264] 62. The immunoconjugate according to embodiment 59, wherein the colorectal cancer is metastatic colorectal cancer.

[0265] 63. The immunoconjugate according to any one of embodiments 59 to 62, wherein the cancer or its cells have increased EGFR expression.

[0266] 64. Use of an immunoconjugate in combination with a chemotherapeutic agent for the preparation of an agent for the treatment of cancer in a subject of need, wherein the immunoconjugate is as defined in any one of embodiments 1 to 58, and the chemotherapeutic agent is as defined in any one of embodiments 1 to 58.

[0267] 65. The use according to embodiment 64, wherein the cancer is selected from pancreatic cancer and colorectal cancer.

[0268] 66. The use according to embodiment 65, wherein the pancreatic cancer is metastatic pancreatic cancer.

[0269] 67. The use according to embodiment 65, wherein the colorectal cancer is metastatic colorectal cancer.

[0270] 68. The use according to any one of embodiments 64 to 67, wherein the cancer or its cells have increased EGFR expression.

[0271] 69. A method for treating cancer in a subject in need, the method comprising administering to the subject (a) an effective amount of an immunoconjugate according to any one of embodiments 1 to 58 and (b) an effective amount of a chemotherapeutic agent according to any one of embodiments 1 to 57.

[0272] 70. The method according to embodiment 69, wherein the immunoconjugate is administered to the subject after administration of the chemotherapeutic drug.

[0273] 71. The method according to embodiment 70, wherein the immunoconjugate is administered to the subject no more than 10 days after administration of the chemotherapeutic drug.

[0274] 72. The method according to any one of embodiments 70 to 71, wherein the immunoconjugate is administered to the subject no more than 3 days after administration of the chemotherapeutic drug.

[0275] 73. The method according to any one of embodiments 69 to 72, wherein the immunoconjugate is administered to the subject two or more times.

[0276] 74. The method according to any one of embodiments 69 to 73, wherein the cancer is selected from pancreatic cancer and colorectal cancer.

[0277] 75. The method according to embodiment 74, wherein the pancreatic cancer is metastatic pancreatic cancer.

[0278] 76. The method according to embodiment 74, wherein the colorectal cancer is metastatic colorectal cancer.

[0279] 77. The method according to any one of embodiments 69 to 76, wherein the cancer or its cells have increased EGFR expression.

[0280] While various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0281] Example

[0282] The following embodiments are shown to provide a complete disclosure and description of how the invention can be made and used to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or close to atmospheric pressure. Standard abbreviations may be used, such as: bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; etc.

[0283] Example 1: Modification and Preparation of Nucleic Acids

[0284] 1.1Fc Modification

[0285] In this disclosure, amino acid modifications (e.g., amino acid substitutions) are made to the interface residues of the human IgG1 Fc domain to obtain the following groups of modifications (as shown in Table 1 below). Chain A is also referred to as Fc9 or the first Fc subunit, while chain B is also referred to as Fc6 or the second Fc subunit:

[0286]

[0287]

[0288] Table 1. Amino acid modification group

[0289] Subsequently, the formation of heterodimeric proteins, including the modification groups listed in Table 1 above, was examined using the ScFv-Fc / Fc system, which will be described in detail below.

[0290] First, the amino acid sequence of the constant region of human immunoglobulin γ1 (IgG1) was obtained from the Uniprot (P01857) database, yielding the amino acid sequence of the wild-type human IgG1-Fc region (SEQ ID NO:30). A polynucleotide fragment encoding wild-type human IgG1-Fc (SEQ ID NO:31, named the Fc gene fragment) was obtained from total RNA of human PBMCs by RT-PCR. A polynucleotide fragment encoding the mouse κIII signal peptide (SEQ ID NO:32) was added to the 5' end of the Fc gene by overlap PCR, and then subcloned into the vector pcDNA4 (Invitrogen, catalog V86220) to obtain a recombinant expression vector for expressing human IgG1-Fc in mammalian cells.

[0291] A nucleic acid molecule encoding the ScFv-Fc fusion protein (SEQ ID NO:33) was synthesized, where ScFv refers to the anti-HER2 single-chain antibody, and the amino acid sequence of the ScFv-Fc fusion protein is shown in SEQ ID NO:34. The ScFv-Fc gene fragment was then subcloned into the vector pcDNA4 (Invitrogen, catalog V86220) to obtain a recombinant expression vector for expressing the ScFv-Fc fusion protein in mammalian cells.

[0292] In some cases, a polypeptide encoding the variable region (VhH) of a camel single-domain antibody is fused to the N-terminus of an Fc gene fragment to obtain a fusion gene fragment encoding the fusion protein VhH-Fc (as shown in SEQ ID NO:36) (as shown in SEQ ID NO:35). This fragment is then subcloned into the vector pcDNA4 (Invitrogen, catalog V86220) to obtain a recombinant expression vector for expressing the fusion protein VhH-Fc in mammalian cells.

[0293] Then, the amino acid modifications listed in Table 1 were introduced into ScFv-Fc (groups 1-17), VhH-Fc (groups 9-12, 14, 15, and 17), and Fc gene fragments (groups 1-8) via overlap PCR. Chain A refers to the Fc subunit in ScFv-Fc, while chain B refers to an independent Fc subunit or the Fc subunit in VhH-Fc. The modified gene fragments were subcloned into the pcDNA4 vector (Invitrogen, catalog V86220) to obtain recombinant expression vectors for expressing the modified ScFv-Fc fusion protein, the modified Fc protein, and the modified VhH-Fc fusion protein in mammalian cells.

[0294] Then, HEK293 cells (ATCC CRL-1573) in suspension culture were transfected with the constructed expression vector and PEI. TM For each group, expression vectors expressing the A chain (ScFv-Fc fusion protein) and expression vectors 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 products was collected, and the expression products, including the corresponding protein heterodimers, were preliminarily purified using Protein A affinity chromatography. Each preliminarily purified expression product included homodimeric proteins ScFv-Fc / ScFv-Fc, homodimeric proteins Fc / Fc (or homodimeric proteins VhH-Fc / VhH-Fc), and heterodimeric proteins ScFv-Fc / Fc (or heterodimeric proteins ScFv-Fc / VhH-Fc), each present in different percentages. Since these proteins (i.e., homodimers and heterodimers) have different molecular weights, their corresponding percentages can be determined based on the corresponding band intensities reflected on a non-reducing SDS-PAGE gel. The intensity was quantified and the results are summarized in Tables 2-5 below.

[0295] Group ScFv-Fc homodimer (%) ScFv-Fc / Fc heterodimer (%) Fc homodimer (%) 1 24 58 18 2 10 70 20 3 25 57 18 4 10 77 13

[0296] Table 2. Percentages of homodimeric and heterodimeric proteins in the expression products.

[0297]

[0298]

[0299] Table 3. Percentages of homodimeric and heterodimeric proteins in the expression products.

[0300]

[0301] Table 4. Percentages of homodimeric and heterodimeric proteins in the expression products.

[0302]

[0303] Table 5. Percentages of homodimeric and heterodimeric proteins in the expression products.

[0304] As can be seen from Tables 2-5 above, all modification groups can promote heterodimer formation very effectively. For illustrative purposes, the modifications in group 10 (modification in chain A: T366W+K409A; modification in chain B: T366S+L368G+Y407A+F405K) are used in the following examples to generate the immunoconjugates or protein mixtures disclosed herein.

[0305] 1.2 Preparation of anti-EGFR (cetuximab)

[0306] 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 / ). Then, a nucleic acid molecule encoding the light chain of cetuximab (Erb-LC) was 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, and a nucleic acid molecule encoding the modified cetuximab heavy chain (referred to as erb-Fc9 in this paper) was synthesized; the corresponding polypeptide encoding it was named Erb-Fc9. The amino acid sequence of Erb-Fc9 is shown in SEQ ID NO:39, while the polynucleotide sequence encoding it is shown in SEQ ID NO:40.

[0307] 1.3 Preparation of (IL10)2-Fc6

[0308] First, the sequence information of human interleukin-10 (IL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and its full-length polynucleotide sequence was obtained. Then, the amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) conforming to the amino acid sequence of the constant region (P01857) of human immunoglobulin γ1 (IgG1) in the Uniprot protein database was obtained. Subsequently, point mutations (T366S, L368G, Y407A, and F405K) were introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. Then, the adapter sequence “(GGGGS)3” (SEQ ID NO: 41) and the hinge region sequence (SEQ ID NO: 60) were added to the N-terminus of Fc6 to obtain adapter-hinge-Fc6. Finally, the corresponding DNA sequence encoding it was designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). Then, the adapter sequence “(GGGGS)3” (SEQ ID NO:41) was added between the two copies of IL10 to obtain (IL10)2. Next, the polynucleotide sequence encoding (IL10)2 was added to the 5' end of the polynucleotide sequence encoding the adapter-hinge-Fc6, thereby obtaining and synthesizing the polynucleotide sequence encoding the fusion protein (IL10)2-Fc6. The amino acid sequence of (IL10)2-Fc6 is shown in SEQ ID NO:42, and the polynucleotide sequence encoding it is shown in SEQ ID NO:43.

[0309] 1.4 Preparation of IL10-Fc

[0310] First, the sequence information of human interleukin-10 (IL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and its full-length polynucleotide sequence was obtained. Then, the amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) conforming to the amino acid sequence of the constant region (P01857) of human immunoglobulin γ1 (IgG1) in the Uniprot protein database was obtained. Next, the adapter sequence “(GGGGS)3” (SEQ ID NO:41) and the hinge region sequence (SEQ ID NO:60) were added to the N-terminus of IgG1-Fc to obtain the adapter-hinge-Fc. Then, the corresponding DNA sequence encoding it was designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding IL10 was added to the 5' end of the polynucleotide sequence encoding the adapter-hinge-Fc, thereby obtaining and synthesizing the polynucleotide sequence encoding the fusion protein IL10-Fc. The amino acid sequence of IL10-Fc is shown in SEQ ID NO:44, while the polynucleotide sequence encoding it is shown in SEQ ID NO:45.

[0311] 1.5 Preparation of Fc9

[0312] The amino acid sequence of human IgG1-Fc was obtained, conforming to the amino acid sequence of the constant region (P01857) of human immunoglobulin γ1 (IgG1) in the Uniprot protein database (i.e., residues 104 to 330 of P01857). Subsequently, point mutations (T366W and K409A) were introduced into the IgG1Fc fragment, and the resulting polypeptide was designated Fc9. The amino acid sequence of Fc9 is shown in SEQ ID NO:17, while the polynucleotide sequence encoding it is shown in SEQ ID NO:46.

[0313] Example 2: Construction of Recombinant Plasmids

[0314] The nucleic acid molecules obtained according to Example 1 (encoding Erb-Fc9, Fc9, Erb-LC (cetuximab light chain), (IL10)2-Fc6, and IL10-Fc) were digested with HindIII and EcoRI (Takara) and then subcloned into the vector pcDNA4 / myc-HisA (Invitrogen, V863-20). The obtained plasmids were verified by sequencing, and the correct recombinant plasmids were named pcDNA4-Erb-Fc9, pcDNA4-Fc9, pcDNA4-Erb-LC, pcDNA4-(IL10)2-Fc6, and pcDNA4-IL10-Fc, respectively.

[0315] Example 3: Expression and purification of immunoconjugates

[0316] Two days prior to transfection, 12 × 600 mL suspensions of HEK293 (ATCC, CRL-1573) were prepared for suspension culture. TM Cells were used for transient transfection at a concentration of 0.8 × 10⁻⁶. 6 Cells were seeded at a density of 1 cell / mL. Two days later, three aliquots of the cell suspension were centrifuged and then resuspended in 600 mL of Freestyle293 medium.

[0317] The recombinant expression vectors obtained from Example 2 were divided into the following groups:

[0318] Group 1: pcDNA4-Erb-Fc9 (200 μg) + pcDNA4-Erb-LC (200 μg) + pcDNA4-(IL10)2-Fc6 (200 μg)

[0319] Group 2: pcDNA4-Fc9(200μg)+pcDNA4-(IL10)2-Fc6(200μg)

[0320] Group 3: pcDNA4-IL10-Fc (200 μg)

[0321] All proteins were produced in transiently transfected 293F cells. In short, FreeStyle 293F cells (Invitrogen) were grown in 293F medium (Invitrogen), transfected with nonlinear plasmid DNA and 293Fectin reagent (Invitrogen), and grown in batches of 80-100 mL / flask at 37°C, 5% CO2 for 6 days in shake flasks. All proteins were purified by one-step protein A chromatography. The quality of each protein was determined by SDS-PAGE and SEC-HPLC. Similarly, the expression and purification results of other immunoconjugates of this application were validated and confirmed by SDS-PAGE.

[0322] The resulting immunoconjugates were named (from group 1 to group 3, respectively): Erb-(IL10)2, Fc9-(IL10)2, and (IL10-Fc)2.

[0323] Figure 1A-1E Immunoconjugates of Erb-(IL10)2, (IL10-Fc)2, and Fc9-(IL10)2 were successfully expressed and purified.

[0324] exist Figure 1AIn the middle, lane 1 is loaded with Erb-(IL10)2 (reduced); lane 2 is loaded with markers; and lane 3 is loaded with Erb-(IL10)2 (non-reduced).

[0325] exist Figure 1B In this process, lane 1 was loaded with (IL10-Fc)2 (raw sample); lane 2 was loaded with (IL10-Fc)2 (flow-through); lane 3 was loaded with (IL10-Fc)2 (elution); lane 4 was loaded with markers; lane 5 was loaded with standard positive control BSA; lane 6 was loaded with blank buffer; lane 7 was blank; and lane 8 was loaded with (IL10-Fc)2 (elution; non-reduction).

[0326] exist Figure 1C In the process, lane 1 was loaded with Fc9-(IL10)2 (original sample); lane 2 was loaded with Fc9-(IL10)2 (flow-through); lane 3 was loaded with Fc9-(IL10)2 (elution); lane 4 was loaded with markers; lane 5 was loaded with standard positive control BSA; lane 6 was loaded with blank buffer; lane 7 was blank; and lane 8 was loaded with Fc9-(IL10)2 (elution; non-reduction).

[0327] Figure 1D The SEC-HPLC results show that the percentage of undesired oligomers in the (IL10-Fc)2 expression product is approximately 27%.

[0328] Figure 1E The SEC-HPLC results show that the percentage of undesired oligomers in the Fc9-(IL10)2 expression product is approximately 3.3%.

[0329] These results demonstrate that the immunoconjugate disclosed herein has been successfully produced. Interestingly, the expression product of Fc9-(IL10)2 contains significantly less of the undesirable oligomers compared to the expression product of (IL10-Fc)2.

[0330] Example 4: Effect of combining chemotherapy with the immunoconjugate of this disclosure

[0331] 4.1 Effects of the immunoconjugate disclosed herein in combination with oxaliplatin

[0332] Female C57BL / 6 mice, aged 6 to 8 weeks, were obtained from the Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China) and housed under specific pathogen-free conditions. All animal use complied with local ethics committee requirements. This study was approved according to the recommendations in the *Guide for the Care and Use of Medical Laboratory Animals* (Ministry of Health of the People's Republic of China, 1998). The mice expressed human EGFR and K+.b The B16-EGFR-SIY melanoma cell line, which binds to the peptide antigen SIYRYYGL (SIY, SEQ ID NO:47), was generated internally and grown in DMEM medium (Gibco Invitrogen) supplemented with 10% (v / v) fetal bovine serum (FBS), 100 units / mL of penicillin, and 100 μg / mL of streptomycin.

[0333] B16-EGFR-SIY melanoma cells (5×10) 5 The tumor was subcutaneously (sc) injected into the flank of mice and allowed to grow for approximately 10 days. The tumor volume was recorded as two vertical diameters (length and width) and calculated as V = ab. 2 / 2, where a and b are the longest and shortest diameters, respectively. Mice were randomly assigned to groups based on tumor size.

[0334] Tumor-carrying mice were divided into several groups, with 5 mice in each group: an isotype control group treated with 0.5 mg / kg human IgG1 (10 μg / mouse); an Erb-(IL10)2 group treated with 0.5 mg / kg (10 μg / mouse) Erb-(IL10)2; an oxaliplatin group treated with 15 mg / kg (300 μg / mouse) oxaliplatin; and an Erb-(IL10)2 + oxaliplatin group treated with 0.5 mg / kg Erb-(IL10)2 and 15 mg / kg oxaliplatin. C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, and injected intraperitoneally with Erb-(IL10)2 on days 7, 10, and 14, respectively; and injected with oxaliplatin via the tail vein on day 7.

[0335] The results are shown in Figure 2A In this study, due to the high toxicity of oxaliplatin, all mice in the oxaliplatin group died after 14 days (see [link]). Figure 2A It can be seen that the combination of Erb-(IL10)2 and oxaliplatin has a synergistic effect in inhibiting tumor growth (see...). Figure 2A ).

[0336] Figure 2D This study shows the effects of Erb-(IL10)2, oxaliplatin, and the combination of Erb-(IL10)2 and oxaliplatin on survival.

[0337] Similarly, mice carrying tumors were divided into several groups, with 5 mice in each group: an isotype control group, in which mice were treated with 0.5 mg / kg human IgG1 (10 μg / mouse); an Fc9-(IL10)2 group, in which mice were treated with 0.5 mg / kg (10 μg / mouse) Fc9-(IL10)2; an oxaliplatin group, in which mice were treated with 15 mg / kg (300 μg / mouse) oxaliplatin; and an Fc9-(IL10)2+oxaliplatin group, in which mice were treated with 0.5 mg / kg Fc9-(IL10)2 and 15 mg / kg (300 μg / mouse) oxaliplatin. C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, and injected intraperitoneally with Fc9-(IL10)2 on days 7, 10, and 14, respectively; and injected with oxaliplatin via the tail vein on day 7.

[0338] The results are shown in Figure 2B In this study, due to the high toxicity of oxaliplatin, all mice in the oxaliplatin group, as well as those in the Fc9-(IL10)2 and oxaliplatin groups, died after 14 days (see [link to study]). Figure 2C Therefore, it is impossible to compare the synergistic effects between the groups.

[0339] Figure 2C This study shows the effects of Fc9-(IL10)2, oxaliplatin, and the combination of Fc9-(IL10)2 and oxaliplatin on survival.

[0340] Example 5: Effect of FOLFOX in combination with the immunoconjugate of this disclosure

[0341] 5.1 Effects of the immunoconjugate of this disclosure in combination with FOLFOX in a pancreatic tumor model

[0342] A pancreatic tumor model was established by subcutaneously inoculating C57BL / 6 mice with pancreatic tumor cells on day 0. The tumor-bearing mice were then divided into several groups: an isotype control group treated with human IgG1; an Erb-(IL10)2 group treated with Erb-(IL10)2; a FOLFOX group treated with FOLFOX; and an Erb-(IL10)2+FOLFOX group treated with both Erb-(IL10)2 and FOLFOX. Erb-(IL10)2 was injected intraperitoneally (ip) and FOLFOX was injected via the tail vein.

[0343] It can be observed that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in inhibiting tumor growth. This suggests that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in the treatment of metastatic pancreatic cancer.

[0344] 5.2 Effects of the immunoconjugate of this disclosure in combination with FOLFOX in a colorectal tumor model

[0345] Seventy C57BL / 6 mice were subcutaneously inoculated with MC38-EGFR-5 colorectal tumor cells on day 0 to establish a colorectal tumor model. The tumor-bearing mice were divided into several groups: an isotype control group treated with human IgG1; an Erb-(IL10)2 group treated with Erb-(IL10)2; a FOLFOX group treated with FOLFOX; and an Erb-(IL10)2+FOLFOX group treated with both Erb-(IL10)2 and FOLFOX. C57BL / 6 mice were subcutaneously inoculated with MC38-EGFR-5 cells on day 0; and intravenously injected with oxaliplatin on day 7, followed by Erb-(IL10)2 8 hours later.

[0346] On day 8, administer calcium formyltetrahydrofolate and 5-FU via intraperitoneal injection. Dosage, route, and schedule are listed in Table 6.

[0347]

[0348]

[0349] Table 6 Application Schedule

[0350] Figure 4A and Figure 4B The combination of Erb-(IL10)2 and FOLFOX showed that it could inhibit tumor growth. Figure 4C The combination of Erb-(IL10)2 and FOLFOX showed that it could prolong survival.

[0351] It can be observed that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in inhibiting tumor growth. This suggests that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in the treatment of metastatic colorectal cancer.

[0352] Example 6: Effect of using fluorouracil in combination with the immunoconjugate of this disclosure

[0353] 6.1 Effects of the immunoconjugate of this disclosure in combination with fluorouracil in a pancreatic tumor model

[0354] A pancreatic tumor model was established by subcutaneously inoculating C57BL / 6 mice with pancreatic tumor cells on day 0. The tumor-carrying mice were then divided into several groups: an isotype control group treated with human IgG1; an Erb-(IL10)2 group treated with Erb-(IL10)2; a 5-FU group treated with 5-FU; and an Erb-(IL10)2+5-FU group treated with both Erb-(IL10)2 and 5-FU. Erb-(IL10)2 was injected intraperitoneally (ip) and 5-FU was injected via the tail vein.

[0355] It can be seen that the combination of Erb-(IL10)2 and 5-FU has a synergistic effect in inhibiting tumor growth. This suggests that the combination of Erb-(IL10)2 and fluorouracil has a synergistic effect in the treatment of metastatic pancreatic cancer.

[0356] 6.2 Effects of the immunoconjugate of this disclosure in combination with fluorouracil in a colorectal tumor model

[0357] A colorectal tumor model was established by subcutaneously inoculating C57BL / 6 mice with MC38-EGFR cells on day 0. The tumor-bearing mice were then divided into several groups: an isotype control group treated with human IgG1; an Erb-(IL10)2 group treated with Erb-(IL10)2; a 5-FU group treated with 5-FU; and an Erb-(IL10)2+5-FU group treated with both Erb-(IL10)2 and 5-FU. Erb-(IL10)2 was injected intraperitoneally (ip) and 5-FU was injected via the tail vein.

[0358] It can be seen that the combination of Erb-(IL10)2 and 5-FU has a synergistic effect in inhibiting tumor growth. This suggests that the combination of Erb-(IL10)2 and fluorouracil has a synergistic effect in the treatment of metastatic colorectal cancer.

[0359] Example 7: Effect of combining oxaliplatin with the immunoconjugate disclosed herein

[0360] 7.1 Effects of the immunoconjugate disclosed herein in combination with oxaliplatin in a pancreatic tumor model

[0361] A pancreatic tumor model was established by subcutaneously inoculating C57BL / 6 mice with pancreatic tumor cells on day 0. The tumor-carrying mice were then divided into several groups: an isotype control group treated with human IgG1; an Erb-(IL10)2 group treated with Erb-(IL10)2; an oxaliplatin group treated with oxaliplatin; and an Erb-(IL10)2 + oxaliplatin group treated with both Erb-(IL10)2 and oxaliplatin. Erb-(IL10)2 was injected intraperitoneally (ip) and oxaliplatin was injected via the tail vein.

[0362] It can be observed that the combination of Erb-(IL10)2 and oxaliplatin has a synergistic effect in inhibiting tumor growth. This suggests that the combination of Erb-(IL10)2 and oxaliplatin has a synergistic effect in the treatment of metastatic pancreatic cancer.

[0363] 7.2 Effects of the immunoconjugate disclosed herein in combination with oxaliplatin in a colorectal tumor model

[0364] A colorectal tumor model was established by subcutaneously inoculating C57BL / 6 mice with MC38-EGFR cells on day 0. The tumor-bearing mice were then divided into several groups: an isotype control group treated with human IgG1; an Erb-(IL10)2 group treated with Erb-(IL10)2; an oxaliplatin group treated with oxaliplatin; and an Erb-(IL10)2 + oxaliplatin group treated with both Erb-(IL10)2 and oxaliplatin. Erb-(IL10)2 was injected intraperitoneally (ip) and oxaliplatin was injected via the tail vein.

[0365] It can be observed that the combination of Erb-(IL10)2 and oxaliplatin has a synergistic effect in inhibiting tumor growth. This suggests that the combination of Erb-(IL10)2 and oxaliplatin has a synergistic effect in the treatment of metastatic colorectal cancer.

[0366] Example 8: Effect of the immunoconjugate of this disclosure in combination with FOLFOX in a melanoma model

[0367] Sixty C57BL / 6 mice were subcutaneously inoculated with melanoma B16-EGFR-5 cells (5 × 10⁻⁶) on day 0. 5A melanoma model was obtained by using 100 cells / mouse. Tumor-carrying mice were divided into several groups: isotype control group, treated with human IgG1; Erb-(IL10)2 group, treated with Erb-(IL10)2; FOLFOX group, treated with FOLFOX; and Erb-(IL10)2+FOLFOX group, treated with both Erb-(IL10)2 and FOLFOX. Dosage, route, and schedule are listed in Table 7. C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-5 cells on day 0; and intravenously injected with oxaliplatin on day 7, followed by Erb-(IL10)2 8 hours later. On day 8, calcium formyltetrahydrofolate and 5-FU were injected intraperitoneally.

[0368]

[0369] Table 7 Application Schedule

[0370] Figure 5A and Figure 5B The combination of Erb-(IL10)2 and FOLFOX showed that it could inhibit tumor growth.

[0371] It can be seen that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in inhibiting tumor growth. This suggests that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in the treatment of melanoma.

[0372] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The invention is not intended to be limited to the specific embodiments provided herein. Although the invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not intended to be construed as limiting. Many variations, modifications, and substitutions will appear to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions shown herein, but depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. Therefore, the invention is also intended to cover any of the said alternatives, modifications, variations, or equivalents. The following embodiments are intended to define the scope of the invention, and methods and structures within the scope of these embodiments and their equivalents are also covered herein. sequence list <110> Shihuida Pharmaceutical Group (Jilin) ​​Co., Ltd. <120> Methods and compositions for cancer treatment <130> 0027‑PA‑038CN <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W <400> 1 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 [[ID=3l]]<210> 2 <211> 227 <212> PRT <213> Artificial Sequence <220> <,223> D356C+T366S+L368A+Y407V+F405K <400> 2 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 3 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W+F405K <400> 3 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 4 <211> 227 <212> PRT <213> Artificial sequence <220> <223> D356C+T366S+L368A+Y407V <400> 4 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 5 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W+K409E <400> 5 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Glu Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 6 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W+K409A <400> 6 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 �5 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 7 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Y349C+T366W+F405K+K360E+Q347E <400> 7 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Glu Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 8 <211> 227 <212> PRT <213> Synthetic sequence <220> <223> D356C+T366S+L368A+Y407V+Q347R <400> 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Arg Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 9 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W+F405K+Q347R <400> 9 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Arg Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser<000115o>210 215 220 Pro Gly Lys 225 <210> 10 <211> 227 <212> PRT <213> Artificial sequence <220> <223> D356C+T366S+L368A+Y407V+K360E+Q347E <400> 10 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 [[ID=do]] Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His It should be noted that there seems to be a misspelling in "15o" in the original text which is translated as "15o" here. It might be "150". 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Glu Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 11 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Y349C+T366W+K409A+K360E+Q347E <400> 11 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Glu Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 21五百一十五220 Pro Gly Lys 225 <210> 12 <211> 227 <212> PRT <213> Artificial Sequence <220> <22三> D356C + T366S + L368A + Y407V + F405K + Q347R <400> 12 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Arg Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 13 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W+K409A+Q347R <400> 13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Arg Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 14 <211> 227 <212> PRT <213> Artificial sequence <220> <223> D356C+T366S+L368A+Y407V+F405K+K360E+Q347E <400> 14 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Glu Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Glu Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 15 <211> 227 <212> PRT <213> Artificial sequence <220> <223> T366W+K409A+K392D <400> 15 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 16 <211> 227 <212> PRT <213> Artificial sequence <220> <223> T366S+L368A+Y407V+D399S+F405K <400> 16 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Ser Ser Asp Gly Ser Phe Lys Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Light 225 <210> 17 <211> 227 <212> PRT <213> artificial sequence <220> <223> T366W+K409A <400> 17 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 18 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> T366S+L368G+Y407A+F405K <400> 18 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Gly Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Ala Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 19 <211> 227 <212> PRT <213> Synthetic sequence <220> <223> T366W+K409A+Y349D <400> 19 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly<0001'494>1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 It should be noted that there seems to be a small formatting issue in the original text where the closing angle bracket in " " is missing a forward slash. This has been corrected in the translation for consistency. Also, " " in the original text seems to be incomplete, and it's translated as is while maintaining the integrity of the overall translation task.Asp Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 20 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> T366S+L368A+Y407V+F405K+E357A <400> 20 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Ala Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 21 <211> 227 <212> PRT <213> Artificial sequence <220> <223> T366W+K409A+Y349D+S354D <400> 21 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Asp Thr Leu Pro Pro Asp Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 22 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> T366W+F405K <400> 22 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 23 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> T366S+L368A+Y407V+K409A <400> 23 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15<{} Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 24​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Ser Ser Asp Gly Ser Phe Lys Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 25 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> T366S+L368A+Y407V+K409A+K392D <400> 25 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 26 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> T366S+L368G+Y407A+K409A <400> 26 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Gly Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Ala Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 27 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> T366W +F405K +Y349D <400> 27 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Asp Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 28 <211> 227 <212> PRT <213> Artificial sequence <220> <223> T366S+L368A+Y407V +K409A +E357A <400> 28 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Ala Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Ala Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 29 <211> 227 <212> PRT <213> Artificial sequence <220> <223> T366W+F405K+Y349D+S354D <400> 29 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Asp Thr Leu Pro Pro Asp Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met l95 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 30 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Amino acids of wild - type human IgG1 - Fc domain <400> 30 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 31 <211> 681 <212> DNA <213> Artificial sequence <220> <223> Fc gene fragment <400> 31 gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg ccccagcgtg ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccgcacccc cgaggtgacc tgcgtggtgg tggacgtgag ccacgagaac cccgaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag ccccgcgagg agcagtacaa cagcacctac cgcgtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga agaccatcag caaggccaag ggccagcccc gcgagcccca ggtgtacacc ctgcccccca gccgcgacga gctgaccaag aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 480 tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 540 gacggcagct tcttcctgta cagcaagctg accgtggaca agagccgctg gcagcagggc 600 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 660 ctgagcctga gccccggcaa g 681 <210> 32 <211> 741 <212> DNA <21३> Artificial sequence <220> <223> Polynucleotide fragment encoding mouse kappaIII signal peptide <400> 32 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60 gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg ccccagcgtg 120 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccgcacccc cgaggtgacc 180 tgcgtggtgg tggacgtgag ccacgagaac cccgaggtga agttcaactg gtacgtggac 240 ggcgtggagg tgcacaacgc caagaccaag ccccgcgagg agcagtacaa cagcacctac 300 It should be noted that in the above translation, "३" in "Artificial sequence" and "0001961" are likely incorrect characters in the original text. If they are typos, they should be corrected before accurate translation.cgcgtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 360 tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga agaccatcag caaggccaag 420 ggccagcccc gcgagcccca ggtgtacacc ctgcccccca gccgcgacga gctgaccaag 480 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 540 tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 600 gacggcagct tcttcctgta cagcaagctg accgtggaca agagccgctg gcagcagggc 660 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 720 ctgagcctga gccccggcaa g 741 <210> 33 <211> 1473 <212> DNA <213> Artificial Sequence <220> <223> ScFv-Fc Fusion Protein Gene <400> 33 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60 gaggtgcagc tgctggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 120 agctgcatcg ccagcggctt caccttcagc agctacccca tgacctgggt gcgccaggcc 180 cccggcaagg gcctggagtg ggtggccagc atcagctacg acggcagcta caagtacaag 240 gccgacagca tgaagggccg cctgaccatc agccgcgaca acagcaagaa caccctgtac 300 ctggagatga acagcctgac cgccgaggac accgccgtgt actactgcgc ccgcaccgcc 360 ttcttcaacg cctacgactt ctggggccag ggcaccctgg tgaccgtgag cagcgccagc 420 accaagggcc ccagcgtggg cggcggcggc agcggcggcg gcggcagcga gatcgtgatg 480 acccagagcc ccgccaccct gagcgtgagc cccggcgagc gcgccaccct gagctgccgc 540 gccagccaga gcgtgcgcag caacctggcc tggtaccagc agaagcccgg ccaggccccc 600 cgcctgctga tctacgccgc cagcacccgc gccaccggca tccccgcccg cttcagcggc 660 agcggcagcg gcaccgagtt caccctgacc atcagcagcc tgcagagcga ggacttcgcc 720 gtgtactact gccagcagta caacgagtgg ttccgcacca gcggccaggg caccaaggtg 780 gagatcaagc gcgacaagac ccacacctgc cccccctgcc ccgcccccga gctgctgggc 840 ggccccagcg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagccgcacc 900 cccgaggtga cctgcgtggt ggtggacgtg agccacgaga accccgaggt gaagttcaac 960 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccgcga ggagcagtac 1020 aacagcacct accgcgtggt gagcgtgctg accgtgctgc accaggactg gctgaacggc 1080 aaggagtaca agtgcaaggt gagcaacaag gccctgcccg cccccatcga gaagaccatc 1140 agcaaggcca agggccagcc ccgcgagccc caggtgtaca ccctgccccc cagccgcgac 1200 gagctgacca agaaccaggt gagcctgacc tgcctggtga agggcttcta ccccagcgac 1260 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac cacccccccc 1320 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagagccgc 1380 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1440 acccagaaga gcctgagcct gagccccggc aag 1473 <210> 34 <211> 471 <212> PRT <213> Synthetic Sequence <220> <223> ScFv-Fc fusion protein <400> 34 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 35 <211> 1121 <212> DNA <213> Artificial sequence <220> <223> Fusion gene fragment encoding the fusion protein VhH-Fc <400> 35 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60 caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 120 tcctgtgcag cctctgaata catctacagt agctactgca tggcctggtt ccgccaggct 180 ccagggaagg agcgcgaggg ggtcgcagtt attgggagtg atggtagcac aagctacgca 240 gactccgtga aaggccgatt caccatctcc aaagacaacg ccaagaacac tctgtatctg 300 caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcggc catcggtggt 360 tactgctacc aaccacccta tgagtaccag tactggggcc aggggaccca ggtcaccgtc 420 tcccagaacc gaaaagcagc gacaagaccc acacctgccc cccctgcccc gcccccgagc 480 tgctgggcgg ccccagcgtg ttcctgttcc cccccaagcc caaggacacc ctgatgatca 540 gccgcacccc cgaggtgacc tgcgtggtgg tggacgtgag ccacgagaac cccgaggtga 600 agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag ccccgcgagg 660 agcagtacaa cagcacctac cgcgtggtga gcgtgctgac cgtgctgcac caggactggc 720 tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga 780 agaccatcag caaggccaag ggccagcccc gcgagcccca ggtgtacacc ctgcccccca 840 gccgcgacga gctgaccaag aaccaggtga gcctgacctg cctggtgaag ggcttctacc 900 ccagcgacat cgccgtggag tgggagagca acggccagcc cgagaacaac tacaagacca 960 ccccccccgt gctggacagc gacggcagct tcttcctgta cagcaagctg accgtggaca 1020 agagccgctg gcagcagggc aacgtgttca gctgcagcgt gatgcacgag gccctgcaca 1080 accactacac ccagaagagc ctgagcctga gccccggcaa g 1121 <210> 36 <211> 354 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein VhH-Fc <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Ile Tyr Ser Ser Tyr 20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Gly Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Ile Gly Gly Tyr Cys Tyr Gln Pro Pro Tyr Glu Tyr Gln Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp 115 120 125 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 130 135 140 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 145 150 155 160 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 165 170 175 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 180 185 190 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 195 200 205 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 210 215 220 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 225 230 235 240 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 245 250 255 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 260 265 270 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 275 280 285 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 290 295 300 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 305 310 315 320 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 325 330 335 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 340 345 350 Gly Lys [[ID=!]]<210> 37 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Erb-LC amino acid sequence <400> 37 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 It should be noted that there seems to be an error in the tag "[[ID=!]]" in the original text. It should probably be something like "". This might cause some issues in a proper context, but I translated it as is based on the provided requirements.Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 38 <211> 642 <212> DNA <213> Artificial sequence <220> <223> Erb-LC gene <400> 38 gacatcctgc tgacccagag ccccgtgatc ctgagcgtga gccccggcga gcgcgtgagc 60 ttcagctgcc gcgccagcca gagcatcggc accaacatcc actggtacca gcagcgcacc 120 aacggcagcc cccgcctgct gatcaagtac gccagcgaga gcatcagcgg catccccagc 180 cgcttcagcg gcagcggcag cggcaccgac ttcaccctga gcatcaacag cgtggagagc 240 gaggacatcg ccgactacta ctgccagcag aacaacaact ggcccaccac cttcggcgcc 300 ggcaccaagc tggagctgaa gcgcaccgtg gccgccccca gcgtgttcat cttccccccc 360 agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagcttcaac cgcggcgagt gc 642 <210> 39 <211> 449 <212> PRT <213> Artificial sequence <220> <223> Erb‑Fc9 amino acid sequence <400> 39 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Ala Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 40 <211> 1347 <212> DNA <213> Artificial sequence <220> <223> Erb-Fc9 gene <400> 40 60. cggtgcagc tgaagcagag cggccccggc ctggtgcagc ccagccagag cctgagcatc acctgcaccg tgagcggctt cagcctgacc aactacggcg tgcactgggt gcgccagagc 120 cccggcaagg gcctggagtg gctgggcgtg atctggagcg gcggcaacac cgactacaac 180 acccccttca ccagccgcct gagcatcaac aaggacaca gcaagagcca ggtgttcttc 300. aagatgaaca gcctgcagag caacgacacc gccatctact actgcgcccg cgccctgacc tactacgact acgagttcgc ctactggggc cagggcaccc tggtgaccgt gagcgccgcc 360 agcactaagg ggccctctgt gtttccactc gccccttcta gcaaaagcac ttccggagga 420 actgccgctc tgggctgtct ggtgaaagat tacttccccg aaccagtcac tgtgtcatgg 480 aactctggag cactgacatc tggagttcac acctttcctg ctgtgctgca gagttctgga 540 ctgtactccc tgtcatctgt ggtcaccgtg ccatcttcat ctctggggac ccagacctac 600 atctgtaacg tgaaccacaa accctccaac acaaaagtgg acaaacgagt cgaaccaaaa tcttgtgaca aaacccacac atgcccaccg tgcccagctc cggaactcct gggcggaccg 720 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​<400> 41 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 42 <211> 582 <212> PRT <213> Artificial Sequence <220> <223> (IL10)2-Fc6 Amino Acid Sequence <400> 42 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 165 170 175 Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly 180 185 190 Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val 195 200 205 Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys 210 215 220 Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu 225 230 235 240 Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu 245 250 255 Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn 260 265 270 Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro 275 280 285 Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn 290 295 300 Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile 305 310 315 320 Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Gly 325 330 335 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro 340 345 350 Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 355 360 365 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 370 375 380 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 385 390 395 400 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 405 410 415 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 420 425 430 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 435 440 445 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 450 455 460 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 465 470 475 480 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 485 490 495 Gln Val Ser Leu Ser Cys Gly Val Lys Gly Phe Tyr Pro Ser Asp Ile 500 505 510 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 515 520 525 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Ala Ser Lys 530 535 540 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 545 550 555 560 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 565 570 575 Ser Leu Ser Pro Gly Lys 580 <210> 43 <211> 1746 <212> DNA <213> The snowstorm <220> <223> (IL10)2‐Fc6 conjugate <400> 43 agccccggcc agggcacaca gtccgagaac agctgcacc actttcccgg caacctgcct aacatgctga gggacctgag ggacgccttc agcagggtga agaccttctt ccagatgaag gaccagctgg atacctgct gctgaaggag agcctgctgg aggacttcaa gggctacctg ggctgccagg ccctgagcga gatgatccag ttctacctgg aggaggtgat gccccaggcc 240 gagaaccagg accccgacat caaggcccac gtgaacagcc tgggcgagaa cctgaagacc 360. ctgaggctga ggctgaggag gtgccacagg ttcctgccct gtgagaacaa atccaaggcc gtggagcagg tgaagaacgc cttcaacaag ctgcaggaa agggcatcta caaggccatg agcgagttcg acatctttat caactatatc gaggcctaca tgacaatgaa gatcaggaac ggcggcggcg gcagcggggc cggcggcagc ggaggaggcg gcagcagccc cggccagggc 540 acacagtccg agaacagctg cacccacttt cccggcaacc tgcctaacat gctgagggac 600 ctgagggacg ccttcagcag ggtgaagacc ttcttccaga tgaaggacca gctggataac 660 ctgctgctga aggagagcct gctggagcac ttcaagggct acctgggctg ccaggccctg 720 agcgagatga tccagttcta cctggaggag gtgatgcccc aggccgagaa ccaggacccc 780 gacatcaagg cccacgtgaa cagcctgggc gagaacctga agaccctgag gctgaggctg 840 aggaggtgcc acaggttcct gccctgtgag aacaaatcca aggccgtgga gcaggtgaag 900 aacgcttca acaagctgca ggaaaagggc atctacaagg ccatgagcga gttcgacatc 960 tttatcaact atatcgaggc ctacatgaca atgaagatca ggaacggcgg cggcggcagc 1020 gggggcggcg gcagcggagg aggcggcagc gagcctaagt ccagcgacaa gacccacacc 1080 tgcccccctt gccccgctcc ggaactcctg ggcggaccgt cagtcttcct cttcccccca 1140 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 1200 gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1260 aatgccaaga caaagccgcg ggaggacag tacaacagca cgtaccgtgt ggtcagcgtc 1320 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1380 aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1440 ccacaggtgt ataccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 1500 agttgcgggg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 1560 cagccggaga acaactacaa gaccacgcct cccgtgttgg actccgacgg ctccttcaag 1620 ctcgccagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1680 tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1740 ggtaaa 1746 <210> 44 <211> 407 <212> PRT <213> Artificial Sequence <220> <223> IL10-Fc Amino Acid Sequence <400> 44 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 165 170 175 Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 180 185 190 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 195 200 205 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 210 215 220 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 225 230 235 240 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 245 250 255 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 260 265 270 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 275 280 285 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 290 295 300 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 305 310 315 320 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 325 330 335 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 340 345 350 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 355 360 365 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 370 375 380 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 385 390 395 400 Leu Ser Leu Ser Pro Gly Lys 405 <210> 45 <211> 1221 <212> DNA <213> Artificial sequence <220> <223> IL10-Fc gene <400> 45 agccccggcc agggcacaca gtccgagaac agctgcaccc actttcccgg caacctgcct 60 aacatgctga gggacctgag ggacgccttc agcagggtga agaccttctt ccagatgaag 120 [[ID=�5]]gaccagctgg ataacctgct gctgaaggag agcctgctgg aggacttcaa gggctacctg 180 ggctgccagg ccctgagcga gatgatccag ttctacctgg aggaggtgat gccccaggcc 240 gagaaccagg accccgacat caaggcccac gtgaacagcc tgggcgagaa cctgaagacc 300 ctgaggctga ggctgaggag gtgccacagg ttcctgccct gtgagaacaa atccaggcc 360 gtggagcagg tgagaacgc cttcacaag ctgcaggaaa agggcatcta caggccatg 420 agcgagttcg acatctttat caactatatc gaggcctaca tgacaatgaa gatcaggaac 480 ggcggcggcg gcagcgggggg cggcggcagc gggaggcg gcagcgagcc taagtccagc 540 gabagaccc acacctgccc cccttgcccc gctccggaac tcctgggcgg accgtcagtc 600 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 660 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 720 ggcgtggagg tgcataatgc cagacaaag ccgcgggagg agcagtaca cagcacgtac 780 cgtgtgtca gcgtcctcac cgtcctgcac caggactggc tgaatggca ggagtacaag 840 tgcaaggtct ccaaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 900 gggcagcccc gagaaccaca ggtgtacacc ctgccccat cccgggatga gctgaccaag 960 aaccaggtca gcctgacctg cctggtcaaa ggctctatc cgccgtggag 1020 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gttggactcc 1080 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1140 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1200 ctctccctgt ctccgggtaa a 1221 <210> 46 <211> 681 <212> DNA <213> Artificial sequence <220> <223> Fc9 gene <400> 46 gacaaaactc acacatgccc accgtgccca gctccggaac tcctgggcgg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccaa gtcgggatga gctgaccaag 420 aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gttggactcc 540 gacggctcct tcttcctcta cagcgcgctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 47 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Kb-binding peptide antigen <400> 47 Ser Ile Tyr Arg Tyr Tyr Gly Leu 1 5 <210> 48 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Cetuximab light chain CDR1 <400> 48 Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His 1 5 10 <210> 49 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Cetuximab light chain CDR2 <400> 49 Tyr Ala Ser Glu Ser Ile Ser 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Cetuximab light chain CDR3 <400> 50 Gln Gln Asn Asn Asn Trp Pro Thr Thr 1 5 <210> 51 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Cetuximab light chain variable region <400> 51 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 52 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Cetuximab heavy chain CDR1 <400> 52 Asn Tyr Gly Val His 1 5 <210> 53 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Cetuximab heavy chain CDR2 <400> 53 Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser 1 5 10 15 <210> 54 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Cetuximab heavy chain CDR3 <400> 54 Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr 1 5 10 <210> 55 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Cetuximab heavy chain variable region <400> 55 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 56 <211> 160 <212> PRT <213> Artificial Sequence <220> <223> IL10 <400> 56 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 <210> 57 <211> 480 <212> DNA <213> Synthetic sequence <220> <223> IL10 gene <400> 57 agccccggcc agggcacaca gtccgagaac agctgcaccc actttcccgg caacctgcct 60 aacatgctga gggacctgag ggacgccttc agcagggtga agaccttctt ccagatgaag 120 gaccagctgg ataacctgct gctgaaggag agcctgctgg aggacttcaa gggctacctg 180 ggctgccagg ccctgagcga gatgatccag ttctacctgg aggaggtgat gccccaggcc 240 gagaaccagg accccgacat caaggcccac gtgaacagcc tgggcgagaa cctgaagacc 300 ctgaggctga ggctgaggag gtgccacagg ttcctgccct gtgagaacaa atccaaggcc 360 gtggagcagg tgaagaacgc cttcaacaag ctgcaggaaa agggcatcta caaggccatg 420 agcgagttcg acatctttat caactatatc gaggcctaca tgacaatgaa gatcaggaac 480 <210> 58 <211> 335 <212> PRT <213> Artificial sequence <220> <223> (IL10)2 <400> 58 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 165 170 175 Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly 180 185 190 Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val 195 200 205 Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys 210 215 220 Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu 225 230 235 240 Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu 245 250 255 Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn 260 265 270 Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro 275 280 285 Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn 290 295 300 Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile 305 310 315 320 Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 325 330 335 <210> 59 <211> 1005 <212> DNA <213> The snowstorm <220> <223> (IL10)2s <400> 59 agccccggcc agggcacaca gtccgagaac agctgcacc actttcccgg caacctgcct aacatgctga gggacctgag ggacgccttc agcagggtga agaccttctt ccagatgaag gaccagctgg atacctgct gctgaaggag agcctgctgg aggacttcaa gggctacctg ggctgccagg ccctgagcga gatgatccag ttctacctgg aggaggtgat gccccaggcc 240 gagaaccagg accccgacat caaggcccac gtgaacagcc tgggcgagaa cctgaagacc 360. ctgaggctga ggctgaggag gtgccacagg ttcctgccct gtgagaacaa atccaaggcc gtggagcagg tgaagaacgc cttcaacaag ctgcaggaa agggcatcta caaggccatg agcgagttcg acatctttat caactatatc gaggcctaca tgacaatgaa gatcaggaac ggcggcggcg gcagcggggc cggcggcagc ggcggcg gcagcagccc cggccagggc 540 acacagtccg agaacagctg cacccacttt cccggcaacc tgcctaacat gctgagggac 600 ctgagggacg ccttcagcag ggtgaagacc ttcttccaga tgaaggacca gctggataac 660 ctgagggacg ccttcagcag ggtgaagacc ttcttccaga tgaaggacca gctggataac 660 ctgctgctga aggagagcct gctggaggac ttcaagggct acctgggctg ccaggccctg 720 ctgctgctga aggagagcct gctggaggac ttcaagggct acctgggctg ccaggccctg 720 agcgagatga tccagttcta cctggaggag gtgatgcccc aggccgagaa ccaggacccc 780 agcgagatga tccagttcta cctggaggag gtgatgcccc aggccgagaa ccaggacccc 780 gacatcaagg cccacgtgaa cagcctgggc gagaacctga agaccctgag gctgaggctg 840 gacatcaagg cccacgtgaa cagcctgggc gagaacctga agaccctgag gctgaggctg 840 aggaggtgcc acaggttcct gccctgtgag aacaaatcca aggccgtgga gcaggtgaag 900 aggaggtgcc acaggttcct gccctgtgag aacaaatcca aggccgtgga gcaggtgaag 900 aacgccttca acaagctgca ggaaaagggc atctacaagg ccatgagcga gttcgacatc 960 aacgccttca acaagctgca ggaaaagggc atctacaagg ccatgagcga gttcgacatc 960 tttatcaact atatcgaggc ctacatgaca atgaagatca ggaac 1005 tttatcaact atatcgaggc ctacatgaca atgaagatca ggaac 1005 <210> 60<210> 60 <211> 5<211> 5 <212> PRT<212> PRT <213> Synthetic Sequence <213> Synthetic Sequence [[ID=二十一]] [[ID=二十二]]<220> <220> [[ID=二十三]] <223>Hinge Region <223>Hinge Region <400> 60 <400> 60 Glu Pro Lys Ser Ser Glu Pro Lys Ser Ser 1 5 1 5

Claims

1. Use of an immunoconjugate in combination with a chemotherapeutic agent for preparing an agent for treating cancer in a subject of need, wherein the immunoconjugate comprises a first member and a second member, wherein the first member comprises a first polypeptide chain and a second polypeptide chain, the second member comprises a third polypeptide chain, the first polypeptide chain being shown in SEQ ID NO:37, the second polypeptide chain being shown in SEQ ID NO:39, and the third polypeptide chain being shown in SEQ ID NO:42, and wherein the chemotherapeutic agent is calcium leucovorin, fluorouracil (5-FU), and oxaliplatin, wherein the cancer is selected from melanoma and colorectal cancer.

2. The use according to claim 1, wherein the colorectal cancer is metastatic colorectal cancer.

3. The use according to any one of claims 1 to 2, wherein the cancer or its cells have increased EGFR expression.

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