Methods and compositions for treating cancer

Abstract

Compositions for treating cancer are provided, which comprise an immunoconjugate in combination with a chemotherapeutic agent. Methods for treating cancer are also provided, which include the use of the compositions.

Description

[Background technology]

[0001] Although immune responses to tumor antigens can be detected (Disis et al. (1997) J. Clin. Oncol. 15:3363-3367), disease-causing malignant cells often fail to elicit an immune response leading to rejection. Studies have demonstrated that the immunogenicity of tumor cells can be enhanced by introducing immunomodulatory molecules, such as cytokines and costimulatory molecules, into tumor cells. However, eradication of residual cancer cells may require targeting widely dispersed micrometastatic tumor deposits, which are unavailable for direct gene transfer. Furthermore, the expression and stability of introduced immunomodulatory molecules are often unsatisfactory. Immunomodulatory factors, such as cytokines, produced by cells of the immune system can directly or indirectly activate cells of the adaptive immune response and play an important role in eliciting protective antitumor immunity. The innate immune system can be triggered by bacterial products or "danger" signals, leading to the release of inflammatory cytokines, such as interleukins.

[0002] Multiple studies have demonstrated that immunomodulators may be useful in exerting antitumor effects in both animal models and cancer patients, but their use is severely limited by the short half-life and systemic toxicity associated with their application.

[0003] In addition to immunotherapy, surgery, chemotherapy, hormone therapy, and radiation therapy are also used to treat cancer (see, for example, Stockdale, 1998, "Principles of Cancer Patient Management," in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). However, surgery may be impossible or unacceptable due to the patient's health condition or advanced stage of disease, and in many cases, cancer cells cannot be completely removed from the patient after surgery. Hormone therapy is rarely used as a single agent, and while it can be effective, it is often used to prevent or delay cancer recurrence after other treatments have largely eliminated the cancer cells. Furthermore, patients can develop drug resistance to chemotherapeutic agents quite rapidly.

[0004] Therefore, new and effective therapeutic approaches for the treatment of cancer remain highly desirable. Summary of the Invention

[0005] The present disclosure provides compositions and methods for treating cancer that involve the use of immunoconjugates in combination with chemotherapeutic agents, and the disclosed inventions have shown significant synergistic effects in cancer treatment.

[0006] In one aspect, the present 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, wherein the first Fc subunit associates with the second Fc subunit to form a dimer, and wherein the one or more interleukins are fused to the Fc domain, and 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 embodiments, the immunoconjugate comprises two or more interleukins.

[0009] In some embodiments, at least two of the two or more interleukins are fused to 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 a peptide linker and / or an immunoglobulin hinge region.

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

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

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

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

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

[0016] In some embodiments, the immunoconjugate further comprises a targeting moiety fused to the Fc domain, wherein the targeting moiety exhibits binding specificity for a tumor antigen.

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

[0018] In some embodiments, the targeting moiety is fused to the Fc domain via a peptide linker and / or an immunoglobulin hinge region.

[0019] In some embodiments, the targeting moiety comprises the antigen-binding domain of an antibody.

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

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

[0022] In some embodiments, the targeting moiety comprises the antigen-binding domain of an anti-EGFR antibody.

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

[0024] In some embodiments, the targeting moiety comprises heavy chain CDRs 1-3 of cetuximab, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, HCDR2 of cetuximab comprises the amino acid sequence set forth in SEQ ID NO: 53, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0025] In some embodiments, the targeting moiety comprises light chain CDRs 1-3 of cetuximab, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0026] In some embodiments, the targeting moiety comprises the heavy chain variable region of cetuximab, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55.

[0027] In some embodiments, the targeting moiety comprises the light chain variable region of cetuximab, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:51.

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

[0029] In some embodiments, the IgG is an IgG1.

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

[0031] In some embodiments, the immune complex is an asymmetric immune complex 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 a second Fc subunit, and wherein the first Fc subunit associates with the second Fc subunit to form a dimer of Fc domains.

[0032] In some embodiments, 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.

[0033] In some embodiments, 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 to the amino-terminal amino acid of a second Fc subunit.

[0034] In some embodiments, the first member further comprises a targeting moiety fused to the first Fc subunit.

[0035] In some embodiments, in the first member, the targeting moiety is fused to the amino terminal amino acid of the first Fc subunit.

[0036] In some embodiments, the first Fc subunit is different from the second Fc subunit and the Fc domain comprises a modification that promotes heterodimerization between the first Fc subunit and the second Fc subunit.

[0037] In some embodiments, the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification.

[0038] In some embodiments, the first modification comprises an amino acid substitution at position T366 and an amino acid substitution at one or more positions selected from the group consisting of Y349, F405, K409, D399, K360, Q347, K392 and S354, where the amino acid positions are determined according to the EU index of the KABAT numbering.

[0039] In some embodiments, the amino acid substitution comprised in the first modification is selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D and T366W, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0040] In some embodiments, the first modification comprises between 2 and 5 amino acid substitutions.

[0041] In some embodiments, the first modification is 1) Y349 and T366, 2) Y349, T366 and F405, 3) Y349, T366 and K409, 4) Y349, T366, F405, K360 and Q347, 5) Y349, T366, F405 and Q347, 6) Y349, T366, K409, K360 and Q347, 7) Y349, T366, K409 and Q347, 8) T366 1) T366, K409 and K392, 2) T366 and K409, 3) T366, K409, Y349 and S354, 4) T366 and F405, 5) T366, F405 and D399, and 6) T366, F405, Y349 and S354, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0042] In some embodiments, the first modification is 1) Y349C and T366W, 2) Y349C, T366W and F405K, 3) Y349C, T366W and K409E, 4) Y349C, T366W and K409A, 5) Y349C, T366W, F405K, K360E and Q347E, 6) Y349C, T366W, F405K and Q347R, 7) Y349C, T366W, K409A, K360E and Q347E, 8) Y349C, T366W, K409A and Q347R, 9) T366W, K409 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 amino acid positions are determined according to the EU index of the KABAT numbering.

[0043] In some embodiments, the second modification comprises amino acid substitutions at positions T366, L368, and Y407, and at one or more positions selected from the group consisting of D356, D399, E357, F405, K360, K392, K409, and Q347, where the amino acid positions are determined according to the EU index of the KABAT numbering.

[0044] In some embodiments, the amino acid substitutions comprised 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 amino acid positions are determined according to the EU index of the KABAT numbering.

[0045] In some embodiments, the second modification comprises 4 to 6 amino acid substitutions.

[0046] In some embodiments, the second modification is 1) D356, T366, L368, Y407, and F405; 2) D356, T366, L368, and Y407; 3) D356, T366, L368, Y407, and Q347; 4) D356, T366, L368, Y407, K360, and Q347; 5) D356, T366, L368, Y407, F405, and Q347; 6) D356, T366, L368, Y407, F405, K360, and Q347; 7) T366, L368, Y40 7) T366, L368, Y407 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 amino acid positions are determined according to the EU index of the KABAT numbering.

[0047] In some embodiments, the second modification is 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, D399 1) T366S, L368A, Y407V, K409A and K392D; 2) T366S, L368G, Y407A and K409A; and 3) T366S, L368A, Y407V, K409A and E357A, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0048] In some embodiments, the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification, the first modification and the second modification being: 1) first modification: Y349 and T366, second modification: D356, T366, L368, Y407 and F405; 2) first modification: Y349, T366 and F405, second modification: D356, T366, L368 and Y407; 3) first modification: Y349, T366 and K409, second modification: D356, T366, L368, Y407 and F405; 4) first modification: Y349, T366, F40 5) first modification: Y349, T366, F405 and Q347, second modification: D356, T366, L368, Y407, K360 and Q347, 6) first modification: Y349, T366, K409, K360 and Q347, second modification: D356, T366, L368, Y407, F405 and Q347, 7) first modification: Y349, T366, K409 and Q347, second modification: D356, T366, L368, Y407, F405, K360 and and Q347, 8) first modification: T366, K409 and K392, second modification: T366, L368, Y407, D399 and F405, 9) first modification: T366 and K409, second modification: T366, L368, Y407 and F405, 10) first modification: T366, K409 and Y349, second modification: T366, L368, Y407, F405 and E357, 11) first modification: T366, K409, Y349 and S354, second modification: T366, L368, Y407, F405 and E357, 12) first modification: T366 and F405, and 15) first modifications: T366, F405, Y349 and S354, second modifications: T366, L368, Y407, K409 and E357, wherein the amino acid positions are selected from the group consisting of: 1) first modifications: T366, F405 and D399, second modifications: T366, L368, Y407, K409 and K392; 2) first modifications: T366, F405 and Y349, second modifications: T366, L368, Y407, K409 and E357;Determined according to the EU index of KABAT numbers.

[0049] In some embodiments, the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification, the first modification and the second modification being selected from the group consisting of: 1) first modification: Y349C and T366W, and second modification: D356C, T366S, L368A, Y407V, and F405K; 2) first modification: Y349C, T366W, and F405K, and second modification: D356C, T366S, L368A, and Y407V; 3) first modification: Y349C, T366W, and K409E, and second modification: D356C, T366S, L368A, Y407V, and F405K; 4) first modification: Y349C, T366W, and K409E, and second modification: D356C, T366S, L368A, Y407V, and F405K; 1) First modification: Y349C, T366W and K409A, Second modification: D356C, T366S, L368A, Y407V and F405K, 5) First modification: Y349C, T366W, F405K, K360E and Q347E, Second modification: D356C, T366S, L368A, Y407V and Q347R, 6) First modification: Y349C, T366W, F405K and Q347R, Second modification: D356C, T366S, L368A, Y407V, K360E and Q347E, 7) First modification: Y349C, T366W, K409A, K360E and Q3 47E, second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) first modification: Y349C, T366W, K409A and Q347R, second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) first modification: T366W, K409A and K392D, second modification: T366S, L368A, Y407V, D399S and F405K; 10) first modification: T366W and K409A, second modification: T366S, L368G, Y407A and F405K; 11) 1) first modification: T366W, K409A and Y349D, second modification: T366S, L368A, Y407V, F405K and E357A; 12) first modification: T366W, K409A, Y349D and S354D, second modification: T366S, L368A, Y407V, F405K and E357A; 13) first modification: T366W and F405K, second modification: T366S, L368A, Y407V and K409A; 14) first modification: T366W, F405K and D399S, second modification: T366S, L368A, Y407V, K409A and K392D;15) first modification: T366W and F405K, second modification: T366S, L368G, Y407A and K409A, 16) first modification: T366W, F405K and Y349D, second modification: T366S, L368A, Y407V, K409A and E357A, 17) first modification: T366W, F405K, Y349D and S354D, second modification: T366S, L368A, Y407V, K409A and E357A, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0050] In some embodiments, the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification, wherein the first modification comprises amino acid substitutions T366W and K409A and the second modification comprises amino acid substitutions T366S, L368G, Y407A and F405K, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0051] In some embodiments, the first member does not include any interleukins.

[0052] In some embodiments, the first Fc subunit comprises the amino acid sequence set forth in any one of SEQ ID NOs:17.

[0053] In some embodiments, the second Fc subunit comprises the amino acid sequence set forth in any one of SEQ ID NOs:18.

[0054] In some embodiments, the interleukin is a human interleukin.

[0055] In some embodiments, the interleukin comprises the amino acid sequence set forth in any one of SEQ ID NOs:56.

[0056] In some embodiments, the immune complex comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 39, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0057] In some embodiments, the first member comprises a first polypeptide chain and a second polypeptide chain, and the second member comprises a third polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:37, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:39, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:42.

[0058] In some embodiments, the first member comprises a first polypeptide chain and the second member comprises a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 17 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0059] In some embodiments, the fluorouracil comprises 5-Fu.

[0060] In some embodiments, the chemotherapeutic agent further comprises folinic acid.

[0061] In some embodiments, the chemotherapeutic agent comprises tetrahydrofolic acid and / or calcium leucovorin.

[0062] In some embodiments, the chemotherapeutic agent comprises a FOLFOX regimen.

[0063] In some embodiments, the fluorouracil and oxaliplatin are not mixed with each other in the composition.

[0064] In another aspect, the present disclosure provides an immunoconjugate for use in the treatment of cancer in combination with a chemotherapeutic agent, wherein the immunoconjugate is as defined in this disclosure and the chemotherapeutic agent is as defined in this disclosure.

[0065] In some embodiments, the cancer is selected from pancreatic cancer and colon cancer.

[0066] In some embodiments, the pancreatic cancer is metastatic pancreatic cancer.

[0067] In some embodiments, the colon cancer is metastatic colon cancer.

[0068] In some embodiments, the cancer or cells thereof have elevated expression of EGFR.

[0069] In another aspect, the present disclosure provides for the use of an immunoconjugate in combination with a chemotherapeutic agent in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the immunoconjugate is as defined in the present disclosure and the chemotherapeutic agent is as defined in the present disclosure.

[0070] In some embodiments, the cancer is selected from pancreatic cancer and colon cancer.

[0071] In some embodiments, the pancreatic cancer is metastatic pancreatic cancer.

[0072] In some embodiments, the colon cancer is metastatic colon cancer.

[0073] In some embodiments, the cancer or cells thereof have elevated expression of EGFR.

[0074] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (a) an effective amount of an immunoconjugate as defined herein; and (b) an effective amount of a chemotherapeutic agent as defined herein.

[0075] In some embodiments, the immunoconjugate is administered to the subject after administration of the chemotherapeutic agent.

[0076] In some embodiments, the immunoconjugate is administered to the subject within 10 days after administration of the chemotherapeutic agent.

[0077] In some embodiments, the immunoconjugate is administered to the subject within three days of administration of the chemotherapeutic agent.

[0078] In some embodiments, the immunoconjugate is administered to the subject more than once.

[0079] In some embodiments, the cancer is selected from pancreatic cancer, melanoma, and colon cancer.

[0080] In some embodiments, the pancreatic cancer is metastatic pancreatic cancer.

[0081] In some embodiments, the colon cancer is metastatic colon cancer.

[0082] In some embodiments, the cancer is melanoma.

[0083] In some embodiments, the cancer or cells thereof have elevated expression of EGFR.

[0084] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present disclosure are shown and described herein. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature and not restrictive. Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0085] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and accompanying drawings (also referred to herein as "figures" and "drawings") that set forth illustrative embodiments, in which the principles of the invention are employed. The accompanying drawings are as follows: [Figures 1A-1E] FIG. 1 shows the results of purification of immunoconjugates of the present disclosure as shown by SDS-PAGE and SEC-HPLC analysis. [Figures 2A-2D] FIG. 1 shows the effect of various chemotherapy regimens in combination with immunoconjugates of the present disclosure. [Figure 3A-3C] FIG. 1 shows an example of an immunoconjugate according to the present disclosure. [Figure 4A-4B] FIG. 1 shows that tumor growth was inhibited by Erb-(IL10)2, FOLFOX, and the combination of Erb-(IL10)2 and FOLFOX. [Figure 4C] FIG. 1 shows that mouse survival was extended by Erb-(IL10)2, FOLFOX, and the combination of Erb-(IL10)2 and FOLFOX. [Figure 5A-5B] FIG. 1 shows that tumor growth was inhibited by Erb-(IL10)2, FOLFOX, and the combination of Erb-(IL10)2 and FOLFOX. DETAILED DESCRIPTION OF THE INVENTION

[0086] Before embodiments of the present disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the present disclosure described herein can be used in practicing the present disclosure. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure.

[0087] 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 to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. In case of conflict, the present patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from this disclosure.

[0088] As used herein, the singular forms "a," "an," and "the" generally include plural referents unless the context clearly dictates otherwise.

[0089] As used herein, the term "immunoconjugate" generally refers to a proteinaceous molecule formed by the binding of one or more antibodies or fragments thereof to one or more second molecules, which may be the same or different and may include, for example, an effector protein.

[0090] As used herein, the term "proteinaceous" generally refers to a substance or molecule that is, relates to, resembles, or is a polypeptide or protein. For example, an immunoconjugate of the present disclosure can be a heterodimeric protein or a heterodimer comprising two or more polypeptides.

[0091] As used herein, the term "heterodimer" generally refers to a molecule (e.g., a proteinaceous molecule) that is 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 comprise polypeptides that differ in the order, number, or type of amino acid residues that form those polypeptides. Each of the two distinct members of a heterodimer may independently comprise one, two, or more units, polypeptide chains, or moieties.

[0092] 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, antigen-binding antibody fragment, bispecific antibody, or other antibody-based molecule or compound. Other examples of targeting moieties can include, but are not limited to, aptamers, avimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, and the like. The terms "targeting moiety" and "binding moiety" are used interchangeably herein.

[0093] As used herein, the term "tumor antigen" generally refers to an antigenic substance within or produced by tumor cells, which may have the ability to elicit an immune response in a host. For example, a tumor antigen may be a protein, polypeptide, peptide, or fragment thereof that constitutes part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. A tumor antigen peptide may be a peptide that is generated as a result of tumor antigen degradation in tumor cells and, when expressed on the cell surface by binding to an HLA molecule, is capable of inducing or activating tumor-specific cytotoxic T lymphocytes. In some embodiments, the term "tumor antigen" may also refer to a biomolecule (e.g., a protein, carbohydrate, glycoprotein, etc.) that is exclusively, preferentially, or differentially expressed on and / or found in association with cancer cells, thereby providing a preferential or specific target for cancer. For example, preferential expression may be preferential expression compared to any other cells in an organism or within a particular region of an organism (e.g., within a particular organ or tissue).

[0094] As used herein, the term "heterodimerization" generally refers to the process of forming heterodimers between two different members (e.g., two non-identical polypeptides), such as by complex formation, association, or aggregation, with or without the formation of a covalent bond between the two different members.

[0095] As used herein, the term "covalent bond" generally refers to a chemical bond formed between atoms by the sharing of electrons. For example, a covalent bond can be polar or non-polar. In some embodiments, the covalent bond is a disulfide bond.

[0096] As used herein, the term "non-covalent pairwise affinity" generally refers to dimerization or heterodimerization sequences that can bind to each other through non-covalent interactions, such as ion pairs, hydrogen bonds, dipole-dipole interactions, charge transfer interactions, π-π interactions, cation-π interactions, van der Waals interactions and dispersion interactions, hydrophobic (lipophilic) interactions, complexation (e.g., complexation of transition metal cations), or a combination of these interactions.

[0097] As used herein, the term "linker" generally refers to a synthetic amino acid sequence that connects or joins two polypeptide sequences, e.g., joins two polypeptide domains. The linker can connect the two amino acid sequences via a peptide bond. In some embodiments, the linkers of the present disclosure connect a biologically active moiety to a second moiety in a linear sequence.

[0098] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers modified by any other procedure, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling moiety. The term can apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term can also include variants on the traditional peptide bond linking the amino acids that make up a polypeptide. For example, "peptide," "polypeptide," and "protein" can be chains of amino acids in which the alpha carbons are linked via peptide bonds. Thus, the terminal amino acid at one end of the chain (the amino terminus) can have a free amino group, and the terminal amino acid at the other end of the chain (the carboxy terminus) can have a free carboxyl group. As used herein, the term "amino terminus" (abbreviated 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 (when involved in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxy terminus" generally refers to the free carboxyl group on the carboxy terminus of a peptide, or the carboxyl group of an amino acid at any other position within the peptide. Peptides can also include essentially any polyamino acid, including, but not limited to, peptidomimetics such as amino acids linked by ether rather than amide bonds.

[0099] As used herein, the term "amino acid" generally refers to either natural and / or unnatural or synthetic amino acids, including, but not limited to, the D or L optical isomers or both, amino acid analogs and peptidomimetics. Standard one-letter or three-letter codes are used to designate amino acids.

[0100] The term "variant," when used in the context of a proteinaceous molecule (e.g., a polypeptide or protein), generally refers to a proteinaceous molecule having sequence homology to a naturally occurring bioactive protein that retains at least some of the therapeutic and / or biological activity of the 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 compared to a reference bioactive protein. In some embodiments, a "variant" may include proteins that have been intentionally modified, for example, by site-directed mutagenesis, synthesis of the encoding gene, insertion, or accidental mutation.

[0101] The terms "linked," "linked," "fused," and "fusion" are used interchangeably herein and generally refer to the joining of two or more chemical elements, sequences, or components, for example, by chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous and in reading phase or in frame. An "in-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a longer, contiguous ORF in a manner that maintains the correct reading frame of the original ORFs. The resulting "fusion polypeptide" is thus a single protein containing two or more fragments corresponding to polypeptides encoded by the original ORFs (these segments are not normally joined in this manner in nature). A "fusion site" refers to the sequence at which two or more fragments are joined to each other. In some cases, the fusion site may be the same sequence as the sequences in the two or more fragments being joined. Optionally, the fusion site can further include a gap segment that is not identical to any of the sequences of the two or more fragments being joined.

[0102] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in the polypeptide, from amino to carboxyl terminus, in which adjacent residues in the sequence are contiguous in the primary structure of the polypeptide. A "subsequence" is a linear sequence forming a portion of a polypeptide that is known to contain additional residues in one or both directions.

[0103] The terms "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

[0104] The terms "gene" and "gene fragment" are used interchangeably herein and generally refer to a polynucleotide containing at least one open reading frame that is capable of encoding a specific protein after being transcribed and translated. A gene or gene fragment can be genomic or cDNA, so long as the polynucleotide contains at least one open reading frame that may cover the entire coding region or a segment thereof. A "fusion gene" is a gene consisting of at least two heterologous polynucleotides linked together.

[0105] As used herein, the term "antibody" generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. As used herein, light chains can be classified as either kappa or lambda. Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Antibodies used in this disclosure can have a structural unit comprising a tetramer. Each tetramer can be composed of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 KD) and one "heavy" chain (approximately 50-70 KD). The N-terminus of each chain can define a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. As used herein, the terms "light chain variable region" (VL) and "heavy chain variable region" (VH) generally refer to these regions of the light and heavy chains, respectively. Antibodies can exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases or expressed de novo. Thus, for example, pepsin can digest an antibody below the disulfide bond in the hinge region to produce F(ab)'2 (a dimer of Fab, itself a light chain linked to VH-CH1 by a disulfide bond). F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)).Although various antibody fragments are defined with respect to the digestion of an intact antibody, one of skill in the art will understand that such Fab' fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody can also include antibody fragments produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodology, including, but not limited to, Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nanobody, unibody, and diabody. In some embodiments, antibodies include, but are not limited to, Fab'2, IgG, IgM, IgA, IgE, and single-chain antibodies, e.g., single-chain Fv (scFv) antibodies in which the variable heavy and variable light chains are linked together (directly or via a peptide linker) to form a contiguous polypeptide.

[0106] As used herein, the term "antigen-binding site" or "binding portion" generally refers to the portion of an antibody that is involved in antigen binding. The antigen-binding site may be formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and / or light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are called "hypervariable regions," which are inserted between more conserved adjacent stretches known as "framework regions" or "FRs." Thus, as used herein, the term "FR" generally refers to the amino acid sequences naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding "surface." This surface may mediate recognition and binding of target antigens. The three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs" and are characterized, for example, by Kabat et al., Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, Md. (1987).

[0107] As used herein, the terms "homology," "homologous," or "sequence identity" generally refer to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program (e.g., Emboss Needle or BestFit) to determine sequence identity, similarity, or homology between two different amino acid sequences, default settings may 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 hybridize under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity compared to the sequences. 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 when sequences of equivalent length are optimally aligned.

[0108] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition sufficient to achieve its intended use, including but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (e.g., in vitro or in vivo), or the subject and condition being treated, such as the subject's weight and age, the severity of the condition, the method of administration, etc., which can be readily determined by one of ordinary skill in the art. The term may also apply to a dose that induces a particular response in target cells, such as target gene induction, proliferation, and / or apoptosis. A particular dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system that carries it.

[0109] The terms "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably herein and refer to an approach to achieving beneficial or desired results, including, but not limited to, a therapeutic effect and / or a prophylactic effect. As used herein, a therapeutic effect generally refers to the eradication or reduction in severity of the underlying disease being treated. A therapeutic effect is also achieved by the eradication, reduction in severity, or reduction in incidence of one or more physiological symptoms associated with the underlying disease, such that an improvement is observed in the subject, even though the subject may still be suffering from the underlying disease. For a prophylactic effect, the composition can be administered to a subject at risk of developing a particular disease or to a subject reporting one or more physiological symptoms of the disease, even if the disease has not been diagnosed.

[0110] As used herein, the term "therapeutic effect" generally encompasses the therapeutic and / or prophylactic effects described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0111] As used herein, "co-administration," "administering in combination," "using in combination," and their grammatical equivalents generally encompass 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 and / or functional in the subject. Co-administration includes simultaneous administration in separate compositions or forms, administration at different times in separate compositions or forms, or administration in a composition in which both agents are present.

[0112] As used herein, the term "agent" generally refers to a biological, pharmaceutical, or chemical compound or other moiety. Non-limiting examples include simple or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, vitamin derivatives, carbohydrates, toxins, chemotherapeutic compounds, or agents capable of generating / emitting radiation.

[0113] As used herein, the term "interleukin" generally refers to a secreted protein or signaling molecule that can promote the development and differentiation of T and / or B lymphocytes and / or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes as well as by monocytes, macrophages, and endothelial cells. As used herein, interleukin (IL) can include IL10. As used herein, the term "interleukin" can include a full-length interleukin or a fragment (e.g., truncated form) or variant thereof that substantially maintains the biological activity of the corresponding wild-type interleukin (e.g., has a biological activity that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the biological activity of the corresponding wild-type interleukin). As used herein, interleukins can be derived from any mammalian species. In some embodiments, the interleukin is derived from a species selected from the group consisting of human, horse, cow, mouse, pig, rabbit, cat, dog, rat, goat, sheep, and non-human primate. In some embodiments, the interleukin can be a mutant form, e.g., with increased or decreased affinity for its receptor.

[0114] As used herein, the term "subject" generally refers to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, goat, rabbit, mouse, rat, or monkey.

[0115] As used herein, the term "EGFR family member" generally refers to a member of the epidermal growth factor receptor family, which may be, for example, ErbB-1 (also known as epidermal growth factor receptor, EGFR).

[0116] As used herein, the term "member" generally refers to a polypeptide, subunit, or moiety that is one component of an immune complex.

[0117] 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 an immunoglobulin heavy chain constant region, or an analog or portion thereof capable of binding to an Fc receptor. As is known, each immunoglobulin heavy chain constant region contains four or five domains. The domains are designated sequentially as CH1-hinge-CH2-CH3 (-CH4). CH4 is present in IgM, which does not have a hinge region. An immunoglobulin heavy chain constant region useful in the present 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, an Fc domain according to the present disclosure consists of the hinge-CH2-CH3 domain.

[0118] As used herein, the term "Fc subunit" generally refers to a component of an Fc domain. For example, an Fc domain may be formed by two or more members, and each member may be considered an Fc subunit.

[0119] As used herein, the term "complexation" 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 complex with a heavy chain to form a targeting moiety.

[0120] As used herein, the term "binding specificity" generally refers to the ability to specifically bind (e.g., immunoreact with) a given target (while not binding, or substantially not binding, to non-targets). Targeting moieties of the present disclosure may be monospecific, comprising one or more binding sites that specifically bind to a target, or multispecific (e.g., bispecific or trispecific), comprising two or more binding sites that specifically bind to the same or different targets.

[0121] As used herein, the terms "associate" or "associated" generally refer to one entity being physically associated with or in contact with another entity. For example, a first member of an immune complex can be "associated" with a second member, either covalently or non-covalently. In some embodiments, the first member of an immune complex associates with the second member through an interface, where the interface is formed by amino acid residues from the first and second members, respectively (i.e., interface residues).

[0122] As used herein, the term "modification" generally refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or any post-translational modification of a polypeptide (e.g., glycosylation). For example, a modification is compared to the sequence of a corresponding wild-type polypeptide. A modification can be a substitution, addition, and / or deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

[0123] As used herein, the term "fusion protein" generally refers to a polypeptide that comprises or alternatively consists of the amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to the amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to the former polypeptide or a domain thereof).

[0124] As used herein, the term "C-terminus" generally refers to the carboxy terminus of a polypeptide.

[0125] As used herein, the term "N-terminus" generally refers to the amino terminus of a polypeptide.

[0126] As used herein, the term "immunoglobulin" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer, consisting of two pairs of identical immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions are both responsible for binding to an antigen, and the constant regions are responsible for the antibody's effector functions. In addition to antibodies, immunoglobulins can exist in a variety of other forms, including, for example, Fv, Fab, Fab', and (Fab')2.

[0127] As used herein, the term "in-frame fusion" generally refers to the joining of two or more open reading frames (ORFs) to form a longer, contiguous ORF in a manner that maintains the exact reading frame of the original ORFs.

[0128] As used herein, the term "amino acid substitution" generally refers to the replacement of one amino acid with another amino acid at a particular position in a polypeptide.

[0129] As used herein, the term "EU Index of Kabat Numbers" generally refers to the index of EU numbers corresponding to amino acid sequences according to Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242.

[0130] The terms "isolated polynucleotide" and "isolated nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, that have been isolated from a natural environment or artificially synthesized.

[0131] As used herein, the term "pharmaceutically acceptable excipient" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0132] The terms "immunogenic cell death" and "immunogenic apoptosis" are used interchangeably herein and generally refer to a form of cell death that induces effective antitumor immune responses, for example, through activation of dendritic cells (DCs) and the resulting activation of specific T cell responses. Immunogenic cell death can be characterized by the secretion of damage-associated molecular patterns (DAMPs). DAMPs can include calreticulin (CRT), heat-shock proteins (HSPs), secreted amphoterin (HMGB1), and ATP. CRTs are normally located in the lumen of the endoplasmic reticulum (ER) and may translocate to the surface of dying cells after induction of immunogenic apoptosis, where they function as an "eat me" signal for professional phagocytes. HSPs include HSP70 and HSP90 and can also translocate to the plasma membrane under stress conditions. HMGB1 is considered a late apoptotic marker, and its release into the extracellular space appears to be necessary for optimal release and presentation of tumor antigens to dendritic cells.12 Once secreted, ATP can function as a "find-me" signal for monocytes, inducing their attraction to sites of apoptosis.

[0133] As used herein, the term "FOLFOX regimen" generally refers to a chemotherapy regimen containing leucovorin calcium (calcium folinate, folinic acid, leucovorin), fluorouracil (5-FU), and oxaliplatin, which can be used to treat advanced and metastatic colorectal cancer. FOLFOX regimens vary in drug dosage and administration schedule and include FOLFOX4, FOLFOX6, modified FOLFOX6 (mFOLFOX6), and FOLFOX7.

[0134] As used herein, the terms "fluorouracil," "5-fluorouracil," or "5-FU" generally refer to antimetabolite fluoropyrimidine analogs of nucleoside pyrimidines used as anti-cancer chemotherapeutic agents.

[0135] As used herein, the term "oxaliplatin" or "eloxatin" generally refers to a cytostatic anti-tumor drug useful in the therapeutic treatment of various types of susceptible cancers and tumors.

[0136] As used herein, the terms "folinic acid," "tetrahydrofolic acid," "leucovorin," "leucovorin calcium," or "calcium leucovorin" refer generally to a chemoprotective agent used in combination with other chemotherapy agents.

[0137] As used herein, the term "upregulated expression" generally refers to higher than normal expression of a product in some conditions. For example, EGFR is upregulated in metastatic pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)) and metastatic colon cancer cells, including wild-type or normal cells.

[0138] Compositions Comprising Immunoconjugates and Chemotherapeutic Agents In one aspect, the present disclosure provides a composition comprising an immunoconjugate and a chemotherapeutic agent. The immunoconjugate can include 1) one or more interleukins and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, where the first Fc subunit associates with the second Fc subunit to form a dimer. The one or more interleukins can be fused to the Fc domain. The chemotherapeutic agent can include fluorouracil and oxaliplatin.

[0139] For immunoconjugates according to the present disclosure, at least one of the one or more interleukins (e.g., IL10) may be fused (e.g., in-frame) to the amino-terminal amino acid of the Fc domain. The immunoconjugate may comprise two or more interleukins. In some embodiments, at least two of the two or more interleukins are fused to the amino-terminal amino acid of the Fc domain. In some embodiments, one or more of the interleukins are fused (e.g., in-frame) to the Fc domain via a peptide linker and / or an immunoglobulin hinge region. In some embodiments, at least two of the two or more interleukins are fused to each other via a peptide linker (e.g., in-frame) to form an interleukin dimer. At least one interleukin dimer may be fused to the amino-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. Thus, in some embodiments, two IL10s may be fused in-frame to each other (e.g., via a peptide linker) to form an IL10 dimer, and then the carboxy terminus of the IL10 dimer may be fused (e.g., in-frame, e.g., via a peptide linker) to the amino-terminal amino acid of the Fc domain.

[0140] The linker may 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 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids), 1 to 15 amino acids (e.g., 1 to 11, 12, 13, 14, 15 amino acids), 1 to 20 amino acids, 1 to 30 amino acids, or more. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:41.

[0141] The immunoconjugate may further comprise a targeting moiety fused to the Fc domain, wherein the targeting moiety may exhibit binding specificity for a tumor antigen. The tumor antigen may be any immunogenic entity or portion thereof that is 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 EGFR family members (such as EGFR).

[0142] The targeting moiety can be fused to the amino-terminal amino acid of the Fc domain. In some embodiments, the targeting moiety is fused to the Fc domain via a peptide linker or an immunoglobulin hinge region.

[0143] The targeting moiety may comprise an antigen-binding domain of an antibody, e.g., 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.

[0144] In some embodiments, the antibody is an anti-EGFR antibody. For example, the anti-EGFR antibody can be cetuximab. In some embodiments, the targeting moiety comprises heavy chain CDR1-3 of cetuximab, light chain CDR1-3 of cetuximab, a heavy chain variable region of cetuximab, a light chain variable region of cetuximab, and / or a light chain of cetuximab. For example, the targeting moiety can be a Fab portion comprising both the heavy chain variable region and the light chain variable region of cetuximab.

[0145] For example, a targeting moiety can comprise a heavy chain CDR having an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that contained in the corresponding heavy chain CDRs 1-3 of cetuximab. Alternatively or additionally, a targeting moiety can comprise a light chain CDR having an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to that contained in the corresponding light chain CDRs 1-3 of cetuximab. For example, a targeting moiety can include a heavy chain variable region having an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding heavy chain variable region of cetuximab. For example, a targeting moiety can include a light chain variable region having an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding light chain variable region of cetuximab. For example, a targeting moiety can include a light chain having an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that contained in the corresponding light chain of cetuximab.

[0146] The heavy chain CDRs 1 to 3 of cetuximab are as shown in SEQ ID NO: 52 (CDR1), SEQ ID NO: 53 (CDR2), and SEQ ID NO: 54 (CDR3), respectively. The light chain CDRs 1 to 3 of cetuximab are as shown in SEQ ID NO: 48 (CDR1), SEQ ID NO: 49 (CDR2), and SEQ ID NO: 50 (CDR3), respectively. The heavy chain variable region of cetuximab is as shown in SEQ ID NO: 55. The light chain variable region of cetuximab is as shown in SEQ ID NO: 51.

[0147] For immunoconjugates according to the present disclosure, the Fc domain may be an IgG Fc domain. The 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).

[0148] In some embodiments, the immune complex is a proteinaceous homodimer consisting of two identical members. Each of the two identical members may contain one or more interleukins (e.g., IL10) fused to an Fc domain subunit (e.g., in-frame via a peptide linker). For example, the carboxy terminus of one or more interleukins may be fused to the amino-terminal amino acid of an Fc subunit. In some embodiments, the carboxy terminus of one interleukin (e.g., IL10) is fused in-frame to the amino-terminal amino acid of one of the two Fc subunits to form one member of the homodimer, and two such identical members associate with each other through interactions between the two Fc subunits to form a homodimer (e.g., as shown in Figure 3A). In some embodiments, two interleukins (e.g., two IL10s) are fused in-frame to each other (e.g., via a peptide linker) to form an interleukin dimer, the carboxy terminus of the interleukin dimer is then fused in-frame to the amino-terminal amino acid of one of the two Fc subunits to form one member of a homodimer, and two identical such members associate with each other via interactions between the two Fc subunits to form the homodimer.

[0149] In some embodiments, the immune complex is a proteinaceous heterodimer comprising a first member and a second member different from the first member, wherein the first member comprises a first Fc subunit, 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.

[0150] In some embodiments, 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.

[0151] In some embodiments, in the second member, at least two of the one or more interleukins are fused to each other to form an interleukin dimer, which is further fused to the amino-terminal amino acid of a second Fc subunit. For example, two IL10s may be fused in-frame to each other (e.g., via a peptide linker to form an IL10 dimer) and then fused in-frame to a second Fc subunit to form the second member of the proteinaceous heterodimer. For example, the carboxy terminus of an IL10 dimer may be fused to the amino-terminal amino acid of a second Fc subunit (e.g., as shown in Figures 3B and 3C).

[0152] For example, the second member of the immune complex can be a fusion protein, in which a second Fc subunit can be fused in-frame to an interleukin. In some embodiments, the carboxy terminus of the interleukin is fused directly or indirectly to the amino terminus of the second Fc subunit to form the fusion protein. In some embodiments, the second Fc subunit is fused in-frame to the interleukin via a peptide linker or an immunoglobulin hinge region.

[0153] A peptide linker according to the present disclosure can be a synthetic amino acid sequence that connects or links two polypeptide sequences, e.g., via a peptide bond. 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 can comprise 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids), 1 to 15 amino acids (e.g., 1 to 11, 12, 13, 14, 15 amino acids), 1 to 20 amino acids, 1 to 30 amino acids, or more. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the linker is resistant to proteolysis or substantially resistant to proteolysis. The hinge region may comprise the amino acid sequence set forth in SEQ ID NO:60.

[0154] In some embodiments, the first member further comprises a targeting moiety fused to the first Fc subunit. For example, the targeting moiety can be fused to the amino-terminal amino acid of the first Fc subunit. For example, the first member can comprise an Fab portion of an antibody, the carboxy terminus of which (e.g., the carboxy terminus of a heavy chain portion such as a CH1 domain or hinge region) is fused to the amino-terminal amino acid of the first Fc subunit (e.g., as shown in Figure 3C).

[0155] In some embodiments, the proteinaceous heterodimer does not include any targeting moiety, for example, the first member may include only the first Fc subunit (e.g., as shown in Figure 3B).

[0156] In some embodiments, the first Fc subunit is the same as the second Fc subunit (eg, a subunit of a wild-type human IgG1 Fc domain).

[0157] In some embodiments, the first Fc subunit differs from the second Fc subunit, and the Fc domain comprises a modification that promotes heterodimerization between the first Fc subunit and the second Fc subunit. For example, the first Fc subunit may comprise a first modification, and the second Fc subunit may comprise 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 each compared to the sequence of its corresponding wild-type Fc domain.

[0158] 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, where the amino acid positions are determined according to the EU index of the KABAT numbering.

[0159] In some embodiments, the first modification comprises an amino acid substitution at position T366 and an amino acid substitution at one or more positions selected from the group consisting of Y349, F405, K409, D399, K360, Q347, K392 and S354, where the amino acid positions are determined according to the EU index of the KABAT numbering.

[0160] For example, the amino acid substitution 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.

[0161] In some embodiments, the first modification comprises between 2 and 5 amino acid substitutions.

[0162] In some embodiments, the first modification is 1) Y349 and T366, 2) Y349, T366 and F405, 3) Y349, T366 and K409, 4) Y349, T366, F405, K360 and Q347, 5) Y349, T366, F405 and Q347, 6) Y349, T366, K409, K360 and Q347, 7) Y349, T366, K409 and Q347, 8) T366 1) T366, K409 and K392, 2) T366 and K409, 3) T366, K409, Y349 and S354, 4) T366 and F405, 5) T366, F405 and D399, and 6) T366, F405, Y349 and S354, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0163] In some embodiments, the first modification is 1) Y349C and T366W, 2) Y349C, T366W and F405K, 3) Y349C, T366W and K409E, 4) Y349C, T366W and K409A, 5) Y349C, T366W, F405K, K360E and Q347E, 6) Y349C, T366W, F405K and Q347R, 7) Y349C, T366W, K409A, K360E and Q347E, 8) Y349C, T366W, K409A and Q347R, 9) T366W, K409 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 amino acid positions are determined according to the EU index of the KABAT numbering.

[0164] In some embodiments, the second modification comprises amino acid substitutions at positions T366, L368, and Y407, and at one or more positions selected from the group consisting of D356, D399, E357, F405, K360, K392, K409, and Q347, where the amino acid positions are determined according to the EU index of the KABAT numbering.

[0165] In some embodiments, the amino acid substitution comprised in the second modification is selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A and Y407V.

[0166] In some embodiments, the second modification comprises an amino acid substitution at positions 4-6.

[0167] In some embodiments, the second modification is 1) D356, T366, L368, Y407, and F405; 2) D356, T366, L368, and Y407; 3) D356, T366, L368, Y407, and Q347; 4) D356, T366, L368, Y407, K360, and Q347; 5) D356, T366, L368, Y407, F405, and Q347; 6) D356, T366, L368, Y407, F405, K360, and Q347; 7) T366, L368, Y40 7) T366, L368, Y407 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 amino acid positions are determined according to the EU index of the KABAT numbering.

[0168] In some embodiments, the second modification is 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, D399 1) T366S, L368A, Y407V, K409A and K392D; 2) T366S, L368G, Y407A and K409A; and 3) T366S, L368A, Y407V, K409A and E357A, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0169] In some embodiments, the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification, the first modification and the second modification being: 1) first modification: Y349 and T366, second modification: D356, T366, L368, Y407 and F405; 2) first modification: Y349, T366 and F405, second modification: D356, T366, L368 and Y407; 3) first modification: Y349, T366 and K409, second modification: D356, T366, L368, Y407 and F405; 4) first modification: Y349, T366, F40 5) first modification: Y349, T366, F405 and Q347, second modification: D356, T366, L368, Y407, K360 and Q347, 6) first modification: Y349, T366, K409, K360 and Q347, second modification: D356, T366, L368, Y407, F405 and Q347, 7) first modification: Y349, T366, K409 and Q347, second modification: D356, T366, L368, Y407, F405, K360 and and Q347, 8) first modification: T366, K409 and K392, second modification: T366, L368, Y407, D399 and F405, 9) first modification: T366 and K409, second modification: T366, L368, Y407 and F405, 10) first modification: T366, K409 and Y349, second modification: T366, L368, Y407, F405 and E357, 11) first modification: T366, K409, Y349 and S354, second modification: T366, L368, Y407, F405 and E357, 12) first modification: T366 and F405, and 15) first modifications: T366, F405, Y349 and S354, second modifications: T366, L368, Y407, K409 and E357, wherein the amino acid positions are selected from the group consisting of: 1) first modifications: T366, F405 and D399, second modifications: T366, L368, Y407, K409 and K392; 2) first modifications: T366, F405 and Y349, second modifications: T366, L368, Y407, K409 and E357;Determined according to the EU index of KABAT numbers.

[0170] In some embodiments, the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification, the first modification and the second modification being selected from the group consisting of: 1) first modification: Y349C and T366W, and second modification: D356C, T366S, L368A, Y407V, and F405K; 2) first modification: Y349C, T366W, and F405K, and second modification: D356C, T366S, L368A, and Y407V; 3) first modification: Y349C, T366W, and K409E, and second modification: D356C, T366S, L368A, Y407V, and F405K; 4) first modification: Y349C, T366W, and K409E, and second modification: D356C, T366S, L368A, Y407V, and F405K; 1) First modification: Y349C, T366W and K409A, Second modification: D356C, T366S, L368A, Y407V and F405K, 5) First modification: Y349C, T366W, F405K, K360E and Q347E, Second modification: D356C, T366S, L368A, Y407V and Q347R, 6) First modification: Y349C, T366W, F405K and Q347R, Second modification: D356C, T366S, L368A, Y407V, K360E and Q347E, 7) First modification: Y349C, T366W, K409A, K360E and Q3 47E, second modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) first modification: Y349C, T366W, K409A and Q347R, second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) first modification: T366W, K409A and K392D, second modification: T366S, L368A, Y407V, D399S and F405K; 10) first modification: T366W and K409A, second modification: T366S, L368G, Y407A and F405K; 11) 1) first modification: T366W, K409A and Y349D, second modification: T366S, L368A, Y407V, F405K and E357A; 12) first modification: T366W, K409A, Y349D and S354D, second modification: T366S, L368A, Y407V, F405K and E357A; 13) first modification: T366W and F405K, second modification: T366S, L368A, Y407V and K409A; 14) first modification: T366W, F405K and D399S, second modification: T366S, L368A, Y407V, K409A and K392D;15) first modification: T366W and F405K, second modification: T366S, L368G, Y407A and K409A, 16) first modification: T366W, F405K and Y349D, second modification: T366S, L368A, Y407V, K409A and E357A, 17) first modification: T366W, F405K, Y349D and S354D, second modification: T366S, L368A, Y407V, K409A and E357A, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0171] In some embodiments, the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification, wherein the first modification comprises amino acid substitutions T366W and K409A and the second modification comprises amino acid substitutions T366S, L368G, Y407A and F405K, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0172] For example, the amino acid sequence of the first Fc subunit can be selected from SEQ ID NO: 17. The amino acid sequence of the interleukin can be as set forth in SEQ ID NO: 56. The amino acid sequence of the second Fc subunit can be selected from SEQ ID NO: 18.

[0173] In some embodiments, the immunoconjugate of the present disclosure is a proteinaceous heterodimer, and the amino acid sequence of the second member can be selected from SEQ ID NO:42.

[0174] In some embodiments, the immune complex may comprise a first member and a second member. The first member may comprise a first polypeptide chain and a second polypeptide chain. The second member may comprise a third polypeptide chain. The first polypeptide chain may comprise a light chain variable region of cetuximab. The second polypeptide chain may comprise a first Fc subunit and a heavy chain variable region of cetuximab. The third polypeptide chain may comprise a second Fc subunit and one or more interleukins. The targeting moiety may comprise a heavy chain variable region of cetuximab and a light chain variable region of cetuximab.

[0175] In some embodiments, the immune complex may comprise a first member comprising a first polypeptide chain, and a second member comprising a second polypeptide chain, wherein the first polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO: 17, and the second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO: 42.

[0176] The different components of the composition may be packaged separately (eg, not mixed with each other prior to administration) or may be premixed and packaged in the same packaging unit.

[0177] The compositions of the present disclosure may be pharmaceutical compositions and may further comprise pharmaceutically acceptable excipients, including, but not limited to, inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers, and adjuvants.

[0178] In some embodiments, the pharmaceutical composition is formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous repository.

[0179] The compositions of the present disclosure may contain therapeutically effective amounts of active agents (e.g., immunoconjugates and chemotherapeutic agents). A therapeutically effective amount is an amount of the subject composition that can prevent and / or (at least partially) cure a disease or disorder (e.g., cancer) and / or any complications thereof in a subject suffering from or at risk of developing the disease or disorder. The specific amount / concentration of the active agent included can vary depending on the method of administration and the needs of the patient, and can be determined based on, for example, the patient's volume, viscosity, and / or weight, etc.

[0180] Cancer Treatment Methods and Uses In another aspect, the present disclosure provides an immunoconjugate for use in the treatment of cancer in combination with a chemotherapeutic agent. The immunoconjugate can include 1) one or more interleukins and 2) an Fc domain consisting of a first Fc subunit and a second Fc subunit, where the first Fc subunit associates with the second Fc subunit to form a dimer. The one or more interleukins can be fused to the Fc domain.

[0181] In another aspect, the present disclosure provides use of an immunoconjugate in combination with a chemotherapeutic agent in the preparation of a medicament for treating cancer in a subject in need thereof. The immunoconjugate can 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. The one or more interleukins can be fused to the Fc domain.

[0182] In a further aspect, the present disclosure provides a method of treating cancer in a subject in need thereof. The method comprises administering to the subject (a) an effective amount of an immunoconjugate described elsewhere in this disclosure, and (b) an effective amount of a chemotherapeutic agent described elsewhere in this disclosure. The immunoconjugate can 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. The one or more interleukins can be fused to the Fc domain.

[0183] Immunoconjugates are as defined elsewhere in this disclosure. For example, immunoconjugates can be included in compositions of the present disclosure.

[0184] The immunoconjugate can be administered to the subject after administration of the chemotherapeutic agent, hi some embodiments, two or more doses of the chemotherapeutic agent are administered to the subject, and the immunoconjugate is administered to the subject after administration of the last (or final) dose of the chemotherapeutic agent.

[0185] For example, 2 to 15 doses (e.g., 2 to 14 doses, 2 to 13 doses, 2 to 12 doses, 2 to 11 doses, 2 to 10 doses, 2 to 9 doses, 2 to 8 doses, 3 to 10 doses, 4 to 10 doses, or 4 to 12 doses) of the chemotherapeutic agent can be administered to the subject, and the immunoconjugate can be administered to the subject after the last (or final) dose of the chemotherapeutic agent.

[0186] The immunoconjugate can be administered to the subject within 10 days (e.g., within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day, or immediately after) after administration of the chemotherapeutic agent (e.g., after administration of the last dose of the chemotherapeutic agent). In some embodiments, the immunoconjugate is administered to the subject 0-7 days (e.g., 0-6 days, 0-5 days, 0-4 days, 0-3 days, 0-2 days, or 0-1 days) after administration of the chemotherapeutic agent.

[0187] The immunoconjugate can be administered to a subject more than once (eg, at least twice, at least three times, at least four or more times).

[0188] The cancer can include a solid tumor. For example, the cancer can be selected from the group consisting of colon cancer, pancreatic cancer, and melanoma.

[0189] In some embodiments, the cancer is in a subject, e.g., a cancer or cancer cell in a human or 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 such mammals can be subjects known in the art as preclinical models of particular diseases or disorders, including solid tumors and / or other cancers (e.g., Talmadge et al., 2007 Am. J. Pathol. 170:793; Kerbel, 2003 Canc. Biol. Therap. 2(4 Suppl 1):S134; Man et al., 2007 Canc. Met. Rev. 26:737; Cespedes et al., 2006 Clin. TransL Oncol. 8:318).

[0190] The present disclosure also includes the following embodiments.

[0191] 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, wherein the first Fc subunit associates with the second Fc subunit to form a dimer, and wherein the one or more interleukins are fused to the Fc domain, and the chemotherapeutic agent comprises fluorouracil and / or oxaliplatin.

[0192] 2. The composition of embodiment 1, wherein at least one of the one or more interleukins is fused to the amino-terminal amino acid of the Fc domain.

[0193] 3. The composition of any one of embodiments 1-2, wherein the immunoconjugate comprises two or more interleukins.

[0194] 4. The composition of embodiment 3, wherein at least two of the two or more interleukins are fused to the amino-terminal amino acid of the Fc domain.

[0195] 5. The composition of 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.

[0196] 6. The composition of any one of embodiments 3-5, wherein at least two of the two or more interleukins are fused to each other via a peptide linker to form an interleukin dimer.

[0197] 7. The composition of embodiment 6, wherein at least one interleukin dimer is fused to the amino-terminal amino acid of the Fc domain.

[0198] 8. The composition of any one of embodiments 3-7, wherein the two or more interleukins are two or more copies of the same interleukin.

[0199] 9. The composition of embodiment 8, wherein the two or more interleukins are two or more copies of IL10.

[0200] 10. The composition of any one of embodiments 1-9, wherein the one or more interleukins include one or more IL10.

[0201] 11. The composition of any one of embodiments 1 to 10, wherein the immunoconjugate further comprises a targeting moiety fused to the Fc domain, wherein the targeting moiety exhibits binding specificity for a tumor antigen.

[0202] 12. The composition of embodiment 11, wherein the targeting moiety is fused to the amino-terminal amino acid of the Fc domain.

[0203] 13. The composition of any one of embodiments 11-12, wherein the targeting moiety is fused to the Fc domain via a peptide linker or an immunoglobulin hinge region.

[0204] 14. The composition of any one of embodiments 11 to 13, wherein the targeting moiety comprises an antigen-binding domain of an antibody.

[0205] 15. The composition of embodiment 14, wherein the antigen-binding domain of the antibody is a Fab portion.

[0206] 16. The composition of any one of embodiments 11 to 15, wherein the tumor antigen is EGFR.

[0207] 17. The composition of any one of embodiments 11 to 16, wherein the targeting moiety comprises the antigen-binding domain of an anti-EGFR antibody.

[0208] 18. The composition of embodiment 17, wherein the anti-EGFR antibody is cetuximab.

[0209] 19. The composition of embodiment 18, wherein the targeting moiety comprises heavy chain CDR1-3 of cetuximab, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 53, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0210] 20. The composition of any one of embodiments 18-19, wherein the targeting moiety comprises light chain CDR1-3 of cetuximab, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0211] 21. The composition of any one of embodiments 18-20, wherein the targeting moiety comprises a heavy chain variable region of cetuximab, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 55.

[0212] 22. The composition of any one of embodiments 18-21, wherein the targeting moiety comprises the light chain variable region of cetuximab, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0213] 23. The composition of any one of embodiments 1 to 22, wherein the Fc domain is an IgG Fc domain.

[0214] 24. The composition of embodiment 23, wherein the IgG is IgG1.

[0215] 25. The composition of embodiment 24, wherein the IgG is human IgG1.

[0216] 26. The composition of any one of embodiments 1 to 25, wherein the immune complex is an asymmetric immune complex 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 a second Fc subunit, and wherein the first Fc subunit associates with the second Fc subunit to form a dimer of Fc domains.

[0217] 27. The composition of 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.

[0218] 28. The composition of any one of embodiments 26-27, 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 to the amino-terminal amino acid of a second Fc subunit.

[0219] 29. The composition of any one of embodiments 26 to 28, wherein the first member further comprises a targeting moiety fused to the first Fc subunit.

[0220] 30. The composition of embodiment 29, wherein in the first member, the targeting moiety is fused to the amino-terminal amino acid of the first Fc subunit.

[0221] 31. The composition of any one of embodiments 1 to 30, wherein the first Fc subunit is different from the second Fc subunit and the Fc domain comprises a modification that promotes heterodimerization between said first Fc subunit and the second Fc subunit.

[0222] 32. The composition of embodiment 31, wherein the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification.

[0223] 33. The composition of embodiment 32, wherein the first modification comprises an amino acid substitution at position T366 and at one or more positions selected from the group consisting of Y349, F405, K409, D399, K360, Q347, K392 and S354, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0224] 34. The composition of embodiment 33, wherein the amino acid substitution comprised in the first modification is selected from the group consisting of Y349C, Y349D, D399S, F405K, K360E, K409A, K409E, Q347E, Q347R, S354D, K392D and T366W, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0225] 35. The composition of any one of embodiments 32 to 34, wherein the first modification comprises 2 to 5 amino acid substitutions.

[0226] 36. The first modification is: 1) Y349 and T366, 2) Y349, T366 and F405, 3) Y349, T366 and K409, 4) Y349, T366, F405, K360 and Q347, 5) Y349, T366, F405 and Q347, 6) Y349, T366, K409, K360 and Q347, 7) Y349, T366, K409 and Q347, 8) T366, K409 and K392, 9) T36 10) T366, K409, Y349 and S354; 11) T366 and F405; 12) T366, F405 and D399; and 13) T366, F405, Y349 and S354, wherein the amino acid positions are determined according to the EU index of KABAT numbers.

[0227] 37. The first modification is: 1) Y349C and T366W, 2) Y349C, T366W and F405K, 3) Y349C, T366W and K409E, 4) Y349C, T366W and K409A, 5) Y349C, T366W, F405K, K360E and Q347E, 6) Y349C, T366W, F405K and Q347R, 7) Y349C, T366W, K409A, K360E and Q347E, 8) Y349C, T366W, K409A and Q347R, 9) T366W, K409A and K392D, 10) T366 10) T366W, K409A and Y349D; 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 amino acid positions are determined according to the EU index of the KABAT numbering.

[0228] 38. The composition of any one of embodiments 32 to 37, wherein the second modification comprises amino acid substitutions at positions T366, L368 and Y407, and at one or more positions selected from the group consisting of D356, D399, E357, F405, K360, K392, K409 and Q347, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0229] 39. The composition of embodiment 39, wherein the amino acid substitution comprised in the second modification is selected from the group consisting of D356C, D399S, E357A, F405K, K360E, K392D, K409A, L368A, L368G, Q347E, Q347R, T366S, Y407A and Y407V, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0230] 40. The composition of any one of embodiments 32 to 39, wherein the second modification comprises 4 to 6 amino acid substitutions.

[0231] 41. The second modification is: 1) D356, T366, L368, Y407, and F405; 2) D356, T366, L368, and Y407; 3) D356, T366, L368, Y407, and Q347; 4) D356, T366, L368, Y407, K360, and Q347; 5) D356, T366, L368, Y407, F405, and Q347; 6) D356, T366, L368, Y407, F405, K360, and Q347; 7) T366, L368, Y407, D399, and F405; 8) T 10) T366, L368, Y407 and K409; 11) T366, L368, Y407, K409 and K392; and 12) T366, L368, Y407, K409 and E357, wherein the amino acid positions are determined according to the EU index of KABAT numbers.

[0232] 42. The second modification is: 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, K360 E 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 10) T366S, L368A, Y407V, K409A and K392D; 11) T366S, L368A, Y407V, K409A and K392D; 12) T366S, L368G, Y407A and K409A; and 13) T366S, L368A, Y407V, K409A and E357A, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0233] 43. The first Fc subunit comprises a first modification, and the second Fc subunit comprises a second modification, the first modification and the second modification being: 1) first modification: Y349 and T366, and second modification: D356, T366, L368, Y407, and F405; 2) first modification: Y349, T366, and F405, and second modification: D356, T366, L368, and Y407; 3) first modification: Y349, T366, and K409, and second modification: D356, T366, L368, Y407, and F405; 4) first modification: Y349, T366, F405, K360, and and Q347, second modification: D356, T366, L368, Y407 and Q347; 5) first modification: Y349, T366, F405 and Q347, second modification: D356, T366, L368, Y407, K360 and Q347; 6) first modification: Y349, T366, K409, K360 and Q347, second modification: D356, T366, L368, Y407, F405 and Q347; 7) first modification: Y349, T366, K409 and Q347, second modification: D356, T366, L368, Y407, F405, K360 and Q347 , 8) first modification: T366, K409 and K392, second modification: T366, L368, Y407, D399 and F405, 9) first modification: T366 and K409, second modification: T366, L368, Y407 and F405, 10) first modification: T366, K409 and Y349, second modification: T366, L368, Y407, F405 and E357, 11) first modification: T366, K409, Y349 and S354, second modification: T366, L368, Y407, F405 and E357, 12) first modification: T366 and F405, second modification: modifications: T366, L368, Y407, and K409, 13) first modification: T366, F405, and D399, second modification: T366, L368, Y407, K409, and K392, 14) first modification: T366, F405, and Y349, second modification: T366, L368, Y407, K409, and E357, and 15) first modification: T366, F405, Y349, and S354, second modification: T366, L368, Y407, K409, and E357, wherein the amino acid positions areThe composition according to any one of embodiments 32 to 42, as determined according to the EU index of KABAT numbers.

[0234] 44. The first Fc subunit comprises a first modification, and the second Fc subunit comprises a second modification, wherein the first modification and the second modification are: 1) first modification: Y349C and T366W, and second modification: D356C, T366S, L368A, Y407V, and F405K; 2) first modification: Y349C, T366W, and F405K, and second modification: D356C, T366S, L368A, and Y407V; 3) first modification: Y349C, T366W, and K409E, and second modification: D356C, T366S, L368A, Y407V, and F405K; 4) first modification: Y349C, T366W and K409A, second modification: D356C, T366S, L368A, Y407V and F405K; 5) first modification: Y349C, T366W, F405K, K360E and Q347E, second modification: D356C, T366S, L368A, Y407V and Q347R; 6) first modification: Y349C, T366W, F405K and Q347R, second modification: D356C, T366S, L368A, Y407V, K360E and Q347E; 7) first modification: Y349C, T366W, K409A, K360E and Q347E; 2) modification: D356C, T366S, L368A, Y407V, F405K and Q347R; 8) first modification: Y349C, T366W, K409A and Q347R; second modification: D356C, T366S, L368A, Y407V, F405K, K360E and Q347E; 9) first modification: T366W, K409A and K392D; second modification: T366S, L368A, Y407V, D399S and F405K; 10) first modification: T366W and K409A; second modification: T366S, L368G, Y407A and F405K; 11) first modification: Modifications: T366W, K409A and Y349D, second modification: T366S, L368A, Y407V, F405K and E357A; 12) first modification: T366W, K409A, Y349D and S354D, second modification: T366S, L368A, Y407V, F405K and E357A; 13) first modification: T366W and F405K, second modification: T366S, L368A, Y407V and K409A; 14) first modification: T366W, F405K and D399S, second modification: T366S, L368A, Y407V, K409A and K392D;44. The composition according to any one of embodiments 32 to 43, comprising a group of amino acid substitutions selected from the group consisting of: 15) first modifications: T366W and F405K, second modifications: T366S, L368G, Y407A and K409A; 16) first modifications: T366W, F405K and Y349D, second modifications: T366S, L368A, Y407V, K409A and E357A; 17) first modifications: T366W, F405K, Y349D and S354D, second modifications: T366S, L368A, Y407V, K409A and E357A, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0235] 45. The composition of embodiment 44, wherein the first Fc subunit comprises a first modification and the second Fc subunit comprises a second modification, wherein the first modification comprises amino acid substitutions T366W and K409A, and the second modification comprises amino acid substitutions T366S, L368G, Y407A, and F405K, wherein the amino acid positions are determined according to the EU index of the KABAT numbering.

[0236] 46. ​​The composition of any one of embodiments 26-45, wherein the first member does not comprise any interleukin.

[0237] 47. The composition of any one of embodiments 1 to 46, wherein the first Fc subunit comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0238] 48. The composition of any one of embodiments 1 to 47, wherein the second Fc subunit comprises an amino acid sequence set forth in any one of SEQ ID NOs: 18.

[0239] 49. The composition of any one of embodiments 1-48, wherein the interleukin is a human interleukin.

[0240] 50. The composition of any one of embodiments 1 to 49, wherein the interleukin comprises an amino acid sequence set forth in any one of SEQ ID NOs: 56.

[0241] 51. The composition of any one of embodiments 1 to 50, wherein the immune complex comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 39, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0242] 52. The composition of any one of embodiments 26 to 50, wherein the first member comprises a first polypeptide chain and a second polypeptide chain, and the second member comprises a third polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39, and the third polypeptide chain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 42.

[0243] 53. The composition of any one of embodiments 26 to 50, wherein the first member comprises a first polypeptide chain, the second member comprises a second polypeptide chain, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 17, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0244] 54. The composition of any one of embodiments 1-53, wherein the fluorouracil comprises 5-Fu.

[0245] 55. The composition of any one of embodiments 1-54, wherein the chemotherapeutic agent further comprises folinic acid.

[0246] 56. The composition of any one of embodiments 1-55, wherein the chemotherapeutic agent comprises tetrahydrofolic acid and / or calcium leucovorin.

[0247] 57. The composition of any one of embodiments 1-56, wherein the chemotherapeutic agent comprises a FOLFOX regimen.

[0248] 58. The composition of any one of embodiments 1 to 57, wherein fluorouracil and oxaliplatin are not mixed with each other in the composition.

[0249] 59. An immunoconjugate for use in the treatment of cancer in combination with a chemotherapeutic agent, wherein the immunoconjugate is as defined in any one of embodiments 1-57, and the chemotherapeutic agent is as defined in any one of embodiments 1-58.

[0250] 60. The immunoconjugate of embodiment 59, wherein the cancer is selected from pancreatic cancer and colon cancer.

[0251] 61. The immunoconjugate of embodiment 59, wherein the pancreatic cancer is metastatic pancreatic cancer.

[0252] 62. The immunoconjugate of embodiment 59, wherein the colon cancer is metastatic colon cancer.

[0253] 63. The immunoconjugate according to any one of embodiments 59 to 62, wherein the cancer or its cells have elevated expression of EGFR.

[0254] 64. Use of an immunoconjugate in combination with a chemotherapeutic agent in the preparation of a medicament for treating cancer in a subject in need thereof, 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.

[0255] 65. The use according to embodiment 64, wherein the cancer is selected from pancreatic cancer and colon cancer.

[0256] 66. The use according to embodiment 65, wherein the pancreatic cancer is metastatic pancreatic cancer.

[0257] 67. The use according to embodiment 65, wherein the colon cancer is metastatic colon cancer.

[0258] 68. The use according to any one of embodiments 64 to 67, wherein the cancer or its cells have elevated expression of EGFR.

[0259] 69. A method of treating cancer in a subject in need thereof, comprising administering to the subject (a) an effective amount of an immunoconjugate as defined in any one of embodiments 1-58, and (b) an effective amount of a chemotherapeutic agent as defined in any one of embodiments 1-57.

[0260] 70. The method of embodiment 69, wherein the immunoconjugate is administered to the subject after administration of the chemotherapeutic agent.

[0261] 71. The method of embodiment 70, wherein the immunoconjugate is administered to the subject within 10 days after administration of the chemotherapeutic agent.

[0262] 72. The method of any one of embodiments 70-71, wherein the immunoconjugate is administered to the subject within 3 days after administration of the chemotherapeutic agent.

[0263] 73. The method of any one of embodiments 69-72, wherein the immunoconjugate is administered to the subject more than once.

[0264] 74. The method of any one of embodiments 69-73, wherein the cancer is selected from pancreatic cancer and colon cancer.

[0265] 75. The method of embodiment 74, wherein the pancreatic cancer is metastatic pancreatic cancer.

[0266] 76. The method of embodiment 74, wherein the colon cancer is metastatic colon cancer.

[0267] 77. The method of any one of embodiments 69 to 76, wherein the cancer or cells thereof have elevated expression of EGFR.

[0268] While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the invention described herein may be employed. [Example]

[0269] The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations can be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acid; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.

[0270] Example 1 Nucleic Acid Modification and Preparation 1.1 Fc modification Amino acid modifications (e.g., amino acid substitutions) were made to interface residues of the human IgG1 Fc domain, resulting in the following group of modifications (as shown in Table 1 below): In this disclosure, chain A is also referred to as Fc9 or the first Fc subunit, and chain B is also referred to as Fc6 or the second Fc subunit.

[0271] [Table 1]

[0272] TIFF0007822049000002.tif165170

[0273] The ScFv-Fc / Fc system was then used to investigate the formation of heterodimeric proteins containing the group of modifications listed in Table 1 above, as described in detail below.

[0274] First, the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region was obtained from the database Uniprot (P01857), and the wild-type human IgG1-Fc region amino acid sequence (SEQ ID NO: 30) was obtained. A polynucleotide fragment encoding wild-type human IgG1-Fc was obtained by RT-PCR from human PBMC total RNA (SEQ ID NO: 31, designated the Fc gene fragment). A polynucleotide fragment encoding the mouse kappa III signal peptide (SEQ ID NO: 32) was added to the 5' end of the Fc gene by overlap PCR and then subcloned into the vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for expressing human IgG1-Fc in mammalian cells.

[0275] A nucleic acid molecule encoding an ScFv-Fc fusion protein (SEQ ID NO: 33) was synthesized, where ScFv refers to an anti-HER2 single-chain antibody, and the amino acid sequence of the ScFv-Fc fusion protein is shown in SEQ ID NO: 34. The ScFv-Fc gene fragment was then subcloned into the vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for expressing the ScFv-Fc fusion protein in mammalian cells.

[0276] In some cases, a polypeptide encoding the variable region of a camelid single-domain antibody (VhH) was fused to the N-terminus of the Fc gene fragment to obtain a fusion gene fragment (shown in SEQ ID NO: 35) encoding the fusion protein VhH-Fc (shown in SEQ ID NO: 36), which was then subcloned into the vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for expressing the fusion protein VhH-Fc in mammalian cells.

[0277] Next, overlap PCR was used to introduce the amino acid modifications shown in Table 1 into ScFv-Fc (groups 1 to 17), VhH-Fc (groups 9 to 12, 14, 15, and 17), and Fc gene fragments (groups 1 to 8), respectively. Here, chain A refers to the Fc subunit of ScFv-Fc, and chain B refers to an independent Fc subunit or the Fc subunit of VhH-Fc. The amino acid-modified gene fragments were subcloned into the vector pcDNA4 (Invitrogen, Cat. V86220) to obtain recombinant expression vectors for expressing the modified ScFv-Fc fusion protein, modified Fc protein, and modified VhH-Fc fusion protein in mammalian cells.

[0278] Next, the constructed expression vectors were transfected into suspension-cultured HEK293 cells (ATCC CRL-1573™) with PEI. For each group, an expression vector expressing the A chain (ScFv-Fc fusion protein) and an expression vector expressing the B chain (Fc protein or VhH-Fc fusion protein) were co-transfected at a 1:1 ratio. After 5–6 days of culture, the supernatant of the transient expression product was collected, and the expression products containing the corresponding protein heterodimers were preliminarily purified using protein A affinity chromatography. Each preliminarily purified expression product contained various ratios of the homodimeric protein ScFv-Fc / ScFv-Fc, the homodimeric protein Fc / Fc (or the homodimeric protein VhH-Fc / VhH-Fc), and the heterodimeric protein ScFv-Fc / Fc (or the heterodimeric protein ScFv-Fc / VhH-Fc). Because the molecular weights of these proteins (i.e., homodimers and heterodimers) are different, their corresponding proportions can be determined according to the corresponding band intensities reflected on non-reducing SDS-PAGE gels. The intensities were quantified, and the results are summarized in Tables 2-5 below.

[0279] [Table 2]

[0280] [Table 3]

[0281] [Table 4]

[0282] [Table 5]

[0283] As can be seen from Tables 2-5 above, all modification groups promoted heterodimer formation very effectively. For illustrative purposes, group 10 modifications (Chain A modifications: T366W+K409A; Chain B modifications: T366S+L368G+Y407A+F405K) were used in the following examples to generate immune complexes or protein mixtures of the present disclosure.

[0284] 1.2 Preparation of anti-EGFR (cetuximab) 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 / ). Next, a nucleic acid molecule encoding the cetuximab light chain (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. Next, point mutations (T366W and K409A) were 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 herein as erb-Fc9) was synthesized. The corresponding polypeptide encoded therein was designated Erb-Fc9. The amino acid sequence of Erb-Fc9 is shown in SEQ ID NO: 39, and the polynucleotide sequence encoding it is shown in SEQ ID NO: 40.

[0285] 1.3 Preparation of (IL10)2-Fc6 First, the sequence information for human interleukin-10 (IL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Next, the amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) was obtained according to the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857) from the protein database Uniprot. Point mutations (T366S, L368G, Y407A, and F405K) were then introduced into the IgG1-Fc fragment, and the resulting polypeptide was designated Fc6. Next, the linker 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 linker-hinge-Fc6. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). A linker sequence "(GGGGS)3" (SEQ ID NO: 41) was then added between two copies of IL10 to obtain (IL10)2. The polynucleotide sequence encoding (IL10)2 was then added to the 5' end of the polynucleotide sequence encoding linker-hinge-Fc6, thereby obtaining and synthesizing a 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.

[0286] 1.4 Preparation of IL10-Fc First, the sequence information for human interleukin-10 (IL10) (P22301) was obtained from the National Center for Biotechnology Information (NCBI), and the full-length polynucleotide sequence encoding it was obtained. Next, the amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) was obtained according to the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857) from the protein database Uniprot. Next, the linker 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 linker-hinge-Fc. The corresponding DNA sequence encoding it was then designed using the online tool DNAworks (helixweb.nih.gov / dnaworks / ). The polynucleotide sequence encoding IL10 was then added to the 5' end of the polynucleotide sequence encoding linker-hinge-Fc, thereby obtaining and synthesizing the polynucleotide sequence encoding the fusion protein IL10-Fc. The amino acid sequence of IL10-Fc is as shown in SEQ ID NO:44, and the polynucleotide sequence encoding it is as shown in SEQ ID NO:45.

[0287] 1.5 Preparation of Fc9 The amino acid sequence of human IgG1-Fc (i.e., residues 104 to 330 of P01857) was obtained according to the amino acid sequence of the human immunoglobulin gamma 1 (IgG1) constant region (P01857) from the protein database Uniprot. Point mutations (T366W and K409A) were then 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, and the polynucleotide sequence encoding it is shown in SEQ ID NO:46.

[0288] Example 2 Construction of recombinant plasmids 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), respectively. The resulting plasmids were verified by sequencing, and the correct recombinant plasmids were designated pcDNA4-Erb-Fc9, pcDNA4-Fc9, pcDNA4-Erb-LC, pcDNA4-(IL10)2-Fc6, and pcDNA4-IL10-Fc, respectively.

[0289] Example 3 Expression and purification of immune complexes Two days before transfection, prepare 12 x 600 mL suspension culture HEK293 (ATCC, CRL-1573™) cells for transient transfection, and inoculate 0.8 x 10 cells. 6 After 2 days, three aliquots of the cell suspension were centrifuged and then resuspended in 600 mL of Freestyle 293 medium.

[0290] The recombinant expression vectors obtained from Example 2 were divided into the following groups:

[0291] Group 1: pcDNA4-Erb-Fc9 (200μg) + pcDNA4-Erb-LC (200μg) + pcDNA4-(IL10)2-Fc6 (200μg) Group 2: pcDNA4-Fc9 (200μg) + pcDNA4-(IL10)2-Fc6 (200μg) Group 3: pcDNA4-IL10-Fc (200μg) All proteins were prepared in transiently transfected 293F cells. Briefly, Freestyle 293F cells (Invitrogen) were grown in 293F medium (Invitrogen), transfected with non-linearized plasmid DNA and 293Fectin reagent (Invitrogen), and grown in shake-flask batches of 80–100 mL per flask at 37°C and 5% CO for 6 days. All proteins were purified by one-step Protein A chromatography. The quality of each protein was measured by SDS-PAGE and SEC-HPLC. Similarly, the expression and purification results of other immunoconjugates in this application were verified and confirmed by SDS-PAGE.

[0292] The immune complexes thus obtained are designated Erb-(IL10)2, Fc9-(IL10)2, and (IL10-Fc)2 (Groups 1 to 3, respectively).

[0293] Figures 1A-1E show that immune complexes of Erb-(IL10)2, (IL10-Fc)2, and Fc9-(IL10)2 were successfully expressed and purified.

[0294] In Figure 1A, lane 1 was loaded with Erb-(IL10)2 (reduced), lane 2 was loaded with marker, and lane 3 was loaded with Erb-(IL10)2 (non-reduced).

[0295] In Figure 1B, lane 1 was loaded with (IL10-Fc)2 (original 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 marker, 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-reduced).

[0296] In Figure 1C, 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 marker, 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-reduced).

[0297] FIG. 1D shows the results of SEC-HPLC, which show that the proportion of undesired oligomers in the (IL10-Fc)2 expression product was approximately 27%.

[0298] FIG. 1E shows the results of SEC-HPLC, which show that the proportion of undesired oligomers in the expression product of Fc9-(IL10)2 was approximately 3.3%.

[0299] These results demonstrate the successful production of the immunoconjugates of the present disclosure. Interestingly, the expression product of Fc9-(IL10)2 contains far fewer undesired oligomers than the expression product of (IL10-Fc)2.

[0300] Example 4 Effect of chemotherapy in combination with immunoconjugates of the present disclosure 4.1 Efficacy of Immunoconjugates According to the Present Disclosure in Combination with Oxaliplatin Six- to eight-week-old female C57BL / 6 mice were obtained from the Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China) and maintained under specific pathogen-free conditions. All animals were used in accordance with the local ethical committee. This study was approved by the recommendations of the Guide for the Care and Use of Laboratory Animals (Ministry of Health, People's Republic of China, 1998). Human EGFR and K b The B16-EGFR-SIY melanoma cell line, which expresses the binding peptide antigen SIYRYYGL (SIY, SEQ ID NO: 47), was generated in-house and grown in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin (Gibco Invitrogen).

[0301] B16‐EGFR‐SIY melanoma cells (5 × 10 5 ) was inoculated subcutaneously (sc) into the flank of mice and allowed to grow for approximately 10 days. Tumor volume was determined by recording two perpendicular diameters (length and width), and V = ab 2 The tumor diameter was calculated as follows: / 2, where a and b are the longest and shortest diameters, respectively. Mice were randomly divided into groups according to tumor size.

[0302] Tumor-bearing mice were divided into several groups, five mice per group: the isotype control group (treated with 0.5 mg / kg human IgG1 (10 μg / mouse), the Erb-(IL10)2 group (treated with 0.5 mg / kg Erb-(IL10)2 (10 μg / mouse), the oxaliplatin group (treated with 15 mg / kg oxaliplatin (300 μg / mouse)), and the 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 intraperitoneally injected with Erb-(IL10)2 on days 7, 10, and 14, respectively, followed by a tail vein injection of oxaliplatin on day 7.

[0303] The results are shown in Figure 2A. Due to the high toxicity of oxaliplatin, all mice in the oxaliplatin group died after 14 days (see Figure 2A). It can be seen that the combination of Erb-(IL10)2 and oxaliplatin exhibited a synergistic effect in suppressing tumor growth (see Figure 2A).

[0304] FIG. 2D shows the effect of Erb-(IL10)2, oxaliplatin, and the combination of Erb-(IL10)2 and oxaliplatin on survival.

[0305] Similarly, tumor-bearing mice were divided into several groups with five mice per 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 Fc9-(IL10)2 (10 μg / mouse), an oxaliplatin group in which mice were treated with 15 mg / kg oxaliplatin (300 μg / mouse), and an Fc9-(IL10)2 + oxaliplatin group in which mice were treated with 0.5 mg / kg Fc9-(IL10)2 and 15 mg / kg oxaliplatin (300 μg / mouse). C57BL / 6 mice were subcutaneously inoculated with B16-EGFR-SIY cells on day 0, and intraperitoneally injected with Fc9-(IL10)2 on days 7, 10, and 14, respectively, and injected with oxaliplatin via the tail vein on day 7.

[0306] The results are shown in Figure 2B. Due to the high toxicity of oxaliplatin, all mice in the oxaliplatin group and the Fc9-(IL10)2 + oxaliplatin group died after 14 days (see Figure 2C). Therefore, the synergistic effects cannot be compared between groups.

[0307] FIG. 2C shows the effect of Fc9-(IL10)2, oxaliplatin, and the combination of Fc9-(IL10)2 and oxaliplatin on survival.

[0308] Example 5 Effect of FOLFOX in Combination with Immunoconjugates of the Present Disclosure 5.1 Efficacy of Immunoconjugates of the Present Disclosure in Combination with FOLFOX in Pancreatic Tumor Models Pancreatic tumor cells were subcutaneously inoculated into C57BL / 6 mice on day 0 to establish a pancreatic tumor model. Tumor-bearing mice were divided into several groups: the isotype control group (mice treated with human IgG1), the Erb-(IL10)2 group (mice treated with Erb-(IL10)2), the FOLFOX group (mice treated with FOLFOX), and the Erb-(IL10)2 + FOLFOX group (mice treated with Erb-(IL10)2 and FOLFOX). Erb-(IL10)2 was injected intraperitoneally, and FOLFOX was injected via the tail vein.

[0309] It can be seen that the combination of Erb-(IL10)2 and FOLFOX exhibited a synergistic effect in tumor growth inhibition, indicating that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in the treatment of metastatic pancreatic cancer.

[0310] 5.2 Efficacy of Immunoconjugates According to the Present Disclosure in Combination with FOLFOX in a Colon Tumor Model Colon tumor models were established by subcutaneously inoculating MC38-EGFR-5 cells into 70 C57BL / 6 mice on day 0. Tumor-bearing mice were divided into several groups: the isotype control group (mice treated with human IgG1), the Erb-(IL10)2 group (mice treated with Erb-(IL10)2), the FOLFOX group (mice treated with FOLFOX), and the Erb-(IL10)2 + FOLFOX group (mice treated with Erb-(IL10)2 and FOLFOX). MC38-EGFR-5 cells were subcutaneously inoculated into C57BL / 6 mice on day 0, and oxaliplatin was injected intravenously on day 7, followed by Erb-(IL10)2 injection 8 hours later. Leucovorin calcium and 5-FU were injected intraperitoneally on day 8. The dose, route of administration, and administration schedule are shown in Table 6.

[0311] [Table 6]

[0312] Figures 4A and 4B show that the combination of Erb-(IL10)2 and FOLFOX can inhibit tumor growth, and Figure 4C shows that the combination of Erb-(IL10)2 and FOLFOX can prolong survival.

[0313] It can be seen that the combination of Erb-(IL10)2 and FOLFOX exhibited a synergistic effect in tumor growth inhibition, indicating that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in the treatment of metastatic colorectal cancer.

[0314] Example 6 Effect of Fluorouracil in Combination with Immunoconjugates of the Present Disclosure 6.1 Efficacy of Immunoconjugates of the Present Disclosure in Combination with Fluorouracil in Pancreatic Tumor Models Pancreatic tumor cells were subcutaneously inoculated into C57BL / 6 mice on day 0 to establish a pancreatic tumor model. Tumor-bearing mice were divided into several groups: the isotype control group (mice treated with human IgG1), the Erb-(IL10)2 group (mice treated with Erb-(IL10)2), the 5-FU group (mice treated with 5-FU), and the Erb-(IL10)2 + 5-FU group (mice treated with Erb-(IL10)2 and 5-FU). Erb-(IL10)2 was injected intraperitoneally, and 5-FU was injected via the tail vein.

[0315] It can be seen that the combination of Erb-(IL10)2 and 5-FU exhibited a synergistic effect in tumor growth inhibition, indicating that the combination of Erb-(IL10)2 and fluorouracil has a synergistic effect in the treatment of metastatic pancreatic cancer.

[0316] 6.2 Efficacy of immunoconjugates according to the present disclosure in combination with fluorouracil in a colon tumor model Colon tumor models were established by subcutaneous inoculation of MC38-EGFR colon tumor cells into C57BL / 6 mice on day 0. Tumor-bearing mice were divided into several groups: the isotype control group (mice treated with human IgG1), the Erb-(IL10)2 group (mice treated with Erb-(IL10)2), the 5-FU group (mice treated with 5-FU), and the Erb-(IL10)2 + 5-FU group (mice treated with Erb-(IL10)2 and 5-FU). Erb-(IL10)2 was injected intraperitoneally, and 5-FU was injected via the tail vein.

[0317] It can be seen that the combination of Erb-(IL10)2 and 5-FU exhibited a synergistic effect in inhibiting tumor growth, indicating that the combination of Erb-(IL10)2 and fluorouracil has a synergistic effect in the treatment of metastatic colorectal cancer.

[0318] Example 7 Effect of Oxaliplatin in Combination with Immunoconjugates of the Present Disclosure 7.1 Efficacy of Immunoconjugates According to the Present Disclosure in Combination with Oxaliplatin in Pancreatic Tumor Models Pancreatic tumor cells were subcutaneously inoculated into C57BL / 6 mice on day 0 to establish a pancreatic tumor model. Tumor-bearing mice were divided into several groups: the isotype control group (mice treated with human IgG1), the Erb-(IL10)2 group (mice treated with Erb-(IL10)2), the oxaliplatin group (mice treated with oxaliplatin), and the Erb-(IL10)2 + oxaliplatin group (mice treated with Erb-(IL10)2 and oxaliplatin). Erb-(IL10)2 was injected intraperitoneally, and oxaliplatin was injected via the tail vein.

[0319] It can be seen that the combination of Erb-(IL10)2 and oxaliplatin exhibited a synergistic effect in tumor growth inhibition, indicating that the combination of Erb-(IL10)2 and oxaliplatin has a synergistic effect in the treatment of metastatic pancreatic cancer.

[0320] 7.2 Efficacy of Immunoconjugates According to the Present Disclosure in Combination with Oxaliplatin in a Colon Tumor Model Colon tumor models were established by subcutaneous inoculation of MC38-EGFR colon tumor cells into C57BL / 6 mice on day 0. Tumor-bearing mice were divided into several groups: the isotype control group (mice treated with human IgG1), the Erb-(IL10)2 group (mice treated with Erb-(IL10)2), the oxaliplatin group (mice treated with oxaliplatin), and the Erb-(IL10)2 + oxaliplatin group (mice treated with Erb-(IL10)2 and oxaliplatin). Erb-(IL10)2 was injected intraperitoneally, and oxaliplatin was injected via the tail vein.

[0321] It can be seen that the combination of Erb-(IL10)2 and oxaliplatin exhibited a synergistic effect in tumor growth inhibition, indicating that the combination of Erb-(IL10)2 and oxaliplatin has a synergistic effect in the treatment of metastatic colorectal cancer.

[0322] Example 8 Efficacy of immunoconjugates according to the present disclosure in combination with FOLFOX in a melanoma model On day 0, melanoma B16-EGFR-5 cells (5 × 10 5 A melanoma model was established by subcutaneously inoculating 60 C57BL / 6 mice with B16-EGFR-5 cells (1000 cells / mouse). Tumor-bearing mice were divided into several groups: the isotype control group (in which mice were treated with human IgG1), the Erb-(IL10)2 group (in which mice were treated with Erb-(IL10)2), the FOLFOX group (in which mice were treated with FOLFOX), and the Erb-(IL10)2 + FOLFOX group (in which mice were treated with Erb-(IL10)2 and FOLFOX). The dose, route of administration, and administration schedule are shown in Table 7. B16-EGFR-5 cells were subcutaneously inoculated into C57BL / 6 mice on day 0, and oxaliplatin was injected intravenously on day 7, followed by Erb-(IL10)2 injection 8 hours later. Leucovorin calcium and 5-FU were injected intraperitoneally on day 8.

[0323] [Table 7]

[0324] Figures 5A and 5B show that the combination of Erb-(IL10)2 and FOLFOX can inhibit tumor growth.

[0325] It can be seen that the combination of Erb-(IL10)2 and FOLFOX showed a synergistic effect in inhibiting tumor growth, indicating that the combination of Erb-(IL10)2 and FOLFOX has a synergistic effect in the treatment of melanoma.

[0326] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein can be used in practicing the present invention. Accordingly, it is intended that the present invention encompass any such alternatives, modifications, variations, or equivalents. The following embodiments define the scope of the present invention, and methods and structures within the scope of these embodiments and their equivalents are intended to be covered thereby.

Claims

1. A composition comprising an immunoconjugate for use in treating pancreatic cancer, melanoma, or colorectal cancer in a subject undergoing chemotherapy, comprising: the immune complex comprises 1) an IL10 dimer and 2) an IgG1 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 carboxy terminus of the IL10 dimer is fused to the amino terminal amino acid of the Fc domain via a peptide linker, and the IL10 dimer is formed from two IL10s fused in-frame to each other via a peptide linker; the first Fc subunit is different from the second Fc subunit, and the Fc domain comprises a modification that promotes heterodimerization between the first Fc subunit and the second Fc subunit; the first Fc subunit comprises a first modification: T366, and K409; and the second Fc subunit comprises a second modification: T366, L368, Y407, and F405, wherein the amino acid positions are determined according to the EU index of KABAT numbering; the immunoconjugate further comprises a targeting moiety fused to the Fc domain, the targeting moiety exhibiting binding specificity for a tumor antigen; the targeting moiety is fused to the amino terminal amino acid of the Fc domain; the targeting moiety comprises a heavy chain variable region of cetuximab, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55; the targeting moiety comprises a light chain variable region of cetuximab, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:51; The chemotherapy comprises administration of calcium leucovorin, fluorouracil (5-FU), and oxaliplatin.

2. 2. The composition of claim 1, wherein the immune complex is an asymmetric immune complex comprising a first member and a second member different from the first member, wherein the first member comprises the first Fc subunit, the second member comprises the 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.

3. the first member further comprises the targeting moiety fused to the first Fc subunit; The composition of claim 2, wherein the targeting moiety is fused to the amino-terminal amino acid of the first Fc subunit.

4. The composition of claim 2 , wherein the first Fc subunit comprises the amino acid sequence set forth in SEQ ID NO:

17.

5. The composition of claim 2 , wherein the second Fc subunit comprises the amino acid sequence set forth in SEQ ID NO:

18.

6. 3. The composition of claim 2, wherein the immune complex comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 39, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

42.

7. 3. The composition of claim 2, 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 the amino acid sequence set forth in SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 39, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

42.

8. 3. The composition of claim 2, wherein the first member comprises a first polypeptide chain, the second member comprises a second polypeptide chain, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 17, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

42.

9. The composition according to any one of claims 1 to 8, wherein the pancreatic cancer is metastatic pancreatic cancer and / or the colon cancer is metastatic colon cancer.

10. The composition of claim 9 , wherein the cancer or cells thereof have elevated expression of EGFR.

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