Modified IgA antibody and method of use

Abstract

Modified IgA antibodies comprising (a) an EGFR-binding domain and (b) an IgA heavy chain constant region are shown herein. Also shown herein are methods for treating cancer in subjects requiring such treatment, comprising the step of administering an effective amount of the modified antibody to the subject.

Description

[Technical Field]

[0001] Applications that are cross-referenced This application claims priority to U.S. Provisional Application No. 63 / 516,428, filed on 28 July 2023, the entire contents of which are incorporated herein by reference.

[0002] Inclusion by referencing the sequence list This application includes a sequence listing filed via the Patent Center. The sequence listing, titled "199828-714601_PCT_SL.xml", was created on 26 July 2024, is 189,604 bytes in size, and is incorporated herein by reference in its entirety.

[0003] This disclosure generally relates to antibodies that bind to the epidermal growth factor receptor (EGFR). [Background technology]

[0004] Monoclonal antibodies that target specific antigens associated with a disease or condition have emerged as attractive therapies for improving disease or condition in human subjects. Over the years, a growing number of monoclonal antibodies (primarily IgG-based antibodies) targeting different tumor antigens have been approved for use in cancer treatment. However, their clinical efficacy and side effects, particularly those associated with IgG-based antibody monotherapy, remain unresolved. Therefore, developing new antibody therapies with increased clinical efficacy and / or reduced incidence / severity of side effects is of great interest. [Overview of the Initiative]

[0005] The engineered antibody described herein comprises (a) an epidermal growth factor receptor (EGFR) binding domain including a heavy chain variable region (VH) domain and a light chain variable region (VL) domain, and (b) an IgA heavy chain constant region having at least one mutation compared to the wild-type immunoglobulin A (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 compared to the corresponding antibody containing the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, increased mechanical stability, or increased circulating half-life. In some embodiments, the VH domain comprises three heavy chain complementarity determining regions (CDR-H), which are: (1) CDR-H1, which comprises one of the amino acid sequences of the heavy chain complementarity determining regions (CDRs) of SEQ ID NOs. 34 to 54 or variants thereof including one to three substitutions, deletions, or insertions; (2) CDR-H2, which comprises one of the amino acid sequences of SEQ ID NOs. 57 to 78 or variants thereof including one to three substitutions, deletions, or insertions; and (3) CDR-H3, which comprises one of the amino acid sequences of SEQ ID NOs. 81 to 102 or variants thereof including one to three substitutions, deletions, or insertions. In some embodiments, the VL domain comprises three light chain complementarity determining regions (CDR-L), which are: (1) CDR-L1, which contains any one of the amino acid sequences of SEQ ID NOs. 105-126 or variants thereof including 1-3 substitutions, deletions, or insertions; (2) CDR-L2, which contains any one of the amino acid sequences of SEQ ID NOs. 129-143 or variants thereof including 1-3 substitutions, deletions, or insertions; and (3) CDR-L3, which contains any one of the amino acid sequences of SEQ ID NOs. 146-166 or variants thereof including 1-3 substitutions, deletions, or insertions.In some embodiments, CDR-H and CDR-L are selected according to one of the combinations shown in Table 9. In some embodiments, the modified antibodies include CDR-H1, which contains an amino acid sequence at least 80% identical to one of the amino acid sequences of SEQ ID NOs. 34-54; CDR-H2, which contains an amino acid sequence at least 80% identical to one of the amino acid sequences of SEQ ID NOs. 57-78; CDR-H3, which contains an amino acid sequence at least 80% identical to one of the amino acid sequences of SEQ ID NOs. 81-102; CDR-L1, which contains an amino acid sequence at least 80% identical to one of the amino acid sequences of SEQ ID NOs. 105-126; CDR-L2, which contains an amino acid sequence identical to one of the amino acid sequences of SEQ ID NOs. 129-143; and CDR-L3, which contains an amino acid sequence at least 80% identical to one of the amino acid sequences of SEQ ID NOs. 146-166. In some embodiments, the modified antibody includes CDR-H1 containing an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 34-54, CDR-H2 containing an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 57-78, CDR-H3 containing an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 81-102, CDR-L1 containing an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 105-126, CDR-L2 containing an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 129-143, and CDR-L3 containing an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 146-166. In some embodiments, the VH domain contains an amino acid sequence that is at least 80% identical to any one of the amino acid sequences listed in Table 5. In some embodiments, the VL domain contains an amino acid sequence that is at least 80% identical to any one of the amino acid sequences listed in Table 7. In some embodiments, the VH domain and VL domain are selected according to one of the combinations shown in Table 8. In some embodiments, the modified antibody includes an IgA light chain constant region having an amino acid sequence at least 80% identical to that of SEQ ID NO: 23. In some embodiments, the IgA heavy chain constant region includes an IgA CH1 region, an IgA CH2 region, and an IgA CH3 region.In some embodiments, at least one mutation is located in the IgA CH1 region and is numbered according to the IMGT scheme as an N45.2 substitution, a P124 substitution, or a combination thereof, compared to a corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, at least one mutation is numbered according to the IMGT scheme as an (a) N45.2 substitution selected from the group consisting of N45.2G and N45.2A, (b) P124R substitution, or (c) any combination thereof, compared to a corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, at least one mutation is located in the IgA CH2 region and is numbered according to the IMGT scheme as an (a) N20 substitution, (b) L21 substitution, (c) T22 substitution, (d) C92 substitution, (e) N120 substitution, (f) I121 substitution, or (g) T122 substitution, compared to a corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, at least one mutation is, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1, numbered according to the IMGT scheme, (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. In some embodiments, at least one mutation is located in the IgA CH3 region and is numbered according to the IMGT scheme, compared to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO: 1, and is either (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) a combination thereof.In some embodiments, at least one mutation is numbered according to the IMGT scheme, compared to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO: 1, and includes: (a) H5 substitutions selected from the group consisting of H5C, H5Y, H5F, H5M and H5W; (b) L7 substitutions selected from the group consisting of L7F, L7Y, L7M, L7W, L7H and L7I; (c) P10C substitutions; (d) T22 substitutions selected from the group consisting of T22V, T22I, T22L and T22A; (e) L79V, L79T, L79A and (f) L79 substitutions selected from the group consisting of L79I; (g) W81 substitutions selected from the group consisting of W81T, W81L, W81A, W81V and W81I; (h) A85.1 substitutions selected from the group consisting of A85.1F, A85.1Y, A85.1M, A85.1W and A85.1H; (i) T86 substitutions selected from the group consisting of T86Y, T86F, T86M, T86W and T86H; (j) I88 substitutions selected from the group consisting of I88L, I88A, I88V and I88T; or (j) any combination thereof. In some embodiments, at least one mutation is (a) an N135Q substitution, (b) a C147 deletion, (c) a Y148 deletion, or (d) any combination thereof, numbered according to the IMGT scheme compared to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO: 1. In some embodiments, at least one mutation is a deletion of P131-Y148, numbered according to the IMGT scheme, compared 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 contains an amino acid sequence that is at least 80% identical to the IgA heavy chain constant region of SEQ ID NO: 3. In some embodiments, the modified antibody is monomeric. In some embodiments, the EGFR binding domain binds to an EGFR polypeptide variant, where the EGFR variant includes EGFRvIII, exon 19 deletion, L858R substitution, C797S substitution, or T790M substitution in exon 21. 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. In some embodiments, the modified antibody has the ability to induce antibody-dependent cell cytotoxicity (ADCC) via immune effector cells. In some embodiments, the immune effector cells are neutrophils, T cells, eosinophils, or macrophages. In some embodiments, the modified antibody is a chimeric antibody, a single-chain antibody, a humanized antibody, a human antibody, a monoclonal antibody, a deimmunized antibody, a bispecific antibody, a multispecific antibody, a polyvalent antibody, or a combination thereof. In some embodiments, the modified antibody is a bispecific antibody. In some embodiments, the modified 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, FcGR3, and 4-1BB.

[0006] Furthermore, modified antibodies are shown herein that include (a) an EGFR-binding domain that binds to domain III or a variant thereof of an epidermal growth factor receptor (EGFR) polypeptide, and (b) an IgA constant domain comprising an IgA heavy chain constant region having at least one mutation compared to a wild-type immunoglobulin A (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 compared to a corresponding antibody comprising a wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, increased mechanical stability, or increased circulating half-life. In some embodiments, the IgA constant domain comprises (a) an IgA heavy chain constant region having the amino acid sequence of SEQ ID NO: 3, and (b) an IgA light chain constant region having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgA constant domain includes an IgA heavy chain constant region comprising IgA CH1, CH2, and CH3 domains, where the IgA heavy chain constant region includes the following mutations, numbered according to the IMGT scheme, compared to the corresponding antibody comprising the wild-type IgA heavy chain constant region of Sequence ID No. 1, respectively: (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. In some embodiments, the EGFR-binding domain binds to an epitope of an EGFR polypeptide or variant thereof containing one of the following EGFR amino acid residues: P349, F352, D355, P362, D355, Q384, P387, Q408, H409, F412, I438, K443, K465, I467, or S468.

[0007] Furthermore, modified antibodies are shown herein that include (a) an EGFR-binding domain that binds to domain II or a variant thereof of an epidermal growth factor receptor (EGFR) polypeptide, and (b) an IgA constant domain comprising an IgA heavy chain constant region having at least one mutation compared to a wild-type immunoglobulin A (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 compared to a wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, increased mechanical stability, or increased circulating half-life. In some embodiments, the IgA constant domain comprises (a) an IgA heavy chain constant region having the amino acid sequence of SEQ ID NO: 3, and (b) an IgA light chain constant region having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IgA constant domain includes an IgA heavy chain constant region comprising IgA CH1, CH2, and CH3 domains, where the IgA heavy chain constant region includes the following mutations, numbered according to the IMGT scheme, compared to the corresponding antibody comprising the wild-type IgA heavy chain constant region of Sequence ID No. 1, respectively: (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.

[0008] Furthermore, modified antibodies comprising (a) an epidermal growth factor receptor (EGFR) binding domain including a heavy chain variable region (VH) domain and a light chain variable region (VL) domain, and (b) an immunoglobulin A (IgA) constant domain including (i) a 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, are also shown herein. 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 comprises the amino acid sequence of SEQ ID NO: 184, and the VL region comprises the amino acid sequence of SEQ ID NO: 209. In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H), which are: (1) CDR-H1 comprising at least one amino acid sequence from the heavy chain complementarity-determining regions (CDRs) of SEQ ID NOs. 34 to 54 or one of their variants including one to three substitutions, deletions, or insertions; (2) CDR-H2 comprising the amino acid sequences of SEQ ID NOs. 57 to 78 or one of their variants including one to three substitutions, deletions, or insertions; and (3) CDR-H3 comprising the amino acid sequences of SEQ ID NOs. 81 to 102 or one of their variants including one to three substitutions, deletions, or insertions. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L), which are: (1) CDR-L1 comprising any one of the amino acid sequences of SEQ ID NOs. 105-126 or variants thereof including 1-3 substitutions, deletions, or insertions; (2) CDR-L2 comprising any one of the amino acid sequences of SEQ ID NOs. 129-143 or variants thereof including 1-3 substitutions, deletions, or insertions; and (3) CDR-L3 comprising any one of the amino acid sequences of SEQ ID NOs. 146-166 or variants thereof including 1-3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected according to any one of the combinations shown in Table 9.

[0009] Furthermore, modified antibodies are shown herein that include (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 IgA heavy chain constant region comprising immunoglobulin A (IgA) CH1, CH2, and CH3 domains, each containing the following mutations numbered according to the IMGT scheme compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of Sequence ID No. 1: (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. In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H), which are: (1) CDR-H1 comprising at least one amino acid sequence from the heavy chain complementarity-determining regions (CDRs) of SEQ ID NOs. 34 to 54 or one of their variants including one to three substitutions, deletions, or insertions; (2) CDR-H2 comprising the amino acid sequences of SEQ ID NOs. 57 to 78 or one of their variants including one to three substitutions, deletions, or insertions; and (3) CDR-H3 comprising the amino acid sequences of SEQ ID NOs. 81 to 102 or one of their variants including one to three substitutions, deletions, or insertions. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L), which are: (1) CDR-L1 comprising any one of the amino acid sequences of SEQ ID NOs. 105-126 or variants thereof including 1-3 substitutions, deletions, or insertions; (2) CDR-L2 comprising any one of the amino acid sequences of SEQ ID NOs. 129-143 or variants thereof including 1-3 substitutions, deletions, or insertions; and (3) CDR-L3 comprising any one of the amino acid sequences of SEQ ID NOs. 146-166 or variants thereof including 1-3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected according to any one of the combinations shown in Table 9.In some embodiments, the IgA heavy chain constant region further includes the following mutations in the CH3 domain, numbered according to the IMGT scheme, compared to a corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1: (a) N135Q substitution, (b) C147 deletion, and (c) Y148 deletion. In some embodiments, the IgA heavy chain constant region further includes deletions of P131-Y148 in the CH3 domain, numbered according to the IMGT scheme, compared to a 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 includes the following mutations in the CH2 domain, numbered according to the IMGT scheme, compared to a corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1: (a) N20 substitution selected from the group consisting of N20G, N20Q, and N20T; (b) L21I substitution; and (c) T22S substitution. In some embodiments, the VH region includes the amino acid sequence of SEQ ID NO: 173, and the VL region includes the amino acid sequence of SEQ ID NO: 211. In some embodiments, the VH region includes the amino acid sequence of SEQ ID NO: 172, and the VL region includes the amino acid sequence of SEQ ID NO: 198. In some embodiments, the VH region includes the amino acid sequence of SEQ ID NO: 182, and the VL region includes the amino acid sequence of SEQ ID NO: 207. In some embodiments, the VH region includes the amino acid sequence of SEQ ID NO: 184, and the VL region includes the amino acid sequence of SEQ ID NO: 209.

[0010] Furthermore, the following are described herein: an engineered epidermal growth factor receptor (EGFR) binding antibody 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.

[0011] Furthermore, EGFR-binding modified antibodies or EGFR-binding functional fragments thereof are shown herein, comprising (a) an epidermal growth factor receptor (EGFR) binding domain including a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region includes the amino acid sequence of SEQ ID NO: 172 and the VL region includes the amino acid sequence of SEQ ID NO: 198, and (b) an immunoglobulin A (IgA) constant domain including 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.

[0012] Furthermore, EGFR-binding modified antibodies or EGFR-binding functional fragments thereof are shown herein, comprising (a) an epidermal growth factor receptor (EGFR) binding domain including a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region includes the amino acid sequence of SEQ ID NO: 182 and the VL region includes the amino acid sequence of SEQ ID NO: 207, and (b) an immunoglobulin A (IgA) constant domain including 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.

[0013] Furthermore, EGFR-binding modified antibodies or EGFR-binding functional fragments thereof are shown herein, comprising (a) an epidermal growth factor receptor (EGFR) binding domain including a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region includes the amino acid sequence of SEQ ID NO: 184 and the VL region includes the amino acid sequence of SEQ ID NO: 209, and (b) an immunoglobulin A (IgA) constant domain including 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.

[0014] Furthermore, pharmaceutical compositions comprising modified antibodies and pharmaceutically acceptable carriers as described herein are also shown herein.

[0015] Furthermore, methods for treating cancer in subjects requiring such treatment are also described herein. In some embodiments, the method includes administering to a subject an effective amount of a modified antibody comprising: (a) an epidermal growth factor receptor (EGFR) binding domain including a heavy chain variable region (VH) domain and a light chain variable region (VL) domain; and (b) an IgA heavy chain constant region having at least one mutation compared to the wild-type immunoglobulin A (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 compared to the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, increased mechanical stability, or increased circulating half-life. In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H), which are: (1) CDR-H1 comprising at least one amino acid sequence from the heavy chain complementarity-determining regions (CDRs) of SEQ ID NOs. 34 to 54 or one of their variants including one to three substitutions, deletions, or insertions; (2) CDR-H2 comprising the amino acid sequences of SEQ ID NOs. 57 to 78 or one of their variants including one to three substitutions, deletions, or insertions; and (3) CDR-H3 comprising the amino acid sequences of SEQ ID NOs. 81 to 102 or one of their variants including one to three substitutions, deletions, or insertions. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L), which are: (1) CDR-L1 comprising any one of the amino acid sequences of SEQ ID NOs. 105-126 or variants thereof including 1-3 substitutions, deletions, or insertions; (2) CDR-L2 comprising any one of the amino acid sequences of SEQ ID NOs. 129-143 or variants thereof including 1-3 substitutions, deletions, or insertions; and (3) CDR-L3 comprising any one of the amino acid sequences of SEQ ID NOs. 146-166 or variants thereof including 1-3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected according to any one of the combinations shown in Table 9.In some embodiments, the VH domain comprises three heavy chain complementarity-determining regions (CDR-H), which are: (1) CDR-H1 comprising at least one amino acid sequence from the heavy chain complementarity-determining regions (CDRs) of SEQ ID NOs. 34 to 54 or one of their variants including one to three substitutions, deletions, or insertions; (2) CDR-H2 comprising the amino acid sequences of SEQ ID NOs. 57 to 78 or one of their variants including one to three substitutions, deletions, or insertions; and (3) CDR-H3 comprising the amino acid sequences of SEQ ID NOs. 81 to 102 or one of their variants including one to three substitutions, deletions, or insertions. In some embodiments, the VL domain comprises three light chain complementarity-determining regions (CDR-L), which are: (1) CDR-L1 comprising any one of the amino acid sequences of SEQ ID NOs. 105-126 or variants thereof including 1-3 substitutions, deletions, or insertions; (2) CDR-L2 comprising any one of the amino acid sequences of SEQ ID NOs. 129-143 or variants thereof including 1-3 substitutions, deletions, or insertions; and (3) CDR-L3 comprising any one of the amino acid sequences of SEQ ID NOs. 146-166 or variants thereof including 1-3 substitutions, deletions, or insertions. In some embodiments, CDR-H and CDR-L are selected according to any one of the combinations shown in Table 9. In some embodiments, the mutations result in one or more of the following compared to the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, increased mechanical stability, or increased cyclic half-life. In some embodiments, the IgA heavy chain constant region comprises IgA CH1, CH2, and CH3 domains, where the IgA heavy chain constant region includes the following mutations, numbered according to the IMGT scheme, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of Sequence ID No. 1: (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.In some embodiments, the IgA heavy chain constant region further includes the following mutations in the CH3 domain, numbered according to the IMGT scheme, compared to a corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1: (a) N135Q substitution, (b) C147 deletion, and (c) Y148 deletion. In some embodiments, the IgA heavy chain constant region further includes deletions of P131-Y148 in the CH3 domain, numbered according to the IMGT scheme, compared to a 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 includes the following mutations in the CH2 domain, numbered according to the IMGT scheme, compared to a corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1: (a) N20 substitution selected from the group consisting of N20G, N20Q, and N20T; (b) L21I substitution; and (c) T22S substitution. In some embodiments, the cancer is a solid tumor carcinoma. 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, 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. In some embodiments, the modified antibody inhibits tumor growth associated with the cancer. In some embodiments, the administration step is subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular, or intracranial administration. In some embodiments, the modified antibody is administered to the subject in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent includes anticancer agents, chemotherapeutic agents, radiotherapy, cytotoxic agents, corticosteroids, immunotherapeutic agents, nutritional supplements, or antioxidants. In some embodiments, the second therapeutic agent is administered before, simultaneously with, or after the administration of the modified antibody. In some embodiments, the subjects are rodents, non-human primates, or humans. In some embodiments, the subjects are rodents, where the effective dose is 1 mg / kg to 25 mg / kg, administered subcutaneously, intravenously, or intraperitoneally twice a week for 35 to 40 days.In some embodiments, the effective dose is administered intravenously at a dose of 25 mg / kg every 7 days for 5 weeks, with the exception of a third dose delivered at 12.5 mg / kg. In some embodiments, the effective dose is administered intravenously twice a week at a dose of 1 mg / kg to 25 mg / kg.

[0016] Furthermore, a method for treating cancer in subjects requiring such treatment is also described herein, the method comprising administering to a subject an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, comprising (a) an epidermal growth factor receptor (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.

[0017] Furthermore, a method for treating cancer in a subject requiring such treatment is also described herein, the method comprising administering to the subject an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, comprising (a) an epidermal growth factor receptor (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.

[0018] Also provided herein is a method of treating cancer in a subject that requires it, the method comprising administering to the subject an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, comprising: (a) an epidermal growth factor receptor (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.

[0019] Also provided herein is a method of treating cancer in a subject that requires it, the method comprising administering to the subject an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, comprising: (a) an epidermal growth factor receptor (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.

[0020] Also provided herein is an isolated nucleic acid encoding the modified antibody described herein.

[0021] Also provided herein is a host cell expressing the modified antibody described herein.

[0022] Also provided herein is a composition comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent comprises any one of the modified antibodies described herein and the second therapeutic agent binds to MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, 4-1BB or a combination thereof.

[0023] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0024] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which illustrates exemplary embodiments in which the principles of the present disclosure are utilized, and the appended drawings, which are as follows.

Brief Description of the Drawings

[0025] [Figure 1] It is a diagram showing the amino acid sequence of a human IgA2 heavy chain having the deposit number UniProt reference number A0A0G2JMB2 (SEQ ID NO: 1). The emphasized amino acids indicate the residues that are modified in some of the embodiments described herein. The underlined sequence indicates the CH3 tail piece, which is partially or completely deleted in some embodiments. [Figure 2] It is a diagram showing the amino acid sequence of a human IgA2 heavy chain having the deposit number UniProt reference number P01877-1 (SEQ ID NO: 2). The emphasized amino acids indicate the residues that are modified in some of the embodiments described herein. The underlined sequence indicates the CH3 tail piece, which is partially or completely deleted in some embodiments. [[ID=*18]] [Figure 3] It is a diagram showing the amino acid sequence of a human IgA2 heavy chain having the deposit number UniProt reference number A0A286YEY5 (SEQ ID NO: 3). The emphasized amino acids indicate the residues that are modified in some of the embodiments described herein. [Figure 4A] ~ [Figure 4D]Figures 4A to 4D show schematic diagrams of a modified anti-EGFR IgA variant and wild-type IgA (IgA2(m1)). Figure 4A is a representation of wild-type (WT) IgA2m1. IgA2m1 contains four N-glycosylation sites within its CH domain, including one glycosylation site in its tailpiece. Figure 4B is a representation of a modified anti-EGFR IgA3.0+ variant. The modified anti-EGFR IgA3.0+ variant is produced by manipulating an anti-EGFR IgA2m1 antibody to contain stabilized heavy and light chain linkages through (i) a CH1-P124R mutation and (ii) the removal of two free cysteine ​​molecules (one of which is mutated to serine (CH2-C92S) and the second is removed by the deletion of the last two amino acids in the tailpiece (CH3-CHS-C147del)). In addition, three N-linked glycosylation sites were removed by substituting the definitive amino acids of the three N-glycosylation motifs. The mutations in the three N-glycosylation motifs include CH1-N45.2G;CH2-N120T-I121L-T122S;CH3-CHS-N135Q. Figure 4C shows a modified anti-EGFR IgA3.0-(IgA3.0min) variant containing a deletion of the entire tailpiece (CH3-CHS-P131-Y148del). The anti-EGFR IgA3.0min variant contains stabilizing heavy and light chain linkage (CH1-P124R mutation), deletion of the entire tailpiece (CH3-CHS-P131-Y148del), lack of two free cysteine ​​molecules, one of which is mutated to serine (CH2-C92S) and the second is a deletion of the tailpiece (CH3-CHS-C147del). In addition, three N-linked glycosylation sites are removed by substituting the definitive amino acids of two N-glycosylation motifs, namely CH1-N45.2G and CH2-N120T-I121L-T122S, as well as the deletion of CH3-CHS-N135Qdel due to the deletion of the tailpiece. Figure 4D shows a representation of a modified anti-EGFR IgA 4.0 variant that contains all the characteristics of anti-EGFR IgA 3.0min and further contains a mutation in the final N-linked glycosylation motif CH2-N20.Therefore, the anti-EGFR IgA4.0 variant contains stabilizing heavy and light chain linkage (CH1-P124R mutation), deletion of the entire tailpiece (CH3-CHS-P131-Y148del), lack of two free cysteine, one of which is mutated to serine (CH2-C92S) and the second is a tailpiece deletion (CH3-CHS-C147del). In addition, the four N-linked glycosylation sites are removed by substituting the definitive amino acid of one of the four N-glycosylation motifs (CH1-N45.2G; CH2-N120T-I121L-T122S; CH3-CHS-N135Q; and one of CH2-N20G, CH2-N20Q, CH2-N20T, or CH2-N20T-L21I-T22S). The anti-EGFR IgA 4.0 variant is a deglycosylated IgA. [Figure 5] This diagram shows the process flow for the manufacturing of anti-EGFR IgA 3.0min DS. [Figure 6] Figures 6A and 6B show gene maps of the vector components contained in the expression plasmids used during the manufacturing of the non-clinical product lot. Figure 6A shows the gene map of the vector components contained in the heavy chain expression plasmid of anti-EGFR IgA 3.0min. Figure 6B shows the gene map of the vector components contained in the light chain expression plasmid of anti-EGFR IgA 3.0min. [Figure 7-1] ~ [Figure 7-3]Figures 7A to 7F show SDS-PAGE gel images of various anti-EGFR IgA 3.0min drug substances (DS) for visualization of size and purity of intermediate products after Capto L antibody capture and size exclusion by SEC, as well as the results of HPLC-SEC profiles of anti-EGFR IgA 3.0min antibodies obtained from production runs. Three types of anti-EGFR IgA 3.0min drug substances (DS) were produced: (1) DS1 contained the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) DS2 contained the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; and (3) DS3 contained the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207. In particular, Figures 7A, 7C, and 7E show reduced (left) and unreduced (right) SDS-PAGE gel images of anti-EGFR IgA 3.0min DS1, anti-EGFR IgA 3.0min DS2, and anti-EGFR IgA 3.0min DS3 for visualization of intermediate products in terms of size and purity after Capto L antibody capture and size exclusion by SEC, respectively. Predicted band sizes are shown for full-length anti-EGFR IgA 3.0min antibody, 150 kDa; and anti-EGFR IgA 3.0min antibody HC and LC, 75 kDa and 25 kDa, respectively. Figures 7B, 7D, and 7F show the HPLC-SEC profiles of anti-EGFR IgA 3.0min DS1, anti-EGFR IgA 3.0min DS2, and anti-EGFR IgA 3.0min DS3 antibodies obtained from production runs, respectively. Antibody purity is shown as a percentage. [Figure 8] This diagram shows the process flowchart for the release test of anti-EGFR IgA 3.0min DP. [Figure 9A] ~ [Figure 9E]Figures 9A to 9E show the results of the binding measurement of four types of anti-EGFR IgA 3.0min DP to EGFR. Briefly, the four variants included (1) anti-EGFR IgA 3.0min DP1 containing the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211, (2) anti-EGFR IgA 3.0min DP2 containing the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198, (3) anti-EGFR IgA 3.0min DP3 containing the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207, and (4) anti-EGFR IgA 3.0min DP4 containing the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Figures 9A to 9D show the results of surface plasmon resonance (SPR) binding measurements of anti-EGFR IgA 3.0min DP1, anti-EGFR IgA 3.0min DP2, anti-EGFR IgA 3.0min DP3, and anti-EGFR IgA 3.0min DP4 to immobilized His-tagged EGFR on a Ni2+-nitrilotriacetate sensor chip, respectively. All measurements were performed on the BiaCore T200 system. Figure 9E shows the results of binding of anti-EGFR IgA 3.0min DP1 to EGFR-expressing A431 and A1207 cell lines, as well as the low-EGFR-expressing D562 cell line, as measured by FACS. Various concentrations of antibody were incubated with cells, followed by the addition of a fluorescently labeled anti-IgA secondary antibody, and detected by flow cytometry. A control without primary antibody was included, with only the addition of the fluorescently labeled anti-IgA secondary antibody to the A431 cell line. [Figure 10A] ~ [Figure 10B]Figures 10A and 10B show the dose-dependent inhibition of EGF binding by each anti-EGFR IgA 3.0min DP. In particular, Figure 10A shows the dose-dependent inhibition of EGF binding by four types of anti-EGFR IgA 3.0min DP, where (1) anti-EGFR IgA 3.0min DP1 contains the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211, (2) anti-EGFR IgA 3.0min DP2 contains the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198, (3) anti-EGFR IgA 3.0min DP3 contains the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207, and (4) anti-EGFR IgA 3.0min DP4 contains the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Figure 10B is an alternative representation of the same data as in Figure 10A for anti-EGFR IgA 3.0min DP1. As shown, mean fluorescence intensity (MFI), a measure of the amount of fluorescently labeled EGF bound to EGFR, was measured using a competitive FACS assay for increasing concentrations of anti-EGFR IgA 3.0min. MFI decreased in a dose-dependent manner. Anti-EGFR IgA 3.0min had no effect on isotype controls, demonstrating specific binding to EGFR. [Figure 11A] ~ [Figure 11E]Figures 11A to 11E show the cell viability results after treatment with each of the four anti-EGFR IgA 3.0min DPs. Briefly, the four variants are: (1) anti-EGFR IgA 3.0min DP1 containing the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211; (2) anti-EGFR IgA 3.0min DP2 containing the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198; (3) anti-EGFR IgA 3.0min DP3 containing the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207; and (4) anti-EGFR IgA 3.0min DP4 containing the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Figures 11A to 11B show the results of CDC (e.g., cell apoptosis) induced by all four anti-EGFR IgA 3.0min DPs. As shown, the cell viability of A431 or human fibroblast cell lines was evaluated using a sulforhodamine B assay kit in the presence of increasing concentrations of each anti-EGFR IgA3.0min DP. The viability of EGFR-highly expressing A431 cells was significantly reduced in a dose-dependent manner by each anti-EGFR IgA3.0min DP. No changes were observed in human fibroblast cell viability. Figures 11C to 11D show alternative representations of the cell viability of A431 or human fibroblast cell lines after treatment with anti-EGFR IgA3.0min DP1, as shown in Figures 11A to 11B. Figure 11E shows an alternative representation of the cell viability assay for anti-EGFR IgA3.0min DP1. [Figure 12A] ~ [Figure 12E]Figures 12A to 12E show ADCC induced by each of the four anti-EGFR IgA 3.0min DPs, which include (1) anti-EGFR IgA 3.0min DP1 containing the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211, (2) anti-EGFR IgA 3.0min DP2 containing the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198, (3) anti-EGFR IgA 3.0min DP3 containing the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207, and (4) anti-EGFR IgA 3.0min DP4 containing the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. Specific cytolysis of the A431 cancer cell line was analyzed as a function of the concentration of each anti-EGFR IgA 3.0min DP using purified neutrophils from any of the three donors (Figure 12A) or using whole blood lysates from which red blood cells have been removed (Figure 12B). Figure 12C is an alternative representation of specific cytolysis of the A431 cancer cell line as a function of the concentration of anti-EGFR IgA 3.0min DP1 using purified neutrophils from the three donors shown in Figure 12A. Similarly, Figure 12D is an alternative representation of specific cytolysis of the A431 cancer cell line as a function of the concentration of anti-EGFR IgA 3.0min DP1 using whole blood lysates from the three donors shown in Figure 12B. Furthermore, the E:T ratio was calculated for the three donors based on the specific lysis of the A431 cancer cell line using neutrophils from the three donors (Figure 12E). [Figure 13A] ~ [Figure 13C]Figures 13A to 13C show the results of in vivo studies of tumor growth in A431- and A549- xenograft tumor model mice. Briefly, the activity of four anti-EGFR IgA 3.0min DPs was tested, and these four anti-EGFR IgA 3.0min DPs included (1) anti-EGFR IgA 3.0min DP1 containing the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211, (2) anti-EGFR IgA 3.0min DP2 containing the VH sequence of SEQ ID NO: 172 and the VL sequence of SEQ ID NO: 198, (3) anti-EGFR IgA 3.0min DP3 containing the VH sequence of SEQ ID NO: 182 and the VL sequence of SEQ ID NO: 207, and (4) anti-EGFR IgA 3.0min DP4 containing the VH sequence of SEQ ID NO: 184 and the VL sequence of SEQ ID NO: 209. The activity of each anti-EGFR IgA 3.0min DP was tested in vivo in xenograft tumor-carrying CD89 Tg NXG mice injected with a low-expression EGFR cancer cell line (A549 - Figure 13A) after twice-weekly administration. Figure 13B is a surrogate representation of the effect of anti-EGFR IgA 3.0min DP1 treatment on low-expression EGFR (A549 - Figure 13B). Figure 13C shows the effect of anti-EGFR IgA 3.0min DP1 treatment on high-expression EGFR (A431 - Figure 13A). Each cancer cell line was transduced with the luciferase gene. PBS-controlled experiments were performed. Longitudinal monitoring of tumor growth and the percentage increase in bioluminescent signaling over time were reported using bioluminescent imaging (BLI). [Figure 14] This figure shows the pharmacokinetic profile of anti-EGFR IgA3.0min DP after a single 3 mg / kg intravenous (IV) or intraperitoneal (IP) administration in immunodeficient NSG mice. Here, anti-EGFR IgA3.0min DP contains the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211. Plasma concentrations of anti-EGFR IgA3.0min DP1 were measured over a 28-day period, and the mean plasma concentration (geometric standard deviation shown by ± bars) was plotted as a function of time. N=4 per dose group. [Figure 15]Figures 15A and 15B show the pharmacokinetic profiles of anti-EGFR IgA3.0min DP administered to non-human primates (NHPs) in either (i) a single 25 mg / kg intravenous (IV) dose (Figure 15A) or (ii) four 25 mg / kg IV doses spaced 7 days apart, followed by a 12.5 mg / kg IV dose (Figure 15B). Anti-EGFR IgA3.0min DP contained the VH sequence of SEQ ID NO: 173 and the VL sequence of SEQ ID NO: 211. [Figure 16] This is a diagram summarizing the overall clinical study design. [Modes for carrying out the invention]

[0026] The following description and examples illustrate embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and is therefore subject to change. Those skilled in the art will recognize that many variations and modifications of the present disclosure exist and are included within its scope.

[0027] All terms are intended to be understood as they would be understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would be commonly understood by those skilled in the art to which this disclosure belongs.

[0028] The section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter being discussed.

[0029] While various characteristics of this disclosure may be described in the context of a single embodiment, the characteristics may also be provided separately or in any appropriate combination. Conversely, while this disclosure may be described in the context of separate embodiments for the purposes of this specification, this disclosure may also be implemented in a single embodiment.

[0030] definition The following definitions supplement the definitions in the art and apply to this application and should not be attributed to any related or unrelated matters, such as any jointly owned patent or application. Any methods and materials similar to or equivalent to those described herein may be used in the practice for testing of this disclosure, but preferred materials and methods are described herein. Accordingly, the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0031] In this application, the use of the singular form includes the plural unless otherwise indicated. It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. In this application, the use of "or" means "and / or" unless otherwise indicated.

[0032] References to “some embodiments,” “embodiments,” “one embodiment,” or “other embodiments” in this specification mean that certain characteristics, structures, or features described in relation to the embodiments are included in at least some embodiments, but not necessarily in all embodiments of the present disclosure.

[0033] Natural amino acids may be referred to herein by the following conventional one- or three-letter abbreviations: 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), and valine (V, Val). Unless otherwise specified, X may represent any amino acid. In some embodiments, X may be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).

[0034] 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 “comprising,” such as “includes” and “include”), and “containing” (and any form of “containing,” such as “contains” and “contain”) are inclusive or unrestricted and do not exclude additional, unenumerated elements or method steps.

[0035] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error for a given value, including a range of up to 10 percent of a given value or within one digit of a given value. Where a particular value is given in this application and claims, unless otherwise indicated, the term “about” should be understood as meaning within an acceptable margin of error for that value.

[0036] As used herein, the term “antibody” means immunoglobulin (Ig), whether naturally occurring or partially or completely synthetically produced, and includes whole antibodies and antibody fragments unless expressly indicated.

[0037] As used herein, "whole 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 form the "hypervariable region" of the antibody, which are responsible for binding to the antigen, and the three CDRs are separated by framework residues, and (ii) a constant region.

[0038] As used herein, the term “complementarity-determining region (CDR)” refers to the amino acid residues in the antibody heavy chain variable region and light chain variable region that are necessary for antigen binding.

[0039] As used herein, the terms "framework residue" or "FR" refer to residues in variable regions other than the CDR.

[0040] As used herein, "heavy chain region" or "heavy chain polypeptide" refers to an antibody moiety containing an N-terminal heavy chain variable (VH) region and a C-terminal heavy chain constant (CH) region, having one or more of the following: a CH1 domain, a hinge, a CH2 domain, a CH3 domain, and a CH3 tailpiece (CS).

[0041] As used herein, the term "hinge" refers to the flexible domain of the heavy chain region that joins the CH1 domain and the CH2 domain, which allows the two N-terminal antigen-binding regions to move independently.

[0042] As used herein, "light chain region" or "light chain polypeptide" refers to an antibody moiety containing an N-terminal light chain variable (VL) region and a C-terminal light chain constant (CL) region. Kappa (κ) and lambda (λ) light chains refer to two major light chain isotypes.

[0043] As used herein, the terms “Fc domain” or “Fc-containing domain” refer to a portion of an antibody or non-antibody that can bind to an Fc receptor and contains a portion of the heavy chain constant (CH) region.

[0044] As used herein, “antibody fragment,” “antibody fragment,” “functional fragment of an antibody,” “antigen-binding moiety,” and their grammatical equivalents are used interchangeably and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen and contain one or more complementarity-determining regions (CDRs).

[0045] A "Fab fragment" refers to a monovalent fragment consisting of VL, VH, CL, and CH1 domains.

[0046] The "F(ab')2" fragment refers to a divalent fragment containing two Fab fragments linked by disulfide bridges in the stalk region.

[0047] An "Fv fragment" refers to a fragment consisting of the VL and VH domains of a single arm of an antibody.

[0048] A "single-chain Fv (scFv)" refers to a monovalent fragment consisting of two domains (i.e., VL and VH) of an Fv fragment, which are joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain.

[0049] A "diabody" refers to a dimer of polypeptide chains, where each polypeptide chain contains a VH linked to a VL by a peptide linker. This peptide linker is too short to allow pairing between the VH and VL of the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to generate a dimer molecule with two functional antigen-binding sites.

[0050] As used herein, the term “monoclonal antibody” refers to an antibody produced by a single clone of a B cell that binds to the same epitope.

[0051] As used herein, the term "polyclonal antibody" refers to an antibody produced by various B cells that binds to different epitopes of the same antigen.

[0052] As used herein, the term "chimeric antibody" refers to an antibody comprising an amino acid sequence derived from two different species or two different sources, and includes synthetic molecules.

[0053] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line transfected with an expression vector (or possibly two or more expression vectors, typically two) containing the coding sequence of the antibody, wherein the coding sequence is not naturally associated with the cell.

[0054] As used herein, the term “recombinant human antibody” means a human antibody prepared, expressed, produced or isolated by recombinant means, and includes (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal with respect to human immunoglobulin genes, or hybridomas prepared therefrom (as further described below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., transfectomas; (c) antibodies isolated from recombinant, combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced or isolated by any other means involved in splicing human immunoglobulin gene sequences to other DNA sequences.

[0055] 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 and that does not exist naturally in the non-human antibody.

[0056] As used herein, the term “antigen” refers to a molecule or part of a molecule that contains a molecule or part of a molecule (epitope) that can interact with a binding site on an antibody or a fragment thereof and can bind to an antibody.

[0057] As used herein, the term “epitope” refers to a region of an antigen that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. Epitopes may be conformational or linear. “Conformational epitopes” are produced by spatially juxtaposed amino acids from various segments of a linear polypeptide chain. “Linear epitopes” are produced by adjacent amino acid residues in a polypeptide chain.

[0058] As used herein, the term “antigen-binding domain” refers to a portion of an antibody or non-antibody that binds to or recognizes an antigen or fragment thereof and includes a heavy chain variable (VH) region and a light chain variable (VL) region.

[0059] As used herein, the term “recognize” refers to the association or binding between an antigen-binding domain and an antigen.

[0060] As used herein, the terms “specifically bind” or “preferentially bind” refer to binding to a target with greater affinity and / or avidity than binding to other polypeptides.

[0061] When used herein, the term "affinity" refers to the equilibrium constant (K) for the dissociation of an antigen and an antigen-binding protein. D It is expressed by the equilibrium constant for the dissociation, and is a measure of the binding strength between the antigen and the antigen-binding domain, K D The lower the value of K, the stronger the binding strength between the antigen and the antigen-binding domain. An antibody or its antigen-binding fragment has a K value for binding to another target or polypeptide. D Affinity K is less than or equal to 1 / 50th of the value. D When a substance binds to a first antigen by a certain value, it is said to be "specific" to the first target or antigen compared to a second target or antigen.

[0062] The term "avidity" refers to the affinity of an antigen-binding domain for an antigen, based on the number of related antigen-binding sites present in the antigen-binding domain.

[0063] As used herein, the term "neutralizing activity" refers to the ability of an antibody or a functional fragment thereof to block the binding of a homologous ligand to a target antigen.

[0064] When used herein, the term "target cell" refers to a cell that can be targeted by the antibodies or functional fragments thereof of this disclosure.

[0065] As used herein, the terms “polynucleotide” and “nucleic acid molecule” are interchangeable and refer to polymers of deoxyribonucleotides, ribonucleotides, modified nucleotides and / or analogs thereof of any length, including DNA and RNA.

[0066] The terms "operably linked" or "transcriptionally regulated" refer to a functional linkage between a regulatory sequence and a nucleic acid sequence that results in the expression of the latter.

[0067] As used herein, the terms “modification” or “mutation” refer to amino acid substitutions, insertions, or deletions in an antibody or fragment thereof compared to the corresponding amino acids in a WT antibody.

[0068] As used herein, the terms “conservative substitution,” “conservative modification,” or “conservative mutation” refer to the substitution of one amino acid by another amino acid that is chemically similar to the first amino acid and can be replaced in the polypeptide structure without significant interference with or alteration of the polypeptide structure or function.

[0069] As used herein, the terms “non-conservative mutation,” “non-conservative modification,” or “non-conservative substitution” refer to the substitution of one amino acid with another amino acid that is not chemically similar to the first amino acid. When the second amino acid substitutes the first amino acid in a non-conservative mutation or substitution, it results in interference with or alteration of the polypeptide structure or function, which includes improvement of the polypeptide structure or function.

[0070] As used herein, the term “improved characteristics” refers to a characteristic associated with one or more modifications of an antibody or fragment thereof that is improved compared to the corresponding unmodified antibody, when referring to one or more modifications of the antibody or fragment thereof.

[0071] 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 fragment thereof having an amino acid sequence corresponding to the amino acid sequence of an antibody or fragment thereof having one or more modifications at each amino acid residue other than one or more modifications.

[0072] When used herein to refer to an improvement in characteristics, the terms “increased stability” or “improved stability” include increased thermal stability and / or reduced aggregation, measured by higher retention of the biological activity (e.g., binding to ADCC, antigen, or FcαR) of the antibody or its functional fragment after an incubation period at a certain temperature compared to the corresponding antibody.

[0073] When used herein to refer to an improvement in properties, the terms “reduction in aggregation” or “reduced aggregation” include, compared to the aggregation shown by the corresponding antibody, a reduction in the aggregation of the antibody or its functional fragment with other antibody molecules and / or other macromolecules, including serum proteins such as albumin.

[0074] When used herein to refer to an improvement in characteristics, the term "biodistribution" refers to the cellular and / or tissue and / or organ distribution of the antibody or functional fragment thereof disclosed herein after administration or delivery to a subject. When used herein, the term "increased biodistribution" generally refers to an increase in the distribution of the IgA antibody or functional fragment thereof disclosed herein at a target site, such as a tumor or tumor cells, compared to administration of either the medium or the corresponding WT IgA antibody.

[0075] As used herein, the terms “in vivo half-life” or “circulating half-life” refer to the time required for half of an antibody or fragment thereof to be removed from circulation when administered to an animal. The term “increased circulating half-life” as used herein when referring to an improvement in characteristics refers to greater persistence of an antibody or fragment thereof in serum or plasma, and / or a longer period required to reduce it to up to half of the measured serum or plasma concentration, compared to administration of either the medium or the corresponding WT IgA antibody.

[0076] The term "Fc receptor-mediated effector cell function" refers to effector functions activated by the binding of immunoglobulins, such as IgA, to Fc receptors on immune effector cells, such as phagocytosis, antibody-dependent cytotoxicity (ADCC), inflammatory mediator release, lysozyme production, and superoxide anion production.

[0077] The term "ADCC activity" refers to the ability of an antibody to induce the lysis of target cells (e.g., cancer cells) via immune effector cells.

[0078] The terms "complement-dependent cytotoxicity" or "CDC" refer to the ability of an antibody to lyse target cells (e.g., cancer cells) in the presence of complement.

[0079] As used herein, the term "glycosylation" refers to the covalent linking of one or more sugar chains to a polypeptide.

[0080] The term “isolated,” as used herein, refers to the separation or modification of a molecule from its native state. A nucleic acid or polypeptide present in a cell and in its native state with coexisting substances (e.g., antibodies or their antigen-binding fragments disclosed herein) is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the cell and coexisting substances is “isolated.”

[0081] As used herein, the term "substantially purified" means that an antibody or functional fragment thereof is substantially free of cellular material, naturally occurring substances, or other contaminants from its cellular or tissue source, or, if chemically synthesized, substantially free of chemical precursors or other chemical substances. As used herein, "substantially free" means a preparation containing less than approximately 30% by dry weight of cellular material, naturally occurring substances, or other contaminants.

[0082] The term "leader sequence" refers to the sequence of amino acid residues located at the N-terminus of a polypeptide that promotes the secretion of polypeptides from mammalian cells.

[0083] The term "label" refers to a compound or composition that is directly or indirectly detectable and directly or indirectly conjugated to an antibody. A label is "directly detectable" if it is detectable on its own (e.g., radioisotope labeling or fluorescent labeling) or if it catalyzes a chemical change in a substrate that produces a detectable change (e.g., enzyme labeling). A label is "indirectly detectable" if it is not detectable on its own but conjugates to another drug that is directly detectable.

[0084] As used herein, the term “fusion protein” refers to a polypeptide comprising the amino acid sequence of an antibody or fragment thereof and the amino acid sequence of a heterogeneous polypeptide (i.e., an unrelated polypeptide).

[0085] As used herein, the terms “cancer,” “tumor,” “proliferative disorder,” “malignant tumor,” or “malignant disease” refer to a physiological condition in mammals characterized by cells having malignant traits. “Malignant traits” include uncontrolled growth, cellular invasion, and metastasis. These malignant traits distinguish cancer from benign tumors that do not typically invasive or metastatic.

[0086] As used herein, the terms “disease,” “disorder,” and “condition” are interchangeable to refer to any change in the state of any part of the body or organ that interrupts or impairs the performance of a function and / or causes symptoms such as discomfort, dysfunction, pain, or even death in the person affected or in contact with the person affected. Disease or disorder may also be related to distemper, pathological condition, illness, disease, disorder, malaise, ill health, complaint or affection.

[0087] As used herein, the terms “need it,” “patient needing it,” and “person needing it” refer, when used in the context of therapeutic or preventive legal action, to a person who has the disease, has been diagnosed with the disease, needs to prevent the disease, or is at risk of developing the disease.

[0088] As used herein, the terms “treat,” “treat,” “medicate,” and “improve” refer to therapeutic and preventive legal measures, the purpose of which is to reduce, alleviate, improve, inhibit, prevent, slow, or halt the progression or severity of a condition associated with a disease or disorder. Treating includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, associated with a disease or disorder. Treatment is “effective” if one or more symptoms or clinical markers are reduced, or if the progression of the disease is reduced or halted.

[0089] As used herein, the term “administer” means the placement of an antibody or fragment thereof into a subject by a method or route that results in at least partial delivery of the antibody or fragment thereof at a desired site. Administration can be carried out by any suitable route that results in effective treatment in the subject.

[0090] The term "combination therapy" refers to medications administered simultaneously or sequentially over substantially the same period of time.

[0091] The term "therapeutic dose" refers to the amount of drug that is effective in achieving the desired therapeutic outcome within a given period of time.

[0092] The term "preventively effective dose" refers to the amount of drug that is effective in achieving the desired preventive outcome within a given period of time.

[0093] When used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and local administration, usually by injection.

[0094] The terms “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally,” as used herein, refer to administrations other than direct administration to a target site, tissue, or organ such as a tumor site, which thus enter the circulatory system of the target and thus undergo metabolism and other similar processes.

[0095] Where used herein, the term "pharmaceutically acceptable" means a compound, material, composition and / or dosage form that is appropriate for contact with or administration to human and animal tissues without excessive toxicity, irritation, allergic reaction or other problem or complication, and that is commensurate with a reasonable benefit-risk ratio, within the bounds of sound medical judgment.

[0096] As used herein, the terms “subject,” “patient,” “individual,” and similar terms are interchangeable and refer to vertebrates, mammals, primates, or humans.

[0097] As used herein, the term “host cell” refers to a specific target cell transfected with a nucleic acid molecule, and its offspring or potential offspring. Such offspring may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur during passage or integration of the nucleic acid molecule into the host cell genome.

[0098] Introduction Modified anti-EGFR IgA antibodies having an IgA skeleton and an anti-EGFR binding domain are described herein. In some embodiments, the modified antibody has one or more of the following: a reduced N-glycosylation profile, removal of a C-terminal residue in the heavy chain constant region, or amino acid substitutions that favor a stabilized monomer form. In some embodiments, the modified IgA antibody targets human EGFR and has an Fc domain that recognizes the FcαRI receptor (CD89) on immune cells, such as neutrophils, macrophages, and eosinophils. Thus, in some embodiments, the modified antibody is involved in the selective recruitment of FcαRI-expressing immune effector cells to EGFR tumor antigens on the cell surface. In some embodiments, the modified antibody selectively recruits neutrophils. Neutrophils have the ability to directly kill tumor cells by releasing reactive oxygen species (ROS) and reactive nitrogen species (RNS). Thus, in some embodiments, the modified antibody induces ADCC in EGFR tumor antigen-expressing cells. Alternatively, in some embodiments, the modified antibody induces direct cytotoxicity of EGFR-positive tumor cells in the absence of immune effector cells by antagonistically binding to EGFR and inhibiting EGF signaling. In addition, in some embodiments, the cytotoxicity induced by the modified antibody enhances T cell activation and attracts pro-inflammatory (M1) macrophages.

[0099] Anti-EGFR IgA antibody Modified anti-EGFR IgA antibodies are disclosed herein. IgA antibodies constitute 15–20% of serum immunoglobulins. On mucosal surfaces, IgA is the most abundant and plays a crucial role in the body's immune response. IgA can be highly versatile and exists in mucosal tissues as monomers, dimers, polymers, and secretory IgA. IgA has two subclasses (IgA1 and IgA2) and can be produced as monomeric, dimeric, and secretory forms. In some embodiments, anti-EGFR IgA antibodies may be monomeric. In some embodiments, anti-EGFR IgA antibodies may contain one or more IgA1 amino acid sequences. In some embodiments, anti-EGFR IgA antibodies may contain one or more IgA2 amino acid sequences. In some embodiments, anti-EGFR IgA antibodies may contain one or more IgA1 amino acid sequences and one or more IgA2 amino acid sequences. IgA1 and IgA2 differ in their hinge region and glycan content. Monomeric IgA1 is the dominant subclass in serum and is readily cleaved by bacterial enzymes, while IgA2 is more resistant to enzymatic degradation and is mainly found in mucosal secretions.

[0100] In some embodiments, the anti-EGFR IgA antibody is an allotype of IgA2 antibody, 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 or Asian IgA2m(2) antibody. In some embodiments, IgA2m(1) also has a superior potential efficacy and safety profile due to its lower glycosylation levels and lack of association with IgA nephropathy compared to IgG1 and IgA1 / IgA2m(2).

[0101] 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 triplicate antibody. In some embodiments, the anti-EGFR IgA antibody may be a multispecific antibody.

[0102] 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 deimmunized antibody, a bispecific antibody, a multispecific antibody, a polyvalent antibody, or a combination thereof. In some embodiments, the anti-EGFR IgA antibody may be a bispecific antibody that binds to EGFR and a second antigen. In some embodiments, the anti-EGFR IgA antibody simultaneously binds to two antigens on the cell surface. In some examples, the binding of the anti-EGFR IgA antibody to two different antigens is sequential. For example, the binding of the anti-EGFR IgA antibody to the first antigen occurs first, thereby limiting the space explored by the second antibody arm. As a result, there may be a significant increase in the local concentration of the second antigen, which can facilitate the binding of the second antibody arm.

[0103] Steady-state region In some embodiments, the anti-EGFR IgA antibody comprises the IgA heavy chain constant region or a functional variant thereof. Table 1 lists exemplary IgA heavy chain constant region amino acid sequences and the nucleotide sequences encoding them. Figures 1-3 show representative amino acid sequences of human IgA2 heavy chains from Table 1. In some embodiments, the IgA heavy chain constant region contains an amino acid sequence that is 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% identical to any one of the amino acid sequences (SEQ ID NOs. 1-9) in Table 1. In some embodiments, the IgA heavy chain constant region is encoded by a nucleotide sequence that is 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% identical to any one of the nucleotide sequences in Table 1. [Table 1] JPEG2026526224000002.jpg245170 JPEG2026526224000003.jpg245170 JPEG2026526224000004.jpg246170 JPEG2026526224000005.jpg118170

[0104] In some embodiments, the IgA heavy chain constant region includes one or more heavy chain constant domains (e.g., a CH1 domain, a CH2 domain, a CH3 domain, or any combination thereof). Table 2 lists the amino acid sequences of exemplary IgA2 CH1 domains, IgA CH2 domains, and IgA CH3 domains. In some embodiments, the IgA heavy chain constant region (e.g., IgA CH1 domain, IgA CH2 domain, and IgA CH3 domain) includes an amino acid sequence that is 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% identical to any of the amino acid sequences in Table 2. In some embodiments, the heavy chain constant region includes one or more of the IgA CH3 domain, IgA CH2 domain, or IgA CH1 domain, or any combination thereof. In some embodiments, the anti-EGFR IgA antibody includes a heavy chain constant region containing one or more amino acids from 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 that includes a heavy chain constant region comprising one or more of the IgA2 CH3 domain, IgA2 CH2 domain, and IgA2 CH1 domain, or any combination thereof. [Table 2]

[0105] In some embodiments, the anti-EGFR IgA antibody includes a light chain constant region (CL). Table 3 lists exemplary IgA light chain constant region amino acid sequences. In some embodiments, the light chain constant region contains an amino acid sequence that is 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% identical to one 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 includes a kappa light chain constant region. [Table 3]

[0106] 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.

[0107] In some embodiments, the anti-EGFR IgA antibody may include at least a portion of the Fc domain. In some embodiments, the anti-EGFR IgA antibody includes a heavy chain constant region comprising the CH3 domain, the CH2 domain, and the CH1 domain. In some embodiments, the anti-EGFR IgA antibody includes a light chain constant region comprising the CL domain.

[0108] In some embodiments, the antibodies or functional fragments thereof shown herein include an IgA heavy chain constant region 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 one of the amino acid sequences of SEQ ID NOs. 21-22 include at least one IgA heavy chain constant domain 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 one of the amino acid sequences of SEQ ID NOs. 21-22. In some embodiments, the antibody or functional fragment thereof shown herein includes an IgA light chain constant domain 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 the amino acid sequence of SEQ ID NO: 23 or 24. In some embodiments, the antibody or functional fragment thereof shown herein includes a variable heavy chain domain from an IgG antibody. In some embodiments, the antibody or functional fragment thereof shown herein includes a variable light chain domain from an IgG antibody.

[0109] Variable region In one embodiment, the antibody or its antigen-binding functional fragment includes a heavy chain variable region (VH) sequence. In some embodiments, the VH includes three CDRs, CDR-H1, CDR-H2, and CDR-H3. Table 4 lists the amino acid sequences of exemplary VH CDRs. Table 5 lists the amino acid sequences of exemplary VH CDRs. [Table 4] [Table 5] JPEG2026526224000010.jpg249170 JPEG2026526224000011.jpg84170

[0110] In some embodiments, the VH sequence contains 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 one of SEQ ID NOs. In some embodiments, the VH 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%, or at least 99% sequence identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the corresponding wild-type antibody amino acid sequence, while retaining the ability to bind to the same antigen as the corresponding wild-type antibody. In some embodiments, one to ten amino acids are substituted, inserted, and / or deleted in any one amino acid sequence of SEQ ID NOs. 169-194. In some embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (e.g., in the FR). The antibody may contain one VH sequence of SEQ ID NOs. 169-194, which includes one or more post-translational modifications of that sequence.

[0111] In some embodiments, VH comprises one, two, or three CDRs selected from (a) CDR-H1 comprising any one amino acid sequence from SEQ ID NOs. 34 to 54 or a variant thereof including one to three substitutions, deletions, or insertions; (b) CDR-H2 comprising any one amino acid sequence from SEQ ID NOs. 57 to 78 or a variant thereof including one to three substitutions, deletions, or insertions; and (c) CDR-H3 comprising any one amino acid sequence from SEQ ID NOs. 81 to 102 or a variant thereof including one to three substitutions, deletions, or insertions.

[0112] In one embodiment, an antibody or its antigen-binding functional fragment is shown, where the antibody or its antigen-binding functional fragment includes a light chain variable region (VL) sequence. In some embodiments, the VL sequence includes three CDRs, CDR-L1, CDR-L2, and CDR-L3. Table 6 lists the amino acid sequences of exemplary VL CDRs. Table 7 lists the amino acid sequences of exemplary VLs. [Table 6] JPEG2026526224000013.jpg118170 [Table 7] JPEG2026526224000015.jpg244170

[0113] In some embodiments, the VL sequence contains 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 one of the amino acid sequences of SEQ ID NOs. In some embodiments, the VL 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%, or at least 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the corresponding wild-type antibody amino acid sequence, while retaining the ability to bind to the same antigen as the corresponding wild-type antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in any one of the amino acid sequences of SEQ ID NOs. 195-219. In some embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (e.g., in the FR). The antibody or its antigen-binding functional fragment may include any one of the VL sequences of SEQ ID NOs. 195-219, which includes post-translational modifications of that sequence.

[0114] In some embodiments, the VL sequence comprises one, two, or three CDRs selected from (a) CDR-L1 comprising any one amino acid sequence from SEQ ID NOs. 105 to 126 or a variant thereof including one to three substitutions, deletions, or insertions; (b) CDR-L2 comprising any one amino acid sequence from SEQ ID NOs. 129 to 143 or a variant thereof including one to three substitutions, deletions, or insertions; and (c) CDR-L3 comprising any one amino acid sequence from SEQ ID NOs. 146 to 166 or a variant thereof including one to three substitutions, deletions, or insertions.

[0115] In one embodiment, an anti-EGFR IgA antibody or an antigen-binding functional fragment thereof is shown, wherein the antibody or the antigen-binding functional fragment comprises VH in any of the embodiments shown above, and VL in any of the embodiments shown above. In some embodiments, the antibody or its antigen-binding functional fragment comprises VH and VL, where 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 one of the amino acid sequences of SEQ ID NOs. 195 to 219, and these may include post-translational modifications of those sequences. In some embodiments, the VH sequence comprises one amino acid sequence from SEQ ID NOs. 169 to 194, and the VL sequence comprises one amino acid sequence from SEQ ID NOs. 195 to 219, and these may include post-translational modifications of those sequences.

[0116] In one embodiment, an anti-EGFR IgA antibody or its antigen-binding functional fragment is shown, wherein the antibody or its antigen-binding functional fragment comprises a combination of VH sequences and VL sequences listed in Table 8, where VH is 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%, and at least 98% of any one of SEQ ID NOs. 169-194. VL contains an amino acid sequence having at least 99% or 100% sequence identity with any one of SEQ ID NOs: 195-219, 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. In some embodiments, the antibody or its antigen-binding functional fragment contains a combination of the VH sequence and VL sequence shown in Table 8. [Table 8]

[0117] In one embodiment, an anti-EGFR IgA antibody or its antigen-binding functional fragment is shown, wherein the antibody or its antigen-binding functional fragment comprises CDR-H3 selected from any of Table 4 or its variants containing 1 to 3 substitutions, deletions, or insertions, and CDR-L3 selected from any of Table 6 or its variants containing 1 to 3 substitutions, deletions, or insertions, wherein the selected CDR-H3 and CDR-L3 are paired according to Table 9. In one embodiment, an antibody or its antigen-binding functional fragment is shown, wherein the antibody or its antigen-binding functional fragment comprises CDR-H2 selected from any of Table 4 or its variants containing 1 to 3 substitutions, deletions, or insertions, and CDR-L2 selected from any of Table 6 or its variants containing 1 to 3 substitutions, deletions, or insertions, wherein the selected CDR-H2 and CDR-L2 are paired according to Table 9. In one embodiment, an antibody or an antigen-binding functional fragment thereof is shown, wherein the antibody or the antigen-binding functional fragment comprises a CDR-H1 selected from any of Table 4 or a variant thereof containing 1 to 3 substitutions, deletions, or insertions, and a CDR-L1 selected from any of 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 embodiment, the disclosure shows an antibody or an antigen-binding functional fragment thereof comprising a VH containing the amino acid sequence of SEQ ID NO: 173 and a VH containing post-translational modifications of those sequences, and a VL containing the amino acid sequence of SEQ ID NO: 211 and a VL containing post-translational modifications of those sequences. In one embodiment, the disclosure shows an antibody or an antigen-binding functional fragment thereof comprising a VH containing the amino acid sequence of SEQ ID NO: 172 and a VH containing post-translational modifications of those sequences, and a VL containing the amino acid sequence of SEQ ID NO: 198 and a VL containing post-translational modifications of those sequences. In one embodiment, the present disclosure provides an antibody or antigen-binding functional fragment thereof comprising a VH containing the amino acid sequence of SEQ ID NO: 182 and a VH containing post-translational modifications of those sequences, and a VL containing the amino acid sequence of SEQ ID NO: 207 and a VL containing post-translational modifications of those sequences.In one embodiment, the present disclosure provides an antibody or antigen-binding functional fragment thereof comprising a VH containing the amino acid sequence of SEQ ID NO: 184 and comprising post-translational modifications of those sequences, and a VL containing the amino acid sequence of SEQ ID NO: 209 and comprising post-translational modifications of those sequences.

[0118] In one embodiment, the disclosure provides an antibody or an antigen-binding functional 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 embodiment, the disclosure provides an antibody or an antigen-binding functional 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 embodiment, the disclosure provides an antibody or an antigen-binding functional 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 embodiment, the disclosure provides an antibody or an antigen-binding functional 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.

[0119] In one embodiment, an anti-EGFR IgA antibody or an antigen-binding functional fragment thereof is shown, wherein the antibody or the antigen-binding functional fragment thereof is: (a) CDR-H1 comprising any one of the amino acid sequences of SEQ ID NOs. 34-54 or variants thereof including 1-3 substitutions, deletions, or insertions; (b) CDR-H2 comprising any one of the amino acid sequences of SEQ ID NOs. 57-78 or variants thereof including 1-3 substitutions, deletions, or insertions; (c) amino acid sequences of SEQ ID NOs. 81-102 or variants thereof including 1-3 substitutions, deletions, or insertions. The present invention includes CDR-H3 containing any one of (d) the amino acid sequences of SEQ ID NOs. 105-126 or any variant thereof containing 1-3 substitutions, deletions, or insertions, CDR-L1 containing any one of the amino acid sequences of SEQ ID NOs. 129-143 or any variant thereof containing 1-3 substitutions, deletions, or insertions, and CDR-L3 containing any one of the amino acid sequences of SEQ ID NOs. 146-166 or any variant thereof containing 1-3 substitutions, deletions, or insertions. In some embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are selected according to any one of the combinations shown in Table 9. [Table 9] JPEG2026526224000018.jpg189170

[0120] IgA antibody modification Anti-EGFR IgA antibodies comprising one or more amino acid insertions, substitutions, and / or deletions are described herein. In some embodiments, the anti-EGFR IgA antibodies disclosed herein are conjugated with one or more therapeutic agents.

[0121] In some embodiments, the amino acid numbering of the anti-EGFR IgA antibodies described herein is indicated according to IMGT unique numbering for C-DOMAIN and C-LIKE-DOMAIN (disclosed in “IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains.” Dev Comp 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 modifications compared to amino acid residues at a selected position in the corresponding WT IgA heavy chain constant region (e.g., WT IgA2 heavy chain constant region) containing the amino acid sequence shown in SEQ ID NO: 1.

[0122] In some embodiments, the anti-EGFR IgA antibodies disclosed herein include deletions of at least four glycosylation sites within the constant region. In some embodiments, the anti-EGFR IgA antibodies disclosed herein include deletions of at least three N-linked glycosylation sites within the constant region of the antibody. In some embodiments, the anti-EGFR IgA antibodies include deletions of at least three N-linked glycosylation sites within the constant region of the antibody and at least one O-linked glycosylation site within the constant region of the antibody. In some embodiments, the anti-EGFR IgA antibodies or functional fragments disclosed herein include 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 antibodies or functional fragments disclosed herein include 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 includes 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 located at amino acid residues within the CH1 domain, CH2 domain, and / or CH3 domain of the IgA1 heavy chain constant region.

[0123] In some embodiments, the anti-EGFR IgA antibody includes a tailpiece deletion. In some embodiments, the anti-EGFR IgA antibody includes one or more modifications, where one or more modifications are located in one or more amino acid residues within the CH1 domain, CH2 domain, and / or CH3 domain of the IgA2 heavy chain constant region. In some embodiments, the IgA CH1 domain includes an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the IgA CH2 domain includes an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the IgA CH3 domain includes an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the anti-EGFR IgA antibodies (e.g., IgA2 antibodies) or functional fragments thereof disclosed herein include deletions of 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, and 3 to 4 C-terminal amino acids. In some embodiments, the C-terminal amino acids include amino acids 131 to 148 of the IgA2 antibody, numbered according to the IMGT scheme.

[0124] In some embodiments, the anti-EGFR IgA antibody is an anti-EGFR IgA2 antibody. In some embodiments, the IgA2 antibody contains deletions of amino acids 131-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 147-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 146-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 145-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 144-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 143-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 142-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 141-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 140-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 139-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 138-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 137-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 136-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 135-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 134-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids 133–148, numbered according to the IMGT scheme.In some embodiments, the IgA2 antibody contains deletions of amino acids 132-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains deletions of amino acids P131-Y148, numbered according to the IMGT scheme.

[0125] In some embodiments, the anti-EGFR IgA antibody contains a mutation in the C-terminal asparagine (N) amino acid. In some embodiments, the mutation is a non-conservative amino acid substitution. In some embodiments, the mutation causes deletion of 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 scheme. In some embodiments, the IgA2 antibody contains the N135Q mutation, numbered according to the IMGT scheme.

[0126] In some embodiments, the anti-EGFR IgA antibodies of this disclosure or their functional fragments exhibit a reduction in glycosylation sites compared to the corresponding WT IgA. From a pharmaceutical standpoint, more glycosylation sites may result in batch-to-batch variability in the product, which may affect safety and efficacy. Therefore, in some embodiments, the modified anti-EGFR IgA antibodies described herein involve a reduction in heterogeneity associated with glycosylation. Alternatively, in some embodiments, the modified anti-EGFR IgA antibodies involve a reduction in the glycosylation profile (Figures 4A-4D). In some embodiments, the reduction in glycosylation is achieved by modifying naturally occurring glycosylation motifs or naturally occurring glycosylation sites containing the amino acid sequence NXT or NXS, for example, amino acid residues near or within an N-linked glycosylation site. In some embodiments, the anti-EGFR IgA antibodies disclosed herein exhibit a reduction in glycosylation 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, compared to the corresponding WT IgA. In some embodiments, the antibodies or functional fragments disclosed herein are partially glycosylated compared to the corresponding WT IgA antibody, for example, less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less. In some embodiments, the anti-EGFR IgA antibodies or their functional fragments disclosed herein are completely glycosylated compared to the corresponding WT IgA antibody.

[0127] In some embodiments, the anti-EGFR IgA antibody or functional fragment thereof disclosed herein includes modifications at at least two naturally occurring glycosylation sites within the IgA heavy chain constant region. In some embodiments, the anti-EGFR IgA antibody or functional fragment thereof disclosed herein includes modifications at at least three naturally occurring glycosylation sites within the IgA heavy chain constant region. In some embodiments, the anti-EGFR IgA antibody or functional fragment thereof disclosed herein includes modifications at at least four naturally occurring glycosylation sites. In some embodiments, the glycosylation site includes an N-linked glycosylation site. In some embodiments, the glycosylation site includes a naturally occurring asparagine residue. In some embodiments, the glycosylation site is located in the CH2 region, the CH3 region and / or the CH1 domain. In some embodiments, the IgA heavy chain constant region includes modifications in CH1-N45.2 or P124, numbered according to the IMGT scheme; CH2-N20, L21, T22, C92, N120, I121 or T122; CH3-N135, C147 or Y148, or combinations thereof. In some embodiments, the IgA heavy chain constant region includes amino acid substitutions such as CH1-N45.2G, N45.2A or P124R, numbered according to the IMGT scheme; CH2-N20G, N20Q, N20T, L21I, T22S, C92S, N120T, I121L or T122S; CH3-N135Q; C147 deletion or Y148 deletion, or combinations thereof. In some embodiments, a deglycosylated antibody or a functional fragment thereof is shown herein. In some embodiments, the deglycosylated antibody includes modifications at all four naturally occurring glycosylation sites within the constant region of the IgA2 heavy chain. In some embodiments, the deglycosylated antibody shown herein includes modifications at 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 scheme.In some embodiments, the deglycosylating antibodies shown herein include modifications at residues CH1-N45.2 and P124; CH2-N20, C92, N120, I121 or T122; and CH3-N135, C147 and Y148, which are numbered according to the IMGT scheme.

[0128] In some embodiments, the deglycosylating antibodies shown herein include modifications at residues CH1-N45.2 or P124, CH2-N20, L21, T22, C92, N120, I121 or T122, numbered according to the IMGT scheme; or deletions of C-terminal CH3 tailpiece residues P131-Y148. In some embodiments, the deglycosylating antibodies shown herein include modifications at residues CH1-N45.2 and P124, CH2-N20, L21, T22, C92, N120, I121 and T122, numbered according to the IMGT scheme; and deletions of C-terminal CH3 tailpiece residues P131-Y148.

[0129] In some embodiments, the antibody or its functional fragment includes modifications in the CH1 domain, including N45.2 substitution or P124 substitution, numbered according to the IMGT scheme; modifications in the CH2 domain, including N20 substitution, L21 substitution, T22 substitution, C92 substitution, N120 substitution, I121 substitution or T122 substitution; or modifications in 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. In some embodiments, the antibody or its functional fragment is modified in the CH1 domain, including N45.2 substitutions, P124R substitutions, or any combination thereof selected from the group consisting of N45.2G and N45.2A numbered according to the IMGT scheme; modifications in the CH2 domain, including N20 substitutions, L21I substitutions, T22S substitutions, C92S substitutions, N120T substitutions, I121L substitutions, T122S substitutions, or any combination thereof selected from the group consisting of N20G, N20Q and N20T; or H5 substitutions selected from the group consisting of H5C, H5Y, H5F, H5M and H5W; L7 substitutions selected from the group consisting of L7F, L7Y, L7M, L7W, L7H and L7I; P10C substitutions. Modifications in the CH3 domain include T22 substitutions selected from the group consisting of T22V, T22I, T22L, and T22A; L79 substitutions selected from the group consisting of L79V, L79T, L79A, and L79I; W81 substitutions selected from the group consisting of W81T, W81L, W81A, W81V, and W81I; A85.1 substitutions selected from the group consisting of A85.1F, A85.1Y, A85.1M, A85.1W, and A85.1H; T86 substitutions selected from the group consisting of T86Y, T86F, T86M, T86W, and T86H; I88 substitutions selected from the group consisting of I88L, I88A, I88V, and I88T; N135Q substitutions; C147 deletions; Y148 deletions; or deletions of P131 to Y148.

[0130] In some embodiments, the IgA heavy chain constant region comprises the IgA CH1, CH2, and CH3 domains, where the IgA heavy chain constant region includes the following mutations, numbered according to the IMGT scheme, compared to the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO: 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. In some embodiments, the IgA heavy chain constant region further includes the following mutations in the CH3 domain, numbered according to the IMGT scheme, compared to the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO: N135Q substitution, C147 deletion, and Y148 deletion; or deletions of P131-Y148 in the CH3 domain. In some embodiments, the IgA heavy chain constant region further includes N20 substitutions, L21I substitutions, and T22S substitutions selected from the group consisting of N20G, N20Q, and N20T, which are numbered according to the IMGT scheme, compared to the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO: 1.

[0131] In some embodiments, the antibody contains a mutation at N45.2, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative mutation at N45.2, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains N45.2G, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the N45.2 amino acid.

[0132] In some embodiments, the antibody contains a P124 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative P124 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains P124R numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the P124 amino acid. In some embodiments, the IgA2 antibody has increased stability compared to an IgA2 antibody that does not have a mutation at the P124 amino acid. In some embodiments, increased stability refers to one or more of the following: reduced glycosylation, reduced aggregation, improved thermal stability, increased mechanical stability, or increased circulating half-life.

[0133] In some embodiments, the antibodies described herein contain C92 mutations numbered according to the IMGT scheme. In some embodiments, the antibodies contain non-conservative C92 mutations numbered according to the IMGT scheme. In some embodiments, the antibodies contain C92S mutations numbered according to the IMGT scheme. In some embodiments, the antibodies exhibit reduced aggregation compared to antibodies without mutations in the C92 amino acid. In some embodiments, the antibodies exhibit reduced aggregation with serum proteins compared to antibodies without mutations in the C92 amino acid. In some embodiments, the antibodies exhibit reduced aggregation in vitro or in vivo compared to antibodies without mutations in the C92 amino acid.

[0134] In some embodiments, the IgA2 antibody contains a C92 mutation according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative C92 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains C92S numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has reduced aggregation compared to an IgA2 antibody without a mutation at the C92 amino acid. In some embodiments, the IgA2 antibody has reduced aggregation with serum proteins compared to an IgA2 antibody without a mutation at the C92 amino acid. In some embodiments, the IgA2 antibody has reduced aggregation in vitro or in vivo compared to an IgA2 antibody without a mutation at the C92 amino acid.

[0135] In some embodiments, the antibody contains an N120 mutation numbered according to the IMGT scheme. In some embodiments, the antibody contains a non-conservative N120 mutation numbered according to the IMGT scheme. In some embodiments, the antibody contains N120T numbered according to the IMGT scheme. In some embodiments, the antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the N120 amino acid.

[0136] In some embodiments, the antibody contains an I121 mutation numbered according to the IMGT scheme. In some embodiments, the antibody contains a non-conservative I121 mutation numbered according to the IMGT scheme. In some embodiments, the antibody contains I121L numbered according to the IMGT scheme. In some embodiments, the antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the I121 amino acid.

[0137] In some embodiments, the antibody contains a T122 mutation numbered according to the IMGT scheme. In some embodiments, the antibody contains a non-conservative T122 mutation numbered according to the IMGT scheme. In some embodiments, the antibody contains T122S numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the T122 amino acid.

[0138] In some embodiments, the IgA2 antibody contains an N120 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative N120 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains N120T numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the N120 amino acid.

[0139] In some embodiments, the IgA2 antibody contains an I121 mutation according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative I121 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains I121L numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the I121 amino acid.

[0140] In some embodiments, the IgA2 antibody contains a T122 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative T122 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains T122S numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the T122 amino acid.

[0141] In some embodiments, the IgA2 antibody contains an N20 mutation according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative N20 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains N20G, N20Q, or N20T, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation in the N20 amino acid.

[0142] In some embodiments, the IgA2 antibody contains an L21 mutation according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative L21 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains L21I numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation at the L21 amino acid.

[0143] In some embodiments, the IgA2 antibody contains a T22 mutation according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative T22 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains T22S mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not have a mutation in the T22S amino acid.

[0144] In some embodiments, the IgA2 antibody contains a C147 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative C147 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a deletion of amino acid C147 numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has reduced aggregation compared to an IgA2 antibody that does not have a mutation at the C147 amino acid.

[0145] In some embodiments, the IgA2 antibody contains a Y148 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a non-conservative Y148 mutation numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody contains a deletion of amino acid Y148 numbered according to the IMGT scheme.

[0146] In some embodiments, the anti-EGFR IgA antibody contains 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 domain of the heavy chain of the IgA constant region. In some embodiments, the IgA2 antibody has an increased circulating half-life compared to an IgA2 antibody that does not contain one or more albumin-binding domains. In some embodiments, the IgA2 antibody contains one or more albumin-binding domains and has a circulating half-life within 1%, 5%, or 10% of the circulating half-life of the corresponding IgG antibody. In some embodiments, the IgA2 antibody contains one or more albumin-binding domains and has a circulating half-life greater than that of the corresponding IgG antibody.

[0147] In some embodiments, one or more mutations or deletions result in an increase or decrease in the circulating half-life of anti-EGFR IgA antibodies. In some embodiments, one or more mutations or deletions result in an increase in the circulating half-life of anti-EGFR IgA antibodies. For example, one or more mutations may increase the serum half-life of anti-EGFR IgA antibodies by up to 21 or 22 days or more in humans. Furthermore, one or more mutations may increase the serum half-life of anti-EGFR IgA antibodies by up to 9 or 10 days or more in mice. In some embodiments, one or more mutations may increase the serum half-life of anti-EGFR IgA antibodies to a level equivalent 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 anti-EGFR IgA antibodies.

[0148] In some embodiments, one or more mutations can increase the serum half-life of anti-EGFR IgA antibodies by at least about 7 days to about 30 days or 31 days or more. In some embodiments, one or more mutations can increase the serum half-life of anti-EGFR IgA antibodies by at least about 7 days. In some embodiments, one or more mutations can increase the serum half-life of anti-EGFR IgA antibodies by up to about 30 days. In some embodiments, one or more mutations can increase the serum half-life of anti-EGFR The serum half-life of IgA antibodies is approximately 7 to 8 days, approximately 7 to 9 days, approximately 7 to 10 days, approximately 7 to 15 days, approximately 7 to 20 days, approximately 7 to 25 days, approximately 7 to 30 days, approximately 8 to 9 days, approximately 8 to 10 days, approximately 8 to 15 days, approximately 8 to 20 days, approximately 8 to 25 days, approximately 8 to 30 days, approximately 9 to 10 days, approximately 9 days The circulating half-life of the anti-EGFR IgA antibody may be increased by approximately 15 days, approximately 9 to approximately 20 days, approximately 9 to approximately 25 days, approximately 9 to approximately 30 days, approximately 10 to approximately 15 days, approximately 10 to approximately 20 days, approximately 10 to approximately 25 days, approximately 10 to approximately 30 days, approximately 15 to approximately 20 days, approximately 15 to approximately 25 days, approximately 15 to approximately 30 days, approximately 20 to approximately 25 days, approximately 20 to approximately 30 days, or approximately 25 to approximately 30 days. In some embodiments, one or more mutations may increase the serum half-life of the anti-EGFR IgA antibody by approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 15 days, approximately 20 days, approximately 25 days, or approximately 30 days. Therefore, in some embodiments, the antibodies or functional fragments thereof disclosed herein exhibit a longer circulating half-life compared to the corresponding WT IgA antibody. In some embodiments, the antibody exhibits a cyclic half-life that is at least about 2%, 5%, 10%, 12%, 15%, 20%, 25%, 50%, 65%, 70%, 75%, 85%, 90%, 95%, 99%, 100%, 150%, and 200% longer than the corresponding WT IgA antibody.

[0149] 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 an anti-EGFR IgA antibody compared to the corresponding IgA antibody result in one or more of the following: reduced glycosylation, reduced aggregation, improved thermal stability, increased mechanical stability, or increased circulating half-life.

[0150] In some embodiments, anti-EGFR IgA antibodies exhibit reduced aggregation. Antibody aggregation is a more general manifestation of physical instability. Protein aggregates generally have reduced activity and, more importantly, greater immunogenic potential due to epitope multiplicity and / or conformational changes. For example, in some embodiments, anti-EGFR IgA antibodies bind to epitopes of EGFR polypeptides or variants thereof containing any one 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-like reactions, such as headache, fever, and chills. Therefore, reducing aggregation in antibody therapeutics is beneficial. In addition, aggregate levels in commercially available intravenous immunoglobulin products are limited to less than 5% based on World Health Organization (WHO) standards. In some embodiments, one or more mutations result in reduced aggregation. In some embodiments, one or more mutations and / or one or more deletions result in reduced aggregation of anti-EGFR IgA antibodies compared to the corresponding IgA antibody that does not contain one or more mutations and / or one or more deletions. In some embodiments, the antibodies or functional fragments thereof disclosed herein exhibit reduced aggregation compared to the corresponding WT IgA antibody. In some embodiments, the antibodies exhibit reduced aggregation 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 the corresponding WT IgA antibody. In some embodiments, the antibodies or functional fragments thereof disclosed herein exhibit reduced aggregation with serum proteins compared to the corresponding WT IgA antibodies.In some embodiments, the antibody exhibits aggregation 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.

[0151] In some embodiments, the anti-EGFR IgA antibodies presented herein have an aggregation level ranging from at least about 0.1% to as much as about 5%. In some embodiments, the IgA antibodies presented herein have an aggregation level ranging from at least about 0.1%. In some embodiments, the IgA antibodies presented herein have an aggregation level ranging from as much as about 5%. In some embodiments, the anti-EGFR IgA antibodies shown herein have aggregation levels in the range of 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 antibodies shown herein have aggregation levels ranging from about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5%.

[0152] The anti-EGFR IgA antibodies disclosed herein may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and 100% hydroxyphenylalanine. This product contains phosphorus-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0153] Methods for substituting or deleting amino acids include, for example, site-directed mutagenesis. Mutagenesis may be performed by synthesizing oligonucleotides having one or more modifications within the sequence of the constant domain of the antibody to be modified. Antibodies of this disclosure (e.g., those containing one or more modifications within the constant region of the IgA heavy chain) may be prepared using any mutagenesis technique known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc. Site-directed mutagenesis enables the production of mutants through the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation, as well as primer sequences of sufficient size on both sides of the traversed deletion junction and a sufficient number of adjacent oligonucleotides to provide sequence complexity for forming a stable double helix. Typically, primers of about 17 to about 75 nucleotides or 76 nucleotides or longer are preferred, with about 10 to about 25 or 26 or more residues on both sides of the junction of the sequences being modified. Several such primers introducing various different mutations at one or more positions may be used to generate a library of mutants.

[0154] Synthetic gene construction involves the in vitro synthesis of a designed polynucleotide molecule encoding the polypeptide of interest. Gene synthesis can be carried out using several techniques, such as multi-microchip-based techniques and similar techniques in which oligonucleotides are synthesized and assembled on photo-programmable microfluidic chips. Single or multiple amino acid substitutions, deletions, and / or insertions can be produced and tested using mutagenesis, recombination, and / or shuffling methods, followed by associated screening techniques. Other methods that may be used include error-prone PCR, phage display, and region-specific mutagenesis.

[0155] Mutagenesis / shuffling methods may be combined with high-throughput automated screening methods to detect the activity of cloned and mutageneised polypeptides expressed by host cells. Mutageneised DNA molecules encoding active polypeptides can be recovered from host cells using standard methods in the art and rapidly sequenced. These methods allow for rapid determination of the importance of individual amino acid residues within the polypeptide.

[0156] Semi-synthetic gene construction is achieved by combining synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or shuffling. A typical semi-synthetic construction is a process that utilizes a synthesized polynucleotide fragment in combination with PCR techniques. Thus, a region of a defined gene may be synthesized de novo, while other regions may be amplified using site-directed mutagenesis primers, or other regions may be subjected to error-prone PCR or non-error-prone PCR amplification. Subsequently, the polynucleotide subsequences can be shuffled.

[0157] Other covalent modifications In some embodiments, anti-EGFR IgA antibodies include one or more covalent modifications. These may be performed, where applicable, by chemical synthesis of the antibody or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies are introduced into the molecule by reacting targeted amino acid residues of the antibody with organic inducers capable of reacting with selected side chains or N- or C-terminal residues.

[0158] Cysteinyl residues are most commonly reacted with haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidemethyl derivatives. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, alpha-bromo-(5-imidozolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercury benzoate, 2-chloromercury-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0159] Since diethyl pyrocarbonate is relatively specific to the histidyl side chain, histidyl residues are derivatized by reaction with this agent at pH 5.5–7.0. Para-bromophenacyl bromide is also useful, and the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. Lydinyl and amino-terminal residues are reacted with succinic acid or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lydinyl residue. Other suitable reagents for derivatization of alpha-amino-containing residues include transaminase-catalyzed reactions with imide esters, such as methyl picoline imidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, methyl isourea, 2,4-pentanedione, and glyoxylate.

[0160] Arginyl residues are modified by reaction with one or more reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires the reaction to be carried out under alkaline conditions due to the high pKa of the guanidine functional group. Furthermore, these reagents can react with lysine groups and arginine epsilon-amino groups.

[0161] Specific modification of tyrosyl residues is also possible, and introducing spectral labeling to tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane is of particular interest. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. To prepare labeled proteins for use in radioimmunoassays, tyrosyl residues are iodized using 125I or 131I. The carboxyl side chain group (aspartyl or glutamyl) is selectively modified by reaction with carbodiimide (RN.dbd.C.dbd.N-R'), where R and R' are different alkyl groups, e.g., 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, the aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues through reaction with ammonium ions.

[0162] Glutaminyl and asparaginyl residues are frequently deamidated to their corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated forms of these residues fall within the scope of the present invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine, and histidine side chains, acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0163] Another type of covalent modification involves chemically or enzymatically coupling a glycoside to an antibody. These procedures are beneficial because they do not require the production of antibodies in host cells that have the glycosylation ability for N- or O-linked glycosylation. Depending on the coupling method used, the sugar(s) may be coupled to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group, e.g., the free sulfhydryl group of cysteine, (d) a free hydroxyl group, e.g., the free hydroxyl group of serine, threonine or hydroxyproline, (e) an aromatic residue, e.g., an aromatic residue of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine.

[0164] The removal of any glycan portion present in an antibody can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of almost all sugars except linked sugars (N-acetylglucosamine or N-acetylgalactosamine), while the antibody remains intact. Enzymatic cleavage of the glycan portion of an antibody can be achieved using various endoglycosidases and exoglycosidases.

[0165] Another type of covalent modification of antibodies involves linking the antibody to one of various non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyol, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylene, or polysaccharide polymers, such as dextran.

[0166] IgA antibody target The anti-EGFR IgA antibodies described herein bind to EGFR expressed by target cells (e.g., cancer cells). In some embodiments, the target cells are human cells. In some embodiments, EGFR is human EGFR.

[0167] EGFR is a transmembrane glycoprotein that promotes cell proliferation in various normal and transformed tissues. Its receptor has several intrinsic ligands, including EGF and transformation growth factor-alpha. Binding of these ligands to the receptor stimulates cell proliferation. Blocking this interaction by means of antibodies targeting the receptor inhibits tumor growth in vivo.

[0168] In some embodiments, an anti-EGFR IgA antibody or a functional fragment thereof comprises one or more modifications disclosed herein within the constant region of the IgA heavy chain, while retaining the ability to specifically bind to an EGFR polypeptide (e.g., human EGFR). Polypeptides and coding nucleic acid sequences of human-derived EGFR and EGFR from several animals are publicly available, for example, from the NCBI website.

[0169] In some embodiments, the anti-EGFR IgA antibody binds to EGFR with a binding affinity (Kd) of less than 0.01 nM. In some embodiments, the anti-EGFR IgA antibody binds to EGFR with a binding affinity (Kd) of approximately 1 μM or more. In some embodiments, the anti-EGFR IgA antibody binds to EGFR with a binding affinity (Kd) in the range of 0.01 nM to 1 μM. In some embodiments, the anti-EGFR IgA antibodies are available in the following concentrations: approximately 0.01 nM to 800 nM, approximately 0.01 nM to 500 nM, approximately 0.01 nM to 300 nM, approximately 0.01 nM to 100 nM, approximately 0.05 nM to 800 nM, approximately 0.05 nM to 500 nM, approximately 0.05 nM to 300 nM, approximately 0.01 nM to 100 nM, approximately 0.1 nM to 800 nM, and approximately 0.1 nM to 500 nM. The antibodies bind to EGFR with binding affinity (Kd) of M, approximately 0.1 nM to approximately 300 nM, approximately 0.1 nM to approximately 100 nM, approximately 5 nM to approximately 800 nM, approximately 5 nM to approximately 500 nM, approximately 5 nM to approximately 300 nM, approximately 5 nM to approximately 100 nM, approximately 10 nM to approximately 800 nM, approximately 10 nM to approximately 500 nM, approximately 10 nM to approximately 300 nM, or approximately 10 nM to approximately 100 nM. In some embodiments, the anti-EGFR IgA antibody binds to EGFR with binding affinity (Kd) of approximately 0.01 nM, approximately 0.05 nM, approximately 0.1 nM, approximately 1 nM, approximately 10 nM, approximately 100 nM, or approximately 200 nM.

[0170] In some embodiments, the anti-EGFR IgA antibodies described herein comprise a Fab domain and an Fc domain, where the Fab domain binds to human EGFR and the Fc domain binds to FcαRI receptors on immune cells (e.g., neutrophils, macrophages, and eosinophils).

[0171] Immunoeffector function of anti-EGFR IgA antibodies Compared to the corresponding WT IgA antibody containing the WT heavy chain constant region, modified anti-EGFR IgA antibodies containing one or more modifications within the heavy chain constant region are shown herein. In some embodiments, anti-EGFR IgA antibodies or functional fragments thereof bind to Fc-alpha receptors (FcαR) expressed on immune effector cells, for example, FcαR for human IgA. FcαR is present on immune effector cells, such as monocytes, macrophages, neutrophils, and other myeloid cells. FcαR may also be found on metamyelocytes, myelocytes, promyelocytes, and some myeloblasts from the bone marrow, for example. Such receptors may also be found on myeloid cell lines, such as U937, PLB985, and HL60 cells. It has also been suggested that FcαR is present on lymphocytes. FcαR expression may be increased by activation of myeloid cells. For example, stimulation of U937 and PLB985 cells with phorbol myristic acetate (PMA) increases the cell surface level of FcαR several times over. Other agents that can increase the surface level of FcαR include calcitriol, 1,25-dihydroxyvitamin D3, and interferon-γ (IFN-γ).

[0172] FcαR has the ability to interact with IgA1 and IgA2 in monomer, dimer, and polymer forms. Therefore, anti-EGFR IgA antibodies or their functional fragments have the ability to activate at least one Fc-receptor-mediated immunoeffector cell function.

[0173] Immune effector cells are cells involved in the effector phase of the immune response, as opposed to the recognition and activation phases of the immune response. Immune effector cells include lymphocytes (e.g., B cells and T cells including cytolytic 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.

[0174] In some embodiments, a modified anti-EGFR IgA antibody or a functional fragment thereof binds to the low-affinity Fc receptor FcαRI (CD89) on immune effector cells. FcαRI interacts with IgA primarily after antibody-antigen recognition, via its Cα1 and Cα2 domains. FcαRI is a 55-75 kDa type I transmembrane receptor consisting of two extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic tail. It is expressed on myeloid cells, such as neutrophils, eosinophils, activated monocytes, granulocytes, a subset of dendritic cells, Kupffer cells, and macrophages. Binding of the modified anti-EGFR IgA antibody to FcαRI mediates effector functions, such as phagocytosis, trogocytosis, oxidative burst, cytokine release, antigen presentation, and ADCC.

[0175] In some embodiments, the anti-EGFR IgA antibodies disclosed herein exhibit an increased binding affinity to the Fc receptor on immune effector cells 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, compared to the corresponding WT IgA or WT IgG.

[0176] In some embodiments, anti-EGFR IgA antibodies or their functional fragments exhibit an increase in at least one effector function compared to the corresponding WT IgA antibody or its functional fragment or the corresponding WT IgG antibody. In some embodiments, anti-EGFR IgA antibodies induce complement-dependent cytotoxicity (CDC). In some embodiments, anti-EGFR IgA antibodies induce polymorphonuclear neutrophil (PMN)-mediated tumor cell lysis. In some embodiments, IgA antibodies activate polymorphonuclear cell (PMN)-mediated ADCC more efficiently than IgG antibodies. In some embodiments, anti-EGFR IgA antibodies induce programmed cell death (PCD) via a caspase-independent pathway. In some embodiments, anti-EGFR IgA antibodies induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0177] In some embodiments, IgA does not bind to B cells, T cells, platelets, and / or red blood cells. For example, in some embodiments, anti-EGFR IgA antibodies may have low immunogenicity.

[0178] ADCC 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., "death") of target cells (e.g., cancer cells). Primary mediator cells may be natural killer (NK) cells and neutrophils. ADCC activity can be directly assessed using an in vitro assay, such as a 51Cr release assay, with peripheral blood mononuclear cells (PBMCs) and / or NK effector cells as described in the examples presented herein. ADCC activity may be expressed as the concentration of antibody at which lysation of target cells occurs by up to half. Accordingly, in some embodiments, the concentrations of the antibodies or antigen-binding functional fragments thereof of the present disclosure, where the solubility level is the same as up to half the solubility level of the wild-type control, are less than or equal to 1 / 2, 1 / 3, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the concentration of the wild-type control itself.

[0179] Neutrophils can be found in the EGFR-positive tumor microenvironment. Therefore, in some embodiments, the anti-EGFR IgA antibodies described herein exhibit neutrophil-mediated ADCC. In some embodiments, anti-EGFR IgA antibodies have a superior ability to recruit neutrophils for antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the corresponding IgG antibodies. In some embodiments, anti-EGFR IgA antibodies require a lower effector:target (E:T) ratio than the corresponding IgG to achieve equivalent levels of ADCC. In some embodiments, neutrophil-mediated ADCC is involved in antibody-mediated trogoptosis, a process in which neutrophils "gnaw" at the cancer cell membrane, leading to loss of membrane integrity and ultimately causing cell death. In some embodiments, neutrophils have the ability to induce direct cytotoxicity, particularly in relation to antibody-mediated targeting, through the release of high levels of ROS or by the release of granular contents. The release of cytotoxic molecules from primary, secondary, and tertiary granules during degranulation can induce apoptotic elimination of cancer cells. In some embodiments, neutrophils have the ability to mediate the death of cancer antigen-loss variants, which are a result of tumor evolution and often interfere with immunotherapy. In addition, neutrophils recruited by anti-EGFR IgA contribute to the recruitment and activation of other immune cells, stimulating adaptive anti-tumor immunity, which further promotes tumor cell elimination.

[0180] In addition, in some embodiments, the anti-EGFR IgA antibodies or functional fragments thereof of this disclosure may exhibit a higher maximum target cell lysis compared to the corresponding wild-type IgA antibodies or WT IgG antibodies. For example, the maximum target cell lysis of the antibodies or functional fragments thereof of this disclosure may be 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or more higher than the maximum of the corresponding WT IgA antibodies or WT IgG antibodies. In some embodiments, the antibodies or functional fragments thereof disclosed herein induce an increase in ADCC compared to the corresponding WT IgG antibodies containing the IgG heavy chain constant region. In some embodiments, 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% compared to the corresponding WT IgG antibody.

[0181] In some embodiments, tumors containing KRAS mutations are susceptible to ADCC-mediated cell death induced by anti-EGFR IgA antibodies 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, anti-EGFR IgA antibodies inhibit tumor growth and reduce tumor size through opsonization and subsequent neutrophil recruitment and ADCC.

[0182] In some embodiments, anti-EGFR IgA antibodies can recruit NK cells and macrophages to kill cells via ADCC. In some embodiments, NK cells, macrophages, and / or neutrophils recruited by modified anti-EGFR IgA antibodies kill opsonized cancer cells by mechanisms including trogocytosis, which results in lytic / necrotic cell death.

[0183] CDC The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that has formed a complex with a congeneral antigen. A CDC assay may be performed to assess complement activation.

[0184] In some embodiments, the anti-EGFR IgA antibodies described herein have the ability to inhibit EGFR, thereby inducing cell cycle arrest and apoptosis in cancer cells.

[0185] In vivo distribution In some embodiments, the anti-EGFR IgA antibodies or functional fragments thereof disclosed herein exhibit increased in vivo distribution compared to the corresponding WT IgA antibodies. Increased in vivo distribution in cells or tissues can be evaluated using a variety of methods, including, but not limited to, nuclear medicine, whole-body autoradiography, micro-autoradiography, fluorescence imaging, cryo-imaging, nano-secondary ion mass spectrometry (nanoSIMS), matrix-assisted laser desorption imaging (MALDI-MS), X-ray imaging, magnetic resonance imaging (MRI), computed tomography (CT), micro-ultrasound single-photon emission CT (SPECT), and positron emission tomography (PET). The increased in vivo distribution of the antibody to the target site includes an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or more compared to the medium or WT IgA antibody.

[0186] Method for producing antibodies In some embodiments, the anti-EGFR IgA antibody is produced in host cells. In some embodiments, the host cell is CHO. In some embodiments, the host cell is SP20. In some embodiments, the host cell is HEK239 host cell. In some embodiments, the HEK293 host cell is HEK293F. In some embodiments, the antibody or its antigen-binding functional fragment is isolated from host cells. In some embodiments, the antibody or its antigen-binding functional fragment is prepared in a cell-free system. Isolated nucleic acid molecules encoding the antibodies, portions, or polypeptides of this disclosure may be recombined with vector DNA (e.g., an expression vector) according to conventional techniques including blunt or adherent termination for ligation, restriction enzyme digestion to provide suitable ends, appropriate adherent end complementation, alkaline phosphatase treatment to avoid undesirable conjugation, and ligation with a suitable ligase. Techniques for such manipulation can be used to construct nucleic acid sequences encoding the antibody molecule or its antigen-binding region. Accordingly, this disclosure shows vectors or expression vectors comprising the isolated nucleic acids shown herein. In one embodiment, the nucleic acid encoding the light chain and the nucleic acid encoding the heavy chain are isolated separately by the procedure outlined above. In one embodiment, the isolated nucleic acids encoding the light chain and the isolated nucleic acids encoding the heavy chain may be inserted into separate expression plasmids or together into the same plasmid, provided that each is under appropriate promoter and translational control.

[0187] When isolated nucleic acid molecules are placed in an expression vector, they are then transfected into host cells that would otherwise not produce immunoglobulin proteins, such as Escherichia coli (E. coli) cells, monkey COS cells, human embryonic kidney 293 cells (e.g., 293E cells), Chinese hamster ovary (CHO) cells, or myeloma cells, to synthesize antibodies or their antigen-binding functional fragments in recombinant host cells. Any available vector can be used. Vector components generally include, but are not limited to, one or more of the following: signal sequences, replication origins, one or more select marker genes, enhancer elements, promoters, and transcription termination sequences.

[0188] Isolated nucleic acid molecules are operably ligated to expression regulatory sequences within vector DNA. An expression regulatory sequence refers to a DNA sequence necessary for the expression of an operably ligated coding sequence in a specific host organism. Appropriate regulatory sequences for prokaryotes include, for example, promoters, may include operator sequences, and may include ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0189] Cells, cell lines, and cell cultures are often used interchangeably, and all such names herein include offspring. Transformants and transformed cells include primary target cells and cultures derived therefrom, regardless of the number of transitions. It is also understood that all offspring may not have exactly the same DNA contents due to intentional or accidental mutations. Mutant offspring with the same function or biological activity as those screened for with respect to the original transformed cells are included. Where a distinct name is intended, it is evident from the context. In alternative embodiments, a suitable coding nucleic acid sequence may be designed according to a universal codon table based on a known amino acid sequence of the immunoglobulin of interest.

[0190] Desired antibody amino acid sequence variants can be prepared by introducing appropriate nucleotide changes into the coding DNA or by peptide synthesis. Such variants include, for example, deletions from and / or insertions of residues in the antibody's amino acid sequence, and / or substitutions of residues. Any combination of deletions, insertions, and substitutions is performed to arrive at the final construct, provided that the final construct possesses the desired characteristics. Furthermore, amino acid changes can alter the post-translational processes of monoclonal antibodies, human antibodies, humanized antibodies, or variant antibodies, such as by changing the number or location of glycosylation sites.

[0191] Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or preparation of variant or non-variant versions of previously prepared antibodies by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0192] Furthermore, this disclosure provides isolated nucleic acid molecules encoding antibodies or antigen-binding functional fragments thereof as described herein, which may be operably ligated to a host cell, a vector, and a nucleic acid-containing regulatory sequence recognized by the host cell, as well as recombinant techniques for antibody production, which may include culturing the host cell to express the nucleic acid, and recovering the antibody from the host cell culture or culture medium.

[0193] For recombinant production of an antibody or its antigen-binding functional fragment, the nucleic acid molecule encoding it may be isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. Therefore, isolated antibodies or their antigen-binding functional fragments are shown herein. In some embodiments, the antibodies or their antigen-binding functional fragments of the Disclosure may be recombinant antibodies. In some embodiments, the recombinant antibody has a glycosylation pattern different from that of an antibody having the same sequence if it were naturally occurring. In one embodiment, the recombinant antibody is expressed in mammalian host cells other than human host cells. Notably, individual mammalian host cells have unique glycosylation patterns.

[0194] In some embodiments, the antibodies of this disclosure or their antigen-binding functional fragments are synthetic. The polypeptides of this disclosure may be purified by isolation / purification methods.

[0195] In one embodiment, a host cell comprising an isolated nucleic acid molecule as described herein, or a vector containing the isolated nucleic acid molecule as described herein, is shown herein. The vector may be a cloning vector or an expression vector. Suitable host cells for cloning or expressing DNA in the vector as described herein are prokaryotes, yeasts, or higher eukaryotes as described above. Suitable prokaryotes for this purpose include bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans and Shigella, as well as bacilli, such as Bacillus subtilis and Bacillus licheniformis (e.g., Bacillus licheniformis 41 P), and Pseudomonas, such as Pseudomonas (P. This includes *E. coli aeruginosa* and the genus *Streptomyces*. One preferred *E. coli* cloning host is *E. coli* 294 (ATCC 31,446), but other strains, such as *E. coli* B, *E. coli* XI776 (ATCC 31,537), and *E. coli* W3110 (ATCC 27,325), are also suitable. These examples are illustrative and not limiting.

[0196] In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for antibody-encoding vectors. Budding yeast (Saccharomyces cerevisiae) or common baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, some other genera, species, and lineages, such as fission yeast (Schizosaccharomyces pombe); Kluyveromyces host species, such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), and Kluyveromyces thermotolerance (K. * thermotolerans* and *K. marxianus*; *yarrowia* (EP 402,226); *Pichia pastors* (EP 183,070); *Candida*; *Trichoderma reesia* (EP 244,234); *Neurospora crassa*; *Schwanniomyces*, e.g., *Schwanniomyces occidentalis*; and filamentous fungi, e.g., *Neurospora*, *Penicillium*, *Tolypocladium* and *Aspergillus*, e.g., pseudofossil *Aspergillus*. Aspergillus nidulans and Aspergillus niger are commonly available and are useful herein.

[0197] Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants, as well as corresponding permissible insect host cells from hosts such as the armyworm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm (Bombyx mori), have been identified. Various virus strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of silkworm NPV, and such viruses may be used as the viruses of this specification according to the present invention, particularly for transfection of armyworm cells.

[0198] Furthermore, plant cell cultures of cotton, corn, potatoes, soybeans, petunias, tomatoes, tobacco, duckweed, and other plant cells can be used as hosts. However, vertebrate cells have attracted the most attention, and the propagation of vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of useful mammalian host cell lines include CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells including -DHFR; monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293, or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-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); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep These include G2, HB 8065); mouse mammary gland tumors (MMT 060562, ATCC CCL51); TRI cells; and human hepatocellular carcinoma cell line (Hep G2).

[0199] Host cells are transformed or transfected with the expression vectors or cloning vectors described above for antibody production and cultured in a conventional nutrient medium modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. In addition, transfected cell lines with novel vectors and numerous copies of transcription units separated by selection markers are particularly useful and preferred for the expression of antibodies described herein.

[0200] For transfection of expression vectors and production of chimeric, humanized, or compound human antibodies as described herein, the recipient cell line may be myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by transfected immunoglobulin nucleic acid sequences and possess mechanisms for immunoglobulin glycosylation. For example, in some embodiments, the recipient cells are recombinant Ig-producing myeloma cells SP2 / 0 (ATCC CRL8287). SP2 / 0 cells produce only immunoglobulins encoded by transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, where the secreted immunoglobulins can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells, e.g., human or non-human B lymphocytes, human or non-human hybridoma cells, or interspecies heterohybridoma cells. Expression vectors having chimeric, humanized, or complex human antibody constructs, or antibody polypeptides as described herein, can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, as well as mechanical means such as electroporation, direct microinjection, and particle guns.

[0201] Yeast offers certain advantages over bacteria in the production of immunoglobulin H and L chains. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies exist that utilize strong promoter sequences and high-copy-number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of a cloned mammalian gene product and secretes a peptide containing the leader sequence (i.e., a pre-peptide). Yeast gene expression systems can be routinely evaluated for the production, secretion, and stability levels of antibody polypeptides or their antigen-binding functional fragment peptides, as well as assembled chimeric, humanized, or compound human antibodies, their functional fragments, and regions. One of a series of yeast gene expression systems can utilize promoters and termination elements of actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast grows in glucose-rich media. Also, known glycolytic genes can provide highly efficient transcriptional regulatory signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Several methods can be used to evaluate the optimal expression plasmid for the expression of immunoglobulin cDNA cloned in yeast. In some embodiments, the plasmid includes a non-integrated plasmid. In some embodiments, the plasmid has the ability to transiently express cDNA.

[0202] Furthermore, bacterial strains can be used as hosts for the production of antibody molecules or their functional fragments as described herein. E. coli K12 strains, e.g., E. coli W3110 (ATCC 27325), bacilli species, enterobacteria, e.g., Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicons and regulatory sequences derived from host cell-compatible species are used in conjunction with these bacterial hosts. The vectors have replication sites and specific genes capable of providing phenotypic selection in transformed cells. Several methods can be employed to evaluate expression plasmids for the production of chimeric, humanized, or compound humanized antibodies and their functional fragments or CDRs encoded by cloned immunoglobulin cDNA in bacteria.

[0203] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including leader peptide removal, H and L chain folding and assembly, antibody molecule glycosylation, and secretion of functional antibody proteins. In addition to the lymphocyte-derived cells described above, mammalian cells that may be useful as hosts for antibody protein production include fibroblast-derived cells, e.g., Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6.RTM. cells (Cru cells); and NSO cells. In some embodiments, specific eukaryotic host cells are selected based on their ability to produce desired post-translational modifications to variable heavy chains and / or variable light chains. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0204] In some embodiments, polypeptides of antibodies or antigen-binding functional fragments thereof disclosed herein can be produced in vivo in animals manipulated or transfected with one or more nucleic acid molecules encoding the polypeptide, according to any suitable method.

[0205] Many vector systems are available for the expression of H and L chain nucleic acid sequences in mammalian cells. Various methods may be followed to obtain a complete H2L2 antibody. As described above, it is possible to co-express the H and L chains in the same cells to achieve intracellular association and linkage of the H and L chains, thereby obtaining a complete tetrameric H2L2 antibody and / or antigen-binding functional fragment peptide. Co-expression can occur by using the same or different plasmids in the same host. Genes for both the H and L chains and / or the CDR3 region peptide may be placed in the same plasmid, which is then transfected into cells to directly select cells that express both chains. Alternatively, cells may be first transfected with a plasmid encoding one chain, e.g., the L chain, and then the resulting cell line may be transfected with an H chain plasmid containing a second selectable marker. Cell lines that produce functional antigen-binding peptide fragments and / or H2L2 molecules via either pathway may be transfected with plasmids encoding additional copies of the peptide, H, L, or H and L chains, along with additional selectable markers, to generate cell lines with improved characteristics such as higher production of assembled H2L2 antibody molecules or improved stability of the transfected cell line.

[0206] In some embodiments, methods and systems for producing humanized antibodies are described herein, prepared by a process comprising the steps of: maintaining a host transformed with a first expression vector encoding the light chain of a humanized antibody and a second expression vector encoding the heavy chain of a humanized antibody under conditions in which each chain is expressed; and isolating a humanized antibody formed by an assembly of the thus expressed chains. The first and second expression vectors may be the same vector. Also described herein are DNA sequences encoding the light or heavy chain of a humanized antibody; expression vectors incorporating the DNA sequences; and a host transformed with the expression vectors. Generating humanized antibodies from the nucleic acid sequences and information described herein can be carried out by those skilled in the art without excessive experimental work. In one method, there are four general steps used to humanize a monoclonal antibody. These include (1) the step of determining the nucleotides and predicted amino acid sequences of the starting antibody light chain and heavy chain variable domains; (2) the step of designing the humanized antibody, i.e., the step of determining which antibody framework regions to use during the humanization process; (3) the actual humanization method / technique; and (4) the transfection and expression of the humanized antibody.

[0207] purification Contaminating components in the natural environment of polypeptides are substances that may interfere with the diagnostic or therapeutic use of polypeptides and may include enzymes, hormones, and other proteinaceous or non-proteinaceous components. In some embodiments, the antibodies of this disclosure or their antigen-binding functional fragments may be purified by appropriate methods. In preferred embodiments, polypeptides are purified (1) to more than 95% by weight, most preferably more than 99% by weight, as 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 by using a spinning cup sequenator; or (3) to a degree of homogeneity as indicated by SDS-PAGE using Coomassie blue or preferably silver staining under reducing or non-reducing conditions. Since at least one component of the natural environment of polypeptides is absent, isolated antibodies include in situ polypeptides in recombinant cells. However, typically, isolated polypeptides are prepared by at least one purification step. In one embodiment, a purified antibody or antigen-binding functional fragment as shown herein is disclosed herein.

[0208] Once expressed, the entire antibody of the present invention, its dimers, individual light and heavy chains, or other immunoglobulin types can be recovered and purified by techniques such as immunoadsorption or immunoaffinity chromatography, chromatography, e.g., HPLC (high-performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. Substantially pure immunoglobulins with at least about 90%–95% homogeneity are beneficial, and immunoglobulins with 98%–99% or higher homogeneity are particularly beneficial for pharmaceutical use. When recombinant techniques are used, antibodies can be produced intracellularly, in the perimembrane space, or secreted directly into a medium containing culture media from a microbial culture. If antibodies are produced intracellularly, as a first step, particulate debris, either from host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.

[0209] Antibody compositions isolated from microorganisms or mammalian cells may be purified, for example, using hydroxyl apatite chromatography, cation or anion (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 the isotype of any 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. The matrix to which the affinity ligand binds is most often agarose, but other matrices are usable. Mechanically stable matrices, such as controlled-pore glass or poly(styrene-divinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, a resin that specifically binds to the CH3 domain may be useful for purification. Other techniques for protein purification, such as fractionation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin bead chromatography on anion or cation exchange resin (e.g., polyaspartate columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation, may also be used depending on the recovered antibody. Once purified partially or to the desired homogeneity, the humanized or compound human antibody can then be used for therapeutic purposes or in the development and implementation of assay procedures, immunofluorescence staining, etc.

[0210] The functional activity of the antibody or antigen-binding functional fragment disclosed herein. Such functional activity includes biological activity and the ability to bind to cancer cell antigens. In addition, a polypeptide having functional activity means that the polypeptide exhibits activity similar to, but not necessarily identical to, the activity of the antibody described herein, including its mature form, as measured in a particular assay, e.g., a biological assay, with or without dose-dependency. Where dose-dependency exists, it does not need to be identical to the dose-dependency of the antibody disclosed herein, but is substantially similar to the dose-dependency of a given activity compared to the antibody described herein (i.e., the candidate polypeptide exhibits greater activity, or about 1 / 25th or less, about 1 / 10th, or about 1 / 3th the activity compared to the antibody described herein).

[0211] Nucleic acid molecules that encode antibodies Using the information provided herein, for example, the nucleic acids and amino acid sequences of antibodies, nucleic acid molecules encoding antibodies or antigen-binding functional fragments thereof can be readily obtained by those skilled in the art. The nucleic acid molecules of this disclosure may exist in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, obtained by cloning, synthetically produced, or any combination thereof, or in the form of DNA, including cDNA and genomic DNA, in no particular limitation. The DNA may be triple-stranded, double-stranded, single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand, known as a sense strand, or it may be an antisense strand, known as an antisense strand.

[0212] Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acid molecules may be isolated or substantially purified by standard techniques, non-limitingly including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others, when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins. The nucleic acids in at least some embodiments of this disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In preferred embodiments, the nucleic acid is a cDNA molecule.

[0213] Another aspect of this disclosure relates to nucleic acid molecules comprising nucleic acid sequences encoding antibody polypeptides or functional fragments thereof or antigen-binding functional fragments 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.

[0214] Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can undergo further manipulation using standard recombinant DNA techniques to convert, for example, the variable region gene to a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operably ligated to another DNA fragment encoding another protein, such as the antibody constant region or a flexible linker. Isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably ligating the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). DNA fragments containing human heavy chain constant region genes can be obtained by standard PCR amplification. The heavy chain constant regions may be the IgA1 or IgA2 constant regions. For Fab fragment heavy chain genes, the DNA encoding VH can be operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0215] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (and Fab light chain gene) by operably ligating the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. DNA fragments containing the human light chain constant region can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is most preferably a kappa constant region.

[0216] To create the scFv gene, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, for example, the amino acid sequence (Gly-4-Ser)3 (SEQ ID NO: 220). Thus, the VH and VL sequences can be expressed as a continuous single-strand protein, with the VL and VH regions joined by the flexible linker.

[0217] Nucleic acid molecules isolated from this disclosure may include: nucleic acid molecules comprising an open reading frame (ORF), which may have one or more introns, for example, at least one identified portion of at least one CDR as CDR1, CDR2 and / or CDR3 of at least one light chain; nucleic acid molecules comprising a coding sequence of a cancer-related antibody or variable region disclosed herein, for example, a variable region of a light chain; and nucleic acid molecules comprising a nucleotide sequence substantially different from the nucleotide sequences described above, but which, due to the degeneracy of genetic coding, still encodes at least one antibody or antigen-binding functional fragment thereof as described herein.

[0218] Nucleic acid molecules comprising nucleic acid sequences encoding one or more chains of an antibody are shown herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding either the heavy chain or the light chain of the antibody. In some embodiments, the nucleic acid molecule comprises both a nucleic acid sequence encoding the heavy chain and a nucleic acid sequence encoding the light chain of the antibody. In some embodiments, the first nucleic acid molecule comprises a first nucleic acid sequence encoding the heavy chain, and the second nucleic acid molecule comprises a second nucleic acid sequence encoding the light chain.

[0219] In some embodiments, the heavy and light chains are expressed as two separate polypeptides from one nucleic acid molecule or from two separate nucleic acid molecules. In some embodiments, such as when the antibody is scFv, a single nucleic acid sequence encodes a single polypeptide containing both the heavy and light chains that are linked together.

[0220] In some embodiments, the nucleic acid sequence encoding the heavy or light chain of the antibody disclosed herein includes a nucleic acid sequence encoding at least one of the CDRs shown herein. In some embodiments, the nucleic acid sequence encoding the heavy or light chain of the antibody disclosed herein includes a sequence encoding at least three of the CDRs shown herein. In some embodiments, the nucleic acid sequence encoding the heavy or light chain of the antibody includes a sequence encoding at least six of the CDRs shown herein. In some embodiments, the nucleic acid sequence encoding the heavy or light chain of the antibody includes a nucleotide sequence encoding a leader sequence, which, when translated, is located at the N-terminus of the heavy or light chain. The leader sequence may be a native heavy or light chain leader sequence, or it may be a different heterogeneous leader sequence. The leader sequence may be cleaved during the export of polypeptides from mammalian cells to form a mature protein. The leader sequences may be native or synthetic, and they may be heterogeneous or homogeneous with respect to the protein to which they are bound.

[0221] In some embodiments, the nucleic acid molecule is a nucleic acid molecule encoding either the amino acid sequence for the variable light chain and variable heavy chain in Tables 5 and 7 of this specification. In some embodiments, the nucleic acid sequence is at least 80% identical, 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% identical to the nucleic acid encoding either the amino acid sequence for the variable light chain and variable heavy chain in Tables 5 and 7 of this specification. In some embodiments, the nucleic acid is a nucleic acid that hybridizes to any one or more of the nucleic acid sequences shown herein. In some embodiments, hybridization is carried out under moderate conditions. In some embodiments, hybridization is carried out under very stringent conditions, such as a first wash at about 42°C for 10 minutes with at least about 6X SSC and 1% SDS and about 20(v / v)% formamide in 0.1X SSC at 65°C, and a second wash at 65°C with 0.2X SSC and 0.1% SDS.

[0222] 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.

[0223] Vectors comprising nucleic acid molecules encoding antibody or antigen-binding functional fragments are shown herein. Vectors comprising nucleic acid molecules encoding heavy chains and / or light chains are also shown. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, and retroviral vectors. In one embodiment, the nucleic acid encoding the light chain and the nucleic acid encoding the heavy chain are isolated separately by the procedure outlined above. In one embodiment, the isolated nucleic acid encoding the light chain and the isolated nucleic acid encoding the heavy chain may be inserted into separate expression plasmids or together into the same plasmid, provided that each is under appropriate promoter and translational control. In some embodiments, the heavy chain and light chain are expressed as a portion of a single polypeptide, for example, when the antibody is scFv.

[0224] In some embodiments, the first vector comprises a nucleic acid molecule encoding a heavy chain, and the second vector comprises 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 masses). In some embodiments, the first and second vectors are transfected into host cells in a molar or mass ratio of 5:1 to 1:5. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the heavy chain-encoding vector and the light chain-encoding vector. In some embodiments, a mass ratio of 1:2 is used for the heavy chain-encoding vector and the light chain-encoding vector. In some embodiments, vectors optimized for polypeptide expression in CHO or CHO-derived cells, or in NSO cells, are selected.

[0225] In one embodiment, the present disclosure describes a method for treating or preventing cancer, comprising the step of administering a nucleic acid molecule wherein the nucleic acid molecule encodes a VH, VL, CDR3 region of VH, or CDR3 region of VL, or an antigen-binding functional fragment thereof. In some embodiments, the nucleic acid molecules disclosed herein are used for gene therapy. Gene therapy refers to treatment carried out by administering expressed or expressible nucleic acids to a subject. In this embodiment, the nucleic acids produce proteins they encode that mediate a preventive or therapeutic effect. Any of the methods for gene therapy available in the art may be used according to embodiments herein.

[0226] The delivery of therapeutic antibodies to suitable cells can be performed ex vivo, in situ, or in vivo by gene therapy using any suitable method, including the use of physical DNA transfer methods (e.g., liposomes or chemical processing) or viral vectors (e.g., adenoviruses, adeno-associated viruses, or retroviruses). For example, for in vivo therapy, nucleic acids encoding the desired antibody may be injected directly into the target, either alone or with a vector, liposome, or precipitate, or in some embodiments, at the site where antibody compound expression is desired. For ex vivo therapy, the target cells are removed, the nucleic acids are introduced into these cells, and the modified cells are returned to the target, either directly or encapsulated in a porous membrane, for example, transplanted into the patient (target). There are various techniques available for introducing nucleic acids into living cells. The technique varies depending on whether the nucleic acids are transferred in vitro to cultured cells or in vivo into the cells of the intended host. Appropriate techniques for the in vitro transfer of nucleic acids into mammalian cells include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and calcium phosphate precipitation. Retroviruses are commonly used vectors for ex vivo delivery of nucleic acids.

[0227] Other in vivo nucleic acid transfer techniques include transfection with viral vectors (e.g., adenovirus, herpes simplex virus type 1, or adeno-associated virus) and lipid-based systems. The nucleic acid and transfection agent may be associated with microparticles. Exemplary transfection agents include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, the quaternary ammonium amphiphilic substance DOTMA ((dioleoyloxypropyl)trimethylammonium bromide, commercially available as Lipofectin by GIBCO-BRL); lipophilic glutamate diesters with pendent-type trimethylammonium heads; metabolizable lipophilic lipids, such as the cationic lipid dioctadecylamide glycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES) Ethanolamylspermine; metabolizable quaternary ammonium salt (DOTB, N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP, N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate), polyethyleneimine (PEI), dioleoyl ester, ChoTB, ChoSC, DOSC);3-beta[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), 1:1 mixture of dioleoylphosphatidylethanolamine (DOPE) / 3-beta[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol-DC-Chol, spermine, spermidine, lipopolyamine, lipophilic polylysine (LPLL), excess phosphatidylcholine / cholesterol with [[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl]dimethylbenzylammonium hydroxide (DEBDA hydroxide, [[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl]dimethylbenzylammonium hydroxide), cetyltrimethylammonium bromide (CTAB, cetyltrimethylammonium Examples of transfection enhancers that increase transfection efficiency include, for example, DEAE-dextran, polybren, lysosomal disruption peptides, chondroitin-based proteoglycans, sulfated proteoglycans, polyethyleneimine, polylysine, integrin-binding peptide CYGGRGDTP (SEQ ID NO: 33), linear dextran xusaccharide, glycerol, cholesteryl groups linked in the 3'-terminal nucleoside linkage of oligonucleotides, lysophosphatide, lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyllysophosphatidylcholine.

[0228] In some situations, it may be desirable to deliver nucleic acids accompanied by agents that direct the vector-containing nucleic acids to target cells. Such agents include antibodies specific to cell surface membrane proteins on target cells, or ligands for receptors on target cells. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis may be used for targeting and / or to promote uptake. Examples of such proteins include capsid proteins and their fragments that are tropic to specific cell types, antibodies to proteins that undergo cyclical internal translocation, and proteins that target intracellular localization and enhance intracellular half-life. In other embodiments, receptor-mediated endocytosis may be used.

[0229] Immunoconjugate The antibodies or their antigen-binding functional fragments disclosed herein may be administered in their “naked” or unconjugated form, or conjugated with a therapeutic agent. In one embodiment, the antibodies or their antigen-binding functional fragments are used as radiosensitizers. In such embodiments, the antibodies or antigen-binding functional fragments are conjugated with a radiosensitizer. In some embodiments, the radiosensitizer is a molecule, preferably a low molecular weight molecule, administered to an animal in a therapeutically effective amount that increases the sensitivity of radiation-sensitized cells to electromagnetic radiation and / or promotes the treatment of diseases treatable by electromagnetic radiation. Diseases treatable by electromagnetic radiation include neoplasms, benign and malignant tumors, and cancerous cells. In some embodiments, electromagnetic radiation and radiation include, but are not limited to, radiation having wavelengths of 10-20 to 100 meters. Preferred embodiments of this disclosure may utilize electromagnetic radiation such as gamma radiation (c10⁻²⁰ to 10⁻¹³ m), X-ray radiation (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).

[0230] Radiosensitizers are known to increase the sensitivity of cancer cells to the toxic effects of electromagnetic radiation. Many cancer treatment protocols currently utilize radiosensitizers activated by X-ray electromagnetic radiation. Examples of X-ray activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU1069, SR4233, E09, RB6145, nicotinamide, 5-bromode-oxyuridine (BUdR, 5-bromode-oxyuridine), 5-iododeoxyuridine (IUdR, 5-iododeoxyuridine), bromode-oxycytidine, fluorodeoxyuridine (FUdR, fluorodeoxyuridine), hydroxyurea, cisplatin, and their therapeutically effective analogs and derivatives.

[0231] Photodynamic therapy (PDT) for cancer uses visible light as a radioactive activator for photodynamic radiosensitizers. Examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine, and their therapeutically effective analogs and derivatives.

[0232] In another embodiment, the antibody may be conjugated to a receptor (e.g., streptavidin) for use in tumor pre-targeting, the antibody-receptor conjugate being administered to the patient, followed by the removal of the unbound conjugate from circulation using a decontamination agent, and then the ligand (e.g., avidin) conjugated to a cytotoxic agent (e.g., radionuclide) being administered.

[0233] This disclosure further illustrates the antibodies described above or their antigen-bindings in detectably labeled forms. Antibodies may be detectably labeled through the use of radioisotopes, affinity labels (e.g., biotin, avidin, etc.), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, etc.), fluorescent, luminescent, or bioluminescent labels (e.g., FITC or rhodamine, etc.), paramagnetic atoms, etc.

[0234] Exemplary therapeutic immunoconjugates include antibodies described herein, conjugated to cytotoxic agents, such as chemotherapeutic agents, toxins (e.g., enzyme-active toxins or fragments thereof of bacterial, fungal, plant, or animal origin), or radioisotopes (i.e., radioconjugates). Fusion proteins are described in further detail below.

[0235] In some embodiments, the antibodies and their antigen-binding functional fragments disclosed herein are therapeutic agents, such as chemotherapeutic cytotoxic agents, such as cell proliferation inhibitors or cell-destroying agents (e.g., paclitaxol, cytochalasin B or diphtheria toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, proca (e.g., tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP, cis-dichlorodiamine Platinum (II) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC, anthramycin)), and antimitotic agents, thrombotic or antiangiogenic agents or radiolabels may be conjugated. In another embodiment, antibodies and their antigen-binding functional fragments disclosed herein are conjugated to detectable substrates, such as enzymes, fluorescent markers, chemiluminescent markers, bioluminescent materials or radioactive materials.In some embodiments, the antibodies and functional antibody fragments disclosed herein are conjugated to toxins (e.g., enzyme-active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), small molecules, siRNA, nanoparticles, targeting agents (e.g., microbubbles), or radioisotopes (i.e., radioconjugates). Such conjugates are referred to herein as “immunoconjugates.” Such immunoconjugates may be used, for example, in diagnostics, theranostics, or targeted methods.

[0236] The enzyme-active toxins and their fragments that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), bitter melon (momordica charantia) inhibitor, curcin, crocin, soapwort (sapaonaria officinalis) inhibitor, geronin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. Various radioisotopes can be used for the production of radioconjugate antibodies. Examples include, but are not limited to, 212Bi, 131I, 131In, 90Y, and 186Re.

[0237] The conjugates of antibodies or their antigen-binding functional fragments described herein with cytotoxic agents include various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP,N-succinimidyl-3-(2-pyridyldithiol) It can be prepared using any of the following: propionate, iminothiolane (IT), bifunctional derivatives of imide esters (e.g., adipimidate HCl), active esters (e.g., disuccinimidyl severate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., tolyene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins may be prepared. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA, 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid) is an exemplary chelating agent for the conjugation of radionuclides to antibodies.

[0238] In other embodiments, an antibody or a portion thereof may be conjugated to a receptor (e.g., streptavidin) for use in tumor pre-targeting, the antibody-receptor conjugate is administered to a subject, the unbound conjugate is removed from circulation using a deconjugate, and then a ligand (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide) is administered. In some embodiments, an antibody or a functional fragment thereof may be conjugated to biotin, and the biotin-conjugated antibody or functional antibody fragment thereof may be further conjugated or linked to a streptavidin-bound or coated agent, such as streptavidin-coated microbubbles, for use, for example, in molecular imaging of angiogenesis.

[0239] Immunoconjugates can be prepared by indirectly conjugating a therapeutic agent to an antibody component. A common method involves reacting an antibody component having an oxidized sugar chain portion with a carrier polymer loaded with multiple drugs, toxins, chelators, boron adducts, or other therapeutic agents having at least one free amine functional group. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0240] The carrier polymer is preferably aminodextran or a polypeptide of at least 50 amino acid residues, but other substantially equivalent polymer carriers can also be used. Preferably, the final immunoconjugate is soluble in aqueous solutions such as mammalian serum for ease of administration and effective targeting for therapeutic use. Therefore, solubilizing functional groups on the carrier polymer improve the serum solubility of the final immunoconjugate. Aminodextran is particularly preferred.

[0241] The process for preparing an immunoconjugate with an aminodextran support typically begins with a dextran polymer, preferably dextran with an average molecular weight of about 10,000 to 100,000. The dextran is reacted with an oxidizing agent to undergo controlled oxidation of a portion of its sugar ring, generating an aldehyde group. This oxidation is conveniently carried out using a glycolytic chemical reagent, such as Na₂O₄, according to conventional procedures.

[0242] Subsequently, the oxidized dextran is reacted with a polyamine, preferably a diamine, more preferably a mono- or polyhydroxydiamine. Suitable amines include ethylenediamine, propylenediamine, or others, such as polymethylenediamine, diethylenetriamine, or similar polyamines, 1,3-diamino-2-hydroxypropane, or others, such as hydroxylated diamines or polyamines. The excess amine relative to the aldehyde group of dextran is used to ensure substantially complete conversion of the aldehyde functional group to the Schiff base group.

[0243] Reducing agents, such as NaBH4, NaBH3CN, and others, are used to reductively stabilize the resulting Schiff base intermediate. The resulting adduct can be purified by passing it through a conventional sizing column to remove the crosslinked dextran. Alternatively, other conventional methods for derivatizing the dextran to introduce amine functional groups, such as reaction with cyanogen bromide and subsequent reaction with a diamine, may be used. Subsequently, the anminodextran is reacted by conventional means with a specific drug, toxin, chelator, immunomodulator, boron adduct, or derivative of another therapeutic agent to be loaded, preferably in an activated form prepared using, for example, dicyclohexylcarbodiimide (DCC) or a water-soluble variant thereof, to form an intermediate adduct.

[0244] Alternatively, polypeptide toxins, such as the burdock antiviral protein or lysine A chain, can be coupled to aminodextran by glutaraldehyde condensation or by the reaction of an activated carboxyl group on the protein with an amine on the aminodextran. Cherators or magnetic resonance enhancers for radioactive metals, such as derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), typically have groups on side chains that can be bonded to a support. Such groups include, for example, benzyl isothiocyanates, which can couple DTPA or EDTA to an amine group on the support. Alternatively, the carboxyl group or amine group on the chelator can be coupled to the support by activation or pre-derivativeization and subsequent coupling.

[0245] Boron adducts, such as carboranes, can be conjugated to antibody components by conventional methods. For example, carboranes can be prepared by carboxyl functional groups on the pendant side chains. The conjugation of such carboranes to a carrier, such as aminodextran, can be achieved by activation of the carboxyl group of the carborane and condensation with an amine on the carrier to produce an intermediate conjugate. Such intermediate conjugates then conjugate to antibody components to produce therapeutically useful immunoconjugates as described below.

[0246] Polypeptide carriers can be used instead of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain, preferably 100 to 5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamic acid or aspartic acid residues. Pendamines of lysine residues, and pendante carboxylates of glutamine and aspartic acid are convenient for conjugating drugs, toxins, immunomodulators, chelators, boron adducts, or other therapeutic agents. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and others, such as serine, which impart desirable solubility properties to the resulting loaded carrier and immunoconjugate.

[0247] Conjugation between the intermediate conjugate and the antibody component is carried out by oxidizing the sugar chain portion of the antibody component and reacting the resulting aldehyde (and ketone) carbonyl with the amine group remaining on the support after loading the drug, toxin, chelator, immunomodulator, boron adduct, or other therapeutic agent. Alternatively, the intermediate conjugate may conjugate to the oxidized antibody component via the amine group introduced into the intermediate conjugate after loading the therapeutic agent. Oxidation is conveniently carried out chemically, for example, with NaI04 or other glycolytic reagents, or enzymatically, for example, with neuraminidase and galactose oxidase. In the case of aminodextran supports, not all of the amines of aminodextran are typically used to load the therapeutic agent. The remaining amines of aminodextran condense with the oxidized antibody component to form a Schiff base adduct, which is then reductively stabilized, usually with a borohydride reducing agent.

[0248] Other immunoconjugates according to the present invention are produced using similar procedures. The polypeptide carrier to be loaded preferably has free lysine residues remaining for condensation with the oxidized sugar chain portion of the antibody component. Carboxyls on the polypeptide carrier can be converted to amines as needed, for example by activation with DCC and reaction with excess diamines.

[0249] The final immunoconjugate is purified using conventional techniques, such as sizing chromatography on Sephacryl S-300, or affinity chromatography using one or more CD84Hy epitopes. Alternatively, the immunoconjugate may be prepared by directly conjugating the antibody component with the therapeutic agent. The general procedure is similar to indirect conjugation, except that the therapeutic agent is directly bound to the oxidized antibody component. It is understood that other therapeutic agents may be substituted for the chelators described herein. Those skilled in the art can devise conjugation schemes without excessive experimental work.

[0250] As a further example, therapeutic agents may be bound to the hinge region of a reduced antibody component via disulfide bond formation. For instance, a tetanus toxoid peptide may be constructed from a single cysteine ​​residue used to bind the peptide to the antibody component. Alternatively, such peptides may be bound to the antibody component using a heterobifunctional crosslinking agent, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP).

[0251] Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolylene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin may be prepared. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclides to antibodies.

[0252] As described above, the sugar chain portion within the Fc region of an antibody can be used to conjugate a therapeutic agent. However, when a functional antibody fragment is used as the antibody component of an immunoconjugate, the Fc region may be absent. Nevertheless, it is possible to introduce the sugar chain portion into the light chain variable region of an antibody or a functional antibody fragment. The modified sugar chain portion is then used to bind a therapeutic agent. In addition, those skilled in the art recognize many possible variations of conjugation methods. For example, the sugar chain portion may be used to bind to polyethylene glycol in order to extend the half-life of an intact antibody or its antigen-binding functional fragment in blood, lymph or other extracellular fluids. Furthermore, it is possible to construct a bivalent immunoconjugate by binding a therapeutic agent to the sugar chain portion and to free sulfhydryl groups. Such free sulfhydryl groups may be located in the hinge region of the antibody component.

[0253] Treatment methods In some embodiments, a method of treating a subject that requires it, the method comprising administering to the subject a therapeutic dose of a therapeutic anti-EGFR IgA antibody described herein, or a pharmaceutical composition comprising the therapeutic anti-EGFR IgA antibody described herein, is disclosed herein. In some embodiments, the subject has cancer, an infectious disease or an autoimmune disease.

[0254] Most studies investigating the potential of IgA antibodies for immunotherapy approaches are based on in vitro experiments. In vivo preclinical studies investigating the therapeutic potential of FcαRI-IgA binding have been conducted using transgenic (Tg) mouse models. This is due to the lack of expression of FcαRI in rodents.

[0255] In some embodiments, IgA exhibits strong pro-inflammatory effector functions, such as induction of oxidative burst, phagocytosis, and ADCC, after binding to FcαRI. In some embodiments, IgA pairs more efficiently with the FcRg chain in the transmembrane domain of FcαRI compared to FcγRI. In some embodiments, the effector functions induced by IgA are stronger when activating FcαRI on polymorphonuclear leukocytes (PMNs) compared to the activation of FcγRI. Thus, in some embodiments, tumor cell killing by a bispecific antibody (bsAb) that binds to both a tumor antigen (e.g., EGFR) and an FcR is more efficient when FcαRI is targeted rather than FcγRI.

[0256] EGFR is expressed in many types of cancer cells, such as HNSCC, CRC, NSCLC, and kidney and other cancers. Therefore, in some embodiments, modified anti-EGFR IgA antibodies that target EGFR are useful in the treatment of any cancer that expresses EGFR (e.g., cancers associated with EGFR expression and / or the expression of EGFR variants having one or more mutations). In some embodiments, EGFR variants include EGFRvIII, exon 19 deletion, L858R substitution, C797S substitution, or T790M substitution in exon 21. In some embodiments, anti-EGFR therapy includes an antibody or small molecule inhibitor that binds to EGFR, prevents its signaling, and results in inhibition of cancer cell proliferation and survival. In some embodiments, anti-EGFR therapy is used to treat several types of cancer, including metastatic CRC, NSCLC, and HNSCC. In some embodiments, anti-EGFR therapy includes the use of a combination therapy comprising at least one anti-EGFR antibody and at least one small molecule inhibitor. In some embodiments, at least one small molecule inhibitor is erlotinib and gefitinib, which reversibly inhibit the EGFR tyrosine kinase domain by competitively binding to ATP. In some embodiments, at least one antibody comprises a modified antibody as described herein. In some embodiments, at least one antibody further comprises cetuximab (chimeric mouse-human IgG1 antibody), nesitumumab (fully humanized IgG1 antibody), matsuzumab (fully humanized IgG1 antibody), and panitumumab (fully humanized IgG2 antibody). All antibodies block ligand binding to the extracellular domain of EGFR, promote receptor translocation, and mediate antibody-mediated and complement-mediated cytotoxicity.

[0257] In cancer treatment, drug resistance is frequently encountered, leading to disease progression and inadequate outcomes. Numerous primary and secondary resistance mechanisms exist for EGFR-TKIs. Primary resistance mechanisms include point mutations in exon 18 of the EGFR gene, deletions or insertions in exon 19, insertions, duplications, and point mutations in exon 20, and point mutations in exon 21 (e.g., L858R substitution). T790M gene mutations are frequently observed in 50%–60% of NSCLC patients with EGFR mutations and are associated with acquired resistance. Furthermore, disease resistance driven by EGFR amplification and KRAS mutations limits the effectiveness of current treatment modes. This drug resistance, along with the low response rates to panitumumab and cetuximab, highlights a significant unmet clinical need for new therapies. Additionally, C797S substitutions at the ATP binding site can lead to drug resistance (e.g., resistance to osimertinib treatment). Therefore, modified antibodies described herein can be used to treat cancers associated with EGFR expression that have any of these mutations.

[0258] In some embodiments, the subject has cancer. In some embodiments, the subject has an inflammatory disorder. In some embodiments, the cancer is associated with the expression of tumor-associated antigens as described herein. In some embodiments, 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-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123, VEGF-R2, or a combination 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 a combination thereof. In some embodiments, an antibody or a functional fragment thereof binds to EGFR, as well as one or more of MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, and 4-1BB.

[0259] 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 hematological malignancy. In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent or refractory cancer.

[0260] In some embodiments, cancer is a solid tumor that expresses EGFR. Exemplary solid tumors include, but are not limited to, anal cancer, appendiceal 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 tumor, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, vulvar cancer, or glioblastoma.

[0261] 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, 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.

[0262] In some embodiments, EGFR-expressing cancers are hematological malignancies. In some embodiments, hematological malignancies include lymphoma, leukemia, myeloma, or B-cell malignancies. In some embodiments, hematological malignancies include lymphoma, leukemia, or myeloma. In some embodiments, exemplary hematological 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, marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, and primary mediastinal large B-cell lymphoma (PMBL). This includes lymphoma, immunoblastic large cell lymphoma, progenitor B lymphoblastic lymphoma, B-cell prelymphoblastic leukemia, lymphoplasmacytic lymphoma, perisplenic zone lymphoma, plasmacytoma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary humoral lymphoma, or lymphomatoid granulomatosis. In some embodiments, hematological malignancies include myeloid leukemia. In some embodiments, hematological malignancies include acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0263] In some embodiments, the anti-EGFR IgA antibody is administered together with one or more additional therapeutic agents. In some embodiments, the anti-EGFR IgA antibody and one or more additional therapeutic agents are administered simultaneously. In some embodiments, the anti-EGFR IgA antibody and one or more additional therapeutic agents are administered sequentially. In some embodiments, the additional therapeutic agents include anticancer agents, chemotherapeutic agents, radiotherapy, cytotoxic agents, corticosteroids, immunotherapy agents, nutritional supplements, antioxidants, or combinations thereof.

[0264] In some embodiments, combination therapy may include one or more antibodies of the present disclosure that are co-formulated and / or co-administered with one or more additional therapeutic agents, such as chemotherapeutic agents or antineoplastic agents, such as cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic agents or cell proliferation inhibitors. Exemplary chemotherapeutic agents include aldesleukin, altretamine, amiphostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC, Examples include, but are not limited to, dacarbazine, dactinomycin, docetaxel, doxorubicin, dronabinol, duocalmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, rebamisol, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine, saproin, tamoxifen, taxol, 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, pacritinib, fedratinib, tofacitinib, oclacitinib, peficitinib, upadacitinib, deucravacitinib, delgocitinib, or combinations thereof. In some embodiments, additional therapeutic agents include KRAS inhibitors. In some embodiments, KRAS inhibitors include sotrasib, adaglasib, or combinations thereof.In some embodiments, additional therapeutic agents are bound to MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, 4-1BB, or a combination thereof.

[0265] In some embodiments, the IgA antibodies described herein are, without limitation, FDA-approved monoclonal antibodies including: rituximab (CD20: chimeric IgG1), trastuzumab (HER2: chimeric IgG1), alemtuzumab (CD52: humanized IgG1), ibritumomab tiuxetan (CD20: mouse IgG1 radiolabeled), tositumomab-I-131 (CD20, mouse, IgG2a, radiolabeled (iodine-131)), cetuximab (EGFR: chimeric (cjimeric), IgG1), bevacizumab (VEGF: humanized, IgG4), panitumumab (EGFR: human IgG2), ofatumumab (CD20: human IgG1), ipilimumab (CTLA-4: human IgG1), and brentiuximab vedotin. It can be combined with other antibodies used in cancer treatment at effective doses, including vedotin (CD30: chimeric, IgG1, drug conjugate), pertuzumab (HER2: humanized IgG1, drug conjugate), adotrastuzumab ematansine (HER2: humanized, IgG1, drug conjugate), obinutuzumab (CD20: humanized and glycol-modified), nivolumab, and pembrolizumab (anti-PD-1).

[0266] In some embodiments, the anti-EGFR IgA antibodies described herein are effective in treating cancer in subjects having EGFR-positive cancer. In some embodiments, subjects include those having either intrinsic or acquired resistance to EGFR-targeted antibodies and tyrosine kinase inhibitors (TKIs). In some embodiments, subjects have cancer that is EGFR-expressing, previously treated, advanced, or metastatic solid tumor cancer. Thus, in some embodiments, subjects are a population of subjects with high medical demands that are not met due to associated inadequate prognosis and a limited number of available effective treatment options. In some embodiments, treatment with the anti-EGFR IgA antibodies described herein overcomes the limitations of EGFR-targeted standard of care (SOC) therapy in current therapeutic medical facilities. In some embodiments, the anti-EGFR IgA antibodies described herein are designed to unleash the full cytotoxic potential of neutrophils, thereby providing a novel treatment option for subjects.

[0267] Dosage Compositions comprising IgA antibodies or their antigen-binding functional fragments for the treatment (including prevention) of diseases (e.g., cancer) are shown herein. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition is administered in an effective amount for the treatment (including prevention) of cancer. In some embodiments, the composition (e.g., antibodies or their antigen-binding functional fragments, or nucleic acid molecules encoding said antibodies or their antigen-binding functional fragments) is administered in an effective amount to enhance the immune response and / or increase T cell activation in a subject. The composition is used for in vivo administration to a subject by any available means, such as parenteral administration. For administration to a subject, the compositions or pharmaceuticals comprising antibodies or their antigen-binding functional fragments described herein may be sterile, which can be readily achieved by filtration through a sterile filtration membrane or other filtration methods. In one embodiment, the composition or pharmaceutical is treated to be free from pyrogens or endotoxins. Testing pharmaceutical compositions or pharmaceuticals for pyrogens or endotoxins, and preparing pharmaceutical compositions or pharmaceuticals that do not contain pyrogens or endotoxins, or preparing pharmaceutical compositions or pharmaceuticals that contain clinically acceptable levels of endotoxins, are well understood by those skilled in the art. Commercially available kits are available for testing pharmaceutical compositions or pharmaceuticals for pyrogens or endotoxins.

[0268] In the methods described herein, the compositions used for in vivo administration, such as parenteral administration, may be sterile, which can be easily achieved by filtration through a sterile filtration membrane or by other filtration methods.

[0269] The IgA antibodies or their antigen-binding functional fragments described herein are formulated, administered, and given in a manner consistent with the principles of good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific subject being treated, the clinical state of the individual subject, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors. The IgA antibodies or their antigen-binding functional fragments may be provided in the therapeutically effective dose disclosed herein. The therapeutically effective dose of a substance / molecule, agonist, or antagonist may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to induce the desired response in the individual. Furthermore, the therapeutically effective dose is the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the substance / molecule, agonist, or antagonist. The therapeutically effective dose may be delivered in one or more doses. The therapeutically effective dose administered is controlled by such considerations and refers to the minimum amount necessary to improve, treat or stabilize cancer, increase the time to progression (duration of progression-free survival), or treat or prevent the development or recurrence of tumors, quiescent tumors, or micrometastases. The antibodies or their antigen-binding functional fragments disclosed herein may be formulated together with one or more additional therapeutic agents currently used to prevent or treat cancer or the risk of developing cancer. The effective dose of such other agents depends on the amount of antibody or its antigen-binding functional fragment present in the formulation, the type of impairment or treatment, and other factors described above. These are generally used by the same route of administration in the same doses used herein, or at about 1-99% of conventionally used doses.

[0270] The antibody dose may vary depending on the age and size of the recipient, the target disease, condition, and route of administration. Preferred doses are typically calculated according to body weight or body surface area. When the antibodies or their antigen-binding functional fragments disclosed herein are used to treat a condition or disease in adult patients, intravenous administration of the antibodies of the present invention in single doses of approximately 0.01 to 20 mg / kg body weight, more preferably 0.02 to 7, 0.03 to 5, or 0.05 to 3 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg body weight may be beneficial. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective doses and schedules for administration may be determined empirically, for example, by monitoring the patient's progression through periodic assessments and adjusting the dose accordingly. Furthermore, interspecies scaling of doses may be performed.

[0271] In some embodiments, the compositions herein may contain a prophylactic effective dose, for example, when administered to subjects at risk of cancer or in the early stages of a disease. Typically, since prophylactic doses are used in subjects before or in the early stages of a disease, prophylactic doses are lower than therapeutic doses.

[0272] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms of unit doses suitable for adapting the dose of the active ingredient. Such dosage forms of unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like.

[0273] Administration may be carried out, for example, by one or more separate doses or by continuous intravenous infusion. Depending on the condition, treatment may be continued for several days or longer, for example, until the cancer is treated. However, other dosage regimens may be useful. In a non-limiting example, the antibodies or their antigen-binding functional fragments disclosed herein may be administered weekly, every other week, or every three weeks in a dose range of approximately 5 mg / kg to approximately 15 mg / kg, including non-limiting doses of 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg. Progress using the methods described herein can be readily monitored by conventional techniques and assays. The duration of treatment using the methods described herein may continue as medically directed or until the desired therapeutic effect (e.g., the effect described herein) is achieved. In some embodiments, administration of one or more antibodies or their antigen-binding functional fragments or compositions described herein is continued for one month, two months, four months, six months, eight months, ten months, one year, two years, three years, four years, five years, ten years, twenty years, or up to several years during the subject's lifetime.

[0274] In some embodiments, the anti-EGFR IgA antibody described herein is administered in doses ranging from 1 mg / kg to 25 mg / kg. In some embodiments, the anti-EGFR IgA antibody described herein is administered in 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.

[0275] Treatment effectiveness The efficacy of a treatment method, for example for cancer, comprising the step of administering the IgA antibodies or their antigen-binding functional fragments or pharmaceutical compositions disclosed herein, can be measured by a variety of endpoints commonly used in evaluating cancer treatment, including, but not limited to, tumor regression, reduction in tumor weight or size, time to progression, survival, progression-free survival, overall response rate, duration of response, and quality of life. The antibodies or their antigen-binding functional fragments disclosed herein may require unique measurements and definitions of the clinical response to the drug. In the case of cancer, a therapeutically effective dose of the antibodies, their antigen-binding functional fragments, or compositions containing them disclosed herein may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that the antibodies or their antigen-binding functional fragments disclosed herein act to prevent proliferation and / or kill existing cancer cells, they may be cell proliferation inhibitory and / or cytotoxic. For cancer treatment, in vivo efficacy can be measured, for example, by assessing survival time, progression-free survival (PFS), response rates (RR), duration of response, and / or quality of life. In some embodiments, the IgA antibodies or their functional fragments disclosed herein inhibit 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 compared to an untreated subject. In some embodiments, the IgA antibodies or functional fragments thereof disclosed herein inhibit tumor engraftment by at least about 2%, 3%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more compared to untreated subjects.In some embodiments, the IgA antibodies or functional fragments thereof disclosed herein induce lysis of tumor cells. In some embodiments, the IgA antibodies or functional fragments thereof disclosed herein induce an increase in lysis of tumor cells of at least about 2%, 3%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more compared to the corresponding WT IgA.

[0276] In other embodiments, methods are provided herein for increasing progression-free survival of a human subject susceptible to or diagnosed with cancer, such as skin cancer, such as melanoma. Time to disease progression is defined as the time from administration of the drug to disease progression or death. In preferred embodiments, the combination therapy of the invention using an antibody or antigen-binding functional fragment thereof disclosed herein 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, 3.5 months, such as 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 various therapeutic agents. The response rate is defined as the percentage of treated subjects that respond to treatment. In one embodiment, the combination therapy described herein using an antibody or antigen-binding functional fragment thereof disclosed herein, such as a recombinant antibody or antigen-binding functional fragment thereof, and one or more chemotherapeutic agents significantly increases the response rate in the group of treated subjects compared to the group treated with chemotherapy alone.

[0277] In some embodiments, the methods described herein include administering to a subject an effective amount of an antibody or antigen-binding functional fragments thereof described herein to alleviate the symptoms of a disease, such as cancer.

[0278] The effective dose, toxicity, and therapeutic efficacy can be determined by standard medical procedures in cell cultures or experimental animals, for example, to determine the LD50 (lethal dose in 50% of the population) and ED50 (therapeutably effective dose in 50% of the population). The dosage may vary depending on the dosage form used and the route of administration utilized. The dose ratio between the toxic effect and the therapeutic effect 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 first be estimated from a cell culture assay. Furthermore, doses that achieve a circulating plasma concentration range (i.e., the concentration of the antibody or its antigen-binding functional fragment) containing an IC50 that achieves the maximum half of the symptom inhibition, as determined in cell cultures or in a suitable animal model, can be formulated in 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 biological assays. The dosage is determined by a physician and, if necessary, can be adjusted to match the observed therapeutic effect.

[0279] Treatment and / or prevention of cancer may include, but is not limited to, alleviating cancer-associated symptoms, inhibiting cancer progression, promoting cancer regression, promoting immune responses, inhibiting tumor growth, inhibiting tumor size, inhibiting metastasis, inhibiting cancer cell growth, inhibiting cancer cell proliferation, or inducing cancer cell death.

[0280] Mode of administration The IgA antibodies or their antigen-binding functional fragments described herein may be administered to a subject in need by any suitable route that provides effective treatment to the subject. In some embodiments, the antibodies or their antigen-binding functional fragments or compositions comprising them described herein are administered to a subject with cancer to be inhibited by any mode of administration, which may include, but not limited to, injection, infusion, drip, and inhalation, delivering the drug systemically or to a desired surface or target. Oral administration forms are also contemplated herein. The antibodies or their antigen-binding functional fragments or compositions comprising them may be administered by injection, which may include, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, cerebral / ventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intracranial, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

[0281] In some embodiments, the antibodies or antigen-binding functional fragments thereof described herein, or compositions comprising them, may be administered intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intraarticularly, synovially, intrathecally, orally, topically, or by inhalation, either by intravenous administration as a bolus or by continuous infusion over a period of time. If significant adverse events or toxicity are associated with the use of the antibodies or antigen-binding functional fragments thereof described herein, or compositions comprising them, topical administration, such as topical administration to a tumor or cancer site where angiogenesis is occurring, is particularly desirable. Furthermore, ex vivo strategies may be used for therapeutic applications in some embodiments. An ex vivo strategy involves transfecting or introducing nucleic acid sequences disclosed herein into cells obtained from a subject. The transfected or introduced cells are then returned to the subject. The cells may be any of a broad 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.

[0282] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein, or compositions comprising them, are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for topical immunosuppressive therapy, intralesional administration.

[0283] Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies or their antigen-binding functional fragments or compositions of the Disclosure are appropriately administered by pulse infusion, particularly in decreasing antibody doses. Preferably, the medication is given by injection, most preferably intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic. In some embodiments, the antibodies or their antigen-binding functional fragments or compositions of the Disclosure are administered locally, for example by direct injection, where permissible from the location of the lesion or tumor, and the injections may be repeated periodically. In some embodiments, the antibodies or their antigen-binding functional fragments or compositions of the Disclosure may also be delivered systemically to a subject or directly to tumor cells, for example, to the tumor or tumor bed after surgical resection of the tumor, for example, to prevent or reduce local recurrence or metastasis of a quiescent tumor or micrometastasis.

[0284] Antibody-targeted sonoporation methods are intended for use in some embodiments of the methods for inhibiting tumors described herein to enhance the efficacy and potency of therapeutic compositions comprising antibodies and their antigen-binding functional fragments as described herein. As used herein, “sonoporation” refers to the use of sound, preferably ultrasonic frequencies, or the interaction of ultrasound with a contrast agent (e.g., stabilized microbubbles) to temporarily alter the permeability of the cell plasma membrane, thereby enabling the uptake of large molecules, such as therapeutic agents. The membrane permeability induced by sonoporation is transient, and after ultrasound exposure, the drug remains trapped within the cell. Sonoporation utilizes the ultrasonic cavitation of microbubbles to enhance the delivery of large molecules.

[0285] Accordingly, in some embodiments of the method, an antibody or its antigen-binding functional fragment, as described herein, mixed with an ultrasound contrast agent, such as microbubbles, may be injected locally or systemically into a subject requiring treatment for cancer, and the ultrasound may be linked to and even focused on a specified area, such as a tumor site, to achieve 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 treatment method that uses high-intensity ultrasound to heat and destroy malignant or pathogenic tissue without damaging healthy tissue on or around it. HIFU may also be used as a means of delivering therapeutic agents, such as antibodies or their functional antibody fragments.

[0286] Furthermore, methods using contrast-enhanced ultrasound (CEUS) are intended for use with antibodies or their antigen-binding functional fragments as described herein. Contrast-enhanced ultrasound (CEUS) refers to the application of ultrasound contrast media and ultrasound contrast agents to traditional medical ultrasound examinations. An ultrasound contrast agent refers to a drug that relies on sound waves being reflected differently from the interface between substances. Various microbubble contrast agents are available for use in the compositions and methods described herein. Microbubbles may differ in their shell structure, gas core structure, and whether they are targeted. Targeted ligands that bind to receptors characteristic of angiogenic disorders may be conjugated to the microbubbles, allowing the microbubble complex to selectively accumulate in the region of interest, e.g., pathological or abnormal tissue. This form of molecular imaging, known as targeted contrast-enhanced ultrasound, generates a strong ultrasound signal only when the targeted microbubbles bind to the region of interest. Targeted contrast-enhanced ultrasound has many applications in both diagnostic medicine and medical treatment. In some embodiments, the antibodies or antigen-binding functional fragments described herein are administered to subjects requiring treatment for cancer or tumors using targeted ultrasound delivery.

[0287] Pharmaceutical compositions and dosage forms In some embodiments, a pharmaceutical composition comprising an anti-EGFR IgA antibody or a functional fragment thereof, as disclosed herein, for administration to a subject, is disclosed herein.

[0288] In some embodiments, the pharmaceutical compositions comprising the anti-EGFR IgA antibody described herein are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the active compound into a preparation that can be used as a pharmaceutical. Appropriate formulation depends on the selected route of administration.

[0289] Pharmaceutical compositions may be manufactured in conventional ways, such as by conventional mixing, dissolution, granulation, sugar coating, wet grinding, emulsification, encapsulation, or compression processes, as merely an example.

[0290] In some embodiments, the composition may also contain one or more pH adjusters or buffers, which include 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-hydroxymethylaminomethane; 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.

[0291] In other embodiments, the composition may also contain one or more salts in amounts required to bring the weight osmolality of the composition into an acceptable range. Such salts include salts having sodium, potassium, or ammonium cations, and chlorides, citrates, ascorbicates, borates, phosphates, bicarbonates, sulfates, thiosulfates, and bisulfite anions. Suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0292] The pharmaceutical compositions described herein are administered by 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, oral, sublingual, or rectal routes. In some embodiments, the pharmaceutical compositions are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intra-articular, intraperitoneal, or intracranial) administration.

[0293] The pharmaceutical compositions described herein may be formulated into any suitable dosage form for oral administration by the individual being treated, including but not limited to aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, etc.; solid oral dosage forms, aerosols, controlled-release formulations, rapid-dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated tablets, capsules, slow-release formulations, sustained-release formulations, pulsatile-release formulations, multi-particle formulations, and immediate-release and controlled-release mixed formulations.

[0294] In some embodiments, the pharmaceutical composition is formulated into capsules. In some embodiments, the pharmaceutical composition is formulated into a solution (e.g., for intravenous administration). In some embodiments, the pharmaceutical composition is formulated as an intravenous infusion. In some embodiments, the pharmaceutical composition is formulated as an injection.

[0295] The pharmaceutical solid dosage forms described herein may contain 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, humectants, plasticizers, stabilizers, penetration enhancers, wetting agents, defoaming agents, antioxidants, preservatives, or one or more combinations thereof.

[0296] In other embodiments, a film coating is provided around the composition using a standard coating technique, for example, the technique described in Remington's Pharmaceutical Sciences, 20th Edition (2000). In some embodiments, the composition is formulated into particles (e.g., for administration by capsule), and some or all of the particles are coated. In some embodiments, the composition is formulated into particles (e.g., for administration by capsule), and some or all of the particles are microencapsulated. In some embodiments, the composition is formulated into particles (e.g., for administration by capsule), and some or all of the particles are not microencapsulated and are not coated.

[0297] In some embodiments, and in the compositions shown herein, one or more preservatives may be included to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0298] In some embodiments, antibodies, functional fragments thereof, or compositions containing them may be administered to subjects in need (e.g., subjects with cancer). In some embodiments, the cancer is a solid tumor or a hematological malignancy. In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is a hematological 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. Similar solid tumors include, but are not limited to, anal cancer, appendiceal 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 tumor, prostate cancer, rectal cancer, skin cancer, stomach 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).

[0299] Antibodies, functional fragments thereof, or compositions thereof may be administered by injection, for example, intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im). One or more such routes may be used. Parenteral administration may be performed, for example, by bolus injection or by slow perfusion over time. Alternatively, or simultaneously, administration may be performed by an oral route. In addition, administration may also be performed by surgical deposition of a bolus or cell pellet, or by placement of a medical device. In one embodiment, a composition of the Disclosure may contain modified cells or host cells expressing the nucleic acid sequences described herein, or a vector containing at least one nucleic acid sequence described herein, in an amount effective to treat or prevent a proliferative disorder. A pharmaceutical composition may contain a target cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline or phosphate-buffered saline; sugars, such as glucose, mannose, sucrose, or dextran or mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; auxiliary agents (such as aluminum hydroxide); and preservatives.

[0300] In some embodiments, defoaming agents may be added during processing to reduce foaming, which may result in aggregation of the aqueous dispersion, bubbles in the finished film, or overall processing defects. Exemplary defoaming agents include silicone emulsion or sorbitan sesquioleate.

[0301] In some embodiments, antioxidants, such as butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium disulfite, and tocopherol, may be added. In some embodiments, antioxidants are added to improve chemical stability if necessary.

[0302] Antioxidants, metal chelating agents, thiol-containing compounds, and other common stabilizers may be beneficial for the formulations described herein. Examples of such stabilizers include, but are not limited to, (a) about 0.5 w / v% to about 2 w / v% glycerol, (b) about 0.1 w / v% to about 1 w / v% methionine, (c) about 0.1 w / v% to about 2 w / v% monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01 w / v% to about 2 w / v% ascorbic acid, (f) 0.003 w / v% to about 0.02 w / v% polysorbate 80, (g) 0.001 w / v% to about 0.05 w / v% polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) polysulfate pentoate and other heparin analogs, (m) divalent cations, such as magnesium and zinc, or (n) combinations thereof.

[0303] In some embodiments, a binder may be added to provide tackiness. Exemplary binders include, for example, alginic acid and its salts; cellulose derivatives, such as carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, ethylcellulose, and microcrystalline cellulose; microcrystalline dextrose; amylose; aluminum magnesium silicate; acidic polysaccharides; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, sugars, such as sucrose, glucose, dextrose, molasses, mannitol, sorbitol, xylitol, and lactose; natural or synthetic rubbers, such as acacia, tragacanth, ghati gum, isapol husk mucus, polyvinylpyrrolidone, larch arabinogalactan, polyethylene glycol, wax, and sodium alginate.

[0304] In some embodiments, a carrier or carrier material may be added. The carrier or carrier material includes any excipient commonly used in compounding and should be selected based on its compatibility with ibrutinib compounds and other compounds disclosed herein, such as anticancer agents, and the release profile characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. Examples of pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, and pregelatinized starch.

[0305] In some embodiments, dispersants may be added to control the diffusion and homogeneity of the drug through a liquid culture medium or granulation or compounding method. These agents may also enhance the effectiveness of the coating or corrosive matrix. Exemplary dispersants include, for example, hydrophilic polymers, electrolytes, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, PEG, polyvinylpyrrolidone (PVP), and sugar-based dispersants such as hydroxypropylcellulose (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), ethylene oxide, and formaldehyde.3-Tetramethylbutyl)-phenol polymer (also known as tyroxapol), poloxamer (e.g., block copolymer of ethylene oxide and propylene oxide), and poloxamine (e.g., tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, polyvinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol (e.g., polyethylene glycol is about 300 to about 6000, or about 3350 to about 4000, or about 7000) This includes sodium carboxymethylcellulose (which may have a molecular weight of approximately 5400), methylcellulose, polysorbate-80, sodium alginate, gums such as tragacanth gum and acacia gum, guar gum, xanthan gum, sugars, cellulosic substances such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol (PVA), alginates, chitosan, and combinations thereof. Plasticizers, such as cellulose or triethylcellulose, may also be used as dispersants. Particularly useful dispersants in liposomal dispersions and self-emulsifying dispersions are dimyristoyl phosphatidylcholine, natural phosphatidylcholine from eggs, natural phosphatidylglycerol from eggs, cholesterol, and isopropyl myristate.

[0306] Furthermore, a combination of one or more corrosion accelerators and one or more diffusion accelerators may be used in this composition.

[0307] In some cases, diluents may be added to dilute the compound of interest before delivery. Diluents may also be used to stabilize compounds, as they can provide a more stable environment. Salts dissolved in buffer solutions (which may also provide pH control or maintenance), including, but not limited to, phosphate-buffered saline solutions, are used as diluents in the art. In some embodiments, diluents increase the bulk of the composition to facilitate compression or create sufficient bulk for homogeneous formulation for capsule filling. Examples of such compounds include lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, calcium hydrogen phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugars, mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrose, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, and bentonite.

[0308] In some cases, fillers containing compounds such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc. may be added.

[0309] In some cases, lubricants or lubricants may be added to prevent, reduce, or inhibit adhesion or friction between materials. Examples of lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl 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 salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, wax, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica, starch, such as corn starch, silicone oil, and surfactants.

[0310] In some cases, plasticizers may be added to soften the microencapsulated material or film coating and reduce its fragility. Suitable plasticizers include, for example, polyethylene glycol, e.g., PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. In some embodiments, the plasticizer may also function as a dispersant or wetting agent.

[0311] In some cases, solubilizers such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycoflor, transcutol, propylene glycol, and dimethyl isosorbide may be added.

[0312] In some cases, stabilizers such as antioxidants, buffers, acids, and preservatives may be added.

[0313] In some cases, a suspending agent may be added, which may be a compound such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (for example, polyethylene glycol may have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, or hydroxymethylcellulose. This includes saturates acetate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth gum and acacia gum, guar gum, xanthan gum, sugars, cellulosic substances such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

[0314] In some cases, surfactants may be added, which include compounds such as sodium lauryl sulfate, sodium doxate, polyethylene glycol sorbitan monostearate or polyoxyethylene sorbitan monooleate, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, and the like. 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 octoxynol 10 and octoxynol 40. In some embodiments, surfactants may be included to improve physical stability or for other purposes.

[0315] In some cases, viscosity improvers may be added, which include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0316] In some cases, humectants may be added, which include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium doxate, sodium oleate, sodium lauryl sulfate, sodium doxate, triacetin, polyoxyethylene sorbitan monooleate, vitamin E TPGS, and ammonium salts.

[0317] Formulations comprising the compositions described herein may contain a pharmaceutically acceptable salt, typically, for example, sodium chloride, preferably at approximately physiological concentrations. Formulations of the present invention may also contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration ranges from 0.1 to 2.0%, typically 0.1 to 2.0 v / v%. Suitable preservatives include, for example, benzyl alcohol, phenol, m-cresol, and methylparaben, with propylparaben being an example of a preservative. Formulations of the present invention may also contain a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

[0318] The compositions described herein may be formulated in solid, liquid, or gel form for the administration of antibodies or their antigen-binding functional fragments to a subject, including (1) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as sterile solutions, suspensions, or sustained-release formulations; (2) topical application, e.g., as creams, ointments, controlled-release patches, or sprays applied to the skin; (3) intravaginally or rectally, e.g., as pessaries, creams, or foams; (4) into the eyes; (5) percutaneously; (6) per mucous membrane; or (7) adapted for use in the nose. In addition, the antibodies or their antigen-binding functional fragments or compositions of this disclosure may be implanted or injected into a patient using a drug delivery system.

[0319] Furthermore, compositions disclosed herein, comprising antibodies or antigen-binding functional fragments as described herein, may contain two or more active compounds necessary for the specific indication being treated, preferably active compounds having complementary activities that do not adversely affect each other. For example, a composition may further contain cytotoxic agents, cytokines, growth inhibitors and / or angiogenesis inhibitors, such as VEGFR antagonists. Such molecules are appropriately present in combination in amounts effective for the intended purpose. The active ingredients of compositions comprising antibodies or antigen-binding functional fragments as described herein may also be encapsulated, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, fine particles, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Pharmaceutical compositions may also be delivered in vesicles, particularly liposomes. Liposomes, including emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, and layered layers, can act 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.

[0320] Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a specified pore size to produce liposomes of the desired diameter. The Fab' fragment of the antibody of the present invention can be conjugated into the liposomes by a disulfide exchange reaction. Chemotherapy agents (e.g., doxorubicin) may be contained within the liposomes.

[0321] In some embodiments, sustained-release preparations may be used. Suitable examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing the antibody or antigen-binding functional fragment of the present disclosure, where the matrix is ​​in the form of a formed article, e.g., a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., microspheres for injection consisting of lactic acid-glycolic acid copolymer and leuprolide acetate, and poly-D-(-)-3-hydroxybutyrate. Polymers, e.g., ethylene-vinyl acetate and lactic acid-glycolic acid, allow for molecular release for more than 100 days, while certain hydrogels release proteins for shorter periods. If encapsulated antibodies remain in the body for an extended period, they may denature or aggregate as a result of exposure to water at 37°C, potentially leading to loss of biological activity and altered immunogenicity. Depending on the mechanism at hand, reasonable measures can be devised for stabilization. For example, if the aggregation mechanism is found to be intermolecular SS bond formation via thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling water content, using appropriate additives, and creating specific polymer matrix compositions. In some embodiments, the pharmaceutical composition may be delivered in a controlled-release system. In one embodiment, a pump may be used. In another embodiment, a polymer material may be used. In yet another embodiment, the controlled-release system may be positioned proximal to the target of the composition and therefore may require only a portion of the systemic dose.

[0322] The pharmaceutical compositions of this disclosure can be delivered, for example, subcutaneously, intravenously, or intraperitoneally by standard needles and syringes. In addition, for subcutaneous delivery, pen delivery devices readily have applications in delivering the pharmaceutical compositions of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of 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 are no replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is depleted from the reservoir, the entire device is discarded. Many reusable pen and auto-injector delivery devices have applications in subcutaneous delivery of the pharmaceutical compositions of the present invention.

[0323] The preparations for injection may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. The preparations for injection may be prepared, for example, by dissolving, suspending, or emulsifying the antibody or salt thereof described above in a conventionally used sterile aqueous or oily medium for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with suitable solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers, such as benzyl benzoate and benzyl alcohol. The injections thus prepared are preferably filled into suitable ampoules.

[0324] The compositions of this disclosure may exist, for example, in the form of granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions. The amount of antibody contained may be about 5 to about 500 mg per unit dose of the dosage form, and in particular, it is preferable that the antibody is contained in about 5 to about 100 mg in the form of an injection and about 10 to about 250 mg in the other dosage forms.

[0325] For oral, oral, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gel caps, and caplets are acceptable solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the present invention or pharmaceutically acceptable salts or tautomers thereof with at least one additive, such as starch or other additives. Suitable additives include sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, starch, agar, alginates, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. Oral dosage forms may contain other components to aid in administration, such as inert 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 fragrances. Tablets and pills may be further treated with appropriate coating materials.

[0326] Liquid dosage forms for oral administration may be pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inert diluent, such as water. In some embodiments, pharmaceutical formulations and pharmaceuticals may be prepared as liquid suspensions or aqueous solutions using, for example, sterile liquids, such as, not limited to, oils, water, alcohols, and combinations thereof. In some embodiments, pharmaceutical compositions may be prepared in lyophilized form. Lyophilized preparations may contain cryoprotective substances containing agents that provide stability to proteins against stress induced by freezing. Examples of cryoprotective substances include polyols, such as mannitol; sugars, such as sucrose; and surfactants, such as polysorbates, poloxamers, or polyethylene glycol. Cryoprotective substances also contribute to the osmotic pressure of the formulation. Pharmaceutically appropriate surfactants, suspending agents, and emulsifiers may be added for oral or parenteral administration.

[0327] As described above, the suspension may contain oil. Examples of such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. The suspension preparation 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 alcohol, such as non-limited ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. In addition, ethers, such as non-limited poly(ethylene glycol), petroleum hydrocarbons, such as mineral oil and petroleum, and water may be used in the suspension formulation.

[0328] For nasal administration, pharmaceutical formulations and pharmaceuticals may be sprays or aerosols containing a suitable solvent and, not limited to, other compounds such as stabilizers, antimicrobial agents, antioxidants, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof. Propellant for aerosol formulations may include compressed air, nitrogen, carbon dioxide, or hydrocarbon-based low-boiling solvents.

[0329] Injectable dosage forms generally include aqueous or oily suspensions that can be prepared using appropriate dispersants or wetting agents and suspending agents. Injectable forms may exist in the form of a solution phase or suspension, prepared with a solvent or diluent. Acceptable solvents or media include sterile water, Ringer's solution, or isotonic aqueous physiological saline solution. Alternatively, sterile oil may be used as a solvent or suspending agent. Preferably, the oil or fatty acid is non-volatile and includes natural or synthetic oils, fatty acids, mono-, di-, or triglycerides.

[0330] For injection, the pharmaceutical formulation and / or pharmaceutical may be a powder suitable for reconstitution with the appropriate solutions described above. Examples of these include, but are not limited to, lyophilized powders, rotary-dried or spray-dried powders, amorphous powders, granules, precipitates, or fine particles. For injection, the formulation may contain stabilizers, pH adjusters, surfactants, bioavailability adjusters, and combinations thereof.

[0331] For rectal administration, pharmaceutical formulations and pharmaceuticals may exist in the form of suppositories, ointments, enemas, tablets, or creams for the release of the compound in the intestine, S-shaped curve, and / or rectum. Rectal suppositories are prepared by mixing one or more compounds of the present invention or pharmaceutically acceptable salts or tautomers of the compounds with an acceptable medium, such as cocoa butter or polyethylene glycol, which exist in a solid phase at normal storage temperatures and in a liquid phase in the body, such as in the rectum, at a temperature suitable for drug release. Oil may also be used in the preparation of soft gelatin-type formulations and suppositories. Water, physiological saline, aqueous dextrose and related sugar solutions, and glycerol may be used in the preparation of suspension formulations, which may also contain suspending agents, such as pectin, carbomer, methylcellulose, hydroxypropylcellulose, or carboxymethylcellulose, as well as buffers and preservatives.

[0332] The concentration of the antibody or its antigen-binding functional fragment in these compositions may vary widely, i.e., less than about 10% by weight, and usually at least about 25% to 75% or as much as 90% by weight, and is selected mainly by fluid volume, viscosity, etc., according to a specific dosage mode selected.

[0333] In another embodiment of the present invention, a manufactured article containing materials useful for treating the diseases, disorders, or conditions described above, including the treatment of cancer, is shown. The manufactured article includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). The active agent in the composition is the antibody of the present invention. A label on or associated with the container indicates that the composition is used to treat a selected condition. The manufactured article may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use. The pharmaceutical compositions and pharmaceuticals described herein are useful in treating cancerous diseases.

[0334] Diagnostic and other uses Methods for using antibodies to detect, diagnose, and monitor diseases, disorders, or conditions associated with antigen expression (increased or decreased expression compared to a normal sample, and / or inappropriate expression, such as the presence of expression in tissues and / or cells that normally lack epitope expression) are described herein. Methods for determining whether a patient will respond to antibody therapy are also described herein.

[0335] In some embodiments, the method includes the step of detecting whether a patient has cells expressing a target antigen using an antibody disclosed herein. In some embodiments, the detection method includes the step of contacting a sample with an antibody or its antigen-binding functional fragment, and determining whether the binding level differs from that of a reference or comparative sample (e.g., control). In some embodiments, the method may be useful for determining whether an antibody or polypeptide described herein is an appropriate treatment for a subject.

[0336] In some embodiments, cells or cell / tissue lysates are contacted with an antibody to determine the binding between the antibody and the cells. If the test cells show binding activity compared to reference cells of the same tissue type, it may indicate that antibody treatment may be beneficial for the subject. In some embodiments, the test cells are cells from human tissue. In some embodiments, the test cells are cells from human blood.

[0337] 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 moieties or labeling groups may be conjugated to the target antibody, selected to meet the requirements of various applications, which are often determined by the availability of assay equipment and suitable immunoassay techniques.

[0338] Suitable labels 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 moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties.

[0339] For diagnostic purposes, antibodies or their antigen-binding functional fragments may be labeled with detectable moieties, including, but not limited to, radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art.

[0340] In some embodiments, the antibody does not need to be labeled, and its presence can be detected using a labeled second antibody bound to a first antibody. The antibodies of the present invention or their antigen-binding functional fragments may be used as affinity purifiers for cancer-associated antigens, or in diagnostic assays for cancer-associated antigen proteins, for example, to detect the expression of cancer-associated antigen proteins in specific cells, tissues, or serum. The antibodies or their antigen-binding functional fragments disclosed herein may also be used in in vivo diagnostic assays. Generally for these purposes, the antibodies are labeled with radionuclides (e.g., ulIn, 99Tc, 14C, 131I, 12sI, 3H, 32p, or 3sS) so that tumors can be located using immunoscintigraphy.

[0341] The antibodies of the present invention can be used in assays, such as competitive binding assays, direct and indirect sandwich assays, such as ELISA, and immunoprecipitation assays. The antibodies can also be used for immunohistochemistry to label tumor samples. For convenience, the antibodies of the present invention may be supplied in a kit, i.e., a set amount of reagents and instructions for use in performing a diagnostic assay. If the antibody is enzyme-labeled, the kit includes the substrate and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers and buffers (e.g., block buffers or lysis buffers). The relative amounts of various reagents can vary widely to provide the concentration of reagents in solution that substantially optimizes the sensitivity of the assay. In particular, the reagents may be supplied as a dry powder, usually a lyophilized dry powder, containing excipients that provide a reagent solution of appropriate concentration upon dissolution.

[0342] kit Furthermore, kits, pharmaceuticals, compositions, and unit dosage forms for use in any of the methods described herein are also shown herein. Kits comprising a therapeutically effective amount of at least one anti-EGFR IgA antibody or its antigen-binding functional fragment disclosed herein are shown herein. In some embodiments, the kit further comprises a second therapeutic agent (e.g., a chemotherapeutic agent). In some embodiments, the antibody or its antigen-binding functional fragment exists in aqueous or lyophilized form. The kit further comprises a diluent or reconstitution solution.

[0343] The kit may comprise one or more containers containing the antibody (or unit dosage form and / or manufactured article). In some embodiments, a unit dose is provided, which comprises a predetermined amount of the antibody-containing composition (e.g., a therapeutically effective dose) with or without one or more additional agents. In some embodiments, such a unit dose is supplied in a single-use pre-filled syringe for injection. In some embodiments, the composition containing the antibody or its antigen-binding functional fragment may contain physiological saline, sucrose, or other buffers, e.g., phosphates, etc.; and / or may be formulated within a stable and effective pH range. In some embodiments, the antibody or its antigen-binding functional fragment may be provided as a lyophilized powder that can be restored upon addition of a suitable liquid, e.g., sterile water. In some embodiments, the antibody or its antigen-binding functional fragment further comprises one or more substances that inhibit protein aggregation, non-limitingly including sucrose and arginine. In some embodiments, the antibody or its antigen-binding functional fragment further comprises heparin and / or proteoglycans.

[0344] In some embodiments, the kit further includes instructions for use in the treatment of cancer according to one of the methods described herein. The kit may further include instructions for the selection or treatment of an appropriate individual. The instructions supplied in the kit are typically written instructions on a label or accompanying document (e.g., a piece of paper included in the kit), but machine-readable instructions (e.g., instructions stored on a magnetic or optical storage disk) are also acceptable. In some embodiments, the kit further includes another therapeutic agent (e.g., an anti-cancer antibody or a chemotherapeutic agent).

[0345] The kit is contained in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar bags or plastic bags). The kit may also provide additional components such as buffers and explanatory information. Therefore, this application also provides manufactured articles containing vials (e.g., sealed vials), bottles, jars, flexible packaging, etc. [Examples] [Examples]

[0346] Modified anti-EGFR IgA design and production method Design of modified anti-EGFR IgA Modified anti-EGFR IgA antibody variants were designed, each possessing an EGFR-binding variable region and a modified IgA constant region. The IgA2(m1) heavy chain gene sequence is the backbone of the modified anti-EGFR IgA variant, with the N-linked glycosylation motif silenced and stabilizing mutations introduced (Table 10; Figures 4A-4D). This results in a series of molecules: anti-EGFR IgA3.0-(min), anti-EGFR IgA3.0+(plus), and anti-EGFR IgA4.0.

[0347] The anti-EGFR IgA3.0+ molecule contains the CH1-P124R mutation, which enables covalent bonding between the heavy and light chains. Two cysteine ​​residues are modified or removed (CH2-C92S;CH3_CHS-C147del_Y148del) to prevent cysteine ​​crosslinking with serum proteins, thus preventing dimeric aggregates and / or complex formation. Furthermore, three N-linked glycosylation motifs are silenced by the substitution of key amino acids within these motifs (CH1-N45.2G;CH2-N120T;CH3_CHS-N135Q).

[0348] The anti-EGFR IgA3.0min molecule contains the same CH1 and CH2 mutations as anti-EGFR IgA3.0+, but in contrast to anti-EGFR IgA3.0+, almost all of the tailpieces are deleted in anti-EGFR IgA3.0min (CH3_CHS-P131-Y148del).

[0349] The anti-EGFR IgA4.0 molecule is created by silencing the sole remaining N-linked glycosylation motif (CH2-N20) through four separate amino acid substitutions, thereby generating four completely deglycosylated IgA2-based molecules. [Table 10]

[0350] Cloning of anti-EGFR IgA3.0 / IgA4.0 To clone anti-EGFR IgA3.0+ and anti-EGFR IgA3.0min molecules, synthetic DNA containing the entire IgA constant region (CH1-CH2-CH3-CHS) was ordered, and cassettes encoding each were cloned into the pEE14.4 vector, and the IgA2(m1) sequence was substituted. The anti-EGFR IgA4.0 molecule was cloned by substituting the CH1-CH2-CH3-CHS region of anti-EGFR IgA3.0min in the pcDNA3.4 vector with gBlocks (IDT) containing the corresponding mutation for IgA4.0.

[0351] production 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 complex formation, separate vectors for anti-EGFR IgA 3.0+, anti-EGFR IgA 3.0min heavy chain, or anti-EGFR IgA 4.0 heavy chain (HC) were mixed with a vector for kappa light chain (LC) and pAdvantage (pAdv; Promega) in an optimal HC:LC:pAdv ratio, and then complexed with either 293fectin or Expifectamine before transfection.

[0352] purification The procedure for purifying anti-EGFR IgA was identical for all anti-EGFR IgA variants. Kappa light chains were captured from clarified and filtered cell culture supernatant using FPCL with a HiTrap KappaSelect column (GE Healthcare), followed by separation by size exclusion chromatography using a HiPrep 26 / 60 Sephacryl S-300 HR column. [Examples]

[0353] Characterization of anti-EGFR IgA variants Binding assay To determine the binding of the modified antibody, the binding of both the variable and Fc portions is evaluated.

[0354] The clarified supernatant of anti-EGFR IgA 3.0min or anti-EGFR IgA 3.0+ from HEK293F production will be tested in EGFR-expressing eukaryotic cells in a FACS binding experiment. The clarified supernatant of anti-EGFR IgA 4.0 from ExpiCHO-S production will be used undiluted in the FACS binding experiment.

[0355] In short, the supernatant is incubated with eukaryotic cells on ice for 1 hour. After washing, the cells are incubated with PE-labeled anti-IgA antibody (Southern Biotech) for 45 minutes. After washing, the cells are fixed with PFA and measured using Canto II (BD). EGFR, which recognizes the control antibody, is added at a concentration of 5-10 μg / mL.

[0356] To determine whether the antibody Fc region is capable of binding to FcαR, anti-EGFR IgA2(m1) and anti-EGFR IgA3.0min (25 μg / mL) are spread onto ELISA plates in carbonate buffer pH 9.0 and left overnight. The plates are blocked with 1% BSA, and then healthy donor CD89-expressing calcein-labeled polymorphonuclear neutrophils (PMNs) are allowed to bind to the plates at 37°C for 45 minutes. Subsequently, binding is determined by comparing the residual signal after each of the two washing steps with the input signal (without washing). An additional control to determine whether the coating concentrations are equal is obtained by staining the ELISA plates overnight with serially diluted anti-EGFR IgA2(m1) and anti-EGFR IgA3.0min antibodies, and then detecting their presence with anti-hIgA-HRP (Southern Biotech).

[0357] ADCC Target cells are loaded with 51Cr (Perkin-Elmer), washed twice, and incubated for 4 hours with serially diluted anti-EGFR IgA antibody and PMN from a healthy donor. Chronium release in the supernatant is measured, and specific lysis is calculated using the following formula: ((experimental cpm - baseline cpm) / (maximum cpm - baseline cpm)) x 100. Maximum lysis is determined by incubating labeled cells with 1.25% TRITON™ (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol), and minimum lysis is determined in the absence of antibody and effector cells. For anti-EGFR IgA 4.0, the undiluted supernatant of ExpiCHO-S production is evaluated.

[0358] thermal stability Thermal stability will be analyzed in a thermal shift assay using Sypro Orange (Life Technologies). A total of 12.5 μg of anti-EGFR IgA antibody, diluted in 25 μL of PBS and 3x SYPRO Orange (final concentration), will be transferred to a white 96-well thin-walled PCR plate (Roche) and sealed with Optical-Quality Sealing Tape (Roche). The plate will be heated in ViiA7 (Roche) from 37°C to 99°C by an exothermic rate of 1.6°C / second and an incubation period of 1 minute at each temperature. Fluorescence will be recorded simultaneously using 490 nm and 575 nm as the excitation and emission wavelengths, respectively.

[0359] To evaluate the functionality of destabilized anti-EGFR IgA antibodies in PBS, each anti-EGFR IgA antibody was incubated in a thermocycle at various temperatures (23°C to 95°C in increasing increments of 4°C and 12°C) for 5 minutes. After incubation, complete medium was added, and the anti-EGFR IgA antibody was used directly in ADCC at a final concentration of 10 μg / mL.

[0360] Glycosylation analysis The PNGase F treatment is performed according to the manufacturer's instructions (NEB). Briefly, the anti-EGFR IgA antibody is first denatured at 100°C for 10 minutes, followed by the addition of NP-40, Glycobuffer, and PNGase F enzyme, and incubated at 37°C for 1 hour. The sample is placed in Laemmli buffer containing 20 mM DTT and run on a 10% Mini Protean TGX SDS-PAGE (Bio-Rad). The gel is stained with InstantBlue (Expedeon) for 10 minutes and rinsed with water.

[0361] Glycan identification and quantification are determined by mass spectrometry. For anti-EGFR IgA2(m1) antibodies, N-glycosylation of the IgA2 antibody released after linkage-specific sialic acid derivatization is analyzed by reflectron-positive mode MALDI-TOF-MS. LC / MS2 was used for anti-EGFR IgA3.0+, anti-EGFR IgA3.0min, and anti-EGFR IgA4.0 variants.

[0362] The antibody was denatured, reduced, alkylated, and then the protein was digested using GluC (Roche, Indianapolis, IN) and trypsin (Sigma-Aldrich, Steinheim, Germany). For this purpose, 10 μg of antibody was added to 100 mM TrisHCl (pH 8.5) (Tris(hydroxymethyl)aminomethane hydrochloride), 5 mM Tris(2-carboxyethyl)-phosphine (TCEP, Tris(2-carboxyethyl)-phosphine, Sigma-Aldrich, Steinheim, Germany), 30 mM chloroacetamide (CAA, chloroacetamide, Sigma-Aldrich, Steinheim, Germany), and 1% sodium deoxycholate (SDC, sodium deoxychelate, Sigma-Aldrich, Steinheim, Germany), and water (MQ) (generated from the Q-POD or Q-Gard1 system (Millipore), used at 18.2 MΩ or higher). This mixture is incubated with GluC in an enzyme:protein ratio of 1:75 w / w at 37°C for 4 hours, followed by incubation with trypsin (1:100 w / w) at 37°C overnight. Then, the sample is added to 0.5% trifluoroic acid (TFA, Sigma-Aldrich, Steinheim, Germany) and the SDC is precipitated by centrifugation at maximum speed for 10 minutes. The supernatant is collected for solid-phase extraction (SPE).

[0363] For SPE, an Oasis μElution HLB 96-well plate (Waters, Wexford, Ireland) positioned on a vacuum manifold is used. The plate is prepared with acetonitrile (ACN, BioSolve Valkenswaard, Netherlands), equilibrated with 0.5% TFA, the supernatant is loaded, washed with 0.5% TFA, and the peptides are eluted with 50% ACN and 0.5% TFA. The recovered eluate is dried by rotary evaporation and reconstituted in 2% formic acid for subsequent LC-MS2 analysis.

[0364] For each digested and desalted sample, 100 ng is analyzed using an Agilent 1290 Infinity HPLC system (Agilent Technologies, Waldbronn, Germany) connected to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a flow splitter to achieve nanoflow. The samples are separated using a 2 cm trap column (100 μm inner diameter, packed with 3 μm ReproSil-Pur C18-AQ; Dr. Maisch GmbH, Ammerbuch-Entlingen, Germany) connected to a 50 cm analytical column (50 μm inner diameter, packed with 2.7 μm Poroshell 120 EC-C18; Agilent Technologies, Amstelveen, Netherlands). Buffer A consists of 0.1% formic acid, and buffer B consists of 0.1% formic acid in 80% ACN. The LC gradient is as follows: 0-5 minutes: 100% A (the rest is B), 5-53 minutes: 87% A to 60% A, 53-58 minutes: 0% A, 58-65 minutes: 100% A.

[0365] Mass spectrometry is performed in positive ion mode by electrospray ionization from a coated fused silica emitter at a spray voltage of 2kV. Each sample is measured in three sets using the same MS1 acquisition method, but with different MS2 methods. For the MS1 scan, the mass range m / z is set to 350-2000, the resolution to 60,000, the AGC target to 400,000, and the maximum injection time to 50 msec. For each of the three MS2 methods, HCD fragmentation (30% normalized collision energy; NCE) is initiated at the highest charge state, the lowest m / z signal is within a 3-second period, and an exclusion time of 30 seconds is used. HCD-based MS2 is recorded with a resolution of 30,000, m / z to 120-4000, the AGC target to 50,000, and a maximum injection time of 50 msec. For MS2 method 1, only HCD fragmentation is performed. For MS2 method 2, detection of oxonium ions at least three times in the HCD spectrum (Hex: 127.0390, 145.0495, 163.0601; HexNAc: 138.0550, 168.0655, 186.0761, 204.0867; PhosphoHex: 243.0264; NeuAc: 274.0921, 292.1027; Composite: 366.1395, 405.0793, 407.1660, 512.1974, 657.2349) triggers stepping-HCD for the same precursor signal, combining the HCD fragments with 10%, 25%, and 40% NCE. Stepping-HCD is recorded with a resolution of 30,000, m / z 120-4000, AGC target of 200,000, and a maximum injection time of 250 msec. For MS2 method 3, oxonium ion detection is performed by activating EThcD (30% additional activation), and this is recorded with a resolution of 30,000, m / z 120-4000, AGC target of 200,000, and a maximum injection time of 250 msec.

[0366] Bottom-up data is interpreted using Byonic v3.3.11 (Protein Metrics Inc.). Raw data is searched for C-terminal cleavage sites in Arg and Lys (trypsin), and Glu and Asp (GluC). Three cleavage errors are tolerated, and a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 20 ppm are used. Cys carbamide methylation is included as a fixed modification, and Met oxidation as a variable modification. For N-glycosylation, 279 compositions are included according to the N-glycan biosynthesis pathway.

[0367] Skyline (v3.7.0.11317) is used for relative quantification. Each peptide found to be glycosylated by Byonic is integrated, containing all major cleavage errors and oxidative variants. For each of these, the 279 glycan compositions listed above are integrated from each LC-MS2 run. The resulting integrated data is then refined to adhere to the following criteria: 1) error of 5 ppm or less relative to theoretical mass, 2) idotp ≥ 0.85 for the theoretical isotope pattern, 3) elution within ±2 minutes of the average retention time of the peptide, and 4) no obvious overlapping isotope patterns. The resulting list of glycopeptides is consistent with the Byonic annotation and is used for further relative quantification. For each of the refined peptide glycotypes, the MS1 region is integrated and mixed with peptides that provide information about the same N-glycosylation site. As an alternative to quantification, the number of peptide spectrum matches (PSMs) is counted for each glycopeptide combination, which provides information about a given glycosylation site.

[0368] To visualize glycan species, we followed the recommendations of the Consortium for Functional Glycomics. We created glycan illustrations using GlycoWorkbench (v2.1 build 146). For native MS analysis, we exchanged the antibody buffer with 150 mM ammonium acetate pH 7.5 by centrifugation at 15,000xg for 10x15 minutes using a Vivaspin 500 30kDa molecular weight cutoff filter (Sartorius Stedim Biotech, Germany). After buffer exchange, we adjusted the antibody concentration to approximately 3 μM with 150 mM ammonium acetate pH 7.5.

[0369] Native MS is performed using an improved Exactive Plus Orbitrap instrument (Thermo Fisher Scientific, Bremen) with an extended mass range (EMR), calibrated with a 25 mg / mL CsI solution. The transport multipole and ion lens voltage settings are manually optimized to obtain excellent transmission in the required m / z range. Electrospray ionization is achieved from a gold-coated glass capillary using a capillary voltage of 1.2 kV, while MS is performed with 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 is further achieved by adding nitrogen to an HCD cell to a gas pressure of approximately 3.7 x 10⁻¹⁰ bar. Mass is calculated from the charge-state distribution by fitting the charge to the lowest standard deviation of the mass (typically resulting in a mass error of less than 1 Da).

[0370] Pharmacokinetic / pharmacodynamic research For pharmacokinetic / pharmacodynamic studies of anti-EGFR IgA antibodies, BALB / cByJ mice (Jackson Laboratory) are intravenously injected with 100 μg (1 mg / mL) of anti-EGFR IgA antibody. Blood is collected at the indicated time points, separated by centrifugation, and serum is collected. ELISA is performed on the diluted serum.

[0371] In vivo distribution Each anti-EGFR IgA antibody is conjugated to a chelator and then radiolabeled. The anti-EGFR IgA antibody is spun through a 30kDa centrifuge filter at 12,000g for 8 minutes. Subsequently, 500 μL of 0.1 M sodium bicarbonate (pH 8.2) is added to the centrifuge filter and spun again at 12,000g for 8 minutes. The centrifuge filter is inverted and transferred to a new tube, and the mixture is centrifuged at 1,000g for 2 minutes to obtain approximately 40 μL. 60 μL of sodium bicarbonate buffer is added to this to make 100 μL. A 20-fold molar excess of p-SCN-Bn-DTPA (2 mg / mL in anhydrous DMSO (1 mg used per 500 μL)) is added, the mixture is vortexed for 30 seconds, and the mixture is incubated at 37°C for 1 hour. Next, the BnDTPA antibody conjugate is passed through a G50 column and eluted in 12 fractions of 100 μL each with 0.5 M MES buffer (pH 5.4). The top 5 most concentrated fractions are pooled and spun through a 30 kDa centrifuge filter at 12,000 g for 8 minutes. 500 μL of 0.5 M MES buffer (pH 5.4) is added to the centrifuge filter and the mixture is centrifuged again at 12,300 g for another 8 minutes. The centrifuge filter is inverted and transferred to a new microcentrifuge tube, and the mixture is centrifuged at 1,000 g for 2 minutes. Protein concentration is measured using Nanodrop.

[0372] To radiolabel the antibody, 150 μL of indium solution (55.5 MBq) is added to 150 μg of antibody in MES buffer in a microcentrifuge tube, and incubated at room temperature for 45 minutes. The reaction mixture is passed through a G50 column and eluted with PBS (pH 7.4). The sample is collected in 16 fractions of 100 μL each (3 drops) in a microcentrifuge tube, the activity is checked, and the fraction with the highest activity is mixed.

[0373] To check the purity of the radiolabeled antibody using an iTLC strip, a total of 2 μL of reaction mixture is dropped onto the iTLC and allowed to dry for 2 minutes. A certain volume of 0.1 M citrate buffer is added to the measurement cylinder and the bottom is covered. The iTLC strip is placed in the measurement cylinder and citrate buffer is drawn up until it reaches approximately 1 cm from the top. The strip is removed and scanned using a radio-TLC. [Examples]

[0374] Manufacturing of anti-EGFR IgA 3.0min drug substance (DS) A plasmid encoding anti-EGFR IgA3.0min was recombinant...

Claims

1. An epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) The VH region is (I) VH complementarity determination region 1 (CDR-H1) comprising any one of the amino acid sequences of SEQ ID NOs. 34 to 54, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) VH complementarity determination region 2 (CDR-H2) comprising any one of the amino acid sequences of SEQ ID NOs. 57-78, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VH complementarity determination region 3 (CDR-H3) comprising any one of the amino acid sequences of SEQ ID NOs. 81 to 102, or any variant thereof including 1 to 3 substitutions, deletions, or insertions. Includes, (ii) The VL region is (I) VL complementarity determination region 1 (CDR-L1) comprising any one of the amino acid sequences of SEQ ID NOs. 105 to 126, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) A VL complementarity determination region 2 (CDR-L2) comprising any one of the amino acid sequences of SEQ ID NOs. 129-143, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VL complementarity determination region 3 (CDR-L3) containing any one of the amino acid sequences of SEQ ID NOs. 146-166, or any variant thereof including one to three substitutions, deletions, or insertions. including, The EGFR binding domain and, (b) An IgA heavy chain constant region having at least one mutation compared to a wild-type immunoglobulin A (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 compared to a corresponding antibody each containing the wild-type IgA heavy chain constant region: reduced glycosylation, reduced dimerization, reduced aggregation, improved thermal stability, or increased circulating half-life. The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

2. (a) CDR-H1 contains one amino acid sequence from SEQ ID NOs. 34 to 54, (b) CDR-H2 contains one amino acid sequence from sequence numbers 57 to 78, (c) CDR-H3 contains one amino acid sequence from sequence numbers 81 to 102, (d) CDR-L1 contains one amino acid sequence from sequence numbers 105 to 126, (e) CDR-L2 contains one of the amino acid sequences of SEQ ID NOs: 129 to 143, (f) CDR-L3 contains one of the amino acid sequences of sequence numbers 146 to 166, The modified antibody according to claim 1.

3. The modified antibody according to claim 1, wherein the VH region contains an amino acid sequence that is at least 80% identical to any one of the amino acid sequences of the VH regions listed in Table 5.

4. The modified antibody according to claim 1, wherein the VL region contains an amino acid sequence that is at least 80% identical to any one of the amino acid sequences of the VL regions listed in Table 7.

5. The modified antibody according to claim 1, wherein the amino acid sequences of the VH region and the VL region follow one of the combinations shown in Table 8.

6. A modified antibody according to claim 1, comprising an IgA light chain constant region having at least 80% the same amino acid sequence as SEQ ID NO:

23.

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

1.

9. At least one mutation is numbered according to the IMGT scheme, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (a) N45.2 substitution selected from the group consisting of N45.2G and N45.2A, (b) P124R substitution, or (c) Any combination of them The modified antibody according to claim 8.

10. At least one mutation is present in the IgA CH2 region, and the mutation is numbered according to the IMGT scheme, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (a) N20 substitution, (b) L21 substitution, (c) T22 substitution, (d) C92 substitution, (e) N120 substitution, (f) Substitution of I121, or (g) T122 substitution The modified antibody according to claim 7.

11. At least one mutation is numbered according to the IMGT scheme, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (a) N20 substitution selected from the group consisting of N20G, N20Q and N20T, (b) L21I substitution, (c) T22S substitution, (d) C92S substitution, (e) N120T replacement, (f) I121L substitution, (g) T122S substitution, or (h) Any combination of them The modified antibody according to claim 10.

12. At least one mutation is present in the IgA CH3 region, and the mutation is numbered according to the IMGT scheme compared to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (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 missing, (l) Y148 deletion, (m) Missing P131-Y148, or (n) combinations of those The modified antibody according to claim 7.

13. At least one mutation is numbered according to the IMGT scheme, compared to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (a) H5 substitution selected from the group consisting of H5C, H5Y, H5F, H5M and H5W, (b) L7 substitution selected from the group consisting of L7F, L7Y, L7M, L7W, L7H and L7I, (c) P10C substitution, (d) T22 substitution selected from the group consisting of T22V, T22I, T22L, and T22A, (e) L79 substitution selected from the group consisting of L79V, L79T, L79A and L79I, (f) W81 substitution selected from the group consisting of W81T, W81L, W81A, W81V and W81I, (g) A85.1 substitution selected from the group consisting of A85.1F, A85.1Y, A85.1M, A85.1W and A85.1H, (h) T86 substitution selected from the group consisting of T86Y, T86F, T86M, T86W and T86H, (i) I88 substitution selected from the group consisting of I88L, I88A, I88V and I88T, or (j) Any combination of them The modified antibody according to claim 12.

14. At least one mutation is numbered according to the IMGT scheme, compared to the corresponding residue in the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (a) N135Q substitution, (b) C147 missing, (c) Y148 missing, or (d) Any combination of them The modified antibody according to claim 12.

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

1.

16. The modified antibody according to claim 1, wherein the IgA heavy chain constant region contains an amino acid sequence that is at least 80% identical to that of the IgA heavy chain constant region of SEQ ID NO:

5.

17. A modified antibody according to claim 1, which is a monomer.

18. The modified antibody according to claim 1, wherein the EGFR-binding domain binds to an EGFR polypeptide variant, and the EGFR variant includes EGFR vIII, exon 19 deletion, L858R substitution, C797S substitution, or T790M substitution in exon 21.

19. The modified antibody according to claim 1, wherein 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.

20. The modified antibody according to claim 1, wherein 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.

21. The modified antibody according to claim 1, wherein 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.

22. The modified antibody according to claim 1, wherein 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.

23. The modified antibody according to claim 1, which can induce antibody-dependent cell-mediated cytotoxicity (ADCC) via immune effector cells.

24. The modified antibody according to claim 23, wherein the immune effector cell is a neutrophil, T cell, eosinophil, or macrophage.

25. A modified antibody according to claim 1, which is a chimeric antibody, a single-chain antibody, a humanized antibody, a human antibody, a monoclonal antibody, a deimmunized antibody, a bispecific antibody, a multispecific antibody, a polyvalent antibody, or a combination thereof.

26. The modified antibody according to claim 25, which is a bispecific antibody.

27. The modified antibody according to claim 26, further comprising 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 epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (a) an EGFR binding domain that binds to domain III of an EGFR polypeptide or a variant thereof, (b) An IgA constant region comprising an IgA heavy chain constant region having at least one mutation compared to a wild-type immunoglobulin A (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 compared to a corresponding antibody comprising the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, or increased circulating half-life. The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

29. The IgA steady-state region is (a) IgA heavy chain constant region having the amino acid sequence of SEQ ID NO: 5, and (b) IgA light chain constant domain having the amino acid sequence of SEQ ID NO: 23 A modified antibody according to claim 28, comprising:

30. The IgA constant domain includes an IgA heavy chain constant region containing IgA CH1, CH2, and CH3 domains, and the IgA heavy chain constant region has the following mutations, numbered according to the IMGT scheme, compared to (g) the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1, (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 A modified antibody according to claim 28, comprising:

31. The modified antibody according to claim 28, wherein the EGFR-binding domain binds to an epitope of an EGFR polypeptide or variant thereof containing one 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 epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (a) an EGFR binding domain that binds to domain II of an EGFR polypeptide or a variant thereof, (b) An IgA constant domain comprising an IgA heavy chain constant region having at least one mutation compared to the wild-type immunoglobulin A (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 compared to a corresponding antibody comprising the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, or increased circulating half-life. The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

33. The IgA steady-state region is (a) IgA heavy chain constant region having the amino acid sequence of SEQ ID NO: 5, and (b) IgA light chain constant domain having the amino acid sequence of SEQ ID NO: 23 A modified antibody according to claim 32, comprising:

34. The IgA constant region includes an IgA heavy chain constant region containing IgA CH1, CH2, and CH3 domains, and the IgA heavy chain constant region is numbered according to the IMGT scheme as follows, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (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 A modified antibody according to claim 32, comprising:

35. An epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) The VH region is (I) VH complementarity determination region 1 (CDR-H1) comprising any one of the amino acid sequences of SEQ ID NOs. 34 to 54, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) VH complementarity determination region 2 (CDR-H2) comprising any one of the amino acid sequences of SEQ ID NOs. 57-78, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VH complementarity determination region 3 (CDR-H3) comprising any one of the amino acid sequences of SEQ ID NOs. 81 to 102, or any variant thereof including 1 to 3 substitutions, deletions, or insertions. Includes, (ii) The VL region is (I) VL complementarity determination region 1 (CDR-L1) comprising any one of the amino acid sequences of SEQ ID NOs. 105 to 126, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) A VL complementarity determination region 2 (CDR-L2) comprising any one of the amino acid sequences of SEQ ID NOs. 129-143, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VL complementarity determination region 3 (CDR-L3) containing any one of the amino acid sequences of SEQ ID NOs. 146-166, or any variant thereof including one to three substitutions, deletions, or insertions. including, The EGFR binding domain and, (b) (i) Heavy chain constant region having the amino acid sequence of SEQ ID NO: 5, (ii) Light chain constant domain having the amino acid sequence of SEQ ID NO: 23 The immunoglobulin A (IgA) constant domain, which includes The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

36. The modified antibody according to claim 35, wherein 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.

37. The modified antibody according to claim 35, wherein 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.

38. The modified antibody according to claim 35, wherein 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.

39. The modified antibody according to claim 35, wherein 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.

40. An epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (a) An EGFR-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) The VH region is (I) VH complementarity determination region 1 (CDR-H1) comprising any one of the amino acid sequences of SEQ ID NOs. 34 to 54, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) VH complementarity determination region 2 (CDR-H2) comprising any one of the amino acid sequences of SEQ ID NOs. 57-78, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VH complementarity determination region 3 (CDR-H3) comprising any one of the amino acid sequences of SEQ ID NOs. 81 to 102, or any variant thereof including 1 to 3 substitutions, deletions, or insertions. Includes, (ii) The VL region is (I) VL complementarity determination region 1 (CDR-L1) comprising any one of the amino acid sequences of SEQ ID NOs. 105 to 126, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) A VL complementarity determination region 2 (CDR-L2) comprising any one of the amino acid sequences of SEQ ID NOs. 129-143, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VL complementarity determination region 3 (CDR-L3) containing any one of the amino acid sequences of SEQ ID NOs. 146-166, or any variant thereof including one to three substitutions, deletions, or insertions. including, The EGFR binding domain and, (b) IgA heavy chain constant region containing immunoglobulin A (IgA) CH1, CH2 and CH3 domains, each having the following mutations, numbered according to the IMGT scheme, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1 (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 IgA heavy chain constant region includes and The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

41. The IgA heavy chain constant region, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1, exhibits the following mutations in the CH3 domain, numbered according to the IMGT scheme: (a) N135Q substitution, (b) C147 deletion, and (c) Y148 missing The modified antibody according to claim 40, further comprising:

42. The modified antibody according to claim 40, wherein the IgA heavy chain constant region further comprises deletions of P131-Y148 in the CH3 domain, which are numbered according to the IMGT scheme compared to the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO:

1.

43. The IgA heavy chain constant region, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1, exhibits the following mutations in the CH2 domain, numbered according to the IMGT scheme: (a) N20 substitution selected from the group consisting of N20G, N20Q and N20T, (b) L21I substitution, and (c) T22S substitution The modified antibody according to claim 42, further comprising:

44. A modified antibody according to any one of claims 40 to 43, wherein 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.

45. A modified antibody according to any one of claims 40 to 43, wherein 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.

46. A modified antibody according to any one of claims 40 to 43, wherein 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.

47. A modified antibody according to any one of claims 40 to 43, wherein 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.

48. An epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (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, (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 The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

49. An epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (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, (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 The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

50. An epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (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, (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 The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

51. An epidermal growth factor receptor (EGFR) binding modified antibody or its EGFR-binding functional fragment, (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, (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 The EGFR-binding modified antibody or its EGFR-binding functional fragment, including the above.

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

53. A method for treating cancer in a subject that requires it, the subject (a) An epidermal growth factor receptor (EGFR) binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, (i) The VH region is (I) VH complementarity determination region 1 (CDR-H1) comprising any one of the amino acid sequences of SEQ ID NOs. 34 to 54, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) VH complementarity determination region 2 (CDR-H2) comprising any one of the amino acid sequences of SEQ ID NOs. 57-78, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VH complementarity determination region 3 (CDR-H3) comprising any one of the amino acid sequences of SEQ ID NOs. 81 to 102, or any variant thereof including 1 to 3 substitutions, deletions, or insertions. Includes, (ii) The VL region is (I) VL complementarity determination region 1 (CDR-L1) comprising any one of the amino acid sequences of SEQ ID NOs. 105 to 126, or any variant thereof including 1 to 3 substitutions, deletions, or insertions, (II) A VL complementarity determination region 2 (CDR-L2) comprising any one of the amino acid sequences of SEQ ID NOs. 129-143, or any variant thereof including 1-3 substitutions, deletions, or insertions, and (III) VL complementarity determination region 3 (CDR-L3) containing any one of the amino acid sequences of SEQ ID NOs. 146-166, or any variant thereof including one to three substitutions, deletions, or insertions. including, The EGFR binding domain and, (b) An IgA heavy chain constant region having at least one mutation compared to a wild-type immunoglobulin A (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 compared to a corresponding antibody each containing the wild-type IgA heavy chain constant region: reduced glycosylation, reduced aggregation, improved thermal stability, or increased circulating half-life. The method comprising the step of administering an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, which contains the above.

54. The IgA heavy chain constant region includes the IgA CH1, CH2, and CH3 domains, and the following mutations, numbered according to the IMGT scheme, are observed when the IgA heavy chain constant region is compared to the corresponding antibody containing the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO:

1. (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 method according to claim 53, including the method described in claim 53.

55. The IgA heavy chain constant region, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1, exhibits the following mutations in the CH3 domain, numbered according to the IMGT scheme: (a) N135Q substitution, (b) C147 deletion, and (c) Y148 missing The method according to claim 53, further comprising:

56. The method according to claim 54, further comprising deletions of P131-Y148 in the CH3 domain, where the IgA heavy chain constant region is numbered according to the IMGT scheme, compared to the corresponding residues in the wild-type IgA heavy chain constant region of SEQ ID NO:

1.

57. The IgA heavy chain constant region, compared to the corresponding antibody containing the wild-type IgA heavy chain constant region of SEQ ID NO: 1, exhibits the following mutations in the CH2 domain, numbered according to the IMGT scheme: (a) N20 substitution selected from the group consisting of N20G, N20Q and N20T, (b) L21I substitution, and (c) T22S substitution The method according to claim 56, further comprising:

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

59. The method according to 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 according to claim 53, wherein the cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.

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

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

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

64. The method according to claim 53, wherein the modified antibody inhibits tumor growth associated with cancer.

65. The method according to claim 53, wherein the administration step is the administration by subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular or intracranial administration.

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

67. The method according to 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 nutritional supplement, or an antioxidant.

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

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

70. The method according to claim 69, wherein the target is a rodent, and the effective dose is 1 mg / kg to 25 mg / kg, administered subcutaneously, intravenously, or intraperitoneally twice a week for 35 to 40 days.

71. The method according to claim 69, wherein the effective dose is administered intravenously at a dose of 25 mg / kg every 7 days for 5 weeks, with the exception of a third dose delivered at 12.5 mg / kg.

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

73. A method for treating cancer in a subject that requires it, the subject (a) An epidermal growth factor receptor (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, (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 The method comprising the step of administering an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, which contains the above.

74. A method for treating cancer in a subject that requires it, the subject (a) An epidermal growth factor receptor (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, (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 The method comprising the step of administering an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, which contains the above.

75. A method for treating cancer in a subject that requires it, the subject (a) An epidermal growth factor receptor (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, (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 The method comprising the step of administering an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, which contains the above.

76. A method for treating cancer in a subject that requires it, the subject (a) An epidermal growth factor receptor (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, (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 The method comprising the step of administering an effective amount of an EGFR-binding modified antibody or an EGFR-binding functional fragment thereof, which contains the above.

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 a modified antibody according to any one of claims 1 to 51.

79. A host cell expressing the modified 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 a modified antibody according to any one of claims 1 to 51, and the second therapeutic agent is conjugated to MET, cMet, CD28, HER2, HER3, IGF-IR, CD3, PD1, PD-L1, VEGFR2, FcGR3, 4-1BB, or a combination thereof.

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