Engineered iga antibodies and methods of use

Abstract

Provided herein is an engineered IgA antibody comprising: (a) an EGFR binding domain and (b) an IgA heavy chain constant region. Further provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the engineered antibody.

Description

[0001] Cross-reference applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 516,428, filed July 28, 2023, the entire contents of which are incorporated herein by reference.

[0003] By referencing and incorporating into the sequence list

[0004] This application includes a sequence list, which has been submitted through the Patent Center. The sequence list, named 199828-714601_PCT_SL.xml, was created on July 26, 2024, and is 189,604 bytes in size, and is incorporated herein by reference in its entirety.

[0005] Invention Field

[0006] This disclosure relates in general to antibodies that bind to the epidermal growth factor receptor (EGFR). Background of the Invention

[0008] Monoclonal antibodies targeting specific antigens associated with diseases or conditions are increasingly becoming a highly attractive therapeutic approach for improving diseases or conditions in human subjects. In recent years, an increasing number of monoclonal antibodies (primarily IgG-based antibodies) targeting various tumor antigens have been approved for cancer treatment. However, their clinical efficacy and side effects, especially those associated with IgG-based antibody monotherapy, remain a concern. Therefore, developing novel antibody therapies with higher clinical efficacy and / or lower incidence / severity of side effects is of great importance. Invention Overview

[0010] This article provides engineered antibodies comprising: (a) an epidermal growth factor receptor (EGFR) binding domain comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain; and (b) an immunoglobulin A (IgA) heavy chain constant region comprising at least one mutation relative to a wild-type IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 1, wherein the mutation results in one or more of the following: reduced glycosylation, reduced aggregation, increased thermal stability, increased mechanical stability, or increased cycling half-life, all relative to a corresponding antibody comprising a wild-type IgA heavy chain constant region. In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H): (1) CDR-H1, comprising any amino acid sequence of at least one heavy chain complementarity-determining region (CDR) of SEQ ID NO: 34-54, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; (2) CDR-H2, comprising any amino acid sequence of SEQ ID NO: 57-78, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; and (3) CDR-H3, comprising any amino acid sequence of SEQ ID NO: 81-102, or a variant thereof containing 1 to 3 substitutions, deletions or insertions. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L): (1) CDR-L1, comprising any amino acid sequence of SEQ ID NO: 105-126, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; (2) CDR-L2, comprising any amino acid sequence of SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; and (3) CDR-L3, comprising any amino acid sequence of SEQ ID NO: 146-166, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected from any combination provided in Table 9.In some embodiments, the engineered antibody comprises: CDR-H1, which comprises an amino acid sequence having at least 80% identity with any amino acid sequence in SEQ ID NO: 34-54; CDR-H2, which comprises an amino acid sequence having at least 80% identity with any amino acid sequence in SEQ ID NO: 57-78; CDR-H3, which comprises an amino acid sequence having at least 80% identity with any amino acid sequence in SEQ ID NO: 81-102; CDR-L1, which comprises an amino acid sequence having at least 80% identity with any amino acid sequence in SEQ ID NO: 105-126; CDR-L2, which comprises an amino acid sequence identical to any amino acid sequence in SEQ ID NO: 129-143; and CDR-L3, which comprises an amino acid sequence having at least 80% identity with any amino acid sequence in SEQ ID NO: 146-166. In some embodiments, the engineered antibody comprises: CDR-H1, which contains an amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 34-54; CDR-H2, which contains an amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 57-78; CDR-H3, which contains an amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 81-102; CDR-L1, which contains an amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 105-126; CDR-L2, which contains an amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 129-143; and CDR-L3, which contains an amino acid sequence identical to any of the amino acid sequences in SEQ ID NO: 146-166. In some embodiments, the VH domain contains an amino acid sequence having at least 80% identity with any of the amino acid sequences in Table 5. In some embodiments, the VL domain contains an amino acid sequence having at least 80% identity with any of the amino acid sequences in Table 7. In some embodiments, the VH and VL domains are selected from any combination provided in Table 8. In some embodiments, the engineered antibody comprises an IgA light chain constant region containing an amino acid sequence having at least 80% identity with SEQ ID NO: 23. In some embodiments, the IgA heavy chain constant region comprises an IgA CH1 region, an IgA CH2 region, and an IgA CH3 region. In some embodiments, the at least one mutation is present in the IgA CH1 region and is an N45.2 substitution, a P124 substitution, or a combination of both, numbered according to the IMGT protocol, with each mutation corresponding to the antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1.In some embodiments, the at least one mutation is: (a) an N45.2 substitution selected from the group consisting of N45.2G and N45.2A; (b) a P124R substitution; or (c) any combination of the two, numbered according to the IMGT protocol, each mutation corresponding to the antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the at least one mutation is present in the IgA CH2 region and is: (a) an N20 substitution; (b) an L21 substitution; (c) a T22 substitution; (d) a C92 substitution; (e) an N120 substitution; (f) an I121 substitution; or (g) a T122 substitution, numbered according to the IMGT protocol, each mutation corresponding to the antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the at least one mutation is: (a) an N20 substitution selected from the group consisting of N20G, N20Q and N20T; (b) an L21I substitution; (c) a T22S substitution; (d) a C92S substitution; (e) an N120T substitution; (f) an I121L substitution; (g) a T122S substitution; or (h) any combination thereof, numbered according to the IMGT scheme, each mutation being relative to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the at least one mutation is present in the IgA CH3 region and is: (a) H5 substitution; (b) L7 substitution; (c) P10 substitution; (d) T22 substitution; (e) L79 substitution; (f) W81 substitution; (g) A85.1 substitution; (h) T86 substitution; (i) I88 substitution; (j) N135 substitution; (k) C147 deletion; (l) Y148 deletion; (m) P131-Y148 deletion; or (n) combinations thereof, numbered according to the IMGT scheme, the mutation being relative to the corresponding residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO: 1.In some embodiments, the at least one mutation is: (a) an H5 substitution selected from the following: H5C, H5Y, H5F, H5M, and H5W; (b) an L7 substitution selected from the following: L7F, L7Y, L7M, L7W, L7H, and L7I; (c) a P10C substitution; (d) a T22 substitution selected from the following: T22V, T22I, T22L, and T22A; (e) an L79 substitution selected from the following: L79V, L79T, L79A, and L79I; (f) a W81 substitution selected from the following: W81T, W81L, W81A, W81V, and W81I; (g) an A85.1 substitution selected from the following: A85.1F, A85.1Y, A85.1M, A85.1W, and A85.1H; (h) The mutation is selected from the following T86 substitutions: T86Y, T86F, T86M, T86W, and T86H; (i) the following I88 substitutions: I88L, I88A, I88V, and I88T; or (j) any combination thereof, numbered according to the IMGT scheme, each mutation corresponding to a corresponding residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO: 1. In some embodiments, the at least one mutation is: (a) N135Q substitution; (b) C147 deletion; (c) Y148 deletion; or (d) any combination thereof, numbered according to the IMGT scheme, the mutation corresponding to a corresponding residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO: 1. In some embodiments, the at least one mutation is a deletion of P131-Y148, numbered according to the IMGT scheme, the mutation corresponding to a corresponding residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO: 1. In some embodiments, the IgA heavy chain constant region comprises an amino acid sequence having at least 80% identity with the IgA heavy chain constant region of SEQ ID NO: 3. In some embodiments, the engineered antibody is a monomer. In some embodiments, the EGFR-binding domain binds to an EGFR peptide variant, wherein the EGFR variant comprises EGFRvIII, exon 19 deletion, L858R substitution in exon 21, C797S substitution, or T790M substitution. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 173, and the VL region comprises the amino acid sequence of SEQ ID NO: 211. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 172, and the VL region comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 182, and the VL region comprises the amino acid sequence of SEQ ID NO: 207.In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 184, and the VL region contains the amino acid sequence of SEQ ID NO: 209. In some embodiments, the engineered antibody is capable of inducing antibody-dependent cytotoxicity (ADCC) through immune effector cells. In some embodiments, the immune effector cells are neutrophils, T cells, eosinophils, or macrophages. In some embodiments, the engineered antibody is a chimeric antibody, a single-chain antibody, a humanized antibody, a human antibody, a monoclonal antibody, a deimmunizing antibody, a bispecific antibody, a multispecific antibody, a multivalent antibody, or a combination thereof. In some embodiments, the engineered antibody is a bispecific antibody. In some embodiments, the engineered antibody further comprises a binding domain that binds to a polypeptide antigen selected from the following: MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, and 4-1BB.

[0011] This document also provides engineered antibodies comprising: (a) an epidermal growth factor receptor (EGFR) binding domain that binds to the III domain of an EGFR peptide or a variant thereof; and (b) an immunoglobulin A (IgA) constant domain comprising an IgA heavy chain constant region having at least one mutation relative to a wild-type IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 1, wherein the mutation results in one or more of the following: decreased glycosylation, decreased aggregation, increased thermal stability, enhanced mechanical stability, or increased cycling half-life, all relative to a corresponding antibody comprising a wild-type IgA heavy chain constant region. In some embodiments, the IgA constant domain comprises: (a) an IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 3; and (b) an IgA light chain constant region having the amino acid sequence SEQ ID NO: 2. In some embodiments, the IgA constant domain comprises an IgA heavy chain constant region containing IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region contains the following mutations: (a) N45.2G substitution in the CH1 domain; (b) P124R substitution in the CH1 domain; (c) C92S substitution in the CH2 domain; (d) N120T substitution in the CH2 domain; (e) I121L substitution in the CH2 domain; and (f) T122S substitution in the CH2 domain, numbered according to the IMGT protocol, each mutation corresponding to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the EGFR binding domain binds to an epitope of an EGFR peptide or a variant thereof, the epitope containing any of the following EGFR amino acid residues: P349, F352, D355, P362, D355, Q384, P387, Q408, H409, F412, I438, K443, K465, I467, or S468.

[0012] This document also provides engineered antibodies comprising: (a) an epidermal growth factor receptor (EGFR) binding domain that binds to the II domain of an EGFR peptide or a variant thereof; and (b) an immunoglobulin A (IgA) constant domain comprising an IgA heavy chain constant region having at least one mutation relative to a wild-type IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 1, wherein the mutation results in one or more of the following: decreased glycosylation, decreased aggregation, increased thermal stability, increased mechanical stability, or increased cycling half-life, all relative to the wild-type IgA heavy chain constant region. In some embodiments, the IgA constant domain comprises: (a) an IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 3; and (b) an IgA light chain constant region having the amino acid sequence SEQ ID NO: 2. In some embodiments, the IgA constant domain comprises an IgA heavy chain constant region containing IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region contains the following mutations: (a) N45.2G substitution in the CH1 domain; (b) P124R substitution in the CH1 domain; (c) C92S substitution in the CH2 domain; (d) N120T substitution in the CH2 domain; (e) I121L substitution in the CH2 domain; and (f) T122S substitution in the CH2 domain, numbered according to the IMGT protocol, each mutation corresponding to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1.

[0013] This document also provides engineered antibodies comprising: (a) an epidermal growth factor receptor (EGFR) binding domain comprising a heavy chain variable region (VH) domain and a light chain variable region (VH) domain; and (b) an immunoglobulin A (IgA) constant domain comprising: (i) a heavy chain constant region having the amino acid sequence SEQ ID NO: 5, and (ii) a light chain constant region having the amino acid sequence SEQ ID NO: 23. In some embodiments, the VH region comprises the amino acid sequence SEQ ID NO: 173, and the VL region comprises the amino acid sequence SEQ ID NO: 211. In some embodiments, the VH region comprises the amino acid sequence SEQ ID NO: 172, and the VL region comprises the amino acid sequence SEQ ID NO: 198. In some embodiments, the VH region comprises the amino acid sequence SEQ ID NO: 182, and the VL region comprises the amino acid sequence SEQ ID NO: 207. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 184, and the VL region contains the amino acid sequence of SEQ ID NO: 209. In some embodiments, the VH domain contains three heavy chain complementarity-determining regions (CDR-H): (1) CDR-H1, containing any amino acid sequence of at least one heavy chain complementarity-determining region (CDR) of SEQ ID NO: 34-54, or containing one to three substitutions, deletions, or insertions thereof; (2) CDR-H2, containing any amino acid sequence of SEQ ID NO: 57-78, or containing one to three substitutions, deletions, or insertions thereof; (3) CDR-H3, containing any amino acid sequence of SEQ ID NO: 81-102, or containing one to three substitutions, deletions, or insertions thereof. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L): (1) CDR-L1, comprising any amino acid sequence of SEQ ID NO: 105-126, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; (2) CDR-L2, comprising any amino acid sequence of SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; and (3) CDR-L3, comprising any amino acid sequence of SEQ ID NO: 146-166, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected from any combination provided in Table 9.

[0014] This article also provides engineered antibodies comprising: (a) an epidermal growth factor receptor (EGFR) binding domain comprising a heavy chain variable region (VH) domain and a light chain variable region (VH) domain; and (b) an immunoglobulin A (IgA) heavy chain constant region comprising IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region comprises the following mutations: (i) N45.2G substitution in the CH1 domain, (ii) P124R substitution in the CH1 domain, (iii) C92S substitution in the CH2 domain, (iv) N120T substitution in the CH2 domain, (v) I121L substitution in the CH2 domain, and (vi) T122S substitution in the CH2 domain, numbered according to the IMGT protocol, each mutation corresponding to the corresponding antibody comprising the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H): (1) CDR-H1, comprising any amino acid sequence of at least one heavy chain complementarity-determining region (CDR) of SEQ ID NO: 34-54, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; (2) CDR-H2, comprising any amino acid sequence of SEQ ID NO: 57-78, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; and (3) CDR-H3, comprising any amino acid sequence of SEQ ID NO: 81-102, or a variant thereof containing 1 to 3 substitutions, deletions or insertions. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L): (1) CDR-L1, comprising any amino acid sequence of SEQ ID NO: 105-126, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; (2) CDR-L2, comprising any amino acid sequence of SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; and (3) CDR-L3, comprising any amino acid sequence of SEQ ID NO: 146-166, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected from any combination provided in Table 9. In some implementations, the IgA heavy chain constant region also includes the following mutations in the CH3 domain: (a) N135Q substitution; (b) C147 deletion; and (c) Y148 deletion, numbered according to the IMGT protocol, with each mutation corresponding to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO:1.In some embodiments, the IgA heavy chain constant region further comprises a P131-Y148 deletion in the CH3 domain, numbered according to the IMGT protocol, corresponding to the antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the IgA heavy chain constant region further comprises the following mutations in the CH2 domain: (a) an N20 substitution selected from the group consisting of N20G, N20Q, and N20T; (b) an L21I substitution; and (c) a T22S substitution, numbered according to the IMGT protocol, each mutation corresponding to the antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 173, and the VL region contains the amino acid sequence of SEQ ID NO: 211. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 172, and the VL region contains the amino acid sequence of SEQ ID NO: 198. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 182, and the VL region contains the amino acid sequence of SEQ ID NO: 207. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 184, and the VL region contains the amino acid sequence of SEQ ID NO: 209.

[0015] This article also provides engineered epidermal growth factor receptor (EGFR) binding antibodies or functional EGFR binding fragments thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173 and the VL region comprises the amino acid sequence of SEQ ID NO: 211; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0016] This article also provides engineered epidermal growth factor receptor (EGFR) binding antibodies or functional EGFR binding fragments thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 172 and the VL region comprises the amino acid sequence of SEQ ID NO: 198; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0017] This article also provides engineered epidermal growth factor receptor (EGFR) binding antibodies or functional EGFR binding fragments thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 182 and the VL region comprises the amino acid sequence of SEQ ID NO: 207; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0018] This article also provides engineered epidermal growth factor receptor (EGFR) binding antibodies or functional EGFR binding fragments thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 184 and the VL region comprises the amino acid sequence of SEQ ID NO: 209; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0019] This article also provides pharmaceutical compositions comprising the engineered antibody described herein and a pharmaceutically acceptable carrier.

[0020] This article also provides methods for treating cancer in subjects in need. In some embodiments, the method comprises administering to the subject an effective amount of an engineered antibody comprising: (a) an epidermal growth factor receptor (EGFR) binding domain comprising a heavy chain variable region (VH) domain and a light chain variable region (VH) domain; and (b) an immunoglobulin A (IgA) heavy chain constant region comprising at least one mutation relative to a wild-type IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 1, wherein the mutation results in one or more of the following: reduced glycosylation, reduced aggregation, increased thermal stability, increased mechanical stability, or increased cyclic half-life, all relative to the wild-type IgA heavy chain constant region. In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H): (1) CDR-H1, comprising any amino acid sequence of at least one heavy chain complementarity-determining region (CDR) of SEQ ID NO: 34-54, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; (2) CDR-H2, comprising any amino acid sequence of SEQ ID NO: 57-78, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; and (3) CDR-H3, comprising any amino acid sequence of SEQ ID NO: 81-102, or a variant thereof containing 1 to 3 substitutions, deletions or insertions. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L): (1) CDR-L1, comprising any amino acid sequence of SEQ ID NO: 105-126, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; (2) CDR-L2, comprising any amino acid sequence of SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; and (3) CDR-L3, comprising any amino acid sequence of SEQ ID NO: 146-166, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected from any combination provided in Table 9. In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H): (1) CDR-H1, comprising any amino acid sequence of at least one heavy chain complementarity-determining region (CDR) of SEQ ID NO: 34-54, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; (2) CDR-H2, comprising any amino acid sequence of SEQ ID NO: 57-78, or a variant thereof containing 1 to 3 substitutions, deletions or insertions; and (3) CDR-H3, comprising any amino acid sequence of SEQ ID NO: 81-102, or a variant thereof containing 1 to 3 substitutions, deletions or insertions.In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L): (1) CDR-L1, comprising any amino acid sequence of SEQ ID NO: 105-126, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; (2) CDR-L2, comprising any amino acid sequence of SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; and (3) CDR-L3, comprising any amino acid sequence of SEQ ID NO: 146-166, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected from any combination provided in Table 9. In some embodiments, the mutation results in one or more of the following: reduced glycosylation, reduced aggregation, increased thermal stability, enhanced mechanical stability, or increased cyclic half-life, all relative to the wild-type IgA heavy chain constant region. In some embodiments, the IgA heavy chain constant region comprises IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region contains the following mutations: (a) N45.2G substitution in the CH1 domain; (b) P124R substitution in the CH1 domain; (c) C92S substitution in the CH2 domain; (d) N120T substitution in the CH2 domain; (e) I121L substitution in the CH2 domain; and (f) T122S substitution in the CH2 domain, numbered according to the IMGT protocol, each mutation corresponding to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the IgA heavy chain constant region also contains the following mutations in the CH3 domain: (a) N135Q substitution; (b) C147 deletion; and (c) Y148 deletion, numbered according to the IMGT protocol, each mutation corresponding to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the IgA heavy chain constant region further comprises a P131-Y148 deletion in the CH3 domain, numbered according to the IMGT protocol, corresponding to the antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the IgA heavy chain constant region further comprises the following mutations in the CH2 domain: (a) an N20 substitution selected from the group consisting of N20G, N20Q, and N20T; (b) an L21I substitution; and (c) a T22S substitution, numbered according to the IMGT protocol, each substitution corresponding to the antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of: lung cancer, head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, renal cancer, mesothelioma, and glioblastoma.In some embodiments, the cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the engineered antibody inhibits cancer-associated tumor growth. In some embodiments, the engineered antibody is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. In some embodiments, the engineered antibody is administered to the subject in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an anticancer agent, a chemotherapeutic agent, radiotherapy, a cytotoxic agent, a corticosteroid, an immunotherapy agent, a dietary supplement, or an antioxidant. In some embodiments, the second therapeutic agent is administered before, simultaneously with, or after the administration of the engineered antibody. In some embodiments, the subject is a rodent, a non-human primate, or a human. In some embodiments, the subjects are rodents, and the effective dose is administered at a dose of 1 mg / kg to 25 mg / kg twice weekly, subcutaneously, intravenously, or intraperitoneally for 35-40 days. In some embodiments, the effective dose is administered intravenously at a dose of 25 mg / kg every seven days for 5 weeks, but a third dose of 12.5 mg / kg. In some embodiments, the effective dose is administered intravenously twice weekly at a dose of 1 mg / kg to 25 mg / kg.

[0021] This article also provides a method for treating cancer in patients in need, comprising administering to a subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR binding fragment thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173 and the VL region comprises the amino acid sequence of SEQ ID NO: 211; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0022] This article also provides a method for treating cancer in a subject in need, comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR binding fragment thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 172 and the VL region comprises the amino acid sequence of SEQ ID NO: 198; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0023] This article also provides a method for treating cancer in a subject in need, comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR binding fragment thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 182 and the VL region comprises the amino acid sequence of SEQ ID NO: 207; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0024] This article also provides a method for treating cancer in a subject in need, comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR binding fragment thereof, comprising: (a) an EGFR binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 184 and the VL region comprises the amino acid sequence of SEQ ID NO: 209; and (b) an immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence of SEQ ID NO: 23.

[0025] This article also provides isolated nucleic acids encoding the engineered antibodies described herein.

[0026] This article also provides host cells expressing the engineered antibodies described herein.

[0027] This document also provides compositions comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent comprises any of the engineered antibodies described herein, and the second therapeutic agent is combined with MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, 4-1BB, or a combination thereof.

[0028] By incorporating via reference

[0029] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, and have the same effect as if each individual publication, patent or patent application were expressly and individually indicated to be incorporated herein by reference.

[0030] Brief description of the attached figures

[0031] The features of this disclosure are described in detail in the appended claims. The features and advantages of this disclosure can be better understood by referring to the following detailed description and drawings of exemplary embodiments utilizing the principles of this disclosure.

[0032] Figure 1 The amino acid sequence of the human IgA2 heavy chain is shown, with UniProt reference number A0A0G2JMB2 (SEQ ID NO: 1). The highlighted amino acid residues in the figure are residues modified in some embodiments described herein. Underlined sequences represent CH3 tails, which are partially or completely omitted in some embodiments.

[0033] Figure 2 The amino acid sequence of the human IgA2 heavy chain is shown, with UniProt reference number P01877-1 (SEQ ID NO: 2). The highlighted amino acid residues in the figure are residues that have been modified in some embodiments described herein. Underlined sequences represent CH3 tails, which are partially or completely omitted in some embodiments.

[0034] Figure 3 The amino acid sequence of the human IgA2 heavy chain is shown, with UniProt accession number A0A286YEY5 (SEQ ID NO: 3). The highlighted amino acid residues in the figure are residues that have been modified in some of the embodiments described herein.

[0035] Figures 4A-4D A schematic diagram of engineered EGFR-resistant IgA variants and wild-type IgA (IgA2(m1)) is shown. Figure 4A This is a schematic diagram of wild-type (WT) IgA2m1. The CH domain of IgA2m1 contains four N-glycosylation sites, one of which is located at its tail. Figure 4B This is a schematic diagram of the engineered anti-EGFR IgA3.0+ (plus) variant. The engineered anti-EGFR IgA3.0+ variant was generated by engineering an anti-EGFR IgA2m1 antibody to include stable heavy and light chain linkages, as follows: (i) a CH1-P124R mutation; (ii) removal of two free cysteine ​​residues, one of which was mutated to a serine residue (CH2-C92S), and the other cysteine ​​residue (CH3-CHS-C147del) was removed by deleting the last two amino acids at the tail. Furthermore, three N-linked glycosylation sites were removed by substituting key amino acids in three N-glycosylation motifs. The mutations in these three N-glycosylation motifs are: CH1-N45.2G; CH2-N120T-I121L-T122S; and CH3-CHS-N135Q. Figure 4C This represents an engineered anti-EGFR IgA3.0-(IgA3.0min) variant containing a complete tail deletion (CH3-CHS-P131-Y148del). This anti-EGFR IgA3.0min variant comprises a stabilized heavy and light chain linker (CH1-P124R mutation), a complete tail deletion (CH3-CHS-P131-Y148del), and the absence of two free cysteine ​​residues, one of which is mutated to serine (CH2-C92S), and the other cysteine ​​residue (CH3-CHS-C147del) is tail-deleted. Furthermore, three N-linked glycosylation sites are removed by substituting two key amino acids in the two N-glycosylation motifs (i.e., CH1-N45.2G and CH2-N120T-I121L-T122S) and by the CH3-CHS-N135Qdel deletion resulting from the tail deletion. Figure 4DA representative diagram of an engineered anti-EGFR IgA4.0 variant is shown, which contains all the features of the anti-EGFR IgA3.0 min variant and further includes a mutation in the last N-linked glycosylation motif CH2-N20. Therefore, the anti-EGFR IgA4.0 variant contains a stable heavy and light chain linker (CH1-P124R mutation), an entire tail deletion (CH3-CHS-P131-Y148del), and the absence of two free cysteine ​​residues, one of which is mutated to serine (CH2-C92S) and the other (CH3-CHS-C147del) is a tail deletion. Furthermore, the four N-linked glycosylation sites are removed by substituting one of the four key amino acids in the four N-glycosylation motifs (CH1-N45.2G; CH2-N120T-I121L-T122S; CH3-CHS-N135Q; and CH2-N20G, CH2-N20Q, CH2-N20T, or CH2-N20T-L21I-T22S). The anti-EGFR IgA4.0 variant is a non-glycosylated IgA.

[0036] Figure 5 The production process of anti-EGFR IgA 3.0min DS is shown.

[0037] Figure 6 A-6B shows the gene map of the vector components contained in the expression plasmids used in the production of non-clinical product batches. Figure 6 A shows the gene map of the vector components contained in the anti-EGFR IgA3.0min heavy chain expression plasmid. Figure 6 B shows the gene map of the vector components contained in the anti-EGFR IgA3.0min light chain expression plasmid.

[0038] Figure 7 A-7F shows SDS-PAGE gel images of various anti-EGFR IgA 3.0min drug substances (DS) to visualize the size and purity of intermediates after Capto L antibody capture and SEC size exclusion, as well as HPLC-SEC analysis results of anti-EGFR IgA 3.0min antibodies obtained from production batches. Three anti-EGFR IgA 3.0min drug substances (DS) were produced: (1) DS1, containing the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) DS2, containing the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; (3) DS3, containing the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207. Specifically, Figure 7 A, 7C, and 7E show the reduced (left) and non-reduced (right) SDS-PAGE gel electrophoresis images of anti-EGFR IgA 3.0min DS1, anti-EGFR IgA 3.0min DS2, and anti-EGFR IgA 3.0min DS3, respectively, to visualize the size and purity of the intermediates after Capto L antibody capture and SEC size exclusion. The expected band sizes are marked in the figures: intact anti-EGFR IgA 3.0min antibody is 150 kDa; the HC and LC values ​​for anti-EGFR IgA 3.0min antibody are 75 kDa and 25 kDa, respectively. Figure 7 B, 7D, and 7F show the HPLC-SEC analysis results of anti-EGFR IgA 3.0 min DS1, anti-EGFR IgA 3.0 min DS2, and anti-EGFR IgA 3.0 min DS3 antibodies obtained from the production batch, respectively. Antibody purity is expressed as a percentage.

[0039] Figure 8 The flowchart for the release test of anti-EGFR IgA 3.0min DP is shown.

[0040] Figures 9A-9E The results of measurements of EGFR binding to four anti-EGFR IgA3.0min DP variants are shown. In summary, the four variants include: (1) anti-EGFR IgA3.0min DP1, which contains the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) anti-EGFR IgA3.0min DP2, which contains the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; (3) anti-EGFR IgA3.0min DP3, which contains the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207; and (4) anti-EGFR IgA3.0min DP4, which contains the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Figures 9A-9D The results showed that the anti-EGFR IgA 3.0 min DP1, anti-EGFR IgA 3.0 min DP2, anti-EGFR IgA 3.0 min DP3, and anti-EGFR IgA 3.0 min DP4 were compared with those immobilized in Ni²⁺. + Surface plasmon resonance (SPR) measurements were performed on His-labeled EGFR on the α-triacetic acid sensor chip. All measurements were performed on a BiaCore T200 system. Figure 9EThe binding results of anti-EGFR IgA 3.0 min DP1 to EGFR-expressing A431 and A1207 cell lines and the D562 cell line with low EGFR expression, as determined by FACS, are shown. Cells were incubated with different concentrations of antibody, followed by the addition of fluorescently labeled anti-IgA secondary antibody, and the results were detected by flow cytometry. A control was included in the experiment, where only fluorescently labeled anti-IgA secondary antibody was added to the A431 cell line without primary antibody.

[0041] Figures 10A-10B The dose-dependent inhibition of EGF binding by each anti-EGFR IgA 3.0 min DP was shown. Specifically, Figure 10A The dose-dependent inhibition of EGF binding by four anti-EGFR IgA 3.0 min DPs is shown, wherein: (1) anti-EGFR IgA 3.0 min DP1 contains the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) anti-EGFR IgA 3.0 min DP2 contains the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; (3) anti-EGFR IgA 3.0 min DP3 contains the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207; and (4) anti-EGFR IgA 3.0 min DP4 contains the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Figure 10B It is an anti-EGFR IgA 3.0min DP1 and Figure 10A Another representation of the same data. As shown in the figure, mean fluorescence intensity (MFI), reflecting the binding amount of fluorescently labeled EGF to EGFR, was measured using a competitive FACS detection method at increasing concentrations of anti-EGFR IgA 3.0 min. MFI decreased in a dose-dependent manner. The isotype control of anti-EGFR IgA 3.0 min showed no effect, indicating specific binding to EGFR.

[0042] Figure 11A-11ECell viability results after treatment with each of the four anti-EGFR IgA 3.0 min DP are shown. In summary, the four variants comprise: (1) anti-EGFR IgA 3.0 min DP1, comprising the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) anti-EGFR IgA 3.0 min DP2, comprising the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; (3) anti-EGFR IgA 3.0 min DP3, comprising the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207; and (4) anti-EGFR IgA 3.0 min DP4, comprising the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Figure 11A-11B The results for CDC (e.g., apoptosis) induced by all four anti-EGFR IgA 3.0 min DP inhibitors are shown. As shown in the figure, cell viability of A431 or human fibroblast cell lines was assessed using a sulforhodamine B assay kit in the presence of each anti-EGFR IgA 3.0 min DP inhibitor at increasing concentrations. Each anti-EGFR IgA 3.0 min DP inhibitor significantly reduced the survival rate of EGFR-overexpressing A431 cells in a dose-dependent manner. No changes in human fibroblast viability were observed. Figure 11C-11D They respectively showed, as Figure 11A-11B This is another representation of cell viability of A431 or human fibroblast cell lines after treatment with anti-EGFRIgA 3.0 min DP1. Figure 11E This shows another representation of cell viability assays for anti-EGFR IgA 3.0 min DP1.

[0043] Figures 12A-12EThe ADCC induced by four anti-EGFR IgA 3.0 min DP sequences are shown. These four anti-EGFR IgA 3.0 min DP sequences include: (1) anti-EGFR IgA 3.0 min DP1, containing the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) anti-EGFR IgA 3.0 min DP2, containing the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; (3) anti-EGFR IgA 3.0 min DP3, containing the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207; and (4) anti-EGFR IgA 3.0 min DP4, containing the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Neutrophils were purified from three donors. Figure 12A ) or by whole blood lysate with red blood cells removed ( Figure 12B The study analyzed the changes in A431 cancer cell line-specific cell lysis with varying concentrations of each anti-EGFR IgA 3.0 min DP. Figure 12C Is it like this? Figure 12A This is another representation of the change in A431 cancer cell line-specific cell lysis with varying anti-EGFR IgA DP1 concentration over 3.0 min, analyzed using purified neutrophils from three donors. Similarly, Figure 12D Is it like this? Figure 12B This is another representation of the change in A431 cancer cell line-specific cell lysis with varying anti-EGFR IgA 3.0 min DP1 concentration, analyzed using whole blood lysates from three donors after the removal of erythrocytes. Furthermore, the E:T ratio of the three donors was calculated based on the specific lysis of the A431 cancer cell line using neutrophils from the three donors. Figure 12E ).

[0044] Figures 13A-13CResults of tumor growth studies in A431 and A549 xenograft mouse models are presented. In summary, we tested the activity of four anti-EGFR IgA3.0min DPs, which include: (1) anti-EGFR IgA3.0min DP1 containing the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) anti-EGFR IgA3.0min DP2 containing the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; (3) anti-EGFR IgA3.0min DP3 containing the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207; and (4) anti-EGFR IgA3.0min DP4 containing the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. In cancer cell lines that expressed low levels of EGFR (A549 - Figure 13A The activity of each anti-EGFR IgA 3.0 min DP was tested in vivo after being administered twice weekly to CD89 Tg NXG xenograft mice. Figure 13B Anti-EGFR IgA 3.0 min DP1 therapy is effective for low expression of EGFR (A549-). Figure 13B Another way to represent the effect of ). Figure 13C The study demonstrated that anti-EGFR IgA 3.0 min DP1 treatment was effective against high expression of EGFR (A431-). Figure 13A The effect of luciferase was investigated. Each cancer cell line was transfected with the luciferase gene. A PBS-mediated control was included. Tumor growth was longitudinally monitored using bioluminescence imaging (BLI), and the percentage increase in bioluminescence signal over time was reported.

[0045] Figure 14 Pharmacokinetic curves of anti-EGFR IgA 3.0 min DP after a single intravenous (IV) or intraperitoneal (IP) administration of 3 mg / kg in immunodeficient NSG mice are shown. This anti-EGFR IgA 3.0 min DP contains the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211. Plasma concentrations of anti-EGFR IgA 3.0 min DP1 were determined over 28 days, and mean plasma concentrations (± geometric standard deviation, expressed as error bars) are plotted as a function of time. n=4 for each dose group.

[0046] Figure 15A-15B shows the pharmacokinetic profiles of anti-EGFR IgA 3.0 min DP in non-human primates (NHP): (i) a single intravenous (IV) administration of 25 mg / kg ( Figure 15 (A) or (ii) four intravenous doses of 25 mg / kg, followed by one intravenous dose of 12.5 mg / kg, 7 days apart. Figure 15 B). This anti-EGFR IgA 3.0 min DP contains the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211.

[0047] Figure 16 The overall clinical study design was summarized. Invention Details

[0049] The following description and examples illustrate embodiments of this disclosure in detail. It should be understood that this disclosure is not limited to the specific embodiments described herein and therefore may vary. Those skilled in the art will recognize that many variations and modifications are possible with respect to this disclosure, all of which are included within its scope.

[0050] All terms used herein are intended to be understood in the same way as those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0051] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0052] Although the various features of this disclosure may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although this disclosure may be described in different embodiments for clarity, it may also be implemented in a single embodiment.

[0053] definition

[0054] The following definitions are supplementary to existing definitions and apply only to this application. They should not be extended to any related or unrelated cases, such as any jointly owned patents or applications. Although any methods and materials similar to or equivalent to those described herein may be used in practice to test the contents of this disclosure, preferred materials and methods are described herein. Therefore, the terminology used herein is for describing particular embodiments only and is not intended to be limiting.

[0055] In this application, unless otherwise expressly stated, the singular forms used include the plural forms. It should be noted that, in this specification, the singular forms “a,” “an,” and “the” all include plural references, unless the context clearly indicates otherwise. In this application, “or” is used to mean “and / or”, unless otherwise specified.

[0056] The terms “some implementation schemes,” “implementation scheme,” “one implementation scheme,” or “other implementation schemes” used in the specification refer to specific features, structures, or characteristics related to an implementation scheme that are included in at least some of the implementation schemes disclosed herein, but not necessarily in all of the implementation schemes disclosed herein.

[0057] In this article, natural amino acids may be represented by their conventional single-letter or three-letter abbreviations, as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine ​​(C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X may represent any amino acid. In some respects, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).

[0058] As used herein, the terms “comprising” (and any form of “comprising” such as “comprise” and “comprises”), “having” (and any form of “having” such as “have” and “has”), “including” (and any form of “including” such as “includes” and “include”), and “containing” (and any form of “containing” such as “contains” and “contain”) are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0059] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error for a given value and include a range of up to 10% of the given value or within an order of magnitude of the given value. If a specific value is described in the application and claims, the term “about” should be assumed to mean within an acceptable margin of error for the given value unless otherwise stated.

[0060] As used herein, the term “antibody” refers to immunoglobulin (Ig), whether naturally occurring or partially or wholly synthetically produced, including complete antibodies and antibody fragments, unless otherwise explicitly stated.

[0061] As used herein, “complete antibody” refers to an antibody consisting of four polypeptides: two heavy chain regions and two light chain regions, each containing: (i) a variable region having three complementarity-determining regions (CDRs) that constitute the “hypervariate region” of the antibody and are responsible for binding the antigen, wherein the three CDRs are separated by framework residues; and (ii) a constant region.

[0062] As used in this article, the term "complementarity-determining region" (CDR) refers to the amino acid residues in the antibody heavy chain variable region and light chain carrier region that are essential for antigen binding.

[0063] As used herein, the term “framework residue” or “FR” refers to residues in the variable region other than the CDR.

[0064] As used herein, “heavy chain region” or “heavy chain polypeptide” refers to the antibody moiety, which includes an N-terminal heavy chain variable (VH) region and a C-terminal heavy chain constant (CH) region, wherein the C-terminal heavy chain constant region has one or more of the following: CH1 domain, hinge, CH2 domain, CH3 domain and CH3 tail (CS).

[0065] As used in this article, the term "hinge" refers to a flexible structural domain in the heavy chain region that connects the CH1 and CH2 domains, allowing the two N-terminal antigen-binding regions to move independently.

[0066] As used herein, "light chain region" or "light chain polypeptide" refers to a portion of an antibody that includes an N-terminal variable (VL) light chain region and a C-terminal constant (CL) light chain region. The kappa ("κ") light chain and lambda ("λ") light chain refer to two main light chain isotypes.

[0067] As used herein, the term "Fc domain" or "Fc-containing domain" refers to a portion of an antibody or non-antibody that is capable of binding to the Fc receptor, including a portion of the heavy chain constant (CH) region.

[0068] As used herein, the terms “fragment of antibody,” “antibody fragment,” “functional fragment of antibody,” “antigen-binding portion,” and their grammatical equivalents are used interchangeably to refer to one or more fragments or portions of an antibody that retain the ability to bind specifically to an antigen and contain one or more complementarity-determining regions (CDRs).

[0069] "Fab fragment" refers to a monovalent fragment composed of VL, VH, CL and CH1 domains.

[0070] The “F(ab')2” fragment refers to a divalent fragment containing two Fab fragments connected by a stem-region disulfide bond.

[0071] "Fv fragment" refers to a fragment composed of the VL and VH domains of a single arm of an antibody.

[0072] "Single-chain Fv (scFv)" refers to a monovalent fragment consisting of two domains (VL and VH) of an Fv fragment connected by a synthetic linker, which enables the synthesis of these two domains into a single polypeptide chain.

[0073] "Diabody" refers to a dimer of polypeptide chains, where each polypeptide chain contains VH and VL, which are linked by a peptide linker. If the linker is too short to allow VH and VL on the same polypeptide chain to pair, it drives the pairing of complementary domains on different VH-VL polypeptide chains, generating a dimer molecule with two functional antigen-binding sites.

[0074] As used in this article, the term "monoclonal antibody" refers to an antibody produced by a single B cell clone that binds to the same epitope.

[0075] As used in this article, the term "polyclonal antibody" refers to an antibody produced by different B cells and bound to different epitopes of the same antigen.

[0076] As used herein, the term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from two different species or two different sources and that comprises a synthetic molecule.

[0077] The term "recombinant antibody" refers to an antibody expressed from cells or cell lines transfected with an expression vector (or possibly more than one expression vector, usually two), the expression vector containing the coding sequence of the antibody, which is not naturally associated with the cell.

[0078] As used herein, the term “recombinant human antibody” means a human antibody prepared, expressed, generated, or isolated by a recombinant manner, including: (a) antibodies isolated from transgenic or transchromosomally transgenic animals (e.g., mice) or hybridomas thus prepared (see below); (b) antibodies isolated from host cells transformed to express human antibodies, such as antibodies isolated from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other method involving splicing human immunoglobulin gene sequences with other DNA sequences.

[0079] As used herein, the term "humanized antibody" refers to a non-human antibody that contains an amino acid sequence derived from a corresponding human antibody, which is not naturally present in non-human antibodies.

[0080] As used herein, the term “antigen” refers to a molecule or part of a molecule that can interact with a binding site of an antibody or a fragment thereof, including molecules or parts of a molecule that can bind to an antibody (epitope).

[0081] As used herein, the term "epitope" refers to a region on an antigen that interacts with a specific antigen-binding site (called a complementary site) in the variable region of an antibody molecule. Epitopes can be conformational or linear. A "conformational epitope" is formed by spatially adjacent amino acids in different segments of a linear polypeptide chain. A "linear epitope" is formed by adjacent amino acid residues in a polypeptide chain.

[0082] As used herein, the term "antigen-binding domain" refers to the portion of an antibody or non-antibody that binds to or recognizes an antigen or fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region.

[0083] As used in this article, the term “recognition” refers to the association or binding between an antigen-binding domain and an antigen.

[0084] As used herein, the terms “specific binding” or “preferential binding” refer to a higher affinity and / or affinity with a target than with other peptides.

[0085] As used in this article, the term "affinity" refers to the equilibrium constant (Kaffinity) for the dissociation of an antigen from an antigen-binding protein. D It measures the binding strength between the antigen and the antigen-binding domain: K D The lower the value, the stronger the binding strength between the antigen and the antigen-binding domain. When the antibody or its antigen-binding fragment binds to the first antigen, the affinity K... D The value is higher than its K value when it binds to another target or peptide. D If the value is at least 50 times lower, the antibody or its antigen-binding fragment is said to be "specific" to the first target or antigen.

[0086] The term "affinity" refers to the affinity of an antigen-binding domain for an antigen, which depends on the number of relevant antigen-binding sites present on the antigen-binding domain.

[0087] As used herein, the term “neutralizing activity” refers to the ability of an antibody or its functional fragment to block the binding of a related ligand to a target antigen.

[0088] As used herein, the term "target cell" refers to a cell that the antibody or functional fragment thereof disclosed herein can target.

[0089] As used herein, the terms “polynucleotide” and “nucleic acid molecule” are used interchangeably and refer to polymers of deoxyribonucleotides, ribonucleotides, modified nucleotides and / or their analogues of any length, including DNA and RNA.

[0090] The term "operable link" or "transcriptional control" refers to a functional link between a regulatory sequence and a nucleic acid sequence that leads to the expression of the latter.

[0091] As used herein, the terms “modification” or “mutation” refer to the substitution, insertion, or deletion of an amino acid in the antibody or a fragment thereof compared to the corresponding amino acid in the WT antibody.

[0092] As used herein, the terms “conservative substitution,” “conservative modification,” or “conservative mutation” refer to the substitution of one amino acid (the first amino acid) in the structure of a polypeptide with another amino acid that has similar chemical properties, and that the substitution can be made with the first amino acid in the polypeptide structure without significantly interfering with or altering the structure or function of the polypeptide.

[0093] As used herein, the terms “non-conservative mutation,” “non-conservative modification,” or “non-conservative substitution” refer to the substitution of one amino acid (the first amino acid) in the structure of a polypeptide by another amino acid (the second amino acid) that has dissimilar chemical properties. When the second amino acid substitutes for the first amino acid in a non-conservative mutation or substitution, it results in a disturbance or alteration of the polypeptide's structure or function, including enhancement of the polypeptide's structure or function.

[0094] As used herein, the term "improved properties" refers to one or more modifications of an antibody or its fragments that improve the properties associated with those one or more modifications compared to the corresponding unmodified antibody.

[0095] As used herein, the terms “corresponding antibody,” “corresponding unmodified antibody,” “corresponding wild-type antibody,” and “corresponding antibody lacking one or more modifications” refer to a wild-type antibody or a fragment thereof having an amino acid sequence that corresponds to the amino acid sequence of an antibody or a fragment thereof having one or more modifications on each amino acid residue other than the one or more modifications mentioned above.

[0096] As used herein, the terms “improved stability” or “enhanced stability” when used to refer to improved properties include improved thermal stability and / or reduced aggregation, as determined by the retention of greater biological activity (e.g., ADCC, binding to antigens, or binding to FcαR) compared to the corresponding antibody after incubation at a certain temperature for a period of time.

[0097] As used herein, the terms “reduced aggregation” or “lower aggregation” when used to refer to an improved property include reduced aggregation of the antibody or its functional fragment with other antibody molecules and / or with other macromolecules, including serum proteins (such as albumin), compared to the aggregation exhibited by the corresponding antibody.

[0098] As used herein, the term "biodistribution," when referring to an improved characteristic, means the distribution of the disclosed antibody or its functional fragment in cells and / or tissues and / or organs after administration or delivery to a subject. As used herein, the term "increased biodistribution" generally refers to an increased distribution of the disclosed IgA antibody or its functional fragment at a target site (e.g., tumor or tumor cells) compared to an administration medium or a corresponding WT IgA antibody.

[0099] As used herein, the terms “in vivo half-life” or “circulating half-life” refer to the time required for half of the antibody or a fragment thereof to be cleared from the circulatory system after administration to an animal. As used herein, the term “increased circulating half-life” when referring to an improved property means greater persistence of the antibody or a fragment thereof in serum or plasma compared to the administered medium or the corresponding WT IgA antibody, and / or a longer time required for the measured maximum serum or plasma concentration to decrease to half.

[0100] The term "Fc receptor-mediated effector cell function" refers to effector functions triggered by the binding of immunoglobulins (such as IgA) to Fc receptors on immune effector cells, such as phagocytosis, antibody-dependent cytotoxicity (ADCC), release of inflammatory mediators, lysozyme production, and superoxide anion production.

[0101] The term "ADCC activity" refers to the ability of an antibody to induce the lysis of target cells (such as cancer cells) through immune effector cells.

[0102] The term "complement-dependent cytotoxicity" or "CDC" refers to the ability of an antibody to lyse target cells (such as cancer cells) in the presence of complement.

[0103] As used in this article, the term "glycosylation" refers to the covalent attachment of one or more carbohydrates to a polypeptide.

[0104] As used herein, the term “separated” means separating or altering a molecule from its native state. Nucleic acids or polypeptides present in cells and coexisting with their native substances (e.g., antibodies or antigen-binding fragments thereof disclosed herein) are not “separated,” but the separation of the same nucleic acid or polypeptide, either partially or completely, from said cells and coexisting substances is “separated.”

[0105] As used herein, the term "substantially purified" means that the antibody or its functional fragment is substantially free of cellular material, naturally occurring coexisting substances, or other contaminants of its cellular or tissue origin, or substantially free of chemical precursors or other chemical substances after chemical synthesis. As used herein, the term "substantially free" means that the dry weight of cellular material, naturally occurring coexisting substances, or other contaminants present in the preparation is less than about 30%.

[0106] The term "leader sequence" refers to the amino acid residue sequence located at the N-terminus of a polypeptide that promotes the secretion of the polypeptide from mammalian cells.

[0107] The term "label" refers to a compound or composition that can be detected directly or indirectly and conjugates directly or indirectly to an antibody. A label is described as "directly detectable" when it is detectable on its own (e.g., radioisotope labeling or fluorescent labeling) or when it catalyzes a chemical change in the substrate that produces a detectable change (e.g., enzyme labeling). A label is described as "indirectly detectable" when it is not detectable on its own but can bind to another directly detectable reagent.

[0108] As used herein, the term "fusion protein" refers to a polypeptide that contains the amino acid sequence of an antibody or a fragment thereof and the amino acid sequence of a heterologous polypeptide (i.e., an unrelated polypeptide).

[0109] As used herein, the terms “cancer,” “tumor,” “proliferative disorder,” “malignant tumor,” or “malignant disease” refer to a mammalian physiological state characterized by malignant features of cells. “Malignant features” include uncontrolled growth, cell invasion, and metastatic formation. These malignant features distinguish cancer from benign tumors, which typically do not invade or metastasize.

[0110] As used herein, the terms “disease,” “symptom,” and “condition” are used interchangeably to refer to any alteration in the state of the body or organs that disrupts or interferes with normal functioning and / or causes discomfort, dysfunction, pain, or even death of the affected person or those in contact with them. Disease or symptom may also be associated with plague, discomfort, minor illness, disease, ailment, disorder, sickness, complaint, or affectation.

[0111] As used in this article, the terms “in need,” “patient in need,” and “subject in need,” in the context of therapeutic or preventative treatment, refer to a person who has a disease, has been diagnosed with a disease, needs to prevent a disease, or is at risk of developing a disease.

[0112] As used herein, the terms “treatment” and “improvement” refer to therapeutic and preventative treatment aimed at reversing, alleviating, improving, inhibiting, preventing, slowing, or stopping the progression or worsening of a condition associated with a disease or symptom. Treatment involves reducing or alleviating at least one adverse reaction or symptom of a condition, disease, or symptom associated with the disease or symptom. Treatment is “effective” if one or more symptoms or clinical indicators are reduced, or if disease progression slows or stops.

[0113] As used herein, the term "administration" refers to placing an antibody or a fragment thereof into a subject by some method or route, thereby delivering the antibody or a fragment thereof at least partially to the target site. Administration can be carried out through any appropriate route that is effective in treating the subject.

[0114] The term "combination therapy" refers to the administration of multiple drugs in a generally concurrent (simultaneous or sequential) manner.

[0115] The term "therapeutic effective dose" refers to the amount of drug that effectively achieves the expected therapeutic effect within a given time period.

[0116] The term "preventive effective dose" refers to the amount of drug that effectively achieves the expected preventive effect within a given time period.

[0117] As used in this article, the term "parenteral administration" refers to administration other than enteral and topical administration, and is usually administered by injection.

[0118] As used in this article, the terms “systemic administration” and “peripheral administration” refer to administration methods in which a drug is administered not directly to a target site, tissue, or organ (such as a tumor site) but into the subject’s circulatory system, where it is affected by metabolism and other similar processes.

[0119] As used herein, the term “pharmaceutically acceptable” means, within reasonable medical judgment, a compound, material, composition, and / or dosage form that is suitable for contact or administration to human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable benefit / risk ratio.

[0120] As used herein, the terms “subject,” “patient,” “individual,” and similar terms are used interchangeably to refer to a vertebrate, mammal, primate, or human.

[0121] As used herein, the term "host cell" refers to a specific target cell transfected with a nucleic acid molecule and its progeny or potential progeny. Due to mutations, environmental influences, or integration of the nucleic acid molecule into the host cell genome that may occur during subsequent passages, the progeny of this cell may not be identical to the parent cell transfected with the nucleic acid molecule.

[0122] introduce

[0123] This article describes engineered anti-EGFR IgA antibodies with an IgA backbone and an anti-EGFR binding domain. In some embodiments, the engineered antibody has one or more of the following: reduced N-glycosylation characteristics, removal of C-terminal residues from the heavy chain constant region, or amino acid substitutions in a monomeric form that facilitate stabilization. In some embodiments, the engineered IgA antibody targets human EGFR, and its Fc domain recognizes the FcαRI receptor (CD89) on immune cells (e.g., neutrophils, macrophages, and eosinophils). Therefore, in some embodiments, the engineered antibody participates in the selective recruitment of FcαR1-expressing immune effector cells to EGFR tumor antigens on the cell surface. In some embodiments, the engineered antibody selectively recruits neutrophils. Neutrophils are capable of directly killing tumor cells by releasing reactive oxygen species (ROS) and reactive nitrogen species (RNS). Therefore, in some embodiments, the engineered antibody can induce ADCC in cells expressing EGFR tumor antigens. Alternatively, in some embodiments, engineered antibodies directly induce cytotoxicity in EGFR-positive tumor cells by antagonistically binding to EGFR and blocking EGF signaling in the absence of immune effector cells. Furthermore, in some embodiments, the cytotoxicity induced by engineered antibodies enhances T cell activation and attracts pro-inflammatory (M1) macrophages.

[0124] Anti-EGFR IgA antibody

[0125] This article discloses engineered anti-EGFR IgA antibodies. IgA antibodies account for 15-20% of serum immunoglobulins. IgA is most abundant on mucosal surfaces and plays an important role in the body's immune response. IgA is highly diverse and can exist in mucosal tissues as monomers, dimers, polymers, and secreted IgA. IgA has two subclasses (IgA1 and IgA2), which can be produced in monomeric, dimeric, and secreted forms. In some embodiments, the anti-EGFR IgA antibody can be a monomer. In some embodiments, the anti-EGFR IgA antibody can contain one or more IgA1 amino acid sequences. In some embodiments, the anti-EGFR IgA antibody can contain one or more IgA2 amino acid sequences. In some embodiments, the anti-EGFR IgA antibody can contain one or more IgA1 amino acid sequences and one or more IgA2 amino acid sequences. IgA1 and IgA2 differ in their hinge regions and carbohydrate content. Monomeric IgA1 is the main subclass in serum and is easily cleaved by bacterial enzymes, while IgA2 is more resistant to enzyme degradation and is mainly found in mucosal secretions.

[0126] In some embodiments, the anti-EGFR IgA antibody is an IgA2 antibody allotype: IgA2m(1), IgA2m(2), or IgA2n. In some embodiments, the IgA2m(1) antibody is a Caucasian IgA2m(1) antibody. In some embodiments, the IgA2m(2) antibody is an African IgA2m(2) antibody or an Asian IgA2m(2) antibody. In some embodiments, IgA2m(1) also has superior potential efficacy and safety profiles due to its lower glycosylation levels and lack of association with IgA nephropathy compared to IgG1 and IgA1 / IgA2m(2).

[0127] In some embodiments, the anti-EGFR IgA antibody is a therapeutic antibody. In some embodiments, the anti-EGFR IgA antibody may be a humanized antibody. In some embodiments, the anti-EGFR IgA antibody may be a chimeric antibody. In some embodiments, the anti-EGFR IgA antibody may be a human antibody. In some embodiments, the anti-EGFR IgA antibody is a recombinant antibody. In some embodiments, the anti-EGFR IgA antibody is a recombinant human antibody. In some embodiments, the anti-EGFR IgA antibody may be a monospecific antibody. In some embodiments, the anti-EGFR IgA antibody may be a bispecific antibody. In some embodiments, the anti-EGFR IgA antibody may be a trispecific antibody. In some embodiments, the anti-EGFR IgA antibody may be a multispecific antibody.

[0128] In some embodiments, the anti-EGFR IgA antibody may be a chimeric antibody, a single-chain antibody, a humanized antibody, a human antibody, a monoclonal antibody, a deimmunizing antibody, a bispecific antibody, a multispecific antibody, a multivalent antibody, or a combination thereof. In some embodiments, the anti-EGFR IgA antibody may be a bispecific antibody that binds to both EGFR and a second antigen. In some embodiments, the anti-EGFR IgA antibody simultaneously binds to two antigens on the cell surface. In some embodiments, the binding of the anti-EGFR IgA antibody to the two different antigens is sequential. For example, the anti-EGFR IgA antibody first binds to the first antigen, thereby limiting the space that the second antibody arm can explore. Therefore, the local concentration of the second antigen can be significantly increased, thereby promoting the binding of the second antibody arm.

[0129] constant region

[0130] In some implementations, the anti-EGFR IgA antibody comprises an IgA heavy chain constant region or a functional variant thereof. Table 1 lists exemplary amino acid sequences of the IgA heavy chain constant region and their encoding nucleotide sequences. Figure 1-3 Three representative amino acid sequences of the human IgA2 heavy chain are provided in Table 1. In some embodiments, the IgA heavy chain constant region comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any amino acid sequence (SEQ ID NO: 1-9) in Table 1. In some embodiments, the IgA heavy chain constant region is encoded by a nucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any nucleotide sequence in Table 1.

[0131] Table 1. Exemplary amino acid sequences of the IgA heavy chain constant region

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] In some embodiments, the IgA heavy chain constant region comprises one or more heavy chain constant domains (e.g., CH1 domain, CH2 domain, CH3 domain, or any combination thereof). Table 2 lists exemplary amino acid sequences of the IgA2 CH1 domain, IgA CH2 domain, and IgA CH3 domain. In some embodiments, the IgA heavy chain constant region (e.g., the IgA CH1 domain, IgA CH2 domain, and IgA CH3 domain) comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any of the amino acid sequences in Table 2. In some embodiments, the heavy chain constant region comprises one or more of the following: the IgA CH3 domain, the IgA CH2 domain, or the IgA CH1 domain, or any combination thereof. In some embodiments, the anti-EGFR IgA antibody includes a heavy chain constant region comprising one or more amino acids of an IgG CH3 domain, an IgG CH2 domain, or an IgG CH1 domain. In some embodiments, the anti-EGFR IgA antibody includes a heavy chain constant region comprising an IgA CH3 domain, an IgA CH2 domain, and an IgA CH1 domain. In some embodiments, the anti-EGFR IgA antibody includes a heavy chain constant region comprising an IgA CH3 domain, an IgA CH2 domain, and an IgG CH1 domain. In some embodiments, the anti-EGFR IgA antibody is an IgA2 antibody comprising a heavy chain constant region containing one or more of the following: an IgA2 CH3 domain, an IgA2 CH2 domain, an IgA2 CH1 domain, or any combination thereof.

[0139] Table 2. Exemplary sequences of IgA heavy chain constant structural domains

[0140]

[0141] In some embodiments, the anti-EGFR IgA antibody comprises a light chain constant region (CL). Table 3 lists exemplary amino acid sequences of the IgA light chain constant region. In some embodiments, the light chain constant region comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any of the amino acid sequences in Table 3 (SEQ ID NO: 23 or SEQ ID NO: 24). In some embodiments, the light chain constant region comprises a κ light chain constant region.

[0142] Table 3. Exemplary sequences of the constant region of the IgA light chain

[0143]

[0144] In some embodiments, the anti-EGFR IgA antibody includes an IgG light chain variable region. In some embodiments, the anti-EGFR IgA antibody includes an IgG heavy chain variable region. In some embodiments, the anti-EGFR IgA antibody includes both an IgG light chain variable region and an IgG heavy chain variable region.

[0145] In some embodiments, the anti-EGFR IgA antibody may contain at least a portion of an Fc domain. In some embodiments, the anti-EGFR IgA antibody includes a heavy chain constant region comprising a CH3 domain, a CH2 domain, and a CH1 domain. In some embodiments, the anti-EGFR IgA antibody includes a light chain constant region comprising a CL domain.

[0146] In some embodiments, the antibodies or functional fragments thereof provided herein comprise an IgA heavy chain constant region having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 4-9. In some embodiments, the antibodies or functional fragments thereof provided herein comprise at least one IgA heavy chain constant domain having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 21-22. In some embodiments, the antibodies or functional fragments thereof provided herein comprise an IgA light chain constant domain having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 23 or 24. In some embodiments, the antibodies or functional fragments thereof provided herein comprise a heavy chain variable domain of an IgG antibody. In some embodiments, the antibodies or functional fragments thereof provided herein comprise a light chain variable domain of an IgG antibody.

[0147] Variable region

[0148] In one aspect, the antibody or its functional antigen-binding fragment comprises a heavy chain variable region (VH) sequence. In some embodiments, the VH comprises three CDRs, namely CDR-H1, CDR-H2, and CDR-H3. Table 4 lists exemplary amino acid sequences of the CDRs of the VH. Table 5 lists exemplary amino acid sequences of the VH CDRs.

[0149] Table 4: Exemplary amino acid sequences of VH CDR

[0150]

[0151] Table 5: Exemplary VH amino acid sequences

[0152]

[0153]

[0154] In some embodiments, the VH sequence has at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences in SEQ ID NO: 169-194. In some embodiments, the VH sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to the corresponding wild-type antibody amino acid sequence comprises substitutions (e.g., conserved substitutions), insertions, or deletions while retaining the ability to bind the same antigen to the corresponding wild-type antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of any of SEQ ID NO: 169-194. In some embodiments, substitution, insertion, or deletion occurs outside the CDR region (e.g., in the FR). Optionally, the antibody comprises the VH sequence of any one of SEQ ID NO: 169-194 and includes one or more post-translational modifications of that sequence.

[0155] In some embodiments, VH comprises one, two, or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of any one of SEQ ID NO: 34-54, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; (b) CDR-H2, comprising the amino acid sequence of any one of SEQ ID NO: 57-78, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; and (c) CDR-H3, comprising the amino acid sequence of any one of SEQ ID NO: 81-102, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions.

[0156] On one hand, an antibody or a functional antigen-binding fragment thereof is provided, wherein the antibody or functional antigen-binding fragment comprises a light chain variable region (VL) sequence. In some embodiments, the VL sequence comprises three CDRs, namely CDR-L1, CDR-L2, and CDR-L3. Table 6 lists exemplary amino acid sequences of VL CDRs. Table 7 lists exemplary VL amino acid sequences.

[0157] Table 6: Exemplary amino acid sequences of CDR-L

[0158]

[0159] Table 7: Exemplary VL amino acid sequences

[0160]

[0161]

[0162] In some embodiments, the VL sequence has at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of any one of SEQ ID NO: 195-219. In some embodiments, the VL sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding wild-type antibody amino acid sequence comprises substitutions (e.g., conserved substitutions), insertions, or deletions while retaining the ability to bind the same antigen to the corresponding wild-type antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of any one of SEQ ID NO: 195-219. In some embodiments, substitution, insertion, or deletion occurs outside the CDR region (e.g., in the FR). Optionally, the antibody or its functional antigen-binding fragment comprises the VL sequence of any one of SEQ ID NO: 195-219 and includes post-translational modifications of that sequence.

[0163] In some embodiments, the VL sequence comprises one, two, or three CDRs selected from: (a) CDR-L1, which comprises an amino acid sequence of any one of SEQ ID NO: 105-126, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; (b) CDR-L2, which comprises an amino acid sequence of any one of SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions; and (c) CDR-L3, which comprises an amino acid sequence of any one of SEQ ID NO: 146-166, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions.

[0164] In one aspect, an anti-EGFR IgA antibody or a functional antigen-binding fragment thereof is provided, wherein the antibody or the functional antigen-binding fragment thereof comprises VH as provided in any of the embodiments described above and VL as provided in any of the embodiments described above. In some embodiments, the antibody or its functional antigen-binding fragment comprises VH and VL, wherein VH comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 195-219, and wherein VL comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 195-219, and optionally comprises post-translational modifications of these sequences. In some embodiments, the VH sequence comprises the amino acid sequence of any one of SEQ ID NO: 169-194, and the VL sequence comprises the amino acid sequence of any one of SEQ ID NO: 195-219, and optionally includes post-translational modifications of these sequences.

[0165] In one aspect, an anti-EGFR IgA antibody or a functional antigen-binding fragment thereof is provided, wherein the antibody or the functional antigen-binding fragment thereof comprises a combination of the VH and VL sequences described in Table 8, wherein the VH comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences in SEQ ID NO: 169-194, and wherein the VL comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences in SEQ ID NO: 195-219. In some embodiments, the antibody or its functional antigen-binding fragment comprises a combination of the VH and VL sequences shown in Table 8.

[0166] Table 8: Exemplary combinations of VH and VL sequences

[0167]

[0168] In one aspect, an anti-EGFR IgA antibody or a functional antigen-binding fragment thereof is provided, wherein the antibody or functional antigen-binding fragment thereof comprises any CDR-H3 selected from Table 4 or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and any CDR-L3 selected from Table 6 or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, wherein the selected CDR-H3 and CDR-L3 are paired according to Table 9. In another aspect, an antibody or a functional antigen-binding fragment thereof is provided, wherein the antibody or functional antigen-binding fragment thereof comprises any CDR-H2 selected from Table 4 or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and any CDR-L2 selected from Table 6 or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, wherein the selected CDR-H2 and CDR-L2 are paired according to Table 9. In one aspect, an antibody or a functional antigen-binding fragment thereof is provided, wherein the antibody or functional antigen-binding fragment thereof comprises any CDR-H1 selected from Table 4 or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and any CDR-L1 selected from Table 6 or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, wherein the selected CDR-H1 and CDR-L1 are paired according to Table 9. In one aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 173 and comprises post-translational modifications of those sequences, and the VL comprises the amino acid sequence of SEQ ID NO: 211 and comprises post-translational modifications of those sequences. In one aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 172 and comprises post-translational modifications of those sequences, and the VL comprises the amino acid sequence of SEQ ID NO: 198 and comprises post-translational modifications of those sequences. In one aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 182 and includes post-translational modifications of those sequences, and the VL comprises the amino acid sequence of SEQ ID NO: 207 and includes post-translational modifications of those sequences. In another aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 184 and includes post-translational modifications of those sequences, and the VL comprises the amino acid sequence of SEQ ID NO: 209 and includes post-translational modifications of those sequences.

[0169] In one aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 38; (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 72; (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 97; (d) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 119; (e) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 140; and (f) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 150. In one aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 37; (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 59; (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 84; (d) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 107; (e) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 131; and (f) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 149. In one aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 45; (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 68; (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 93; (d) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 115; (e) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 137; and (f) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 157.In one aspect, this disclosure provides an antibody or a functional antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 46; (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 70; (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 95; (d) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 117; (e) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 129; and (f) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 159.

[0170] On one hand, an anti-EGFR IgA antibody or a functional antigen-binding fragment thereof is provided, wherein the antibody or the functional antigen-binding fragment thereof comprises: (a) CDR-H1, comprising any amino acid sequence of SEQ ID NO: 34-54, or containing one to three variants thereof with substitutions, deletions, or insertions; (b) CDR-H2, comprising any amino acid sequence of SEQ ID NO: 57-78, or containing one to three variants thereof with substitutions, deletions, or insertions; (c) CDR-H3, comprising any amino acid sequence of SEQ ID NO: 81-102, or containing one to three variants thereof with substitutions, deletions, or insertions; (d) CDR-L1, comprising any amino acid sequence of SEQ ID NO: 105-126, or containing one to three variants thereof with substitutions, deletions, or insertions; (e) CDR-L2, comprising any amino acid sequence of SEQ ID NO: 129-143, or containing one to three variants thereof with substitutions, deletions, or insertions; and (f) CDR-L3 comprises any amino acid sequence of SEQ ID NO: 146-166, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions. In some embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are selected from any combination provided in Table 9.

[0171] Table 9: Exemplary Combinations of CDRs

[0172]

[0173] IgA antibody modification

[0174] The anti-EGFR IgA antibodies described herein comprise one or more amino acid insertions, substitutions, and / or deletions. In some embodiments, the anti-EGFR IgA antibodies disclosed herein are conjugated to one or more therapeutic agents.

[0175] In some embodiments, the amino acid numbering of the anti-EGFR IgA antibody described herein is based on the unique numbering of the C-domain and C-like domain by IMGT (as disclosed in "IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains." DevComp Immunol. 2005;29(3):185-203, the entire contents of which are incorporated herein by reference). In some embodiments, the amino acid modifications disclosed herein are relative to amino acid residues at selected positions in the corresponding WT IgA heavy chain constant region (e.g., the WT IgA2 heavy chain constant region) containing the amino acid sequence listed in SEQ ID NO: 1.

[0176] In some embodiments, the anti-EGFR IgA antibody disclosed herein contains the deletion of at least four glycosylation sites within the constant region. In some embodiments, the anti-EGFR IgA antibody disclosed herein contains the deletion of at least three N-linked glycosylation sites within the antibody constant region. In some embodiments, the anti-EGFR IgA antibody contains the deletion of at least three N-linked glycosylation sites and at least one O-linked glycosylation site within the antibody constant region. In some embodiments, the anti-EGFR IgA antibody or functional fragment disclosed herein contains one or more modifications within the IgA heavy chain constant region corresponding to the modifications disclosed in Table 10. In some embodiments, the anti-EGFR IgA antibody or functional fragment disclosed herein contains one or more modifications within the IgA1 heavy chain constant region corresponding to the modifications in Table 10. In some embodiments, the anti-EGFR IgA antibody or functional fragment disclosed herein contains one or more modifications within the IgA2 heavy chain constant region corresponding to the modifications in Table 10. In some embodiments, one or more modifications are made to amino acid residues located in the CH1, CH2, and / or CH3 domains of the IgA1 heavy chain constant region.

[0177] In some embodiments, the anti-EGFR IgA antibody includes a missing tail. In some embodiments, the anti-EGFR IgA antibody includes one or more modifications, wherein said one or more modifications are located in one or more amino acid residues within the CH1, CH2, and / or CH3 domains of the IgA2 heavy chain constant region. In some embodiments, the IgA CH1 domain includes an amino acid sequence having at least about 80%, 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the IgA CH2 domain includes an amino acid sequence having at least about 80%, 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the IgA CH3 domain includes an amino acid sequence having at least about 80%, 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the anti-EGFR IgA antibody (e.g., IgA2 antibody) or its functional fragments disclosed herein contain the deletion of C-terminal amino acids 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, and 3-4. In some embodiments, the C-terminal amino acids include amino acids 131-148 of the IgA2 antibody, numbered according to the IMGT protocol.

[0178] In some embodiments, the anti-EGFR IgA antibody is an anti-EGFR IgA2 antibody. In some embodiments, the IgA2 antibody contains a deletion of amino acids 131-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 147-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 146-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 145-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 144-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 143-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 142-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 141-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 140-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 139-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 138-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 137-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 136-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 135-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 134-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 133-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids 132-148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acids P131-Y148, numbered according to the IMGT protocol.

[0179] In some embodiments, the anti-EGFR IgA antibody contains a mutation in the C-terminal asparagine (N) amino acid. In some embodiments, this mutation is a non-conserved amino acid substitution. In some embodiments, the mutation deletes the glycosylation site of the C-terminal asparagine (N) amino acid of IgA. In some embodiments, the IgA2 antibody contains the N135 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved N135 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains the N135Q mutation, numbered according to the IMGT protocol.

[0180] In some embodiments, the anti-EGFR IgA antibody or its functional fragment thereof disclosed herein exhibits reduced glycosylation compared to the corresponding WT IgA. From a pharmaceutical perspective, more glycosylation sites can lead to batch-to-batch variability in the product, which may affect safety and efficacy. Therefore, in some embodiments, the engineered anti-EGFR IgA antibody described herein has reduced glycosylation heterogeneity. Alternatively, in some embodiments, the engineered anti-EGFR IgA antibody has a reduced glycosylation profile (…). Figures 4A-4D In some embodiments, glycosylation reduction is achieved by modifying amino acid residues near or within naturally occurring glycosylation motifs or naturally occurring glycosylation sites (e.g., N-linked glycosylation sites containing amino acid sequences NXT or NXS). In some embodiments, the anti-EGFR IgA antibodies disclosed herein exhibit glycosylation reduction of at least about 2%, at least 5%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 50%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or more relative to the corresponding WT IgA. In some embodiments, the antibodies or functional fragments disclosed herein are partially glycosylated relative to the corresponding WT IgA antibodies, for example, less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or lower. In some embodiments, the anti-EGFR IgA antibodies disclosed herein or their functional fragments are fully glycosylated relative to the corresponding WT IgA antibodies.

[0181] In some embodiments, the anti-EGFR IgA antibody or functional fragment thereof disclosed herein includes modification with at least two naturally occurring glycosylation sites in the constant region of the IgA heavy chain. In some embodiments, the anti-EGFR IgA antibody or functional fragment thereof disclosed herein includes modification with at least three naturally occurring glycosylation sites in the constant region of the IgA heavy chain. In some embodiments, the anti-EGFR IgA antibody or functional fragment thereof disclosed herein includes modification with at least four naturally occurring glycosylation sites. In some embodiments, the glycosylation sites include N-linked glycosylation sites. In some embodiments, the glycosylation sites include naturally occurring asparagine residues. In some embodiments, the glycosylation sites are located in the CH2, CH3, and / or CH1 domains. In some embodiments, the IgA heavy chain constant region comprises modifications located at CH1-N45.2 or P124, CH2-N20, L21, T22, C92, N120, I121 or T122, CH3-N135, C147 or Y148, or combinations thereof, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions, wherein the amino acid substitutions are CH1-N45.2G, N45.2A or P124R, CH2-N20G, N20Q, N20T, L21I, T22S, C92S, N120T, I121L or T122S, CH3-N135Q, C147 deletion or Y148 deletion, or combinations thereof, numbered according to the IMGT scheme. In some embodiments, this document provides non-glycosylated antibodies or functional fragments thereof. In some embodiments, the non-glycosylated antibody comprises modifications at all four naturally occurring glycosylation sites in the constant region of the IgA2 heavy chain. In some embodiments, the non-glycosylated antibody provided herein comprises modifications at the following residues: CH1 - N45.2 and P124, CH2 - N20, L21, T22, C92, N120, I121, and T122, and CH3 - N135, C147, and Y148, numbered according to the IMGT protocol. In some embodiments, the non-glycosylated antibody provided herein comprises modifications at the following residues: CH1 - N45.2 and P124, CH2 - N20, C92, N120, I121, or T122, and CH3 - N135, C147, and Y148, numbered according to the IMGT protocol.

[0182] In some embodiments, the non-glycosylated antibodies provided herein comprise modifications at the following residues: CH1 - N45.2 or P124, CH2 - N20, L21, T22, C92, N120, I121 or T122, or deletion of C-terminal CH3 tail residues P131-Y148, numbered according to the IMGT protocol. In some embodiments, the non-glycosylated antibodies provided herein comprise modifications at the following residues: CH1 - N45.2 and P124, CH2 - N20, L21, T22, C92, N120, I121 and T122, and deletion of C-terminal CH3 tail residues P131-Y148, numbered according to the IMGT protocol.

[0183] In some embodiments, the antibody or its functional fragment contains modifications to the CH1 domain, including N45.2 substitution or P124 substitution; modifications to the CH2 domain, including N20 substitution, L21 substitution, T22 substitution, C92 substitution, N120 substitution, I121 substitution or T122 substitution; or modifications to the CH3 domain, including H5 substitution, L7 substitution, P10 substitution, T22 substitution, L79 substitution, W81 substitution, A85.1 substitution, T86 substitution, I88 substitution, N135 substitution, C147 deletion, Y148 deletion or P131-Y148 deletion; numbering follows the IMGT protocol. In some embodiments, the antibody or its functional fragment comprises modifications to the CH1 domain, including N45.2 substitution selected from the group consisting of N45.2G and N45.2A, P124R substitution, or any combination thereof; modifications to the CH2 domain, including N20 substitution selected from the group consisting of N20G, N20Q, and N20T, L21I substitution, T22S substitution, C92S substitution, N120T substitution, I121L substitution, T122S substitution, or any combination thereof; or modifications to the CH3 domain, including H5 substitution selected from the group consisting of H5C, H5Y, H5F, H5M, and H5W, L7 substitution selected from the group consisting of L7F, L7Y, L7M, L7W, L7H, and L7I, P10C substitution, and T22 substitution. Replace T22 in the group consisting of V, T22I, T22L, and T22A; replace L79 in the group consisting of L79V, L79T, L79A, and L79I; replace W81 in the group consisting of W81T, W81L, W81A, W81V, and W81I; replace A85.1 in the group consisting of A85.1F, A85.1Y, A85.1M, A85.1W, and A85.1H; replace T86 in the group consisting of T86Y, T86F, T86M, T86W, and T86H; replace I88 in the group consisting of I88L, I88A, I88V, and I88T; replace N135Q; C147 is missing; Y148 is missing; or P131-Y148 is missing; numbering follows the IMGT scheme.

[0184] In some embodiments, the IgA heavy chain constant region comprises IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region contains the following mutations: N45.2G substitution in the CH1 domain, P124R substitution in the CH1 domain, C92S substitution in the CH2 domain, N120T substitution in the CH2 domain, I121L substitution in the CH2 domain, and T122S substitution in the CH2 domain, numbered according to the IMGT protocol, with each mutation corresponding to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, the IgA heavy chain constant region also contains the following mutations in the CH3 domain: N135Q substitution, C147 deletion, and Y148 deletion; or P131-Y148 deletion in the CH3 domain, numbered according to the IMGT protocol, corresponding to the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some implementations, the IgA heavy chain constant region also includes the following mutations in the CH2 domain: N20 substitution selected from the group consisting of N20G, N20Q and N20T; L21I substitution; and T22S substitution, numbered according to the IMGT scheme, each mutation corresponding to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO: 1.

[0185] In some embodiments, the antibody contains the N45.2 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved N45.2 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains N45.2G, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without the N45.2 amino acid mutation.

[0186] In some embodiments, the antibody contains a mutation in P124, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved mutation in P124, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains P124R, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without a mutation in the P124 amino acid. In some embodiments, the IgA2 antibody has increased stability compared to an IgA2 antibody without a mutation in the P124 amino acid. In some embodiments, increased stability refers to one or more of the following: reduced glycosylation, reduced aggregation, increased thermal stability, increased mechanical stability, or increased circulating half-life.

[0187] In some embodiments, the antibody described herein contains a mutation in C92, numbered according to the IMGT protocol. In some embodiments, the antibody contains a non-conserved mutation in C92, numbered according to the IMGT protocol. In some embodiments, the antibody contains C92S, numbered according to the IMGT protocol. In some embodiments, the antibody exhibits reduced aggregation compared to antibodies without mutations in the C92 amino acid. In some embodiments, the antibody exhibits reduced aggregation with serum proteins compared to antibodies without mutations in the C92 amino acid. In some embodiments, the antibody exhibits reduced aggregation in vitro or in vivo compared to antibodies without mutations in the C92 amino acid.

[0188] In some embodiments, the IgA2 antibody contains a mutation in C92, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved mutation in C92, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains C92S, numbered according to the IMGT protocol. In some embodiments, the aggregation of this IgA2 antibody is reduced compared to an IgA2 antibody without a mutation in C92 amino acids. In some embodiments, the aggregation of this IgA2 antibody with serum proteins is reduced compared to an IgA2 antibody without a mutation in C92 amino acids. In some embodiments, the aggregation of this IgA2 antibody is reduced in vitro or in vivo compared to an IgA2 antibody without a mutation in C92 amino acids.

[0189] In some embodiments, the antibody contains a mutation in N120, numbered according to the IMGT protocol. In some embodiments, the antibody contains a non-conserved mutation in N120, numbered according to the IMGT protocol. In some embodiments, the antibody contains N120T, numbered according to the IMGT protocol. In some embodiments, the antibody has a longer circulating half-life compared to IgA2 antibodies without mutations in the N120 amino acid.

[0190] In some embodiments, the antibody contains a mutation in I121, numbered according to the IMGT protocol. In some embodiments, the antibody contains a non-conserved mutation in I121, numbered according to the IMGT protocol. In some embodiments, the antibody contains I121L, numbered according to the IMGT protocol. In some embodiments, the antibody has a longer circulating half-life compared to IgA2 antibodies without mutations in the I121 amino acid.

[0191] In some embodiments, the antibody contains a mutation in T122, numbered according to the IMGT protocol. In some embodiments, the antibody contains a non-conserved mutation in T122, numbered according to the IMGT protocol. In some embodiments, the antibody contains T122S, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without a mutation in the T122 amino acid.

[0192] In some embodiments, the IgA2 antibody contains an N120 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved N120 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains N120T, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without an N120 amino acid mutation.

[0193] In some embodiments, the IgA2 antibody contains a mutation in I121, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved mutation in I121, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains I121L, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without a mutation in the I121 amino acid.

[0194] In some embodiments, the IgA2 antibody contains a T122 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved T122 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains T122S, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without a T122 amino acid mutation.

[0195] In some embodiments, the IgA2 antibody contains an N20 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved N20 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains N20G, N20Q, or N20T, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without an N20 amino acid mutation.

[0196] In some embodiments, the IgA2 antibody contains an L21 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved L21 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains L21I, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without an L21 amino acid mutation.

[0197] In some embodiments, the IgA2 antibody contains a T22 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved T22 mutation, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains T22S, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody without a T22S amino acid mutation.

[0198] In some embodiments, the IgA2 antibody contains a mutation in C147, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved mutation in C147, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of the amino acid C147, numbered according to the IMGT protocol. In some embodiments, the aggregation of this IgA2 antibody is reduced compared to IgA2 antibodies without a mutation in the C147 amino acid.

[0199] In some embodiments, the IgA2 antibody contains a mutation in Y148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a non-conserved mutation in Y148, numbered according to the IMGT protocol. In some embodiments, the IgA2 antibody contains a deletion of amino acid Y148, numbered according to the IMGT protocol.

[0200] In some embodiments, the anti-EGFR IgA antibody comprises one or more albumin-binding domains. In some embodiments, one or more albumin-binding domains are fused to the light or heavy chain of the IgA constant region. In some embodiments, one or more albumin-binding domains are fused to the heavy chain of the IgA constant region. In some embodiments, one or more albumin-binding domains are fused to the C-terminal region of the CH3 region of the heavy chain of the IgA constant region. In some embodiments, the IgA2 antibody has a longer circulating half-life compared to an IgA2 antibody that does not contain one or more albumin-binding domains. In some embodiments, the IgA2 antibody comprises one or more albumin-binding domains and has a circulating half-life of 1%, 5%, or 10% of that of the corresponding IgG antibody. In some embodiments, the IgA2 antibody comprises one or more albumin-binding domains and has a circulating half-life greater than that of the corresponding IgG antibody.

[0201] In some embodiments, one or more mutations or deletions result in an increase or decrease in the circulating half-life of the anti-EGFR IgA antibody. In some embodiments, one or more mutations or deletions result in an increase in the circulating half-life of the anti-EGFR IgA antibody. For example, one or more mutations can increase the serum half-life of the anti-EGFR IgA antibody in humans to up to 21 days or longer. Furthermore, one or more mutations can increase the serum half-life of the anti-EGFR IgA antibody in mice to up to 9 days or longer. In some embodiments, one or more mutations can increase the serum half-life of the anti-EGFR IgA antibody to a level comparable to that of immunoglobulin G (IgG) molecules. In some embodiments, one or more mutations or deletions result in a decrease in the circulating half-life of the anti-EGFR IgA antibody.

[0202] In some embodiments, one or more mutations can increase the serum half-life of the anti-EGFR IgA antibody by at least about 7 days to about 30 days or longer. In some embodiments, one or more mutations can increase the serum half-life of the anti-EGFR IgA antibody by at least about 7 days. In some embodiments, one or more mutations can increase the serum half-life of the anti-EGFR IgA antibody by up to about 30 days. In some embodiments, one or more mutations can increase the serum half-life of the anti-EGFR IgA antibody by at least about 7 days. The serum half-life of IgA antibodies increases by approximately 7 days to approximately 8 days, approximately 7 days to approximately 9 days, approximately 7 days to approximately 10 days, approximately 7 days to approximately 15 days, approximately 7 days to approximately 20 days, approximately 7 days to approximately 25 days, approximately 7 days to approximately 30 days, approximately 8 days to approximately 9 days, approximately 8 days to approximately 10 days, approximately 8 days to approximately 15 days, approximately 8 days to approximately 20 days, approximately 8 days to approximately 25 days, approximately 8 days to approximately 30 days, approximately 9 days to approximately 10 days, approximately 9 days to approximately 15 days, approximately 9 days to approximately 20 days, approximately 9 days to approximately 25 days, approximately 9 days to approximately 30 days, approximately 10 days to approximately 15 days, approximately 10 days to approximately 20 days, approximately 10 days to approximately 25 days, approximately 10 days to approximately 30 days, approximately 15 days to approximately 20 days, approximately 15 days to approximately 25 days, approximately 15 days to approximately 30 days, approximately 20 days to approximately 25 days, approximately 20 days to approximately 30 days, or approximately 25 days to approximately 30 days. In some embodiments, one or more mutations may increase the serum half-life of the anti-EGFR IgA antibody by about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days. Therefore, in some embodiments, the antibodies or functional fragments thereof disclosed herein have a longer circulating half-life compared to the corresponding WT IgA antibodies. In some embodiments, the circulating half-life of said antibodies is at least about 2%, 5%, 10%, 12%, 15%, 20%, 25%, 50%, 65%, 70%, 75%, 85%, 90%, 95%, 99%, 100%, 150%, and 200% higher than that of the corresponding WT IgA antibodies.

[0203] In some embodiments, anti-EGFR IgA antibodies exhibit increased stability. In some embodiments, one or more mutations (e.g., insertions, substitutions, and / or deletions) result in increased stability of the anti-EGFR IgA antibody compared to the corresponding IgA antibody that does not contain one or more mutations. Therefore, in some embodiments, one or more mutations (e.g., insertions, substitutions, and / or deletions) in the anti-EGFR IgA antibody result in one or more of the following: decreased glycosylation, reduced aggregation, increased thermal stability, increased mechanical stability, or increased cyclic half-life compared to the corresponding IgA antibody.

[0204] In some implementations, anti-EGFR IgA antibodies exhibit reduced aggregation. Antibody aggregation is a more common manifestation of physical instability. Protein aggregates are generally less active and, more importantly, have greater immunogenic potential due to epitope diversity and / or conformational changes. For example, in some implementations, anti-EGFR IgA antibodies bind to an epitope of an EGFR polypeptide or a variant thereof, the epitope containing any of the following EGFR amino acid residues: P349, F352, D355, P362, D355, Q384, P387, Q408, H409, F412, I438, K443, K465, I467, or S468. Immunoglobulin aggregates are known to cause severe renal failure and anaphylactic reactions, such as headache, fever, and chills. Therefore, reducing the aggregation of antibody therapeutics is advantageous. Furthermore, according to World Health Organization (WHO) standards, the aggregation level of commercially available intravenous immunoglobulin products is limited to less than 5%. In some implementations, one or more mutations lead to reduced aggregation. In some embodiments, one or more mutations and / or deletions result in reduced aggregation of anti-EGFR IgA antibodies compared to corresponding IgA antibodies that do not contain one or more mutations and / or one or more deletions. In some embodiments, the antibodies disclosed herein or their functional fragments exhibit reduced aggregation compared to corresponding WT IgA antibodies. In some embodiments, the aggregation of said antibodies is reduced by at least about 2%, at least 5%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 50%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, and at least 200% compared to corresponding WT IgA antibodies. In some embodiments, the antibodies disclosed herein or their functional fragments exhibit reduced aggregation with serum proteins compared to corresponding WT IgA antibodies. In some implementations, the aggregation of the antibody is reduced by at least 2%, at least 5%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 50%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, and at least 200% compared to the corresponding WT IgA antibody.

[0205] In some embodiments, the anti-EGFR IgA antibody provided herein has an aggregation level ranging from at least about 0.1% to at most about 5%. In some embodiments, the IgA antibody provided herein has an aggregation level ranging from at least about 0.1%. In some embodiments, the IgA antibody provided herein has an aggregation level ranging from at most about 5%. In some embodiments, the anti-EGFR IgA antibody provided herein has aggregation levels ranging from about 0.1% to about 0.5%, about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 3%, about 0.1% to about 4%, about 0.1% to about 5%, about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 0.5% to about 5%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 3% to about 4%, about 3% to about 5%, or about 4% to about 5%. In some embodiments, the IgA antibody provided herein has aggregation levels ranging from about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5%.

[0206] The anti-EGFR IgA antibodies disclosed herein may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- Carboxylic acids, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0207] Methods for performing amino acid substitutions or deletions include, for example, site-directed mutagenesis. Mutagenesis can be achieved by synthesizing oligonucleotides that have one or more modifications in the constant domain sequence of the antibody to be modified. The antibodies disclosed herein (e.g.) ，One or more modifications containing the IgA heavy chain constant region can be prepared using any mutagenesis method known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, rearrangement, etc. Site-specific mutagenesis allows the generation of mutants by using a specific oligonucleotide sequence encoding the DNA sequence of the desired mutation and a sufficient number of adjacent oligonucleotides that provide primer sequences of sufficient length and sequence complexity to form stable double strands on both sides of the deleted linker region. Typically, primers of about 17 to about 75 nucleotides or longer are preferred, wherein about 10 to about 25 or more residues on both sides of the sequence linker region are altered. Multiple such primers can be used to introduce various different mutations at one or more sites to construct mutant libraries.

[0208] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode target peptides. Gene synthesis can be performed using various techniques, such as multi-microchip-based techniques and similar methods, where oligonucleotides are synthesized and assembled on optically programmable microfluidic chips. Substitutions, deletions, and / or insertions of single or multiple amino acids can be performed and tested using mutagenesis, recombination, and / or rearrangement methods and subsequent screening procedures. Other methods available include error-prone PCR, phage display, and regional targeted mutagenesis.

[0209] Mutagenesis / rearrangement methods can be combined with high-throughput automated screening methods to detect the activity of clonal mutagenic peptides expressed in host cells. The mutagenic DNA molecules encoding the active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods enable the rapid determination of the importance of individual amino acid residues within the peptide.

[0210] Semi-synthetic gene construction is achieved by combining synthetic gene construction with / or site-directed mutagenesis, and / or random mutagenesis and / or rearrangement methods. A typical method for semi-synthetic construction utilizes synthetic polynucleotide fragments in conjunction with PCR technology. Specific regions of the gene can be synthesized de novo, other regions can be amplified using site-specific mutagenesis primers, and still others can be amplified using error-prone or non-error-prone PCR. Subsequently, the polynucleotide subsequences can be rearranged.

[0211] Other covalent modifications

[0212] In some embodiments, anti-EGFR IgA antibodies contain one or more covalent modifications. These modifications can be prepared by chemical synthesis or, where applicable, by enzymatic or chemical cleavage of the antibody. Other types of antibody covalent modifications are introduced into the molecule by reacting the target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chain or N-terminal or C-terminal residues.

[0213] Cysteine ​​residues most commonly react with haloacetic esters (and corresponding amines) such as chloroacetic acid or chloroacetamide to generate carboxymethyl or carboxamide methyl derivatives. Cysteine ​​residues are also derivatized by reactions with bromotrifluoroacetone, α-bromo-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0214] Histidine residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5–7.0 because this reagent is relatively specific to the histidine side chain. p-Bromobenzoylmethyl bromide can also be used; the reaction is preferably carried out in a 0.1 M sodium dimethylarsinate solution at pH 6.0. Lysidine residues and amino-terminal residues react with succinic acid or other carboxylic anhydrides. Derivatization with these reagents has the effect of reversing the charge of the lysidine residue. Other reagents suitable for derivatizing α-amino residues include imide esters such as methyl pyridinium imide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylates.

[0215] Arginyl residues are modified by reacting with one or more reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidinyl functional group, the derivatization of arginine residues requires alkaline conditions. Furthermore, these reagents can also react with the lysine group and the ε-amino group of arginine.

[0216] Specific modifications to tyrosyl residues are possible, with particular interest in introducing spectral labeling via reactions with aromatic diazo compounds or tetranitromethane. Most commonly, O-acetyltyrosyl substances and 3-nitro derivatives are generated using N-acetimidazole and tetranitromethane, respectively. Tyrosyl residues are iodinated with 125I or 131I to prepare labeled proteins for radioimmunoassay. Carboxyl side groups (aspartic or glutamic) are selectively modified by reacting with carbodiimides (RN. dbd.C.dbd.N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholino-4-ethyl)carbodiimide or 1-ethyl-3-(4-aza-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartic and glutamic residues are converted to asparagine and glutamine residues by reacting with ammonium ions.

[0217] Glutamine and asparagine residues are typically deamidated to yield the corresponding glutamine and asparagine residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated forms of these residues are within the scope of this invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the α-amino groups of the lysine, arginine, and histidine side chains, acetylation of the N-terminal amino group, and amidation of any C-terminal carboxyl group.

[0218] Another type of covalent modification involves chemically or enzymatically coupling glycosides to antibodies. The advantage of these methods is that they do not require antibody production in host cells with the ability to glycosylate for N- or O-linked glycosylation. Depending on the coupling method employed, the sugar can be linked to: (a) arginine and histidine; (b) a free carboxyl group; (c) a free thiol group, such as the thiol group of cysteine; (d) a free hydroxyl group, such as the hydroxyl group of serine, threonine, or hydroxyproline; (e) an aromatic residue, such as an aromatic residue of phenylalanine, tyrosine, or tryptophan; or (f) an amide group of glutamine.

[0219] The removal of any sugar moieties present on the antibody can be achieved chemically or enzymatically. Chemical deglycosylation requires exposing the antibody to the compound trifluoromethanesulfonic acid or its equivalent. This treatment results in the cleavage of most or all glycosyl groups, except for the linking sugar (N-acetylglucosamine or N-acetylglucosamine), while maintaining the integrity of the antibody. Enzymatic cleavage of sugar moieties on the antibody can be achieved using various endoglucosidases and exoglucosidases.

[0220] Another covalent modification of antibodies involves linking the antibody to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylene polyols, polyoxyethylene sorbitol, polyoxyethylene glucose, polyoxyethylene glycerol, polyoxyalkylene, or polysaccharide polymers (such as dextran).

[0221] IgA antibody target

[0222] The anti-EGFR IgA antibody described herein binds to EGFR expressed on target cells (e.g., cancer cells). In some embodiments, the target cells are human cells. In some embodiments, the EGFR is human EGFR.

[0223] EGFR is a transmembrane glycoprotein that promotes cell growth in a variety of normal and transformed tissues. This receptor has several natural ligands, including EGF and transforming growth factor-α. Ligand binding to the receptor stimulates cell proliferation. Blocking this interaction with antibodies targeting this receptor has inhibited tumor growth in vivo.

[0224] In some embodiments, the anti-EGFR IgA antibody or a functional fragment thereof includes one or more modifications to the IgA heavy chain constant region disclosed herein, while retaining the ability to specifically bind to an EGFR peptide (e.g., human EGFR). Peptides and encoding nucleic acid sequences of human and various animal EGFRs are publicly available, for example, from the NCBI website.

[0225] In some embodiments, the binding affinity (Kd) of the anti-EGFR IgA antibody to EGFR is less than 0.01 nM. In some embodiments, the binding affinity (Kd) of the anti-EGFR IgA antibody to EGFR is greater than about 1 µM or more. In some embodiments, the binding affinity (Kd) of the anti-EGFR IgA antibody to EGFR is in the range of 0.01 nM to 1 µM. In some embodiments, the binding affinity (Kd) of the anti-EGFR IgA antibody to EGFR is about 0.01 nM to about 800 nM, about 0.01 nM to about 500 nM, about 0.01 nM to about 300 nM, about 0.01 nM to about 100 nM, about 0.05 nM to about 800 nM, about 0.05 nM to about 500 nM, about 0.05 nM to about 300 nM, about 0.01 nM to about 100 nM, about 0.1 nM to about 800 nM, about 0.1 nM to about 500 nM, about 0.1 nM to about 300 nM, about 0.1 nM to about 100 nM, about 5 nM to about 800 nM, about 5 nM to about 500 nM, about 5 nM to about 300 nM, about 5 nM to about 100 nM, about 10 nM to about 800 nM. nM, about 10 nM to about 500 nM, about 10 nM to about 300 nM, or about 10 nM to about 100 nM. In some embodiments, the binding affinity (Kd) of the anti-EGFR IgA antibody to EGFR is about 0.01 nM, about 0.05 nM, about 0.1 nM, about 1 nM, about 10 nM, about 100 nM, or about 200 nM.

[0226] In some embodiments, the anti-EGFR IgA antibody described herein comprises a Fab domain and an Fc domain, wherein the Fab domain binds to human EGFR and the Fc domain binds to the FcαRI receptor on immune cells (e.g., neutrophils, macrophages, and eosinophils).

[0227] Immune effector function of anti-EGFR IgA antibodies

[0228] This article provides engineered anti-EGFR IgA antibodies whose heavy chain constant region has one or more modifications relative to the corresponding WT IgA antibody containing the WT heavy chain constant region. In some embodiments, the anti-EGFR IgA antibody or a functional fragment thereof binds to an Fcα receptor (FcαR) expressed on immune effector cells, such as the FcαR of human IgA. FcαR is present on immune effector cells, such as monocytes, macrophages, neutrophils, and other myeloid cells. FcαR is also present on postmyeloid cells, myeloid cells, promyelocytes, and some medulloblasts, such as cells derived from bone marrow. Furthermore, such receptors are also present on myeloid cell lines (e.g., U937, PLB985, and HL60 cells). Studies have shown that FcαR is also present on lymphocytes. Myeloid cell activation can increase FcαR expression. For example, stimulation of U937 and PLB985 cells with phorbol myristate acetate (PMA) can increase FcαR levels on the cell surface several times. Other agents that can increase FcαR surface levels include calcitriol, 1,25-dihydroxyvitamin D3, and interferon-γ (IFN-γ).

[0229] FcαR can interact with IgA1 and IgA2 in monomeric, dimeric, and polymeric forms. Therefore, anti-EGFRIgA antibodies or functional fragments thereof can trigger at least one Fc receptor-mediated immune effector cell function.

[0230] Immune effector cells are cells that participate in the effector phase of the immune response, distinct from the recognition and activation phases. Immune effector cells include lymphocytes (such as B cells and T cells, including cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Immune effector cells can phagocytose target antigens, target cells, or microorganisms. They can also lyse target cells or microorganisms.

[0231] In some implementations, engineered anti-EGFR IgA antibodies or functional fragments thereof bind to the low-affinity Fc receptor FcαRI (CD89) on immune effector cells. FcαRI primarily interacts with IgA via its Cα1 and Cα2 domains after antibody-antigen recognition. FcαRI is a 55-75 kDa type I transmembrane receptor composed of two extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic tail. It is expressed in myeloid cells, such as neutrophils, eosinophils, activated monocytes, granulocytes, dendritic cell subsets, Kupffer cells, and macrophages. The binding of engineered anti-EGFR IgA antibodies to FcαRI mediates various effector functions, such as phagocytosis, trogocytosis, oxidative burst, cytokine release, antigen presentation, and ADCC.

[0232] In some embodiments, the binding affinity of the anti-EGFR IgA antibody disclosed herein to the Fc receptor on immune effector cells is increased by at least about 2%, at least 5%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 50%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or more.

[0233] In some embodiments, the anti-EGFR IgA antibody or a functional fragment thereof exhibits enhanced effector function at least one compared to the corresponding WT IgA antibody or a functional fragment thereof or the corresponding WT IgG antibody. In some embodiments, the anti-EGFR IgA antibody induces complement-dependent cytotoxicity (CDC). In some embodiments, the anti-EGFR IgA antibody induces polymorphonuclear neutrophil (PMN)-mediated tumor cell lysis. In some embodiments, the IgA antibody triggers polymorphonuclear cell (PMN)-mediated ADCC more effectively than the IgG antibody. In some embodiments, the anti-EGFR IgA antibody induces programmed cell death (PCD) via a caspase-independent pathway. In some embodiments, the anti-EGFR IgA antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0234] In some implementations, IgA does not bind to B cells, T cells, platelets, and / or erythrocytes. For example, in some implementations, anti-EGFR IgA antibodies may have low immunogenicity.

[0235] ADCC

[0236] ADCC is a cell-mediated reaction in which antigen-nonspecific cytotoxic / immune effector cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to the surface of target cells, subsequently leading to the lysis (i.e., “killing”) of the target cells (e.g., cancer cells). The primary mediators can be natural killer (NK) cells and neutrophils. ADCC activity can be directly assessed using in vitro assays, such as a 51Cr release assay using peripheral blood mononuclear cells (PBMCs) and / or NK effector cells, as described in the examples provided herein. ADCC activity can be expressed as the antibody concentration that causes target cell lysis to reach the half-maximum lysis value. Therefore, in some embodiments, the concentration of the antibody of this disclosure or its antigen-binding functional fragment (at which the lysis level is the same as the half-maximum lysis level of the wild-type control) is at least 2, 3, 5, 10, 20, 50, or 100 times lower than the concentration of the wild-type control itself.

[0237] Neutrophils can be present in the EGFR-positive tumor microenvironment. Therefore, in some embodiments, the anti-EGFR IgA antibody described herein exhibits neutrophil-mediated ADCC. In some embodiments, the anti-EGFR IgA antibody has a stronger ability to recruit neutrophils for antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the corresponding IgG antibody. In some embodiments, the anti-EGFR IgA antibody requires a lower effector cell to target cell (E:T) ratio to achieve ADCC levels comparable to those of the corresponding IgG. In some embodiments, neutrophil-mediated ADCC involves antibody-mediated trogoptosis. This is a process in which neutrophils “gnaw” at the cancer cell membrane, leading to loss of membrane integrity and ultimately cell death. In some embodiments, neutrophils can induce direct cytotoxicity, particularly in antibody-mediated targeting, by releasing high levels of ROS or unloading granule contents. During degranulation, cytotoxic molecules released from primary, secondary, and tertiary granules can induce apoptotic elimination of cancer cells. In some implementations, neutrophils can mediate the killing of cancer antigen loss variants, which are a result of tumor evolution and often hinder immunotherapy. Furthermore, neutrophils recruited by anti-EGFR IgA can help recruit and activate other immune cells and stimulate adaptive anti-tumor immunity, thereby further promoting tumor cell clearance.

[0238] Furthermore, in some embodiments, the anti-EGFR IgA antibody or functional fragment thereof disclosed herein may exhibit a higher maximum target cell lysis rate compared to the corresponding wild-type IgA antibody or WT IgG antibody. For example, the maximum target cell lysis rate of the antibody or functional fragment thereof disclosed herein may be 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or more higher than that of the corresponding WT IgA antibody or the corresponding WT IgG antibody. In some embodiments, the antibody or functional fragment thereof disclosed herein may induce increased ADCC compared to the corresponding WT IgG antibody containing the IgG heavy chain constant region. In some embodiments, the ADCC is increased by at least 2%, at least 5%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 50%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, and at least 200% relative to the corresponding WT IgG antibody.

[0239] In some embodiments, tumors containing KRAS mutations are sensitive to ADCC-mediated cell killing induced by the anti-EGFR IgA antibody described herein. Effective induction of ADCC depends on the interaction between the Fc region of the therapeutic antibody bound to target cells and the Fc receptor on the surface of immune effector cells. In some embodiments, the anti-EGFR IgA antibody acts as an opsonin, subsequently recruiting neutrophils and mediating ADCC, thereby inhibiting tumor growth and reducing tumor volume.

[0240] In some implementations, anti-EGFR IgA antibodies can recruit NK cells and macrophages for cell killing via ADCC. In some implementations, NK cells, macrophages, and / or neutrophils recruited by engineered anti-EGFR IgA antibodies kill opsonized cancer cells through mechanisms involving cytokinesis, thereby leading to lytic / necrotic cell death.

[0241] CDC

[0242] The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule conjugated with the relevant antigen (e.g., an antibody). To assess complement activation, a CDC assay can be performed.

[0243] In some implementations, the anti-EGFR IgA antibody described herein can inhibit EGFR, thereby causing cell cycle arrest and apoptosis in cancer cells.

[0244] Biological distribution

[0245] In some embodiments, the anti-EGFR IgA antibody or its functional fragment disclosed herein exhibits increased biodistribution relative to the corresponding WTIgA antibody. Various methods can be used to assess the biodistribution in cells or tissues, including but not limited to: nuclear medicine, whole-body autoradiography, microautoradiography, phosphorus imaging, cryo-imaging, nano-secondary ion mass spectrometry (nanoSIMS), matrix-assisted laser desorption / sorption spectroscopy (MALDI-MS), X-ray imaging, magnetic resonance imaging (MRI), computed tomography (CT), micro-ultrasound single-photon emission computed tomography (SPECT), positron emission tomography (PET), etc. Increased biodistribution of the antibody at the target site includes increases of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or more compared to the mediator or WT IgA antibody.

[0246] Antibody preparation methods

[0247] In some embodiments, the anti-EGFR IgA antibody is prepared in host cells. In some embodiments, the host cell is a CHO cell. In some embodiments, the host cell is an SP20 cell. In some embodiments, the host cell is a HEK 239 host cell. In some embodiments, the HEK 293 host cell is a HEK 293 F cell. In some embodiments, the antibody or its functional antigen-binding fragment is isolated from the host cell. In some embodiments, the antibody or its functional antigen-binding fragment is prepared in a cell-free system. According to conventional techniques, isolated nucleic acid molecules encoding antibodies, fragments, or polypeptides of this disclosure can be recombined with vector DNA (e.g., an expression vector), including blunt or staggered terminal ends for ligation, restriction enzyme digestion to provide suitable ends, filling with sticky ends as needed, alkaline phosphatase treatment to avoid unnecessary ligation, and ligation using a suitable ligase. Such manipulation techniques can be used to construct nucleic acid sequences encoding antibody molecules or their antigen-binding regions. Therefore, this disclosure provides a vector or expression vector comprising the isolated nucleic acid described herein. In one embodiment, the nucleic acid encoding the light chain and the nucleic acid encoding the heavy chain are isolated by the methods described above. In one implementation, separate nucleic acids encoding the light chain and the heavy chain can be inserted into different expression plasmids or co-inserted into the same plasmid, provided that they are each under appropriate promoter and translational control.

[0248] After the isolated nucleic acid molecules are placed in an expression vector, they can be transfected into host cells, such as *E. coli* cells, simian COS cells, human embryonic kidney 293 cells (e.g., 293E cells), Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins, thereby enabling the synthesis of antibodies or their functional antigen-binding fragments in the recombinant host cells. Any available vector can be used. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selectable marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0249] Isolated nucleic acid molecules are operatively ligated to expression control sequences in vector DNA. Expression control sequences are DNA sequences necessary for the expression of operatively ligated coding sequences in a specific host organism. Control sequences suitable for prokaryotes include, for example, promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0250] Cells, cell lines, and cell cultures are generally used interchangeably, and all such names herein include progeny. Transformants and transformed cells include primary guest cells and their derived cultures, regardless of passage number. Furthermore, it should be understood that the DNA content of all progeny may not be entirely identical due to intentional or unintentional mutations. Mutant progeny with the same function or biological activity as those screened in the initially transformed cells are also included. If a different name is required, the context will clearly indicate this. In another embodiment, a suitable coding nucleic acid sequence can be designed based on a universal codon table and the known amino acid sequence of the target immunoglobulin.

[0251] Amino acid sequence variants of desired antibodies can be prepared by introducing appropriate nucleotide alterations into the coding DNA or through peptide synthesis. These variants include, for example, deletions and / or insertions and / or substitutions of residues in the antibody's amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct possesses the desired properties. Amino acid alterations can also modify the post-translational modification process of monoclonal antibodies, human antibodies, humanized antibodies, or variant antibodies, such as changing the number or location of glycosylation sites.

[0252] Nucleic acid molecules encoding antibody amino acid sequence variants can be prepared by a variety of methods. These methods include, but are not limited to, isolation from natural sources (for naturally occurring amino acid sequence variants) or preparation via oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant antibodies.

[0253] This disclosure also provides isolated nucleic acid molecules (as described herein) encoding antibodies or functional antigen-binding fragments thereof, which are optionally operably linked to host cells, vectors, and host cell-recognized regulatory control sequences containing said nucleic acids, and recombinant techniques for producing said antibodies, which may include culturing said host cells to express said nucleic acids, and optionally recovering said antibodies from said host cell cultures or culture media.

[0254] To recombinantly produce antibodies or their functional antigen-binding fragments, nucleic acid molecules encoding them can be isolated and inserted into a reproducible vector for further cloning (DNA amplification) or expression. Therefore, isolated antibodies or their functional antigen-binding fragments are provided herein. In some embodiments, the antibodies or their functional antigen-binding fragments disclosed herein may be recombinant antibodies. In some embodiments, the glycosylation pattern of the recombinant antibody differs from that of antibodies with the same sequence in nature. In one embodiment, the recombinant antibody is expressed in mammalian host cells that are not human host cells. It is noteworthy that different mammalian host cells have unique glycosylation patterns.

[0255] In some embodiments, the antibody or its functional antigen-binding fragment of the present disclosure is synthetic. The polypeptide of the present disclosure can be purified by isolation / purification methods.

[0256] In one aspect, this document provides a host cell comprising the isolated nucleic acid molecules described herein, or a vector comprising the isolated nucleic acid molecules described herein. The vector may be a cloning vector or an expression vector. Suitable host cells for cloning or expressing the DNA in the vector herein are prokaryotic, yeast, or higher eukaryotic cells as described above. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive bacteria, such as Enterobacteriaceae, including Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella; as well as Bacilli bacteria (e.g., B. subtilis and B. licheniformis), such as B. licheniformis 41P. Pseudomonas bacteria (such as *Pseudomonas aeruginosa*) and Streptomyces bacteria. A preferred *E. coli* cloning host is *E. coli* 294 (ATCC 31446), but other strains, such as *E. coli* B, *E. coli* Xl 776 (ATCC 31537), and *E. coli* W3110 (ATCC 27325), are also suitable. These examples are for reference only and are not intended to be limiting.

[0257] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are readily available and applicable to this study, such as *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces*, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12424), *Kluyveromyces bulgaricus* (ATCC 16045), *Kluyveromyces wickeramii* (ATCC 24178), *Kluyveromyces waltii* (ATCC 56500), *Kluyveromyces drosophilarum* (ATCC 36906), *Kluyveromyces thermotolerans*, and *Kluyveromyces marxianus*; *Yarrowia* (EP 402226); and *Pichia pastors* (EP 402226). 183070); Candida; Trichoderma reesia (EP 244234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as hosts of Neurospora, Penicillium, Tolypocladium and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.

[0258] Examples of invertebrate cells include plant and insect cells. Various baculovirus strains and variants, as well as corresponding susceptible insect host cells from the host, have been identified, such as those from the fall armyworm (Spodoptera frugiperda, caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Several viral strains for transfection are publicly available, such as the L-1 variant of the California silver-striped moth (Autographa californica) NPV and the Bm-5 strain of the silkworm NP7 strain. These viruses can be used as the viruses described in this invention, and are particularly suitable for transfection of fall armyworm cells.

[0259] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, duckweed, and other plant cells can also serve as hosts. However, vertebrate cells have generated the greatest interest, and the culture and proliferation of vertebrate cells (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary / -DHFR; and SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (subclones for growth in suspension culture of 293 or 293 cells); young hamster kidney cells (BHK, ATCC CCL 10); mouse Support cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VER0-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); and human lung cells (W138, ATCC CCL 1442). 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; and human hepatocellular carcinoma cell line (Hep G2).

[0260] Host cells, after being transformed or transfected with the aforementioned expression or cloning vectors, are used for antibody production and cultured in conventional culture media, which need to be appropriately adjusted to induce promoters, screen transformants, or amplify genes encoding target sequences. Furthermore, novel vectors and transfected cell lines containing multiple copies of transcription units separated by selectable markers are particularly useful and preferred for antibody expression, as described herein.

[0261] For the transfection and production of chimeric, humanized, or recombinant human antibodies using the expression vectors described herein, the recipient cell line can be myeloma cells. Myeloma cells are capable of synthesizing, assembling, and secreting immunoglobulins encoded by the transfected immunoglobulin nucleic acid sequence and possess the mechanism for immunoglobulin glycosylation. For example, in some embodiments, the recipient cell is a recombinant Ig-producing myeloma cell line SP2 / 0 (ATCC #CRL 8287). SP2 / 0 cells produce only immunoglobulins encoded by the transfected gene. Myeloma cells can be grown in cell culture or in the peritoneum of mice to obtain the secreted immunoglobulins from the ascites fluid. Other suitable recipient cells include lymphoid cells, such as human or non-human B lymphocytes, human or non-human hybridoma cells, or interspecies heterologous hybridoma cells. The expression vectors described in this article, which carry chimeric, humanized, or composite human antibody constructs or antibody peptides, can be introduced into suitable host cells through a variety of appropriate methods, including transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and biochemical methods such as the application of polycations (e.g., diethylaminoethyl (DEAE) dextran), as well as mechanical methods such as electroporation, direct microinjection, and microbulb bombardment.

[0262] Compared to bacteria, yeast has certain advantages in the production of immunoglobulin H and L chains. Yeast undergoes post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies utilizing strong promoter sequences and high-copy-number plasmids exist for the production of desired proteins in yeast. Yeast recognizes the leader sequence of cloned mammalian gene products and secretes peptides with the leader sequence (i.e., propeptides). Yeast gene expression systems can be routinely used to evaluate the production, secretion, and stability of antibody peptides or their functional antigen-binding fragments, as well as assembled chimeric, humanized, or composite human antibodies, their functional fragments, and regions. A range of yeast gene expression systems can be utilized by introducing promoter and terminator elements from active expression genes encoding glycolytic enzymes, which produce large quantities of glycolytic enzymes when yeast is grown in glucose-rich media. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, promoter and terminator signals from phosphoglycerate kinase (PGK) genes can be utilized. Many methods can be employed to evaluate the optimal expression plasmids for expressing cloned immunoglobulin cDNA in yeast. In some embodiments, the plasmid contains a non-integrating plasmid. In some implementations, the plasmid can transiently express cDNA.

[0263] Bacterial strains can also serve as hosts for the production of the antibody molecules or their functional fragments described herein. These include *Escherichia coli* K12 strains (such as *E. coli* W3110 (ATCC 27325)), *Bacillus* species, *Enterobacteria* species (such as *Salmonella typhimurium* or *Serratia marcescens*), and various *Pseudomonas* species. Plasmid vectors containing replicons and control sequences (derived from species compatible with the host cell) are used in conjunction with these bacterial hosts. The vectors carry replication sites and specific genes capable of phenotypic selection in transformed cells. Various methods can be employed to evaluate the ability of expression plasmids to produce chimeric, humanized, or recombinant humanized antibodies and their functional fragments encoded by cloned immunoglobulin cDNA or CDR in bacteria.

[0264] Host mammalian cells can be cultured in vitro or in vivo. Mammalian cells can perform post-translational modifications on immunoglobulin molecules, including removal of leader peptides, folding and assembly of H and L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins. In addition to the lymphoid-derived cells described above, other mammalian cells that can be used as hosts for antibody protein production include fibroblast-derived cells, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Exemplary eukaryotic cells that can be used to express peptides include, but are not limited to: COS cells (including COS 7 cells), 293 cells (including 293-6E cells), CHO cells (including CHO-S and DG44 cells), PER.C6.RTM. cells (Crucell), and NSO cells. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications on variable heavy chains and / or variable light chains. For example, in some embodiments, peptides produced by CHO cells have a higher level of sialylation than the same peptides produced by 293 cells.

[0265] In some embodiments, the antibodies disclosed herein or polypeptides of their functional antigen-binding fragments can be produced in animals engineered or transfected with one or more nucleic acid molecules encoding the polypeptide by any suitable method. 。

[0266] Currently, various vector systems are available for expressing H-chain and L-chain nucleic acid sequences in mammalian cells. Complete H2L2 antibodies can be obtained using different methods. As mentioned above, H-chains and L-chains can be co-expressed in the same cell, achieving intracellular binding and ligation of the H-chain and L-chain to form a complete tetrameric H2L2 antibody and / or a functional antigen-binding peptide fragment. Co-expression can be achieved by using the same or different plasmids in the same host cell. The genes for the H-chain, L-chain, and / or CDR3 region peptide can be placed in the same plasmid, and then the plasmid can be transfected into cells to directly select cells expressing both chains. Alternatively, cells can be transfected first with a plasmid encoding one chain (e.g., the L-chain), and then the resulting cell line can be transfected with an H-chain plasmid containing a second selection marker. Cell lines that produce functional antigen-binding peptide fragments and / or H2L2 molecules through any of the above pathways can be transfected with plasmids encoding additional copies of peptides, H chains, L chains, or H chains plus L chains, and combined with other selection markers, thereby generating cell lines with enhanced properties, such as higher yields of assembled H2L2 antibody molecules or enhanced stability of transfected cell lines.

[0267] In some respects, this document provides methods and systems for producing humanized antibodies, the preparation of which includes: maintaining a host transformed with a first expression vector encoding a humanized antibody light chain and a second expression vector encoding a humanized antibody heavy chain under conditions that allow each chain to be expressed, and isolating the humanized antibody assembled from these expression chains. The first and second expression vectors may be the same vector. This document also provides a DNA sequence encoding the humanized antibody light or heavy chain; an expression vector containing the DNA sequence; and a host cell transformed with the expression vector. Those skilled in the art can utilize the nucleic acid sequences and information provided herein to produce humanized antibodies without extensive experimentation. In one method, the humanization of a monoclonal antibody comprises four general steps. These steps are: (1) determining the nucleotide and predicted amino acid sequences of the variable domains of the starting antibody light and heavy chains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use in the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody.

[0268] purification

[0269] The contaminant components in its natural environment refer to substances that may interfere with the diagnostic or therapeutic use of the peptide, and may include enzymes, hormones, and other protein or non-protein components. In some embodiments, the antibody or its functional antigen-binding fragment disclosed herein may be purified by suitable methods. In a preferred embodiment, the peptide is purified to: (1) greater than 95% (by weight), most preferably greater than 99% (by weight), when determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence when using a rotary cup sequencer; or (3) showing homogeneity when passed by SDS-PAGE under reducing or non-reducing conditions and stained with Coomassie brilliant blue or preferably silver staining. The isolated antibody contains the peptide in situ within recombinant cells, since at least one component of the peptide's natural environment will be absent. However, typically, the isolated peptide is prepared by at least one purification step. In one aspect, a purified antibody or functional antigen-binding fragment, as provided herein, is disclosed.

[0270] After expression, the complete antibody, its dimer, single light and heavy chains, or other immunoglobulin forms described in this invention can be recovered and purified by chromatographic methods such as immunoadsorption or immunoaffinity chromatography, high-performance liquid chromatography (HPLC), ammonium sulfate precipitation, gel electrophoresis, or any combination of these methods. Essentially pure immunoglobulins with at least about 90% to 95% homogeneity are advantageous, for example, immunoglobulins with 98% to 99% or higher homogeneity, especially suitable for pharmaceutical applications. When using recombinant technology, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium, including from microbial cultures. If antibodies are produced intracellularly, the first step is to remove particulate debris, such as host cell fragments or lysed fragments, for example by centrifugation or ultrafiltration.

[0271] Antibody compositions isolated from microbial or mammalian cells can be purified using techniques such as hydroxyapatite chromatography, cation exchange chromatography, avian exchange chromatography, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fe domain present in the antibody. Protein A can be used to purify antibodies based on human Y1, Y2, or Y4 heavy chains. Protein G is recommended for all mouse isotypes and human Y3. Agarose is the most commonly used matrix for affinity ligand attachment, but other matrices can also be used. Mechanically stable matrices, such as controlled-pore glass or poly(styrene-divinylbenzene), allow for faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, purification can be performed using resins that specifically bind to the CH3 domain. Depending on the antibody to be recovered, other protein purification techniques can also be employed, such as ion exchange column fractionation, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin bead chromatography, anion or cation exchange resin chromatography (e.g., polyaspartic acid column), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation. After purification (partial purification or purification to the desired homogeneity), humanized antibodies or composite human antibodies can be used for treatment or development and for conducting detection procedures, immunofluorescence staining, etc.

[0272] This document discloses the functional activity of an antibody or functional antigen-binding fragment. Such functional activity includes biological activity and the ability to bind to cancer cell antigens. Furthermore, a functionally active peptide is defined as a peptide exhibiting activity similar to, but not necessarily identical to, the activity measured in a specific assay (e.g., bioassay) of the antibody described herein (including mature forms), regardless of the presence of dose dependence. If dose dependence exists, its dose dependence need not be identical to that of the disclosed antibody, but should be substantially similar to the dose dependence of the antibody at a given activity compared to the antibody described herein (i.e., the candidate peptide will exhibit higher activity relative to the antibody described herein, or a reduction in activity not exceeding about 25-fold, about 10-fold, or about 3-fold).

[0273] Nucleic acid molecules encoding antibodies

[0274] Using the information provided herein, such as the nucleic acid and amino acid sequences of antibodies, those skilled in the art can readily obtain nucleic acid molecules encoding antibodies or functional antigen-binding fragments thereof. The nucleic acid molecules disclosed herein may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or synthesis, or any combination thereof. DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand (also known as a sense strand) or an antisense strand (also known as an antisense strand).

[0275] Nucleic acids can be present in intact cells, cell lysates, or in partially or substantially purified forms. Nucleic acid molecules are isolated or substantially purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) using standard techniques (including, but not limited to, alkali / SDS treatment, CsCl strip centrifugation, column chromatography, agarose gel electrophoresis, etc.). Nucleic acids according to at least some embodiments of this disclosure can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0276] Another aspect of this disclosure relates to nucleic acid molecules comprising a nucleic acid sequence encoding an antibody polypeptide or a functional fragment thereof or a functional antigen-binding fragment thereof as described herein. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a modified IgA heavy chain constant region. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding an antibody light chain variable region polypeptide.

[0277] Once the DNA fragments encoding the VH and VL segments are obtained, these fragments can be further modified using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these modifications, the DNA fragment encoding VL or VH can be operatively ligated to another DNA fragment encoding a different protein (e.g., an antibody constant region or a flexible linker). Isolated DNA encoding the VH region can be operatively ligated to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3) to convert it into a full-length heavy chain gene. DNA fragments containing human heavy chain constant region genes can be obtained via standard PCR amplification. The heavy chain constant region can be either IgA1 or IgA2. For Fab fragment heavy chain genes, the DNA encoding VH can be operatively ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0278] By operatively linking DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). DNA fragments containing the human light chain constant region can be obtained by standard PCR amplification. The light chain constant region can be either a κ or λ constant region, but a κ constant region is preferred.

[0279] To create the scFv gene, a DNA fragment encoding VH and VL is operatively linked to another fragment encoding a flexible adapter (e.g., encoding the amino acid sequence (Gly-4-Ser)3 (SEQ ID NO: 220)), such that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are linked by a flexible adapter.

[0280] Nucleic acid molecules isolated from this disclosure may include: nucleic acid molecules containing open reading frames (ORFs) that optionally have one or more introns, such as, but not limited to, at least one specific portion of at least one CDR, such as CDR1, CDR2 and / or CDR3 of at least one light chain; nucleic acid molecules containing the coding sequence or variable region (e.g., light chain variable region) of the cancer-associated antibodies disclosed herein; and nucleic acid molecules containing nucleotide sequences substantially different from the above-described nucleic acid molecules, but which, due to the degeneracy of the genetic code, still at least encode the antibodies described herein or their functional antigen-binding fragments.

[0281] This document provides nucleic acid molecules comprising nucleic acid sequences encoding one or more chains of an antibody. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an antibody heavy chain or a light chain. In some embodiments, the nucleic acid molecule comprises both a nucleic acid sequence encoding an antibody heavy chain and a nucleic acid sequence encoding an antibody light chain. In some embodiments, a first nucleic acid molecule comprises a first nucleic acid sequence encoding a heavy chain, and a second nucleic acid molecule comprises a second nucleic acid sequence encoding a light chain.

[0282] In some implementations, the heavy and light chains can be expressed from a single nucleic acid molecule or from two separate nucleic acid molecules, forming two separate polypeptides. In some implementations, such as when the antibody is scFv, a single nucleic acid sequence encodes a single polypeptide containing a heavy and light chain linked together.

[0283] In some embodiments, the nucleic acid sequence encoding the antibody heavy or light chain disclosed herein comprises a nucleic acid sequence encoding at least one CDR provided herein. In some embodiments, the nucleic acid sequence encoding the antibody heavy or light chain disclosed herein comprises a sequence encoding at least three CDRs provided herein. In some embodiments, the nucleic acid sequence encoding the antibody heavy or light chain comprises a sequence encoding at least six CDRs provided herein. In some embodiments, the nucleic acid sequence encoding the antibody heavy or light chain comprises a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain after translation. The leader sequence may be a native heavy or light chain leader sequence or another heterologous leader sequence. The leader sequence may be cleaved as the polypeptide is exported from mammalian cells to form a mature protein. The leader sequence may be native or synthetic and may be heterologous or homologous to the protein to which it is linked.

[0284] In some embodiments, a nucleic acid molecule refers to a nucleic acid molecule encoding any amino acid sequence of the variable light chain and variable heavy chain in Tables 5 and 7 of this document. In some embodiments, a nucleic acid sequence is a sequence having at least 80% identity with a nucleic acid encoding any amino acid sequence of the variable light chain and variable heavy chain in Tables 5 and 7 of this document, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity. In some embodiments, a nucleic acid is a nucleic acid that hybridizes with any one or more nucleic acid sequences provided herein. In some embodiments, hybridization is performed under mild conditions. In some embodiments, hybridization is performed under very stringent conditions, for example: at least about 6X SSC and 1% SDS, 65°C, first washing with 0.1X SSC containing about 20% (v / v) formamide at about 42°C for 10 minutes, then washing with 0.2X SSC and 0.1% SDS at 65°C.

[0285] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecules are placed in an expression vector suitable for expression in selected host cells.

[0286] This invention provides vectors comprising nucleic acid molecules encoding antibody or functional antigen-binding fragments described herein. This invention also provides vectors comprising nucleic acid molecules encoding heavy and / or light chains. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In one embodiment, the nucleic acid encoding the light chain and the nucleic acid encoding the heavy chain are separated separately by the methods described above. In one embodiment, the separated nucleic acids encoding the light chain and the heavy chain can be inserted into separate expression plasmids or co-inserted into the same plasmid, provided they are each under suitable promoter and translational control. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, for example, when the antibody is scFv.

[0287] In some embodiments, the first vector contains a nucleic acid molecule encoding a heavy chain, and the second vector contains a nucleic acid molecule encoding a light chain. In some embodiments, the first and second vectors are transfected into host cells in similar amounts (e.g., similar molar amounts or similar mass amounts). In some embodiments, the molar ratio or mass ratio of the first and second vectors transfected into host cells is 5:1 to 1:5. In some embodiments, the mass ratio of the vector encoding the heavy chain to the vector encoding the light chain is 1:1 to 1:5. In some embodiments, the mass ratio of the vector encoding the heavy chain to the vector encoding the light chain is 1:2. In some embodiments, a vector optimized for peptide expression in CHO or CHO-derived cells or NSO cells is selected.

[0288] In one aspect, this disclosure provides a method of treating or preventing cancer, comprising administering a nucleic acid molecule, wherein said nucleic acid molecule encodes a CDR3 region of VH, VL, or VH, or a CDR3 region of VL, or a functional antigen-binding fragment thereof. In some embodiments, the nucleic acid molecules disclosed herein are used for gene therapy. Gene therapy refers to treatment performed by administering an expressed or expressible nucleic acid to a subject. In this embodiment, said nucleic acid produces a protein it encodes that mediates a preventive or therapeutic effect. Any gene therapy method available in the art can be used according to the embodiments described herein.

[0289] Delivering therapeutic antibodies to appropriate cells can be achieved ex vivo, in situ, or in vivo via gene therapy, using any suitable method, including physical DNA transfer methods (e.g., liposomes or chemical treatment) or viral vectors (e.g., adenovirus, adeno-associated virus, or retrovirus). For example, for in vivo therapy, nucleic acids encoding the desired antibody (alone or in combination with a vector, liposome, or precipitate) can be directly injected into the subject; in some embodiments, they can be injected into the site where the antibody compound needs to be expressed. For ex vivo therapy, cells from the subject are removed, nucleic acids are introduced into these cells, and then the modified cells are either directly or, for example, encapsulated in a porous membrane to be implanted into the patient (subject) before being reinfused into the subject. Several techniques are currently available for introducing nucleic acids into living cells. These techniques vary depending on whether the nucleic acids are transferred to cells cultured in vitro or to cells of the target host in vivo. Techniques suitable for in vitro transfer of nucleic acids to mammalian cells include liposomes, electroporation, microinjection, cell fusion, DEAE-glucan, and calcium phosphate precipitation. Retroviruses are commonly used ex vivo nucleic acid delivery vectors.

[0290] Other in vivo nucleic acid transfer techniques include transfection using viral vectors (e.g., adenovirus, herpes simplex virus type I, or adeno-associated virus) and lipid-based systems. Nucleic acids and transfection reagents may optionally be bound to microparticles. Exemplary transfection reagents include calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, quaternary amphiphilic DOTMA ((dioleoyloxypropyl)trimethylammonium bromide, sold by GIBCO-BRL as Lipofectin); a lipophilic glutamate diester with a pendant trimethylammonium head; metabolizable parental lipids, such as cationic lipids dioctamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamine spermine (DPPES); metabolizable quaternary ammonium salts (DOTB, N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium sulfate (DOTAP), polyethyleneimine (PEI), dioleoyl ester, ChoTB, ChoSC, DOSC); 3β-[N-(N',N'-dimethylamino] [Ethylene]-carbamoyl]cholesterol (DC-Chol), a 1:1 mixture of dioleoylphosphatidylethanolamine (DOPE) / 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol-DC-Chol, spermine, spermidine, lipopolyamine, lipophilic polylysine (LPLL), [[(1,1,3,3-tetramethylbutyl)cresyloxy]ethoxy]ethyl]dimethylbenzylammonium hydroxide (DEBDA hydroxide) with excess phosphatidylcholine / cholesterol, a mixture of hexadecyltrimethylammonium bromide (CTAB) / DOPE, lipophilic glutamate diester (TMAG) with DOPE, CTAB, DEBDA, didecylammonium bromide (DDAB), and a mixture of stearamine with phosphatidylethanolamine, and oligogalactosyl lipids. Exemplary transfection enhancers that improve transfection efficiency include, for example, DEAE-glucan, polyglucan, lysosomal disrupting peptide, chondroitin-based proteoglycan, sulfated proteoglycan, polyethyleneimine, polylysine, integrin-binding peptide CYGGRGDTP (SEQ ID NO: 33), linear glucan nonaglycone, glycerol, cholesterol groups linked to the 3' end nucleoside linker of oligonucleotides, lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyllysophosphatidylcholine.

[0291] In some cases, it may be necessary to use an agent that directs the delivery of nucleic acid-containing vectors to target cells. Such agents include specific antibodies against cell surface membrane proteins on the target cells, or ligands against receptors on the target cells. When using liposomes, proteins that bind to cell surface membrane proteins associated with endocytosis can be used for targeting and / or promoting uptake. Examples of such proteins include: cell type-specific capsid proteins and fragments thereof, antibodies against proteins that are internalized during the cell cycle, and proteins that target intracellular localization and prolong intracellular half-life. In other embodiments, receptor-mediated endocytosis may also be used.

[0292] Immunoconjugates

[0293] The antibodies or functional antigen-binding fragments thereof disclosed herein may be administered in a "naked" or unconjugated form, or conjugated with a therapeutic agent. In one embodiment, the antibody or functional antigen-binding fragment thereof is used as a radiosensitizer. In these embodiments, the antibody or functional antigen-binding fragment is conjugated with a radiosensitizer. In some embodiments, the radiosensitizer is a molecule, preferably a low molecular weight molecule, administered to animals at a therapeutically effective dose to increase the sensitivity of cells to be radiosensitized to electromagnetic radiation and / or promote the treatment of diseases that can be treated with electromagnetic radiation. Diseases that can be treated with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancer cells. In some embodiments, the electromagnetic radiation and radiation include, but are not limited to, radiation with wavelengths from 10-20 to 100 meters. Preferred embodiments of this disclosure may employ, for example, the following electromagnetic radiation: gamma rays (10⁻²⁰ to 10⁻¹³ m), X-rays (10⁻¹² to 10⁻⁹ m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and microwave radiation (1 mm to 30 cm).

[0294] Radiation sensitizers are known to enhance the sensitivity of cancer cells to the toxic effects of electromagnetic radiation. Many current cancer treatment regimens utilize radiation sensitizers activated by X-ray electromagnetic radiation. Examples of X-ray-activated radiation sensitizers include, but are not limited to, the following: metronidazole, misotronidazole, desmethylmisotronidazole, pimonitrile, ethamidazole, nimozolomide C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and their therapeutically effective analogues and derivatives.

[0295] Photodynamic therapy (PDT) for cancer utilizes visible light as a radiation activator of photosensitizers. Examples of photodynamic radiation sensitizers include, but are not limited to: hematoporphyrin derivatives, benzoporphyrin derivatives, NPe6, styrinoporphyrin (SnET2), pheophytin-a, bacterial chlorophyll-a, naphthylphthalocyanine, phthalocyanine, zinc phthalocyanine, and their therapeutically effective analogues and derivatives.

[0296] In another embodiment, the antibody may be conjugated to a receptor (e.g., streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, and then unbound conjugates are removed from circulation using a scavenger, followed by the administration of a ligand (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide).

[0297] This disclosure also provides detectable labeled forms of the above-described antibodies or their antigen-binding. Antibodies can be detectably labeled using radioisotopes, affinity labels (e.g., biotin, avidin, etc.), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, etc.), fluorescent or luminescent or bioluminescent labels (e.g., FITC or rhodamine, etc.), paramagnetic atoms, etc.

[0298] Exemplary therapeutic immunoconjugates comprise antibodies described herein conjugated to cytotoxic agents (e.g., chemotherapeutic agents, toxins (e.g., bacterial, fungal, plant or animal-derived enzyme-active toxins or fragments thereof) or radioisotopes (i.e., radioconjugates)). Fusion proteins will be described in detail below.

[0299] In some embodiments, the antibodies disclosed herein and their functional antigen-binding fragments may be conjugated to therapeutic agents, such as chemotherapeutic cytotoxic agents, such as cell inhibitors or cell killers (e.g., paclitaxel, cytochalasin B, or diphtheria toxin, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine). Doxorubicin, daunorubicin, dihydroxyanthrone, mitoxantrone, sclerosomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., dichloromethyldiethylamine, thiotepa phenytobutylammonium), and thiotepa phenytobutylammonium. The disclosed antibodies include chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiaminoplatin(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., daunorubicin (formerly known as actinomycin), bleomycin, styrosinase, and amiodarone (AMC)), and antimitotic agents, thrombotic agents, or antiangiogenic agents or radiolabeled substances. In another embodiment, the antibodies and functional antigen-binding fragments disclosed herein are conjugated to a detectable substrate (e.g., an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent material, or a radiolabeled material). In some embodiments, the antibodies and functional antibody fragments disclosed herein are conjugated to toxins (e.g., enzyme-active toxins or fragments thereof derived from bacteria, fungi, plants, or animals), small molecules, siRNA, nanoparticles, targeting agents (e.g., microvesicles), or radioisotopes (i.e., radioconjugates). Such conjugates are referred to herein as “immunoconjugates.” These immunoconjugates can be used, for example, in diagnostic, therapeutic, or targeted approaches.

[0300] Available enzymatically active toxins and their fragments include diphtheria toxin A chain, the non-bound active fragment of diphtheria toxin, exotoxin A chain (from *Pseudomonas aeruginosa*), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, *Aleurites fordii* protein, dianthin protein, *Phytolaca americana* protein (PAPI, PAPII, and PAP-S), *Momordica charantia* inhibitor, curcin, crotin, *Sapaonaria officinalis* inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. Various radioisotopes can be used to produce radioconjugated antibodies. Examples include, but are not limited to, 212 Bi, 131 I, 131 In, 90Y, and 186Re.

[0301] The antibodies or their functional antigen-binding fragments described herein, conjugates with cytotoxic agents, can be prepared using any of a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyldithio)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imine esters (e.g., dimethyl diimide adipate HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazide compounds (e.g., bis(p-diazoniumbenzoyl)hexamethylenediamine), diazo compound derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and biactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared. Carbon-14 labeled l-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides with antibodies.

[0302] In other embodiments, the antibody or a portion thereof may be conjugated to a receptor (e.g., streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to a subject, unbound conjugates are then removed from circulation using a scavenger, and a ligand (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide) is administered. In some embodiments, the antibody or a functional fragment thereof may be conjugated to biotin, and the biotin-conjugated antibody or its functional antibody fragment may be further conjugated or linked to a streptavidin-bound or streptavidin-coated agent (e.g., streptavidin-coated microvesicles) for molecular imaging, such as angiogenesis.

[0303] Immunoconjugates can be prepared by indirectly conjugating a therapeutic agent to an antibody component. A common method involves reacting an antibody component with an oxidized sugar moiety with a carrier polymer having at least one free amino group and loaded with various drugs, toxins, chelating agents, boron additives, or other therapeutic agents. This reaction generates an initial Schiff base (imine) linker, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0304] The carrier polymer is preferably glycosaminoglycan or a polypeptide of at least 50 amino acid residues, but other substantially equivalent polymer carriers may also be used. Preferably, the final immunoconjugate is soluble in an aqueous solution (e.g., mammalian serum) to facilitate administration and effective targeted therapy. Therefore, the solubilizing function on the carrier polymer will improve the serum solubility of the final immunoconjugate. In particular, glycosaminoglycan is preferred.

[0305] Methods for preparing immunoconjugates with aminodextran carriers typically begin with dextran polymers, advantageously dextrans with an average molecular weight of about 10,000-100,000. The dextran is reacted with an oxidizing agent to achieve controlled oxidation of a portion of its sugar ring to generate an aldehyde group. Oxidation is conveniently achieved according to conventional procedures using saccharification chemicals (e.g., NaIO4).

[0306] The oxidized dextran is then reacted with a polyamine (preferably a diamine, and more preferably a monohydroxy diamine or polyhydroxy diamine). Suitable amines include ethylenediamine, propylenediamine or other similar polymethylene diamines, diethylenetriamine or similar polyamines, 1,3-diamino-2-hydroxypropane or other similar hydroxylated diamines or polyamines, etc. An excess of amine relative to the dextran aldehyde group is used to ensure that the aldehyde functional group is substantially completely converted into a Schiff base group.

[0307] The resulting Schiff base intermediate is reduced and stabilized using reducing agents such as NaBH4, NaBH3CN, etc. The resulting adduct can be purified by conventional-sized column chromatography to remove the cross-linked dextran. Other conventional methods can also be used to introduce amine functional groups by derivatizing the dextran, such as reacting with cyanogen bromide followed by a diamine. The aminodextran is then reacted with an activated derivative (preferably a carboxyl-activated derivative) of the specific drug, toxin, chelating agent, immunomodulator, boron additive, or other therapeutic agent to be loaded, prepared by conventional methods (e.g., using dicyclohexylcarbodiimide (DCC) or its water-soluble variant) to form an intermediate adduct.

[0308] Optionally, peptide toxins such as pokeweed antiviral protein or ricin A chain can be coupled to glycosaminoglycans via glutaraldehyde condensation or by reacting an activated carboxyl group on the protein with an amine on the glycosaminoglycan. Chelating agents of radioactive metals or magnetic resonance enhancers, such as derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), typically have groups on their side chains that can be attached to the carrier. Such groups include, for example, benzyl isothiocyanate, through which DTPA or EDTA can be coupled to the amine group of the carrier. Optionally, the carboxyl or amine group on the chelating agent can be coupled to the carrier through activation or pre-derivatization and subsequent coupling.

[0309] Boron additives, such as carboranes, can be attached to antibody components using conventional methods. For example, carboranes can be prepared using carboxyl functional groups on their side chains. The attachment of such carboranes to a carrier, such as an glycosaminoglycan, can be achieved by activating the carboxyl groups of the carborane and condensing them with an amine on the carrier to generate an intermediate conjugate. This intermediate conjugate is then attached to the antibody component to produce a therapeutically useful immunoconjugate, as described below.

[0310] Peptide carriers can be used instead of glycosaminoglycans, but the peptide carrier should have at least 50 amino acid residues in its chain, preferably 100-5000 amino acid residues. At least some of these amino acids should be lysine, glutamic acid, or aspartic acid residues. The side amines of lysine residues and the side carboxylates of glutamine and aspartic acid facilitate attachment to drugs, toxins, immunomodulators, chelating agents, boron additives, or other therapeutic agents. Examples of suitable peptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and other amino acids such as serine (to impart the desired solubility to the resulting loading carrier and immunoconjugate).

[0311] The conjugation of intermediate conjugates to antibody components is achieved by oxidizing the sugar moiety of the antibody component and reacting the resulting aldehyde (and ketone) carbonyl group with an amino group retained on the carrier after loading the drug, toxin, chelating agent, immunomodulator, boron additive, or other therapeutic agent. Alternatively, the intermediate conjugate can be attached to the oxidized antibody component via an amino group introduced into the intermediate conjugate after loading the therapeutic agent. Oxidation is conveniently achieved chemically (e.g., with NaIO4 or other sugar-degrading agents) or enzymatically (e.g., with neuraminidase and galactose oxidase). In the case of glycosaminoglycans, not all amines of the glycosaminoglycan are typically used for loading the therapeutic agent. The remaining amines of the glycosaminoglycan condense with the oxidized antibody component to form a Schiff base adduct, and are then reduced and stabilized, typically using a borohydride reducing agent.

[0312] Similar procedures are used to generate other immunoconjugates of the present invention. The loaded polypeptide carrier preferably has residual free lysine residues for condensation with the oxidized sugar portion of the antibody component. If desired, the carboxyl group on the polypeptide carrier can be converted to an amine, for example, by activation with DCC and reaction with an excess of diamine.

[0313] The final immunoconjugates were purified using conventional techniques, such as size chromatography on a Sephacryl S-300 or affinity chromatography using one or more CD84Hy epitopes. Alternatively, the immunoconjugates could be prepared by directly conjugating the antibody component to a therapeutic agent. The general procedure is similar to indirect conjugation methods, except that the therapeutic agent is directly attached to the oxidized antibody component. It should be understood that other therapeutic agents can replace the chelating agents described herein. Those skilled in the art will be able to design conjugation schemes without excessive experimentation.

[0314] As a further illustration, the therapeutic agent can be attached to the hinge region of the reduced antibody component via disulfide bond formation. For example, a tetanus toxoid peptide can be constructed using a single cysteine ​​residue for attaching the peptide to the antibody component. Alternatively, such a peptide can be attached to the antibody component using a heterobifunctional cross-linking agent, such as N-succinyl 3-(2-pyridyl dithio)propionate (SPDP) .

[0315] Antibody-cytotoxic agent conjugates are prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyldithio)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imine esters (e.g., dimethyl diimide adipate HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazide compounds (e.g., bis(p-diazoniumbenzoyl)hexamethylenediamine), diazo compound derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and biactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared. Carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclides with antibodies.

[0316] As described above, the sugar moiety in the Fc region of an antibody can be used to conjugate therapeutic agents. However, if a functional antibody fragment is used as the antibody component of an immunoconjugate, the Fc region may be absent. Instead, the sugar moiety can be introduced into the light chain variable region of the antibody or functional antibody fragment. The engineered sugar moiety is then used to attach the therapeutic agent. Furthermore, those skilled in the art will appreciate many possible variations in the conjugation method. For example, the sugar moiety can be used to attach polyethylene glycol to increase the half-life of the intact antibody or its functional antigen-binding fragment in blood, lymph, or other extracellular fluids. Additionally, divalent immunoconjugates can be constructed by attaching a therapeutic agent to a sugar moiety and a free thiol group. Such a free thiol group can be located in the hinge region of the antibody component.

[0317] Treatment

[0318] In some embodiments, the present invention discloses a method of treating a subject in need, comprising administering to the subject a therapeutic dose of the therapeutic anti-EGFR IgA antibody described herein or a pharmaceutical composition comprising the therapeutic anti-EGFR IgA antibody described herein. In some embodiments, the subject suffers from cancer, an infectious disease, or an autoimmune disease.

[0319] Most studies exploring the potential of IgA antibodies in immunotherapy are based on in vitro experiments. However, preclinical in vivo studies on the therapeutic potential of FcαRI-IgA binding are conducted using transgenic (Tg) mouse models. This is because rodents lack FcαRI expression.

[0320] In some embodiments, IgA binding to FcαRI can exert a potent pro-inflammatory effect, such as inducing oxidative burst, phagocytosis, and ADCC. In some embodiments, IgA forms a more efficient pairing with the FcRg chain in the transmembrane domain of FcαRI compared to FcγRI. In some embodiments, IgA-induced effector function is stronger when FcαRI on polymorphonuclear leukocytes (PMNs) is activated compared to FcγRI activation. Therefore, in some embodiments, tumor cell killing is more efficient when targeting FcαRI rather than FcγRI with bispecific antibodies (bsAbs) that simultaneously bind to tumor antigens (e.g., EGFR) and FcR.

[0321] EGFR is expressed in a variety of cancer cell types, such as HNSCC, CRC, NSCLC, renal cell carcinoma, and other cancers. Therefore, in some embodiments, engineered anti-EGFR IgA antibodies targeting EGFR can be used to treat any cancer expressing EGFR (e.g., cancers associated with EGFR expression and / or expression of EGFR variants with one or more mutations). In some embodiments, the EGFR variant comprises EGFRvIII, exon 19 deletion, L858R substitution in exon 21, C797S substitution, or T790M substitution. In some embodiments, anti-EGFR therapy comprises an antibody or small molecule inhibitor that binds to EGFR and blocks its signaling, thereby inhibiting the growth and survival of cancer cells. In some embodiments, anti-EGFR therapy is used to treat multiple types of cancer, including metastatic CRC, NSCLC, and HNSCC. In some embodiments, anti-EGFR therapy comprises a combination therapy using at least one anti-EGFR antibody and at least one small molecule inhibitor. In some embodiments, the at least one small molecule inhibitor includes erlotinib and gefitinib, which reversibly inhibit the EGFR tyrosine kinase domain by competitively binding to ATP. In some embodiments, at least one antibody includes the engineered antibody described herein. In some embodiments, at least one antibody also includes cetuximab (a chimeric mouse-human IgG1 antibody), netutuzumab (a fully human IgG1 antibody), mateuzumab (a fully human IgG1 antibody), and panitumumab (a fully human IgG2 antibody). All antibodies block the binding of the ligand to the extracellular domain of EGFR, promote receptor internalization, and mediate antibody- and complement-mediated cytotoxicity.

[0322] Drug resistance is a common problem in cancer treatment, leading to disease progression and poor prognosis. EGFR-TKIs exhibit various primary and secondary resistance mechanisms. Primary resistance mechanisms include point mutations in exon 18, deletions or insertions in exon 19, insertions, duplications, and point mutations in exon 20, and point mutations in exon 21 (e.g., the L858R mutation). The T790M gene mutation is prevalent in 50-60% of EGFR-mutant NSCLC patients and is associated with acquired resistance. EGFR amplification and KRAS mutation-driven disease resistance also limit the efficacy of existing treatments. This resistance, coupled with the low response rates of panitumumab and cetuximab, highlights a significant unmet clinical need for novel therapies. Furthermore, C797S mutations at the ATP-binding site can also lead to resistance (e.g., resistance to osimertinib). Therefore, the engineered antibody described in this article could be used to treat cancers associated with EGFR expression and any of these mutations.

[0323] In some implementations, the subject has cancer. In some implementations, the subject has an inflammatory disease. In some implementations, the cancer is associated with the expression of tumor-associated antigens described herein. In some implementations, the cancer is associated with the expression of CD47, CD20, GD2, CD38, CD19, EGFR, HER2, PD-L1, CD25, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER3, folate-binding protein, GD3, IL-13R-α2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123, VEGF-R2, or combinations thereof. In some embodiments, cancer is associated with the expression of EGFR, MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, 4-1BB, or combinations thereof. In some embodiments, an antibody or a functional fragment thereof binds to EGFR and one or more of MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, and 4-1BB.

[0324] In some embodiments, the cancer is a metastatic cancer expressing EGFR. In other embodiments, the cancer is a recurrent or refractory cancer. In some embodiments, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent or refractory cancer.

[0325] In some implementations, cancer is a solid tumor that expresses EGFR. Exemplary solid tumors include, but are not limited to: anal cancer; appendix cancer; bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; brain tumor; breast cancer; cervical cancer; colon cancer; cancer of unknown primary origin (CUP); esophageal cancer; eye cancer; fallopian tube cancer; gastrointestinal cancer; glioblastoma (e.g., glioma); head and neck cancer; kidney cancer; liver cancer; lung cancer; medulloblastoma; melanoma; mesothelioma; oral cancer; ovarian cancer; pancreatic cancer; parathyroid disease; penile cancer; pituitary adenoma; prostate cancer; rectal cancer; skin cancer; stomach cancer; testicular cancer; pharyngeal cancer; thyroid cancer; uterine cancer; vaginal cancer; vulvar cancer; or glioblastoma.

[0326] In some embodiments, the cancer is selected from lung cancer, head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, kidney cancer, mesothelioma, and glioblastoma. In some embodiments, the cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer.

[0327] In some implementations, the cancer expressing EGFR is a hematologic malignancy. In some implementations, the hematologic malignancy includes lymphoma, leukemia, myeloma, or B-cell malignancy. In some implementations, exemplary hematologic malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, and nodal marginal zone lymphoma. The following are considered hematologic malignancies: marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-stage B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis. In some implementations, hematologic malignancies include myeloid leukemia. In some implementations, hematologic malignancies include acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0328] In some embodiments, the anti-EGFR IgA antibody is administered together with one or more other therapeutic agents. In some embodiments, the anti-EGFR IgA antibody is administered concurrently with one or more other therapeutic agents. In some embodiments, the anti-EGFR IgA antibody is administered sequentially with one or more other therapeutic agents. In some embodiments, the other therapeutic agents include anticancer agents, chemotherapeutic agents, radiotherapy, cytotoxic agents, corticosteroids, immunotherapeutic agents, dietary supplements, antioxidants, or combinations thereof.

[0329] In some embodiments, combination therapy may include one or more antibodies of this disclosure co-formulated and / or co-administered with one or more additional therapeutic agents, such as chemotherapeutic agents or antitumor agents, such as cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic agents or cell inhibitors. Exemplary chemotherapy agents include, but are not limited to, aldesleukin, hexamethicone, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dextrin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon-alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol acetate, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, and paclitaxel. TM The treatment includes pilucarpine, prochlorperazine, saproin, tamoxifen, paclitaxel, topotecan hydrochloride, vinblastine, vincristine, and vinorelbine tartrate. In some embodiments, additional therapeutic agents include Janus kinase (JAK) inhibitors. In some embodiments, JAK inhibitors include ruxolitinib, paclitinib, fildratinib, tofacitinib, occratinib, perfiltinib, utpatinib, declatinib, delgotinib, or combinations thereof. In some embodiments, additional therapeutic agents include KRAS inhibitors. In some embodiments, KRAS inhibitors include sotorasiib, adagraxib, or combinations thereof. In some embodiments, additional therapeutic agents are combined with MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, 4-1BB, or combinations thereof.

[0330] In some implementations, the IgA antibodies described herein may be used in combination with effective doses of other antibodies already used for cancer treatment, including but not limited to the following FDA-approved monoclonal antibodies: rituximab (CD20: chimeric IgG1), trastuzumab (HER2: chimeric IgG1), alemtuzumab (CD52: humanized IgG1), ibritumomabtiuxetan (CD20: radiolabeled mouse IgG1), tocetuzumab-I-131 (CD20, mouse, IgG2a, radiolabeled (iodine-131)), and cetuximab (EGFR). Chimeric, IgG1), bevacizumab (VEGF: humanized, IgG4), panitumumab (EGFR: human IgG2), oftatumab (CD20: human IgG1), ipilimumab (CTLA-4: human IgG1), brentuximab velituximab (CD30: chimeric, IgG1, drug conjugate), pertuzumab (HER2: humanized IgG1, drug conjugate), trastuzumab emtansine (HER2: humanized IgG1, drug conjugate), oxantuzumab (CD20: humanized and glycosylated engineered), nivolumab, and pembrolizumab (anti-PD-1), etc.

[0331] In some embodiments, the anti-EGFR IgA antibody described herein is effective in treating cancer in patients with EGFR-positive cancer. In some embodiments, the subjects include those with inherent or acquired resistance to EGFR-targeting antibodies and tyrosine kinase inhibitors (TKIs). In some embodiments, the subjects have previously treated advanced or metastatic solid tumors expressing EGFR. Therefore, in some embodiments, these subjects represent a population with a high degree of unmet medical need due to poor prognosis and limited effective treatment options. In some embodiments, the anti-EGFR IgA antibody treatment described herein overcomes the limitations of current standard-of-care (SOC) EGFR-targeted therapies. In some embodiments, the anti-EGFR IgA antibody described herein is designed to fully exploit the cytotoxic potential of neutrophils, thereby providing a novel treatment option for these subjects.

[0332] dose

[0333] This document provides compositions comprising an IgA antibody or an antigen-binding fragment thereof for the treatment (including prevention) of diseases (e.g., cancer). In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition is administered in an amount effective in treating (including preventing) cancer. In some embodiments, the composition (e.g., an antibody or a functional antigen-binding fragment thereof, or a nucleic acid molecule encoding said antibody or its antigen-binding fragment) is administered in an amount effective in enhancing the immune response of a subject and / or increasing T-cell activation in the subject. The composition will be administered to the subject by any available method, such as parenteral administration. For administration to a subject, the composition or pharmaceutical comprising the antibody or its functional antigen-binding fragment described herein may be sterile, which can be readily achieved by filtration through a sterile filter membrane or other filtration methods. In one embodiment, the composition or pharmaceutical has been treated to be pyrogen-free or endotoxin-free. Testing pharmaceutical compositions or pharmaceuticals for pyrogens or endotoxins, and preparing pyrogen-free or endotoxin-free pharmaceutical compositions or pharmaceuticals, or preparing pharmaceutical compositions or pharmaceuticals with clinically acceptable levels of endotoxins, are well known to those skilled in the art. Commercial kits are available for testing pharmaceutical compositions or pharmaceuticals for pyrogens or endotoxins.

[0334] In the methods described herein, the composition for in vivo administration (e.g., parenteral administration) can be sterile, which can be readily achieved by filtration through a sterile filter membrane or other filtration methods.

[0335] The IgA antibodies or functional antigen-binding fragments thereof described herein are formulated, administered, and applied in accordance with good medical practice. Factors considered in the context of this document include the specific condition to be treated, the specific subject to be treated, the individual subject's clinical condition, the cause of the condition, the site of delivery, the method of administration, the schedule of administration, and other factors. The IgA antibodies or functional antigen-binding fragments thereof disclosed herein may be provided in therapeutically effective amounts. The therapeutically effective amount of a substance / molecule, agonist, or antagonist may vary depending on factors such as, for example, an individual's disease state, age, sex, and weight, and the ability of the substance / molecule, agonist, or antagonist to elicit the desired response in the individual. The therapeutically effective amount is also the amount by which any toxic or adverse effects of the substance / molecule, agonist, or antagonist are exceeded by the therapeutically beneficial effects. The therapeutically effective amount may be delivered in a single or multiple administration. The therapeutically effective amount to be administered will be governed by considerations that refer to the minimum amount required to improve, treat, or stabilize cancer; increase the time until progression (duration of progression-free survival); or treat or prevent the occurrence or recurrence of tumors, dormant tumors, or micrometastases. The antibodies or their functional antigen-binding fragments disclosed herein may optionally be formulated with one or more other therapeutic agents currently used for the prevention or treatment of cancer or the risk of developing cancer. The effective amount of such other agents depends on the amount of the antibody or its functional antigen-binding fragment present in the formulation, the type of disease or treatment, and other factors discussed above. These are typically used at approximately 1% to 99% of the same dosage and route of administration as previously used herein or at doses previously employed.

[0336] The dosage of the antibody can vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. Preferred dosages are typically calculated based on body weight or body surface area. When the antibodies disclosed herein or their functional antigen-binding fragments are used to treat conditions or diseases in adult patients, it may be advantageous to administer the antibodies of the invention intravenously in a single dose of about 0.01 mg / kg to about 20 mg / kg body weight, more preferably about 0.02 mg / kg to about 7 mg / kg, about 0.03 mg / kg to about 5 mg / kg, or about 0.05 mg / kg to about 3 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, or about 15 mg / kg body weight. The frequency and duration of treatment can be adjusted according to the severity of the condition. Effective dosages and regimens for administration can be determined empirically; for example, patient progression can be monitored through periodic evaluation, and the dosage adjusted accordingly. Furthermore, interspecies scaling of the dosage can be performed.

[0337] In some embodiments, the compositions herein may include a preventatively effective amount, for example, when administered to a subject at risk of cancer or in an early stage of the disease. Typically, because the preventative dose is administered to the subject before or in an early stage of the disease, the preventative dose is lower than the therapeutic dose.

[0338] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared in dosage forms in unit doses suitable for the dosage of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0339] Administration can be, for example, by single or multiple administrations alone, or by continuous infusion. Depending on the situation, treatment may continue for repeated administrations over several days or longer until, for example, cancer is treated. However, other dosing regimens may also be useful. In one non-limiting example, the antibodies or functional antigen-binding fragments thereof disclosed herein are administered at doses ranging from about 5 mg / kg to about 15 mg / kg, including but not limited to 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg once weekly, once every two weeks, or once every three weeks. Progress using the methods described herein can be readily monitored using conventional techniques and assays. The duration of therapy using the methods described herein will continue for as long as medically indicated, or until the desired therapeutic effect is achieved (e.g., those described herein). In some embodiments, administration of one or more antibodies or functional antigen-binding fragments or compositions thereof described herein may continue for 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or up to the subject's lifetime.

[0340] In some embodiments, the anti-EGFR IgA antibody described herein is administered at a dose of 1 mg / kg to 25 mg / kg. In some embodiments, the anti-EGFR IgA antibody described herein is administered as a single dose of 25 mg / kg. In some embodiments, the anti-EGFR IgA antibody described herein is administered twice daily at a dose of 11 mg / kg.

[0341] Therapeutic effects

[0342] For example, the efficacy of treatments for cancer (including administration of the IgA antibody or its functional antigen-binding fragment or pharmaceutical composition disclosed herein) can be measured by various endpoints commonly used to evaluate cancer treatment, including but not limited to tumor regression, shrinkage of tumor weight or size, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, and quality of life. The antibodies or their functional antigen-binding fragments disclosed herein may require unique measurements and definitions of clinical response to the drug. In the case of cancer, a therapeutically effective amount of the antibody disclosed herein, its functional antigen-binding fragment, or a composition containing such antibody or its functional antigen-binding fragment can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow down and preferably stop) the invasion of cancer cells into peripheral organs; inhibit (i.e., to some extent slow down and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with the disease to some extent. To some extent, the antibodies or their functional antigen-binding fragments disclosed herein exert an effect of inhibiting the growth of existing cancer cells and / or killing existing cancer cells; it can be cytoseptic and / or cytotoxic. For cancer therapies, in vivo efficacy can be measured, for example, by evaluating the duration of survival, the duration of progression-free survival (PFS), the response rate (RR), the duration of the response, and / or quality of life. In some embodiments, the IgA antibody or functional fragment thereof disclosed herein inhibits tumor growth by at least about 2%, 3%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, relative to treatment-naïve subjects. In some embodiments, the IgA antibody or functional fragment thereof disclosed herein inhibits tumor transplantation by at least about 2%, 3%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, relative to treatment-naïve subjects. In some embodiments, the IgA antibody or functional fragment thereof disclosed herein induces cell lysis of tumor cells. In some implementations, the IgA antibody or functional fragment thereof disclosed herein induces an increase in tumor cell lysis of at least about 2%, 3%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more relative to the corresponding WT IgA.

[0343] In other embodiments, methods are described herein for increasing progression-free survival in human subjects susceptible to or diagnosed with cancer (e.g., skin cancer, such as melanoma). The time until disease progression is defined as the time from drug administration until disease progression or death. In a preferred embodiment, the combination therapy of the present invention, using an antibody disclosed herein or a functional antigen-binding fragment thereof, and one or more chemotherapeutic agents, can significantly increase progression-free survival by at least about 1 month, 1.2 months, 2 months, 2.4 months, 2.9 months, or 3.5 months, for example, from about 1 to about 5 months, compared to treatment with chemotherapy alone. In another embodiment, the methods described herein can significantly increase the response rate in a group of human subjects susceptible to or diagnosed with cancer treated with multiple therapeutic agents. The response rate is defined as the percentage of treated subjects who respond to treatment. In one embodiment, the combination therapy described herein, using an antibody disclosed herein or a functional antigen-binding fragment thereof (e.g., a recombinant antibody or a functional antigen-binding fragment thereof) and one or more chemotherapeutic agents, significantly increases the response rate in the treated subject group compared to the group treated with chemotherapy alone.

[0344] In some implementations, the methods described herein include administering an effective amount of the antibody described herein or a functional antigen-binding fragment thereof to a subject to alleviate symptoms of a disease such as cancer.

[0345] Effective doses, toxicity, and therapeutic efficacy can be determined in cell culture or laboratory animals using standard pharmaceutical procedures, such as determining the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population). Dosage can vary depending on the dosage form used and the route of administration employed. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be initially estimated based on cell culture assays. Furthermore, doses can be formulated in animal models to achieve a range of circulating plasma concentrations including the IC50 (i.e., the concentration of the antibody or its functional antigen-binding fragment), which achieves half-maximal inhibition of symptoms determined in cell culture or in appropriate animal models. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dose can be monitored by appropriate bioassays. Dosage can be determined by a physician and adjusted as needed to suit the observed therapeutic effect.

[0346] Treatment and / or prevention of cancer include, but are not limited to, alleviating cancer-related symptoms, inhibiting cancer progression, promoting cancer regression, promoting immune response, inhibiting tumor growth, inhibiting tumor size, inhibiting metastasis, inhibiting cancer cell growth, inhibiting cancer cell proliferation, or inducing cancer cell death.

[0347] Application method

[0348] The IgA antibody or its functional antigen-binding fragment described herein may be administered to a subject in need via any appropriate route to produce an effective treatment in the subject. In some embodiments, the antibody or its functional antigen-binding fragment described herein, or a composition containing the antibody or its functional antigen-binding fragment, may be administered to a subject with cancer to be inhibited via any administration modality, including but not limited to systemic delivery or delivery to a target surface or target, such administration modalities may include, but are not limited to, injection, infusion, instillation, and inhalation. Oral administration is also included herein. The antibody or its functional antigen-binding fragment described herein, or a composition containing the antibody or its functional antigen-binding fragment, may be administered by injection, including but not limited to intravenous, intramuscular, intraarticular, intrathecal, intracapsular, intraorbital, intracapsular, intraorbital, intracardiac, intravascular, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intracranial, intraspinal, intracerebral, and intrasternal injection and infusion.

[0349] In some embodiments, the antibody described herein or its functional antigen-binding fragment, or a composition containing the antibody or its functional antigen-binding fragment, may be administered as a bolus via intravenous administration or by continuous infusion over a period of time via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Topical administration, for example, to a tumor or cancer site where angiogenesis is occurring, is particularly desirable if extensive side effects or toxicity are associated with the use of the antibody described herein or its functional antigen-binding fragment, or a composition containing the antibody or its functional antigen-binding fragment. In some embodiments, ex vivo strategies may also be used for therapeutic application. Ex vivo strategies involve transfecting or transducing cells obtained from a subject with the nucleic acid sequences disclosed herein. The transfected or transduced cells are then returned to the subject. Cells can be any of a wide range of types, including, but not limited to, hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

[0350] In some embodiments, the antibody disclosed herein or its functional antigen-binding fragment, or a composition comprising the antibody or its functional antigen-binding fragment, is administered by any suitable method, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and, if local immunosuppressive therapy is desired, intralesional administration is also included.

[0351] Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies of this disclosure or their functional antigen-binding fragments or compositions are suitably administered via pulse infusion, particularly with decreasing doses of the antibody. Preferably, administration is by injection, most preferably by intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. In some embodiments, the antibodies of this disclosure or their functional antigen-binding fragments or compositions are administered locally, for example, by direct injection when the location of the symptom or tumor permits, and the injections can be repeated periodically. In some embodiments, the antibodies of this disclosure or their functional antigen-binding fragments or compositions may also be delivered systemically to the subject or directly to tumor cells, for example, to the tumor or to the tumor bed after surgical resection of the tumor, in order to prevent or reduce local recurrence or metastasis, such as of dormant tumors or micrometastases.

[0352] To enhance the efficacy and potential of therapeutic compositions comprising the antibodies and their functional antigen-binding fragments provided herein, some embodiments of antibody-targeted sonopooration are envisioned for use in the tumor-suppressing methods described herein. As used herein, “sonic cavitation” refers to the use of sound, preferably at ultrasound frequencies, or the interaction of ultrasound with a contrast agent (e.g., stabilizing microbubbles), to temporarily alter the permeability of the cell membrane, thereby allowing the uptake of macromolecules, such as therapeutic agents. The membrane permeability induced by sonopooration is temporary, allowing the agent to be trapped within the cell after ultrasound exposure. Sonopooration utilizes the acoustic cavitation of microbubbles to enhance the delivery of macromolecules.

[0353] Therefore, in some embodiments of the method, the antibody or its functional antigen-binding fragment described herein, mixed with an ultrasound contrast agent such as microbubbles, can be injected locally or systemically into a subject requiring cancer treatment, and the ultrasound can be coupled and even focused onto a defined area (e.g., the tumor site) for targeted delivery. In some embodiments, the method uses focused ultrasound to achieve targeted delivery. As used herein, HIFU or “high-intensity focused ultrasound” refers to a non-invasive therapeutic method that uses high-intensity ultrasound to heat and destroy malignant or pathogenic tissue without damaging overlapping or surrounding healthy tissue. HIFU can also be used as a method for delivering therapeutic agents such as antibodies or their functional antibody fragments.

[0354] The use of contrast-enhanced ultrasound (CEUS) is also envisioned for use with the antibodies or their functional antigen-binding fragments described herein. CEUS refers to the application of ultrasound contrast media and contrast agents to conventional medical ultrasound examinations. Contrast agents are reagents that rely on different modes of sound wave reflection from interfaces between substances. Various microbubble contrast agents can be used with the compositions and methods described herein. Microbubbles can vary in their shell composition, gas core composition, and whether they are targeted. Targeting ligands that bind to characteristic receptors of angiogenesis disorders can be conjugated to microbubbles, allowing the microbubble complex to selectively accumulate in regions of interest, such as diseased or abnormal tissue. This form of molecular imaging, known as targeted ultrasound contrast, will only produce a strong ultrasound signal when the targeted microbubble binds to the region of interest. Targeted ultrasound contrast has numerous applications in medical diagnosis and treatment. In some embodiments, targeted ultrasound delivery is used to administer the antibodies or their functional antigen-binding fragments described herein to subjects requiring cancer or tumor treatment.

[0355] Pharmaceutical compositions and dosage forms

[0356] In some embodiments, this document discloses pharmaceutical compositions comprising the anti-EGFRIgA antibody or a functional fragment thereof disclosed herein for administration to a subject.

[0357] In some embodiments, pharmaceutical compositions comprising the anti-EGFR IgA antibody described herein are formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants) that facilitate the processing of the active compound into an article suitable for pharmaceutical use. A suitable formulation depends on the chosen route of administration.

[0358] The pharmaceutical composition is optionally prepared in a conventional manner, for example, by way of example only, through conventional methods of mixing, dissolving, granulation, forming sugar-coated pellets, grinding, emulsifying, encapsulating, embedding or compressing.

[0359] In some embodiments, the composition may further include one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethyl)aminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0360] In other embodiments, the composition may further comprise one or more salts in an amount required to bring the osmolality of the composition within an acceptable range. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0361] The pharmaceutical compositions described herein may be administered via any suitable route of administration, including but not limited to oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intra-articular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal administration. In some embodiments, the pharmaceutical compositions are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intra-articular, intraperitoneal, or intracranial) administration.

[0362] The pharmaceutical compositions described herein are formulated in any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, pastes, suspensions, etc., for oral administration by an individual to be treated, solid oral dosage forms, aerosols, controlled-release formulations, rapidly soluble formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated pills, capsules, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations, and mixtures of immediate-release and controlled-release formulations.

[0363] In some embodiments, the pharmaceutical composition is formulated as a capsule. In some embodiments, the pharmaceutical composition is formulated as a solution (e.g., for intravenous administration). In some embodiments, the pharmaceutical composition is formulated as an infusion. In some embodiments, the pharmaceutical composition is formulated as an injection.

[0364] The solid dosage forms of medicines described herein optionally include the compounds described herein and one or more pharmaceutically acceptable additives, such as compatible carriers, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, humectants, defoamers, antioxidants, preservatives, or one or more combinations thereof.

[0365] In other aspects, standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th edition (2000), are used to provide a film coating around the composition. In some embodiments, the composition is formulated as granules (e.g., for administration via capsules), and some or all of the granules are coated. In some embodiments, the composition is formulated as granules (e.g., for administration via capsules), and some or all of the granules are microencapsulated. In some embodiments, the composition is formulated as granules (e.g., for administration via capsules), and some or all of the granules are not microencapsulated and are not coated.

[0366] In some embodiments, the compositions provided herein may also contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as merfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, hexadecyltrimethylammonium bromide, and hexadecylpyridine chloride.

[0367] In some embodiments, the antibody, its functional fragment, or a composition comprising the antibody or its functional fragment may be administered to a subject in need (e.g., a subject with cancer). In some embodiments, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is recurrent or refractory cancer. In some embodiments, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, anal cancer, appendix cancer, biliary tract cancer (i.e., bile duct cancer), bladder cancer, brain tumors, breast cancer, cervical cancer, colon cancer, cancer of unknown primary origin (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, glioblastoma (e.g., glioma), head and neck cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, mesothelioma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary adenoma, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, vulvar cancer, or glioblastoma. In some embodiments, leukemia may be, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML).

[0368] Antibodies, their functional fragments, or combinations thereof may be administered by injection, such as intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im). One or more of these routes may be used. Parenteral administration may be, for example, by bolus injection or gradual perfusion over time. Optionally, or simultaneously, administration may be by oral route. Furthermore, administration may also be by surgical implantation of cell clumps or cell particles, or by placement of a medical device. In one embodiment, the compositions of this disclosure may comprise an amount of engineered cells or host cells expressing the nucleic acid sequences described herein, or a vector containing at least one of the nucleic acid sequences described herein, effective in treating or preventing proliferative conditions. Pharmaceutical compositions may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0369] In some implementations, defoamers may be added to reduce foam generated during processing, which can lead to coagulation of aqueous dispersions, air bubbles in the finished film, or overall impact on processing. Exemplary defoamers include silicone emulsions or sorbitan sesquoleate.

[0370] In some embodiments, antioxidants such as butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite, and tocopherol may be added. In some embodiments, antioxidants enhance chemical stability when needed.

[0371] The formulations described in this article may benefit from antioxidants, metal chelators, thiol compounds, and other general stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparin analogues, (m) divalent cations such as magnesium and zinc ions; or (n) combinations thereof.

[0372] In some embodiments, an adhesive may be added to impart cohesiveness. Exemplary adhesives include, for example, alginate and its salts; cellulose derivatives such as carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, and microcrystalline cellulose; microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acids; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; crosslinked povidone; povidone; starch; pregelatinized starch; tragacanth gum; dextrin; sugars such as sucrose, glucose, dextrose, molasses, mannitol, sorbitol, xylitol, and lactose; natural or synthetic gums such as gum arabic, tragacanth gum, solanum, mucilage of isapol husks, polyvinylpyrrolidone, larch arabinogalactan, polyethylene glycol, waxes, sodium alginate, etc.

[0373] In some embodiments, a carrier or carrier substance may be added. The carrier or carrier material includes any excipient commonly used in pharmaceuticals and should be selected based on compatibility with the compounds disclosed herein (e.g., ibrutinib and compounds of anticancer agents) and the release profile characteristics of the desired dosage form. Exemplary carrier substances include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Pharmaceutically compatible carrier substances may include, but are not limited to, gum arabic, gelatin, colloidal silica, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurine, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sodium saccharide stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc.

[0374] In some implementations, dispersants may be added to control the diffusion and uniformity of the drug in liquid media, granulation methods, or mixing methods. These agents also contribute to improving the effectiveness of coatings or aggressive matrices. Exemplary dispersants include, for example, hydrophilic polymers, electrolytes, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, PEG, polyvinylpyrrolidone (PVP), and carbohydrate-based dispersants such as hydroxypropyl cellulose (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (e.g., HPMC K100, HPMC K4M, HPMC...). K15M and HPMCK100M), sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymers with ethylene oxide and formaldehyde polymers (also known as tyloxacillin), poloxamer (e.g., block copolymers of ethylene oxide and propylene oxide); poloxamer (e.g., cis-propylene oxide and ethylene oxide ester copolymers). The following are listed as examples of polyvinylpyrrolidone (PVP) compounds: tetrafunctional block copolymers added to ethylenediamine, polyvinylpyrrolidone K12, PPVK K17, PPVK K25 or PPVK K30, PPVK / vinyl acetate copolymer (S-630), polyethylene glycol (e.g., polyethylene glycol having a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methyl cellulose, polysorbate-80, sodium alginate, gums such as gum arabic and gum arabic, guar gum, xanthan gums including xanthan gum. Gum), sugars, cellulose (e.g., sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose), polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol (PVA), alginate, chitosan, and combinations thereof. Plasticizers such as cellulose or triethyl cellulose can also be used as dispersants. Particularly useful dispersants in liposome dispersions and self-emulsifying dispersions are dimyristoyl phosphatidylcholine, natural phosphatidylcholine derived from eggs, natural phosphatidylglycerol derived from eggs, cholesterol, and isopropyl myristate.

[0375] In the compositions of the present invention, a combination of one or more erosion facilitators and one or more diffusion facilitators may also be used.

[0376] In some cases, a diluent may be added to dilute the target compound before delivery. Diluents can also be used to stabilize compounds because they provide a more stable environment. Salts dissolved in buffer solutions (which can also provide pH control or maintenance) are used as diluents in the art, including but not limited to phosphate-buffered saline solutions. In some embodiments, the diluent increases the volume of the composition to facilitate compression or to generate sufficient volume for uniform blending for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dicalcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar; mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; monobasic base calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextran derivatives; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

[0377] In some cases, fillers may be added, including combinations of lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextran, dextran anhydride derivatives, dextran anhydride, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[0378] In some cases, lubricants or flow aids may be added to prevent, reduce, or inhibit the adhesion or friction of substances. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearoyl fumarate, hydrocarbons such as mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil, higher fatty acids and their alkali metal and alkaline earth metal (e.g., aluminum, calcium, magnesium, zinc) salts, stearic acid, sodium stearate, glycerol, talc, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxy polyethylene glycol, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium dodecyl sulfate or sodium dodecyl sulfate, colloidal silica, starch such as corn starch, silicone oil, surfactants, etc.

[0379] In some cases, plasticizers may be added to soften microencapsulated materials or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. In some embodiments, plasticizers may also function as dispersants or wetting agents.

[0380] In some cases, solubilizers may be added, such as triacetin, triethyl citrate, ethyl oleate, ethyl octanoate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-600, tetraethylene glycol, transcutol, propylene glycol, and isosorbide dimethyl ether, etc.

[0381] In some cases, stabilizers, such as antioxidants, buffers, acids, and preservatives, can be added.

[0382] In some cases, suspending agents may be added, including compounds such as polyvinylpyrrolidone (PVP), for example, PPVP K12, PPVP K17, PPVP K25, or PPVP K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol having a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methyl cellulose, etc. Ingredients include: hydroxypropyl methylcellulose, hydroxymethyl cellulose acetate stearate, polysorbate-80, hydroxyethyl cellulose, sodium alginate, gums such as gum arabic and gum arabic, guar gum, xanthan gums including xanthan gum, sugars, cellulose (e.g., sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose), polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, etc.

[0383] In some cases, surfactants may be added, including compounds such as sodium dodecyl sulfate, sodium docusate, polyethylene glycol monostearate or polyoxyethylene monooleate sorbitol, triacetin, vitamin ETPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, etc. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octylphenyl polyol 10 and octylphenyl polyol 40. In some embodiments, surfactants may be included to enhance physical stability or for other purposes.

[0384] In some cases, thickeners may be added, including, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, hydroxypropyl methylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

[0385] In some cases, wetting agents may be added, including compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, glyceryl triacetate, polyoxyethylene sorbitan monooleate, vitamin E TPGS, ammonium salts, etc.

[0386] Optionally, formulations comprising the compositions described herein contain a pharmaceutically acceptable salt, typically, for example, sodium chloride, and preferably at approximately physiological concentrations. Optionally, formulations of the present invention may contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration ranges from 0.1% to 2.0%, typically v / v. Suitable preservatives include, for example, benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben. Optionally, formulations of the present invention may contain a pharmaceutically acceptable surfactant at a concentration of 0.005% to 0.02%.

[0387] The compositions described herein can be specifically formulated for administering antibodies or their functional antigen-binding fragments to subjects in solid, liquid, or gel form, including those suitable for: (1) parenteral administration, e.g., as a sterile solution, suspension, or sustained-release formulation via subcutaneous, intramuscular, intravenous, or epidural injection; (2) topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin; (3) intravaginal or rectal administration, e.g., as a vaginal suppository, cream, or foam; (4) ocular administration; (5) transdermal administration; (6) mucosal administration; or (7) nasal administration. Furthermore, the antibodies or their functional antigen-binding fragments or compositions disclosed herein can be implanted into a patient or injected using a drug delivery system.

[0388] The compositions disclosed herein comprising the antibodies or functional antigen-binding fragments described herein may also contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other, depending on the specific indications to be treated. For example, the compositions may also contain cytotoxic agents, cytokines, growth inhibitors, and / or angiogenesis inhibitors, such as VEGFR antagonists. Such molecules are suitably present in a combination of amounts effective for the intended purpose. The active ingredient of a composition comprising the antibodies or their functional antigen-binding fragments described herein may also be encapsulated, for example, in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules) prepared by coascervation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microparticles, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. The pharmaceutical compositions may also be delivered in vesicles, particularly liposomes. Liposomes include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, and sheet-like layers, and can be used as a medium for targeting M-CSF antibodies to specific tissues and for increasing the half-life of compositions. Various methods can be used to prepare liposomes.

[0389] Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with defined pore sizes to obtain liposomes with the desired diameter. The Fab' fragment of the antibody of the present invention can be conjugated to the liposomes via a disulfide exchange reaction. A chemotherapeutic agent (e.g., doxorubicin) is optionally included in the liposomes.

[0390] In some embodiments, sustained-release articles may be used. Suitable examples of sustained-release articles include semi-permeable matrices of solid hydrophobic polymers containing antibodies or functional antigen-binding fragments of the present disclosure, wherein the matrix is ​​in the form of molded articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres of lactic acid-glycolic acid copolymer and leucrobenzide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable sustained molecular release for more than 100 days, some hydrogels release proteins over shorter time periods. When encapsulated antibodies are held in vivo for extended periods, they can denature or aggregate due to exposure to moisture at 37°C, leading to loss of biological activity and possible immunogenicity alterations. Based on the mechanism involved, rational strategies for stabilization can be designed. For example, if the aggregation mechanism is found to be the formation of intermolecular S-S bonds through thiodisulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing from an acidic solution, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. In some embodiments, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric substance can be used. In yet another embodiment, the controlled release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose.

[0391] The pharmaceutical compositions disclosed herein can be delivered subcutaneously, intravenously, or intraperitoneally using, for example, standard needles and syringes. Furthermore, for subcutaneous delivery, pen delivery devices are readily adaptable for delivering the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. After all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, a disposable pen delivery device is pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition in the reservoir is depleted, the entire device is discarded. Many reusable pen and auto-injector delivery devices are suitable for subcutaneous delivery of the pharmaceutical compositions of the present invention.

[0392] Injectable products can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable products can be prepared, for example, by dissolving, suspending, or emulsifying the antibodies or their salts described above in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other excipients, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include, for example, sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The prepared injection is preferably filled in a suitable ampoule.

[0393] The compositions disclosed herein may be in the following forms: for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions. The amount of the aforementioned antibody contained in a unit dose may be from about 5 mg to about 500 mg per dosage form; particularly in injectable forms, it is preferred to contain from about 5 mg to about 100 mg of the aforementioned antibody, and for other dosage forms, it contains from about 10 mg to about 250 mg of the aforementioned antibody.

[0394] For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, soft capsules, and capsule sheets as solid dosage forms are acceptable. These can be prepared, for example, by mixing one or more compounds of the present invention or their pharmaceutically acceptable salts or tautomers with at least one additive, such as starch or other additives. Suitable additives are sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, starch, agar, alginate, chitosan, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. Optionally, oral dosage forms may contain other ingredients that facilitate administration, such as inactive diluents, or lubricants such as magnesium stearate, or preservatives such as parabens or sorbic acid, or antioxidants such as ascorbic acid, tocopherol, or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, or aroma enhancers. Tablets and pills may be further processed with suitable coating materials.

[0395] Liquid dosage forms for oral administration can be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, and may contain inactive diluents such as water. In some embodiments, pharmaceutical formulations and drugs may be prepared as liquid suspensions or aqueous solutions using, for example, sterile liquids such as, but not limited to, oils, water, alcohols, and combinations thereof. In some embodiments, pharmaceutical compositions may be prepared in lyophilized form. Lyophilized formulations may contain cryoprotectants, including agents that provide stability to proteins against cryo-induced stress. Examples of cryoprotectants include polyols such as mannitol, and sugars such as sucrose, as well as surfactants such as polysorbates, poloxamer, or polyethylene glycol. Cryoprotectants also contribute to the tonicity of the formulation. Pharmaceutically suitable surfactants, suspending agents, and emulsifiers may be added for oral or parenteral administration.

[0396] As described above, the suspension may contain oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. The suspension may also contain fatty acid esters, such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. The suspension formulation may contain alcohols, such as, but not limited to, ethanol, isopropanol, cetyl alcohol, glycerol, and propylene glycol. Ethers, such as, but not limited to, polyethylene glycol, petroleum hydrocarbons, such as mineral oil and petrolatum; and water may also be used in the suspension formulation.

[0397] For nasal administration, the pharmaceutical formulation and the drug can be a spray or aerosol containing a suitable solvent and optional other compounds, such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof. Propellants used in aerosol formulations can include compressed air, nitrogen, carbon dioxide, or low-boiling-point hydrocarbon-based solvents.

[0398] Injectable dosage forms typically comprise aqueous or oily suspensions, which can be prepared using suitable dispersants or wetting agents and suspending agents. The injectable form can be in the form of a solution or suspension prepared with a solvent or diluent. Acceptable solvents or media include sterile water, Ringer's solution, or isotonic saline solutions. Optionally, sterile oil can be used as a solvent or suspending agent. Preferably, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, monoglycerides, diglycerides, or triglycerides.

[0399] For injection, the pharmaceutical formulation and / or the drug may be a powder suitable for reconstitution with a suitable solution as described above. Examples of such powders include, but are not limited to, freeze-dried, rotary-dried, or spray-dried powders, amorphous powders, granules, precipitates, or microparticles. For injection, the formulation may optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0400] For rectal administration, pharmaceutical formulations and drugs can be in the form of suppositories, ointments, enemas, tablets, or creams for the release of compounds in the intestine, sigmoid colon, and / or rectum. Rectal suppositories are prepared by mixing one or more compounds of the present invention, or pharmaceutically acceptable salts or tautomers of such compounds, with an acceptable medium such as cocoa butter or polyethylene glycol. The rectal suppositories are in a solid phase at normal storage temperatures and in a liquid phase at temperatures suitable for release of the drug in vivo, such as in the rectum. Oils can also be used to prepare soft gelatin-type formulations and suppositories. Water, saline, aqueous dextran and related sugar solutions, as well as glycerin, can be used to prepare suspension formulations, which may also contain suspending agents such as pectin, carbomer, methylcellulose, hydroxypropyl cellulose, or carboxymethyl cellulose, as well as buffers and preservatives.

[0401] The concentration of the antibody or its functional antigen-binding fragment in these compositions can vary considerably, from less than about 10% by weight, typically at least about 25% to as high as 75% or 90%, and will be selected primarily by fluid volume, viscosity, etc., depending on the specific administration method chosen.

[0402] In another embodiment of the invention, a preparation comprising a substance that can be used to treat the aforementioned diseases, conditions, or illnesses, including those for the treatment of cancer, is provided. The preparation includes a container and labeling. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed from various materials, such as glass or plastic. The container contains a composition effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The active agent in the composition is an antibody of the present invention. Labeling on or associated with the container indicates that the composition is intended to treat the selected condition. The preparation may also include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextran solution. The preparation may also include other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. The pharmaceutical compositions and medicaments described herein can be used to treat cancerous diseases.

[0403] Diagnostic and other uses

[0404] This article provides methods for using antibodies to detect, diagnose, and monitor diseases, symptoms, or conditions associated with antigen expression (increased or decreased expression relative to normal samples, and / or inappropriate expression, such as expression in tissues and / or cells that typically lack epitope expression). This article also provides methods for determining whether a patient will respond to antibody therapy.

[0405] In some embodiments, the method includes detecting whether a patient has cells expressing a target antigen using an antibody disclosed herein. In some embodiments, the detection method includes contacting a sample with an antibody disclosed herein or a functional antigen-binding fragment thereof, and determining whether the binding level differs from the binding level of a reference or comparative sample (e.g., a control). In some embodiments, the method can be used to determine whether the antibody or peptide described herein is an appropriate treatment for a subject.

[0406] In some embodiments, cells or cell / tissue lysates are contacted with an antibody, and the binding between the antibody and the cells is determined. When the test cells show binding activity compared to reference cells of the same tissue type, this can indicate that the subject will benefit from treatment with the antibody. In some embodiments, the test cells are derived from human tissue. In some embodiments, the test cells are derived from human blood.

[0407] Various methods can be used to detect specific antibody-antigen binding. Exemplary immunoassays that can be performed include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). Indicator portions or labeling groups may be attached to the test antibody and are selected to meet the needs of various uses of the method, typically determined by the availability of the assay equipment and compatible immunoassay procedures.

[0408] Suitable markers include, but are not limited to, radionuclides (e.g., 125I, 131I, 35S, 3H, or 32P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-galactosidase), fluorescent portions or proteins (e.g., luciferin, rhodamine, phycoerythrin, GFP, or BFP), or luminescent portions.

[0409] For diagnostic purposes, antibodies or their functional antigen-binding fragments may be labeled with a detectable portion, which includes, but is not limited to, radioactive isotopes, fluorescent markers, and various enzyme-substrate markers known in the art.

[0410] In some embodiments, the antibody does not need to be labeled, and its presence can be detected using a labeled secondary antibody bound to the primary antibody. The antibodies of the present invention, or functional antigen-binding fragments thereof, can be used as affinity purifiers for cancer-associated antigens, or for diagnostic assays of cancer-associated antigen proteins, such as detecting the expression of cancer-associated antigen proteins in specific cells, tissues, or serum. The antibodies or functional antigen-binding fragments thereof disclosed herein can also be used for in vivo diagnostic assays. Typically, for these purposes, the antibodies are labeled with radionuclides (e.g., 111In, 99Tc, 14C, 131I, 125I, 3H, 32p, or 35S) so that tumors can be located using immunoscintigraphy.

[0411] The antibodies of this invention can be used in assays such as competitive binding assays, direct and indirect sandwich assays (e.g., ELISA), and immunoprecipitation assays. The antibodies can also be used in immunohistochemistry to label tumor samples. For convenience, the antibodies of this invention can be provided in a kit (i.e., a package combination of a predetermined amount of reagents and instructions for performing the diagnostic assay). When the antibody is labeled with an enzyme, the kit will contain the substrate and cofactor required by the enzyme (e.g., a substrate precursor that can detect chromophores or fluorophores). Additionally, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents can vary considerably to provide a concentration of reagent solution that substantially optimizes the assay sensitivity. In particular, the reagents can be provided as a dry powder (typically in lyophilized form) including excipients that, upon dissolution, provide a reagent solution with an appropriate concentration.

[0412] Reagent test kit

[0413] This document also provides kits, pharmaceuticals, compositions, and unit dosage forms for any of the methods described herein. One kit is provided that contains a therapeutically effective amount of at least one anti-EGFR IgA antibody or a functional antigen-binding fragment thereof disclosed herein. In some embodiments, the kit further contains a second therapeutic agent (e.g., a chemotherapeutic agent). In some embodiments, the antibody or its functional antigen-binding fragment is in aqueous or lyophilized form. The kit also contains a diluent or reconstitution solution.

[0414] The kit may include one or more containers containing antibodies (or unit dosage forms and / or preparations). In some embodiments, unit doses are provided, wherein the unit dose contains a predetermined amount of an antibody-containing composition (e.g., a therapeutically effective amount), with or without one or more additional agents. In some embodiments, such unit doses are provided in single-use, pre-filled syringes for injection. In some embodiments, the composition containing an antibody or a functional antigen-binding fragment thereof may contain saline, sucrose, etc.; buffers, such as phosphates, etc.; and / or be formulated within a stable and effective pH range. In some embodiments, the antibody or a functional antigen-binding fragment thereof may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid, such as sterile water. In some embodiments, the antibody or a functional antigen-binding fragment thereof also contains one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. In some embodiments, the antibody or a functional antigen-binding fragment thereof also contains heparin and / or proteoglycans.

[0415] In some embodiments, the kit also includes instructions for use in treating cancer according to any of the methods described herein. The kit may also include descriptions of selection suitable for an individual or treatment. Instructions provided with the kit are typically written instructions on a label or packaging insert (e.g., paper pages included with the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. In some embodiments, the kit also includes another therapeutic agent (e.g., an anticancer antibody or chemotherapy agent).

[0416] The kit is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kit may optionally include additional components, such as buffers and explanatory information. Therefore, this application also provides preparations including vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0417] Example

[0418] Example 1 - Design and manufacturing method of engineered anti-EGFR IgA

[0419] Engineering design for EGFR IgA resistance

[0420] Engineered anti-EGFR IgA antibody variants were designed, each with an EGFR-binding variable region and modifications. IgA constant region.

[0421] The gene sequence of the IgA2(m1) heavy chain is the backbone of the engineered anti-EGFR IgA variant, in which the N-linked glycosylation motif is silenced and a stabilizing mutation is introduced (Table 10). Figures 4A-4DThis resulted in a series of molecules: anti-EGFR IgA3.0-(min), anti-EGFR IgA3.0+(plus), and anti-EGFR IgA4.0.

[0422] The anti-EGFR IgA 3.0+ molecule contains the CH1-P124R mutation, which allows covalent bonding between the heavy and light chains. Two cysteine ​​residues are modified or removed (CH2-C92S; CH3_CHS-C147del_Y148del) to prevent the formation of cysteine ​​bridges with serum proteins, thereby inhibiting dimer aggregation and / or complex formation. Furthermore, three N-linked glycosylation motifs are silenced by substituting key amino acids in these motifs (CH1-N45.2G; CH2-N120T; CH3_CHS-N135Q).

[0423] The anti-EGFR IgA3.0min molecule contains the same mutations in CH1 and CH2 as the anti-EGFR IgA3.0+ molecule, but unlike the anti-EGFR IgA3.0+ molecule, the anti-EGFR IgA3.0min molecule lacks almost the entire tail (CH3_CHS-P131-Y148del).

[0424] The anti-EGFR IgA4.0 molecules were generated by silencing the only remaining N-linked glycosylation motif (CH2-N20) through the substitution of four independent amino acids, resulting in four completely glycosylated IgA2-based molecules.

[0425] Table 10. List of engineered EGFR IgA mutations, namely IgA3.0+, IgA3.0min, and IgA4.0, relative to wild-type (WT) IgA2(m1).

[0426]

[0427] Clonal anti-EGFR IgA3.0 / IgA4.0

[0428] To clone anti-EGFR IgA3.0+ and anti-EGFR IgA3.0min molecules, synthetic DNA covering the entire IgA constant region (CH1-CH2-CH3-CHS) was ordered, and the cassettes encoding them were cloned into the pEE14.4 vector, replacing the IgA2(m1) sequence. Cloning of the anti-EGFR IgA4.0 molecule was performed by replacing the CH1-CH2-CH3-CHS region of the anti-EGFR IgA3.0min molecule in the pcDNA3.4 vector with gBlocks (IDTs) containing the corresponding IgA4.0 mutation.

[0429] Production

[0430] HEK293F cells were used to produce anti-EGFR IgA 3.0+ and anti-EGFR IgA 3.0min, while ExpiCHO-S cells were used to produce anti-EGFR IgA 4.0. For DNA complexation, individual vectors of anti-EGFR IgA 3.0+, anti-EGFR IgA 3.0min heavy chains, or anti-EGFR IgA 4.0 heavy chains (HC), along with vectors of κ light chains (LC) and pAdvantage (pAdv; Promega), were mixed at the optimal HC:LC:pAdv ratio and complexed with 293fectin or Expifectamine prior to transfection.

[0431] purification

[0432] The purification procedure for anti-EGFR IgA is the same for all anti-EGFR IgA variants, which is performed as follows: the kappa light chain is captured from the clarified and filtered cell culture supernatant using a HiTrap KappaSelect column (GE Healthcare) via FPCL, and then separated by size exclusion chromatography using a HiPrep 26 / 60 Sephacryl S-300 HR column.

[0433] Example 2 - Characterization of anti-EGFR IgA variants

[0434] Combined measurement

[0435] To determine the binding of engineered antibodies, the binding of the variable and Fc moieties was evaluated.

[0436] In the FACS binding assay, clear supernatant from HEK293F containing either anti-EGFR IgA 3.0 min or anti-EGFR IgA 3.0+ was tested on EGFR-expressing eukaryotic cells. Undiluted clear supernatant from ExpiCHO-S containing anti-EGFR IgA 4.0 was used for the FACS binding assay.

[0437] In short, incubate the supernatant with eukaryotic cells on ice for 1 hour. After washing, incubate the cells with PE-labeled anti-IgA antibody (Southern Biotech) for 45 minutes. After washing, fix the cells with PFA and analyze on a Canto II (BD) spectrometer. Add EGFR recognition control antibody at a concentration of 5-10 µg / mL.

[0438] To determine whether the antibody Fc region could bind to FcαR, anti-EGFR IgA2 (m1) and anti-EGFR IgA3.0 min (25 µg / mL) were coated overnight in pH 9.0 carbonate buffer in an ELISA plate. The plate was blocked with 1% BSA, and then healthy donor polymorphonuclear neutrophils (PMNs) expressing CD89 and labeled with calcein were allowed to bind to the plate at 37°C for 45 min. Binding was then determined by comparing the residual signal with the input signal (unwashed) after every two washing steps. An additional control was performed to verify that the coating concentrations were equal by staining the ELISA plate overnight with serially diluted anti-EGFR IgA2 (m1) and anti-EGFR IgA3.0 min antibodies and then detecting the presence of antibodies with anti-hIgA-HRP (Southern Biotech).

[0439] ADCC

[0440] Target cells were loaded with 51Cr (Perkin-Elmer), washed twice, and incubated for 4 hours with serially diluted anti-EGFR IgA antibody and healthy donor PMN. Chromium release in the supernatant was measured, and specific lysis was calculated using the following formula: ((experimental cpm – basal cpm) / (maximum cpm – basal cpm)) x 100. Maximum lysis was determined by reacting labeled cells with 1.25% TRITON. TM Incubation with (2-[4-(2,4,4-trimethylpentyl-2-yl)phenoxy]ethanol) was used to determine minimal lysis in the absence of antibodies and effector cells. Undiluted supernatants produced by ExpiCHO-S were evaluated for anti-EGFR IgA4.0.

[0441] thermal stability

[0442] Thermal stability was analyzed using Sypro Orange (Life Technologies) in a thermal drift assay. A total of 12.5 μg of anti-EGFR IgA antibody diluted in 25 μL PBS and 3× Sypro Orange (final concentration) was transferred to a white 96-well thin-walled PCR plate (Roche) and sealed with optical-grade sealing tape (Roche). The plate was placed in a ViiA7 (Roche) and heated from 37°C to 99°C at a heating rate of 1.6°C / s, incubating for 1 min in increments of 1°C. Fluorescence intensity was recorded simultaneously, using an excitation wavelength of 490 nm and an emission wavelength of 575 nm.

[0443] To evaluate the functionality of destabilized anti-EGFR IgA antibodies in PBS, each anti-EGFR IgA antibody was incubated for 5 minutes in a thermal cycler at different temperatures (4°C, 23°C to 95°C, in increments of 12°C). After incubation, complete culture medium was added, and the anti-EGFR IgA antibody was directly used for ADCC at a final concentration of 10 μg / mL.

[0444] Glycosylation analysis

[0445] Perform PNGase F treatment according to the manufacturer's instructions (NEB). In short, denature the anti-EGFR IgA antibody first at 100°C for 10 minutes, then add NP-40, Glycobuffer, and PNGase F enzyme, and incubate at 37°C for 1 hour. Add the sample to Laemmli buffer containing 20 mM DTT and run on a 10% Mini Protean TGX SDS-PAGE (Bio-Rad). Stain the gel with Instant Blue (Expedeon) for 10 minutes and wash with water.

[0446] The identification and quantification of glycans were determined by mass spectrometry. For anti-EGFR IgA2(m1) antibodies, the N-glycosylation of IgA2 antibodies released after specific sialic acid derivatization was analyzed by reflectance positive mode MALDI-TOF-MS. For anti-EGFR IgA3.0+, anti-EGFR IgA3.0min, and anti-EGFR IgA4.0 variants, LC / MS2 was used.

[0447] The antibody was denatured, reduced, and alkylated, and then subjected to proteolytic digestion using GluC (Roche (Indianapolis, IN)) and trypsin (Sigma-Aldrich (Steinheim, Germany)). For this purpose, 10 μg of antibody was added to 100 mM Tris-HCl (pH 8.5) (tris(hydroxymethyl)aminomethane hydrochloride), 5 mM Tris(2-carboxyethyl)-phosphine (TCEP, Sigma-Aldrich (Steinheim, Germany)), 30 mM chloroacetamide (CAA, Sigma-Aldrich (Steinheim, Germany)), and 1% sodium deoxycholate (SDC, Sigma-Aldrich (Steinheim, Germany)), and water (MQ) (generated by a Q-POD or Q-Gard 1 system (Millipore), running at ≥ 18.2 MΩ). The mixture was incubated with GluC at a 1:75 w / w enzyme:protein ratio at 37°C for 4 h, followed by incubation with trypsin (1:100 w / w) at 37°C overnight. SDC was then precipitated by adding 0.5% trifluoroacetic acid (TFA, Sigma-Aldrich (Steinheim, Germany)) and centrifuging at maximum speed for 10 min. The supernatant was collected for solid-phase extraction (SPE).

[0448] For SPEs, Oasis µElution HLB 96-well plates (Waters, Wexford, Ireland) were used in a vacuum manifold. The plates were conditioned with acetonitrile equilibrated with 0.5% TFA (ACN, BioSolve Valkenswaard, The Netherlands), the supernatant was loaded, washed with 0.5% TFA, and the peptides were eluted with 50% ACN and 0.5% TFA. The recovered eluent was dried by rotary evaporation and reconstituted in 2% formic acid for subsequent LC-MS2 analysis.

[0449] For each digested and desalted sample, 100 ng of sample was analyzed using an Agilent 1290 Infinity HPLC system (Agilent Technologies, Waldbronn, Germany), equipped with a splitter for nanoflow and coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were separated on a 2 cm trapping column (100 μm inner diameter, packed with 3 μm ReproSil-Pur C18-AQ; Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) connected to a 50 cm analytical column (50 μm inner diameter, packed with 2.7 μm Poroshell 120 EC-C18; Agilent Technologies, Amstelveen, The Netherlands). Buffer A consisted of 0.1% formic acid, and buffer B consisted of 80% ACN containing 0.1% formic acid. The LC gradients are as follows: 0-5 min: 100% A (the rest is B), 5-53 min: 87% A to 60% A, 53-58 min: 0% A, 58-65 min: 100% A.

[0450] Mass spectrometry was performed in positive ion mode, with electrospray ionization from a coated fused silica emitter at a spray voltage of 2 kV. Each sample was measured in triplicate, using the same MS1 acquisition method but different MS2 methods. For MS1 scans, the mass range was set to m / z 350 to 2000, the resolution to 60,000, the AGC target to 400,000, and the maximum injection time to 50 ms. For each of the three MS2 methods, HCD fragmentation (30% normalized collision energy; NCE) was initiated at the highest charge state, the lowest m / z signal within a 3 s period, using a 30 s exclusion time. MS2 of HCD was recorded at a resolution of 30,000, m / z 120 to 4000, an AGC target to 50,000, and a maximum injection time of 50 ms. For MS2 method 1, only HCD fragmentation was performed. For MS2 method 2, at least three oxonium ions (Hex: 127.0390, 145.0495, 163.0601; HexNAc: 138.0550, 168.0655, 186.0761, 204.0867; PhosphoHex: 243.0264; NeuAc: 274.0921, 292.1027; complex: 366.1395, 405.0793, 407.1660, 512.1974, 657.2349) are detected in the HCD spectrum to trigger a stepping HCD of the same precursor signal, combining the HCD fragment with 10%, 25%, and 40% NCE. Stepped HCD recorded at a resolution of 30,000, m / z from 120 to 4000, an AGC target of 200,000, and a maximum injection time of 250 ms. For MS2 Method 3, oxonium ion detection triggered EThCD (30% supplemental activation), which recorded at a resolution of 30,000, m / z from 120 to 4000, an AGC target of 200,000, and a maximum injection time of 250 ms.

[0451] Bottom-up data were interpreted using Byonic v3.3.11 (Protein Metrics Inc.). Raw data were retrieved using C-terminal cleavage sites at Arg and Lys (trypsin) and Glu and Asp (GluC). Cleavage with up to 3 deletions was allowed using a precursor quality tolerance of 10 ppm and a fragment quality tolerance of 20 ppm. Cys carbamidomethylation was included as a fixed modification, and Met oxidation as a variable modification. For N-glycosylation, 279 compositions were included according to the N-glycan biosynthetic pathway.

[0452] Relative quantification was performed using Skyline (v3.7.0.11317). For this purpose, each glycosylated peptide identified by Byonic was integrated, including all major miscleavages and oxidative variants. For each of these, the aforementioned 279 glycan compositions were integrated from each LC-MS2 run. The resulting integrations were then selected to meet the following criteria: 1) error from theoretical mass ≤ 5 ppm, 2) idotp with a theoretical isotopic pattern ≥ 0.85, 3) elution within ± 2 min of the peptide's mean retention time, and 4) no significant overlapping isotopic patterns. The resulting list of glycopeptides was consistent with Byonic's annotations and was further used for relative quantification. For each selected peptide glycoform, the MS1 region was integrated, and peptides reported at the same N-glycosylation site were grouped. As an alternative quantification method, the number of peptide match (PSM) counts for each glycopeptide combination reported at a given glycosylation site was counted.

[0453] For visualization of glycan species, the recommendations of the Consortium for Functional Glycomics were followed. Glycan cartoons were constructed using GlycoWorkbench (v2.1 build146). For native MS analysis, a Vivaspin 500 30kDa molecular weight cutoff filter (Sartorius Stedim Biotech, Germany) was used. Antibody was buffer-exchanged to 150mM ammonium acetate pH 7.5 by centrifugation at 15,000×g for 10×15 min. After buffer exchange, the antibody concentration was adjusted to approximately 3μM with 150mM ammonium acetate pH 7.5.

[0454] Natural MS was performed on a modified Exactive Plus Orbitrap instrument (Thermo Fisher Scientific, Bremen) with extended mass range (EMR), calibrated using 25 mg / mL CsI solution. Voltage settings for the transport multipole and ion lens were manually optimized to provide good transport over the desired m / z range. Electrospray ionization was achieved using a gold-plated glass capillary at a capillary voltage of 1.2 kV, while MS was run at a source fragmentation of 80 V, a source temperature of 250 °C, a collision energy of 80 V, and a resolution of 35,000 at m / z 200. Desolvation of molecules was further achieved by adding nitrogen to the HCD cell to achieve a pressure of approximately 3.7 × 10⁻¹⁰ bar. Mass was calculated from the charge state distribution by fitting the charge against the mass to the lowest standard deviation (typically resulting in a mass error of less than 1 Da).

[0455] Pharmacokinetic / Pharmacodynamic Studies

[0456] For pharmacokinetic / pharmacodynamic studies of the anti-EGFR IgA antibody, BALB / cByJ mice (Jackson Laboratory) were intravenously injected with 100 µg (1 mg / mL) of the anti-EGFR IgA antibody. Blood was collected at specified time points, centrifuged to separate and collect serum. The diluted serum was then subjected to ELISA.

[0457] Biological distribution

[0458] Prior to radiolabeling, the anti-EGFR IgA antibody was conjugated with a chelating agent. The anti-EGFR IgA antibody was centrifuged at 12,000g for 8 min through a 30 kDa centrifuge filter. Subsequently, 500 μL of 0.1 M sodium bicarbonate (pH 8.2) was added to the centrifuge filter, and centrifuged again at 12,000g for 8 min. The centrifuge filter was inverted into a new tube and centrifuged at 1,000g for 2 min to obtain approximately 40 μL. 60 μL of sodium bicarbonate buffer was added to this to obtain 100 μL. A 20-fold molar excess of p-SCN-Bn-DTPA (2 mg / mL in dry DMSO (1 mg used in 500 μL)) was added, and the mixture was vortexed for 30 seconds and incubated at 37°C for 1 hour. Next, the BnDTPA-antibody conjugate was run through a G50 column and eluted in 100 μL fractions of 0.5 M MES buffer (pH 5.4) to obtain 12 fractions. The five most concentrated fractions were combined and centrifuged at 12,000g for 8 min through a 30kDa centrifuge filter. 500 μL of 0.5M MES buffer (pH 5.4) was added to the centrifuge filter, and the sample was centrifuged again at 12,300g for 8 min. The centrifuge filter was then inverted into a new microcentrifuge tube and centrifuged at 1,000g for 2 min. Protein concentration was measured using Nanodrop.

[0459] To radiolabel the antibody, 150 μL of indium solution (55.5 MBq) was added to 150 μg of antibody in MES buffer in a microcentrifuge tube, and the mixture was incubated at room temperature for 45 min. The reaction mixture was run through a G50 column and eluted with PBS (pH 7.4). Sixteen fractions of the sample were collected in 100 μL fractions (3 drops) in microcentrifuge tubes, the activity was checked, and the highest-performing fractions were combined.

[0460] To check the purity of the radiolabeled antibody using an iTLC strip, pipette a total of 2 μL of the reaction mixture onto the iTLC strip and allow it to dry for 2 minutes. Add a volume of 0.1 M citrate buffer to the measuring cylinder, covering the bottom. Place the iTLC strip in the measuring cylinder, allowing the citrate buffer to be drawn upwards until approximately 1 cm from the top. Remove the strip and scan using radiometric TLC.

[0461] Example 3 - Production of anti-EGFR IgA 3.0 min drug substance (DS)

[0462] The plasmid encoding anti-EGFR IgA3.0min was recombinantly produced in Chinese hamster ovary (CHO) cells. The resulting anti-EGFR IgA3.0min drug substance 1 (DS1) was purified and formulated into an anti-EGFR IgA3.0min drug product (DP) in buffer. The anti-EGFR IgA3.0min contains the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211. In short, the DNA plasmid encoding anti-EGFR IgA3.0min was transfected into CHO-K1 cells, and the anti-EGFR IgA3.0min antibody was expressed, purified, and characterized. The antibody was then exchanged for formulation buffer with buffer. The production process of anti-EGFR IgA3.0min is summarized as follows: Figure 5 As shown.

[0463] Design of anti-EGFR IgA 3.0min amino acid sequence

[0464] The anti-EGFR IgA3.0min has an IgA2 Fc backbone modified as described in Example 1 and an anti-EGFR Fv region. This anti-EGFR Fv region comprises a heavy chain variable region (VH) sequence (SEQ ID NO: 173) and a light chain variable region (VL) sequence (SEQ ID NO: 211). Furthermore, the anti-EGFR IgA3.0min antibody also contains a 19-amino acid signal peptide (MGWSCIILFLVATATGVHS (SEQ ID NO: 221)) and a 20-amino acid signal peptide (METDTLLLWVLLLWVPGSTG (SEQ ID NO: 167)) located at the N-terminus of the heavy and light chain constant regions, respectively, to facilitate efficient transport, folding, assembly, and post-translational modification. The final anti-EGFR IgA3.0min antibody is expected to produce a protein with a molecular weight of 141.4 kDa.

[0465] Cloning and transfection of expression vector into CH...

Claims

1. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) The EGFR binding domain contains a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (i) The VH region includes: (I) VH complementarity-determining region 1 (CDR-H1), which contains any amino acid sequence in SEQ ID NO: 34-54, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VH complementarity-determining region 2 (CDR-H2), comprising any amino acid sequence in SEQ ID NO: 57-78, or containing one to three variants thereof with substitutions, deletions, or insertions, and (III) VH complementarity-determining region 3 (CDR-H3), which contains any amino acid sequence in SEQ ID NO: 81-102, or contains one to three variants thereof with substitutions, deletions, or insertions. (ii) The VL region includes: (I) VL complementarity-determining region 1 (CDR-L1), which contains any amino acid sequence in SEQ ID NO: 105-126, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VL complementarity-determining region 2 (CDR-L2), which contains any amino acid sequence in SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and (III) VL complementarity-determining region 3 (CDR-L3), comprising any amino acid sequence in SEQ ID NO: 146-166, or containing one to three variants thereof with substitutions, deletions, or insertions, and (b) Immunoglobulin A (IgA) heavy chain constant region, which contains at least one mutation relative to the wild-type IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 1, wherein the mutation results in one or more of the following relative to the corresponding antibody containing the wild-type IgA heavy chain constant region: reduced glycosylation, reduced dimerization, reduced aggregation, increased thermal stability, or increased cyclic half-life.

2. The engineered antibody according to claim 1, wherein: (a) CDR-H1 contains any of the amino acid sequences in SEQ ID NO: 34-54; (b) CDR-H2 contains any of the amino acid sequences in SEQ ID NO: 57-78; (c) CDR-H3 contains any of the amino acid sequences in SEQ ID NO: 81-102; (d) CDR-L1 contains any of the amino acid sequences in SEQ ID NO: 105-126; (e) CDR-L2 contains any of the amino acid sequences in SEQ ID NO: 129-143; and (f) CDR-L3 contains any of the amino acid sequences in SEQ ID NO: 146-166.

3. The engineered antibody according to claim 1, wherein the VH region comprises an amino acid sequence having at least 80% identity with any of the VH region amino acid sequences listed in Table 5.

4. The engineered antibody according to claim 1, wherein the VL region comprises an amino acid sequence having at least 80% identity with any of the VL region amino acid sequences listed in Table 7.

5. The engineered antibody according to claim 1, wherein the amino acid sequence of the VH region and the amino acid sequence of the VL region are any combination provided in Table 8.

6. The engineered antibody according to claim 1, wherein the engineered antibody comprises an IgA light chain constant region, the IgA light chain constant region comprising an amino acid sequence having at least 80% identity with SEQ ID NO:

23.

7. The engineered antibody according to claim 1, wherein the IgA heavy chain constant region comprises an IgA CH1 domain, an IgA CH2 domain, and an IgA CH3 domain.

8. The engineered antibody according to claim 7, wherein the at least one mutation is present in the IgA CH1 region and is an N45.2 substitution, a P124 substitution, or a combination thereof, numbered according to the IMGT scheme, and each mutation is relative to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO:

1.

9. The engineered antibody according to claim 8, wherein the at least one mutation is: (a) Replace N45.2 with N45.2 from the group consisting of N45.2G and N45.2A; (b) P124R replacement; or (c) Any combination thereof; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

10. The engineered antibody according to claim 7, wherein the at least one mutation is present in the IgA CH2 region, and is: (a) N20 is substituted; (b) L21 replacement; (c) T22 is substituted; (d) C92 substitution; (e) Replaced by N120; (f) I121 substitution; or (g) T122 substitution; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

11. The engineered antibody according to claim 10, wherein the at least one mutation is: (a) Replace N20 with N20 from the group consisting of N20G, N20Q and N20T; (b) L21I replacement; (c) Replaced by T22S; (d) C92S replacement; (e) Replaced by N120T; (f) I121L replacement; (g) T122S substitution; or (h) Any combination thereof; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

12. The engineered antibody according to claim 7, wherein the at least one mutation is present in the IgA CH3 region, and is: (a) H5 substitution; (b) L7 replacement; (c) P10 is substituted; (d) T22 replacement; (e) L79 is replaced; (f) W81 replaces; (g) A85.1 substitute; (h) T86 replacement; (i) I88 replaces; (j) N135 is replaced; (k) C147 missing; (l) Y148 missing; (m) P131-Y148 missing; or (n) Their combinations; The numbering was performed according to the IMGT protocol, with each mutation corresponding to a residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

13. The engineered antibody according to claim 12, wherein the at least one mutation is: (a) H5 substitutions selected from the following: H5C, H5Y, H5F, H5M and H5W; (b) L7 substitutions selected from the following: L7F, L7Y, L7M, L7W, L7H and L7I; (c) P10C substitution; (d) T22 substitution selected from the following: T22V, T22I, T22L and T22A; (e) The L79 substitutes selected from the following: L79V, L79T, L79A and L79I; (f) The following W81 substitutes are selected: W81T, W81L, W81A, W81V and W81I; (g) A85.1 substitution selected from the following: A85.1F, A85.1Y, A85.1M, A85.1W and A85.1H; (h) T86 substitutions selected from the following: T86Y, T86F, T86M, T86W and T86H; (i) I88 substitutes selected from the following: I88L, I88A, I88V, and I88T; or (j) Any combination thereof; The numbering was performed according to the IMGT protocol, with each mutation corresponding to a residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

14. The engineered antibody according to claim 12, wherein the at least one mutation is: (a) Replaced by N135Q; (b) C147 is missing; (c) Y148 is missing; or (d) Any combination thereof; The numbering was performed according to the IMGT protocol, with each mutation corresponding to a residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

15. The engineered antibody according to claim 12, wherein the at least one mutation is a P131-Y148 deletion, numbered according to the IMGT protocol, the mutation corresponding to the corresponding residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

16. The engineered antibody according to claim 1, wherein the IgA heavy chain constant region comprises an amino acid sequence having at least 80% identity with the IgA heavy chain constant region of SEQ ID NO:

5.

17. The engineered antibody according to claim 1, wherein the engineered antibody is a monomer.

18. The engineered antibody of claim 1, wherein the EGFR binding domain binds to an EGFR peptide variant, wherein the EGFR variant comprises EGFRvIII, exon 19 deletion, L858R substitution in exon 21, C797S substitution, or T790M substitution.

19. The engineered antibody according to claim 1, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173, and the VL region comprises the amino acid sequence of SEQ ID NO:

211.

20. The engineered antibody according to claim 1, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 172, and the VL region comprises the amino acid sequence of SEQ ID NO:

198.

21. The engineered antibody according to claim 1, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 182, and the VL region comprises the amino acid sequence of SEQ ID NO:

207.

22. The engineered antibody according to claim 1, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 184, and the VL region comprises the amino acid sequence of SEQ ID NO:

209.

23. The engineered antibody of claim 1, wherein the engineered antibody is capable of inducing antibody-dependent cytotoxicity (ADCC) through immune effector cells.

24. The engineered antibody according to claim 23, wherein the immune effector cells are neutrophils, T cells, eosinophils or macrophages.

25. The engineered antibody according to claim 1, wherein the engineered antibody is a chimeric antibody, a single-chain antibody, a humanized antibody, a human antibody, a monoclonal antibody, a deimmunizing antibody, a bispecific antibody, a multispecific antibody, a multivalent antibody, or a combination thereof.

26. The engineered antibody according to claim 25, wherein the engineered antibody is a bispecific antibody.

27. The engineered antibody of claim 26, wherein the engineered antibody further comprises a binding domain that binds to a polypeptide antigen selected from the group consisting of MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, CD47, FcGR3, and 4-1BB.

28. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain that binds to the III domain of an EGFR peptide or a variant thereof; and (b) Immunoglobulin A (IgA) constant region, which, relative to the wild-type IgA heavy chain constant region having the amino acid sequence of SEQ ID NO: 1, comprises an IgA heavy chain constant region having at least one mutation, wherein the mutation results in one or more of the following relative to the corresponding antibody containing the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, increased thermal stability, or increased cyclic half-life.

29. The engineered antibody of claim 28, wherein the IgA constant region comprises: (a) The IgA heavy chain constant region having the amino acid sequence of SEQ ID NO: 5; (b) An IgA light chain constant domain having the amino acid sequence SEQ ID NO:

23.

30. The engineered antibody of claim 28, wherein the IgA constant domain comprises an IgA heavy chain constant region, the IgA heavy chain constant region comprises IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region comprises the following mutation: (a) N45.2G substitution in the CH1 domain; (b) P124R substitution in the CH1 domain; (c) C92S substitution in the CH2 domain; (d) N120T substitution in the CH2 domain; (e) I121L substitution in the CH2 domain; and (f) T122S substitution in the CH2 domain; (g) Numbering is performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

31. The engineered antibody of claim 28, wherein the EGFR binding domain binds an epitope of an EGFR polypeptide or a variant thereof, the epitope comprising any of the following EGFR amino acid residues: P349, F352, D355, P362, D355, Q384, P387, Q408, H409, F412, I438, K443, K465, I467, or S468.

32. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain that binds to domain II of an EGFR peptide or a variant thereof; and (b) An immunoglobulin A (IgA) constant domain comprising an IgA heavy chain constant region having at least one mutation relative to a wild-type IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 1, wherein the mutation results in one or more of the following relative to the corresponding antibody containing the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, increased thermal stability, or increased cyclic half-life.

33. The engineered antibody of claim 32, wherein the IgA constant region comprises: (a) The IgA heavy chain constant region having the amino acid sequence SEQ ID NO: 5; and (b) An IgA light chain constant domain having the amino acid sequence SEQ ID NO:

23.

34. The engineered antibody of claim 32, wherein the IgA constant region comprises an IgA heavy chain constant region, the IgA heavy chain constant region comprising IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region comprises the following mutation: (a) N45.2G substitution in the CH1 domain; (b) P124R substitution in the CH1 domain; (c) C92S substitution in the CH2 domain; (d) N120T substitution in the CH2 domain; (e) I121L substitution in the CH2 domain; and (f) T122S substitution in the CH2 domain; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

35. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (i) The VH region includes: (I) VH complementarity-determining region 1 (CDR-H1), which contains any amino acid sequence in SEQ ID NO: 34-54, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VH complementarity-determining region 2 (CDR-H2), comprising any amino acid sequence in SEQ ID NO: 57-78, or containing one to three variants thereof with substitutions, deletions, or insertions, and (III) VH complementarity-determining region 3 (CDR-H3), which contains any amino acid sequence in SEQ ID NO: 81-102, or contains one to three variants thereof with substitutions, deletions, or insertions. (ii) The VL region includes: (I) VL complementarity-determining region 1 (CDR-L1), which contains any amino acid sequence in SEQ ID NO: 105-126, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VL complementarity-determining region 2 (CDR-L2), which contains any amino acid sequence in SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and (III) VL complementarity-determining region 3 (CDR-L3), comprising any amino acid sequence in SEQ ID NO: 146-166, or containing one to three variants thereof with substitutions, deletions, or insertions; and (b) Immunoglobulin A (IgA) constant domain, which includes: (i) the heavy chain constant region having the amino acid sequence of SEQ ID NO: 5; and (ii) A light chain constant domain having the amino acid sequence of SEQ ID NO:

23.

36. The engineered antibody according to claim 35, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173, and the VL region comprises the amino acid sequence of SEQ ID NO:

211.

37. The engineered antibody according to claim 35, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 172, and the VL region comprises the amino acid sequence of SEQ ID NO:

198.

38. The engineered antibody according to claim 35, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 182, and the VL region comprises the amino acid sequence of SEQ ID NO:

207.

39. The engineered antibody according to claim 35, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 184, and the VL region comprises the amino acid sequence of SEQ ID NO:

209.

40. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (i) The VH region includes: (I) VH complementarity-determining region 1 (CDR-H1), which contains any amino acid sequence in SEQ ID NO: 34-54, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VH complementarity-determining region 2 (CDR-H2), comprising any amino acid sequence in SEQ ID NO: 57-78, or containing one to three variants thereof with substitutions, deletions, or insertions, and (III) VH complementarity-determining region 3 (CDR-H3), which contains any amino acid sequence in SEQ ID NO: 81-102, or contains one to three variants thereof with substitutions, deletions, or insertions. (ii) The VL region includes: (I) VL complementarity-determining region 1 (CDR-L1), which contains any amino acid sequence in SEQ ID NO: 105-126, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VL complementarity-determining region 2 (CDR-L2), which contains any amino acid sequence in SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and (III) VL complementarity-determining region 3 (CDR-L3), comprising any amino acid sequence in SEQ ID NO: 146-166, or containing one to three variants thereof with substitutions, deletions, or insertions, and (b) Immunoglobulin A (IgA) heavy chain constant region comprising IgA CH1, CH2, and CH3 domains, wherein the IgA heavy chain constant region contains the following mutations: (i) N45.2G substitution in the CH1 domain, (ii) P124R substitution in the CH1 domain (iii) C92S substitution in the CH2 domain (iv) N120T substitution in the CH2 domain (v) I121L substitution in the CH2 domain, and (vi) T122S substitution in the CH2 domain; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

41. The engineered antibody according to claim 40, wherein the IgA heavy chain constant region further comprises the following mutation in the CH3 domain: (a) Replaced by N135Q; (b) C147 missing; and (c) Y148 missing; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

42. The engineered antibody according to claim 40, wherein the corresponding residues in the IgA heavy chain constant region relative to the wild-type IgA heavy chain constant region of SEQ ID NO: 1 are further included by a P131-Y148 deletion in the CH3 domain, numbered according to the IMGT protocol.

43. The engineered antibody according to claim 42, wherein the IgA heavy chain constant region further comprises the following mutation in the CH2 domain: (a) Replace N20 with N20 from the group consisting of N20G, N20Q and N20T; (b) L21I replacement; and (c) Replaced by T22S; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

44. The engineered antibody according to any one of claims 40 to 43, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173, and the VL region comprises the amino acid sequence of SEQ ID NO:

211.

45. The engineered antibody according to any one of claims 40 to 43, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 172, and the VL region comprises the amino acid sequence of SEQ ID NO:

198.

46. ​​The engineered antibody according to any one of claims 40 to 43, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 182, and the VL region comprises the amino acid sequence of SEQ ID NO:

207.

47. The engineered antibody according to any one of claims 40 to 43, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 184, and the VL region comprises the amino acid sequence of SEQ ID NO:

209.

48. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173 and the VL region comprises the amino acid sequence of SEQ ID NO: 211; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

49. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 172 and the VL region comprises the amino acid sequence of SEQ ID NO: 198; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

50. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 182 and the VL region comprises the amino acid sequence of SEQ ID NO: 207; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

51. An engineered epidermal growth factor receptor (EGFR) binding antibody or its functional EGFR-binding fragment, comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 184 and the VL region comprises the amino acid sequence of SEQ ID NO: 209; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

52. A pharmaceutical composition comprising the engineered antibody according to any one of claims 1 to 51, and a pharmaceutically acceptable carrier.

53. A method of treating cancer in a subject in need, the method comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR-binding fragment thereof, said engineered epidermal growth factor receptor (EGFR) binding antibody or functional EGFR-binding fragment thereof comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (i) The VH region includes: (I) VH complementarity-determining region 1 (CDR-H1), which contains any amino acid sequence in SEQ ID NO: 34-54, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VH complementarity-determining region 2 (CDR-H2), comprising any amino acid sequence in SEQ ID NO: 57-78, or containing one to three variants thereof with substitutions, deletions, or insertions, and (III) VH complementarity-determining region 3 (CDR-H3), which contains any amino acid sequence in SEQ ID NO: 81-102, or contains one to three variants thereof with substitutions, deletions, or insertions. (ii) The VL region includes: (I) VL complementarity-determining region 1 (CDR-L1), which contains any amino acid sequence in SEQ ID NO: 105-126, or contains one to three variants thereof with substitutions, deletions, or insertions. (II) VL complementarity-determining region 2 (CDR-L2), which contains any amino acid sequence in SEQ ID NO: 129-143, or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and (III) VL complementarity-determining region 3 (CDR-L3), which contains any amino acid sequence in SEQ ID NO: 146-166, or contains one to three variants thereof with substitutions, deletions or insertions; as well as (b) Immunoglobulin A (IgA) heavy chain constant region, which contains at least one mutation relative to the wild-type IgA heavy chain constant region having the amino acid sequence of SEQ ID NO: 1, wherein the mutation results in one or more of the following relative to the corresponding antibody containing the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, increased thermal stability, or increased cyclic half-life.

54. The method of claim 53, wherein the IgA heavy chain constant region comprises IgA CH1, CH2, and CH3 domains, and wherein the IgA heavy chain constant region comprises the following mutations: (a) N45.2G substitution in the CH1 domain; (b) P124R substitution in the CH1 domain; (c) C92S substitution in the CH2 domain; (d) N120T substitution in the CH2 domain; (e) I121L substitution in the CH2 domain; and (f) T122S substitution in the CH2 domain; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the corresponding antibody containing the corresponding residue in the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

55. The method of claim 53, wherein the IgA heavy chain constant region further comprises the following mutation in the CH3 domain: (a) Replaced by N135Q; (b) C147 missing; and (c) Y148 missing; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

56. The method of claim 54, wherein the IgA heavy chain constant region further comprises a deletion of P131-Y148 in the CH3 domain relative to the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO: 1, numbered according to the IMGT scheme.

57. The method of claim 56, wherein the IgA heavy chain constant region further comprises the following mutation in the CH2 domain: (a) Replace N20 with N20 from the group consisting of N20G, N20Q and N20T; (b) L21I replacement; and (c) Replaced by T22S; The numbering was performed according to the IMGT protocol, with each mutation corresponding to the antibody containing the constant region of the wild-type IgA heavy chain of SEQ ID NO:

1.

58. The method of claim 53, wherein the cancer is a solid tumor cancer.

59. The method of claim 53, wherein the cancer is selected from the group consisting of: lung cancer, head and neck cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, kidney cancer, mesothelioma, and glioblastoma.

60. The method of claim 53, wherein the cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

61. The method of claim 53, wherein the cancer is colorectal cancer.

62. The method of claim 53, wherein the cancer is squamous cell carcinoma of the head and neck.

63. The method of claim 53, wherein the cancer is non-small cell lung cancer.

64. The method of claim 53, wherein the engineered antibody inhibits cancer-related tumor growth.

65. The method of claim 53, wherein the administration is subcutaneous, intravenous, transdermal, intraperitoneal, oral, intramuscular, or intracranial.

66. The method of claim 53, wherein the engineered antibody is administered in combination with the second therapeutic agent to the subject.

67. The method of claim 66, wherein the second therapeutic agent comprises an anticancer agent, a chemotherapeutic agent, a radiotherapy agent, a cytotoxic agent, a corticosteroid, an immunotherapy agent, a dietary supplement, or an antioxidant.

68. The method of claim 53, wherein the second therapeutic agent is administered before, simultaneously with, or after the administration of the engineered antibody.

69. The method of claim 53, wherein the subject is a rodent, a non-human primate, or a human.

70. The method of claim 69, wherein the subject is a rodent, and wherein the effective amount is administered at a dose of 1 mg / kg to 25 mg / kg, twice weekly subcutaneously, intravenously or intraperitoneally for 35-40 days.

71. The method of claim 69, wherein the effective dose is administered intravenously at a dose of 25 mg / kg every seven days for five weeks, except for a third dose delivered at 12.5 mg / kg.

72. The method of claim 69, wherein the effective amount is administered intravenously twice weekly at a dose of 1 mg / kg to 25 mg / kg.

73. A method of treating cancer in a subject of need, the method comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR-binding fragment thereof, said engineered epidermal growth factor receptor (EGFR) binding antibody or functional EGFR-binding fragment thereof comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 173 and the VL region comprises the amino acid sequence of SEQ ID NO: 211; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

74. A method of treating cancer in a subject of need, the method comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR-binding fragment thereof, said engineered epidermal growth factor receptor (EGFR) binding antibody or functional EGFR-binding fragment thereof comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 172 and the VL region comprises the amino acid sequence of SEQ ID NO: 198; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

75. A method of treating cancer in a subject of need, the method comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR-binding fragment thereof, said engineered epidermal growth factor receptor (EGFR) binding antibody or functional EGFR-binding fragment thereof comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 182 and the VL region comprises the amino acid sequence of SEQ ID NO: 207; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

76. A method of treating cancer in a subject of need, the method comprising administering to the subject an effective amount of an engineered epidermal growth factor receptor (EGFR) binding antibody or a functional EGFR-binding fragment thereof, said engineered epidermal growth factor receptor (EGFR) binding antibody or functional EGFR-binding fragment thereof comprising: (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 184 and the VL region comprises the amino acid sequence of SEQ ID NO: 209; and (b) Immunoglobulin A (IgA) constant domain comprising a heavy chain constant region having the amino acid sequence SEQ ID NO: 5 and a light chain constant domain having the amino acid sequence SEQ ID NO:

23.

77. The method according to any one of claims 73 to 76, wherein the cancer is colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, or a combination thereof.

78. An isolated nucleic acid encoding an engineered antibody according to any one of claims 1 to 51.

79. A host cell expressing the engineered antibody according to any one of claims 1 to 51.

80. A composition comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent comprises an engineered antibody according to any one of claims 1 to 51, and the second therapeutic agent is combined with MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, 4-1BB, or a combination thereof.

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