Engineered IgA antibodies and methods of use - Patents.com

Abstract

Provided herein are engineered antibodies, pharmaceutical compositions, and methods of use comprising modified IgA heavy chain constant regions. The engineered antibodies described herein comprise one or more amino acid substitutions or deletions in the constant region of the IgA domain. Also provided herein are methods of treating disorders, including cancer, by administering the engineered IgA antibodies described herein. In some embodiments, the IgA heavy chain constant region of the antibodies described herein comprises non-conservative amino acid substitutions at amino acid residues N45.2, N114, N135, and N15.2, numbered according to the IMGT scheme.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 824,864, filed March 27, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Monoclonal antibodies of IgG isotype targeting tumor antigens have proven to be an effective treatment for various cancers. Over the years, an increasing number of monoclonal antibodies targeting different tumor antigens have been approved for use in cancer treatment. However, their clinical efficacy and side effects, especially as monotherapy, are still insufficient. Therefore, the goal is to develop new antibody treatments with increased clinical efficacy, novel targeting modalities or modes of action, and / or reduced number and severity of side effects. Summary of the Invention [Means for solving the problem]

[0003] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an immunoglobulin A (IgA) heavy chain constant region, wherein the IgA heavy chain constant region comprises an IgA CH2 region and an IgA CH3 region, and wherein the IgA heavy chain constant region comprises an alteration of at least two naturally occurring glycosylation sites compared to a corresponding wild-type (WT) IgA heavy chain constant region, wherein each of the at least two naturally occurring glycosylation sites is present within the IgA CH2 region or the IgA CH3 region.

[0004] In some embodiments, the at least two naturally occurring glycosylation sites are two naturally occurring N-linked glycosylation sites. In some embodiments, one or more of the at least two naturally occurring glycosylation sites comprises a naturally occurring asparagine (N) amino acid residue that is altered in the engineered antibody disclosed herein compared to the corresponding wild-type IgA. In some embodiments, the alteration comprises an amino acid substitution or deletion at one or both of the at least two naturally occurring glycosylation sites. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution. In some embodiments, the IgA heavy chain constant region comprises an amino acid substitution at i.N114 and N135, ii.N114 and N15.2, or iii.N135 and N15.2 compared to the corresponding WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises, relative to a corresponding WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme: i. an N114T amino acid substitution and an N135Q amino acid substitution; ii. an N114T amino acid substitution and an amino acid substitution selected from the group consisting of N15.2G, N15.2Q, and N15.2T; or iii. an N135Q amino acid substitution and an amino acid substitution from the group consisting of N15.2G, N15.2Q, and N15.2T.

[0005] In some embodiments, the IgA heavy chain constant region comprises at least three altered naturally occurring glycosylation sites compared to the corresponding wild-type IgA. In some embodiments, the at least three naturally occurring glycosylation sites are three N-linked glycosylation sites. In some embodiments, the three naturally occurring glycosylation sites each comprise an asparagine (N) amino acid residue that is altered in the antibodies described herein.

[0006] In some embodiments, the modification is an amino acid substitution or deletion. In some embodiments, the amino acid substitution is a non-conservative substitution. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions at N114, N135, and N15.2 compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions selected from the group consisting of: i. an N114T amino acid substitution; ii. an N135Q amino acid substitution; and iii. an N15.2G, N15.2Q, and N15.2T compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0007] In some embodiments, the IgA heavy chain constant region of an antibody described herein further comprises an IgA CH1 region. In some embodiments, the heavy chain constant region comprises at least one naturally occurring N-linked glycosylation site in the IgA CH2 region, at least one naturally occurring glycosylation site in the IgA CH3 region, and at least one naturally occurring glycosylation site in the IgA CH1 region, compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions at i.N45.2, N114, and N135, or ii.N45.2, N15.2, and N135, compared to a wild-type IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0008] In some embodiments, the IgA heavy chain constant region comprises, relative to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme: i. an N45.2G amino acid substitution, an N114T amino acid substitution, and an N135Q amino acid substitution; or ii. an N45.2G amino acid substitution, an N135Q amino acid substitution, and an amino acid substitution selected from the group consisting of N15.2G, N15.2Q, and N15.2T.

[0009] In some embodiments, the heavy chain constant region of an antibody described herein comprises an alteration of at least two naturally occurring N-linked glycosylation sites in the IgA CH2 region and at least one naturally occurring glycosylation site in the IgA CH1 region compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions at i.N45.2, N114, and N15.2G, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region described herein comprises an amino acid substitution selected from the group consisting of i.N45.2G, ii.N114T, and iii.N15.2G, N15.2Q, and N15.2T, numbered according to the IMGT scheme, compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1.

[0010] In some embodiments, the antibodies or functional fragments thereof described herein exhibit a longer circulating half-life compared to a corresponding WT IgA antibody. In some embodiments, the antibodies or functional fragments thereof exhibit reduced aggregation compared to a corresponding WT IgA antibody. In some embodiments, the antibodies or functional fragments thereof exhibit reduced aggregation with serum proteins compared to a corresponding WT IgA antibody. In some embodiments, the antibodies or functional fragments thereof induce increased antibody-dependent cell-mediated cytotoxicity (ADCC) compared to a comparable antibody comprising an IgG heavy chain constant region. In some embodiments, the antibodies or functional fragments thereof exhibit increased thermal stability compared to a corresponding WT IgA antibody. In some embodiments, the antibodies or functional fragments thereof exhibit reduced glycosylation compared to a corresponding WT IgA antibody.

[0011] In some embodiments, the IgA heavy chain constant region exhibits increased affinity binding to FcαR expressed on immune effector cells compared to a corresponding wild-type IgA antibody. In some embodiments, the IgA heavy chain constant region comprises at least four naturally occurring glycosylation sites modified compared to a corresponding wild-type IgA. In some embodiments, the at least four naturally occurring glycosylation sites are four naturally occurring N-linked glycosylation sites. In some embodiments, the at least four naturally occurring glycosylation sites each comprise a naturally occurring asparagine (N) amino acid residue. In some embodiments, the heavy chain constant region comprises at least two naturally occurring N-linked glycosylation sites in the IgA CH2 region, at least one naturally occurring N-linked glycosylation site in the IgA CH3 region, and at least one naturally occurring N-linked glycosylation site in the IgA CH1 region modified compared to a corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions at amino acid residues N45.2, N114, N135, and N15.2 compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0012] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises non-conservative amino acid substitutions at amino acid residues N45.2, N114, N135, and N15.2, numbered according to the IMGT scheme.

[0013] In some embodiments, the IgA heavy chain constant region comprises, relative to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme, an amino acid substitution selected from the group consisting of: i. N45.2G, ii. N114T, iii. N135Q, and iv. N15.2G, N15.2Q, and N15.2T. In some embodiments, the antibody or functional fragment thereof exhibits a longer circulating half-life compared to a corresponding IgA antibody comprising at least one naturally occurring N-linked glycosylation site. In some embodiments, the antibody or functional fragment thereof induces increased antibody-dependent cellular cytotoxicity (ADCC) compared to a corresponding equivalent antibody comprising an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the antibody or functional fragment thereof exhibits increased thermal stability compared to a corresponding IgA antibody comprising at least one naturally occurring N-linked glycosylation site. In some embodiments, the antibody or functional fragment thereof exhibits reduced glycosylation compared to a corresponding IgA antibody comprising at least one naturally occurring N-linked glycosylation site. In some embodiments, the IgA heavy chain constant region exhibits increased affinity binding to FcαR expressed on immune effector cells compared to a corresponding IgA antibody that contains at least one naturally occurring N-linked glycosylation site.

[0014] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises a modification of at least one naturally occurring cysteine ​​(C) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the modification is an amino acid substitution or deletion of at least one naturally occurring cysteine ​​(C) amino acid residue. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue. In some embodiments, the at least one naturally occurring cysteine ​​amino acid residue is C147 or C86 compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a C86S amino acid substitution compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a deletion of C147 compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation compared to a corresponding WT IgA antibody or functional fragment thereof, hi some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation with serum proteins compared to a corresponding WT IgA construct.

[0015] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises an alteration of at least two naturally occurring cysteine ​​(C) amino acid residues compared to the corresponding wild-type IgA. In some embodiments, the alteration comprises an amino acid substitution of one or both of the at least two naturally occurring cysteine ​​(C) amino acid residues. In some embodiments, the alteration comprises a deletion of one or both of the at least two naturally occurring cysteine ​​(C) amino acid residues.

[0016] In some embodiments, the IgA heavy chain constant region comprises an amino acid substitution of one of the at least two naturally occurring cysteine ​​(C) amino acid residues and a deletion of one of the at least two naturally occurring cysteine ​​(C) amino acid residues. In some embodiments, the at least two naturally occurring cysteine ​​amino acid residues are C147 and C86, as compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, as numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a deletion of C147, as compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, as numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises an amino acid substitution of C86, as compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, as numbered according to the IMGT scheme.

[0017] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises an amino acid substitution of the naturally occurring C86 amino acid residue and a deletion of the naturally occurring C147 amino acid residue compared to a wild-type IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a C86S amino acid substitution compared to a wild-type IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation compared to a corresponding wild-type IgA antibody. In some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation with serum proteins compared to a corresponding wild-type IgA antibody.

[0018] In some embodiments, the IgA heavy chain constant region of the antibody described herein comprises a modification of at least one naturally occurring tyrosine (Y) amino acid residue compared to a corresponding wild-type IgA. In some embodiments, the modification is an amino acid substitution or deletion of at least one naturally occurring tyrosine (Y) amino acid residue. In some embodiments, the amino acid substitution is a non-conservative amino acid mutation of at least one naturally occurring tyrosine (Y) amino acid residue compared to a WT IgA antibody. In some embodiments, the at least one tyrosine residue is Y148 compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, amino acid Y148 is deleted compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation compared to a corresponding WT IgA antibody. In some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation with serum proteins compared to a corresponding WT IgA antibody.

[0019] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises a modification of at least one naturally occurring threonine (T) amino acid residue compared to a corresponding wild-type IgA antibody. In some embodiments, the modification comprises an amino acid substitution or deletion of at least one naturally occurring threonine (T) amino acid residue compared to a corresponding wild-type IgA antibody. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution of at least one naturally occurring threonine (T) amino acid residue. In some embodiments, the at least one naturally occurring threonine amino acid residue is T116 or T16 compared to a wild-type IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation compared to a corresponding wild-type IgA antibody. In some embodiments, the antibody or functional fragment thereof exhibits reduced aggregation with serum proteins compared to a corresponding wild-type IgA antibody. In some embodiments, the IgA heavy chain constant region comprises a modification of at least two naturally occurring threonine (T) amino acid residues compared to a corresponding wild-type IgA. In some embodiments, the modification comprises an amino acid substitution or deletion of one or both of at least two naturally occurring threonine (T) amino acid residues. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution of one or both of at least two naturally occurring threonine (T) amino acid residues. In some embodiments, the at least two naturally occurring threonine (T) amino acid residues are T116 or T16 relative to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a T116S amino acid substitution relative to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a T16S amino acid substitution relative to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0020] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises a modification of at least one naturally occurring isoleucine (I) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the modification comprises an amino acid substitution or deletion of at least one naturally occurring isoleucine (I) amino acid residue. In some embodiments, the amino acid substitution comprises a non-conservative amino acid substitution of at least one naturally occurring isoleucine (I) amino acid residue. In some embodiments, the at least one naturally occurring isoleucine (I) residue is I115 compared to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises an I115L amino acid substitution compared to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a modification of at least one naturally occurring leucine (L) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the modification comprises an amino acid substitution or deletion of at least one naturally occurring leucine (L) amino acid residue. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution of at least one naturally occurring leucine (L) amino acid residue. In some embodiments, the at least one naturally occurring leucine (L) residue is L15.3 compared to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises an L15.3I amino acid substitution compared to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0021] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises an alteration of at least one naturally occurring proline (P) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant region comprises an amino acid substitution or deletion of at least one naturally occurring proline (P) amino acid residue. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution of at least one naturally occurring proline (P) amino acid residue. In some embodiments, the at least one naturally occurring proline (P) residue is P124 compared to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a P124R amino acid substitution compared to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the antibody or functional fragment thereof exhibits increased stability compared to the corresponding WT IgA antibody. In some embodiments, the antibody or functional fragment thereof exhibits increased stability between the heavy and light chains compared to the corresponding WT IgA antibody. In some embodiments, the antibody or functional fragment thereof has a covalent linkage between the heavy and light chains. In some embodiments, the antibody or functional fragment thereof comprises a disulfide bond between a cysteine ​​(C) amino acid residue in the heavy chain and a cysteine ​​(C) amino acid residue in the light chain. In some embodiments, the IgA heavy chain constant region comprises an amino acid sequence selected from any one of SEQ ID NOs: 16-21.

[0022] In some embodiments, the antibodies described herein comprise an altered IgA heavy chain region of allotype IgA2m(1) or IgA2m(2). In some embodiments, the antibody is an allotype Caucasian IgA2m(1) antibody. In some embodiments, the IgA heavy chain constant region comprises a modification of the C-terminal IgA tailpiece compared to the corresponding wild-type IgA. In some embodiments, the modification comprises a deletion of 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 12, 10, 8, 6, 4, or 2 amino acids at the C-terminus. In some embodiments, the modification comprises a deletion of 18 amino acids at the C-terminus. In some embodiments, the modification comprises a deletion of C-terminal amino acid residues 131-148 compared to a wild-type IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises a deletion of C-terminal amino acid residues P131 to Y148 compared to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0023] In some embodiments, the IgA heavy chain constant region of an antibody described herein comprises an IgA1 constant region comprising an IgA1 CH2 region and an IgA1 CH3 region. In some embodiments, the IgA1 constant region further comprises an IgA1 CH1 region. In some embodiments, the IgA heavy chain constant region comprises an IgA2 constant region comprising an IgA2 CH2 region and an IgA2 CH3 region. In some embodiments, the IgA2 constant region further comprises an IgA2 CH1 region. In some embodiments, the antibody or functional fragment thereof exhibits a circulating half-life within 1%, 5%, 10%, 20%, or 30% of the circulating half-life of a corresponding antibody comprising an antigen-binding domain and an IgA heavy chain constant region comprising an IgG CH2 region and an IgG CH3 region.

[0024] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA2 CH3 region, wherein the IgA2 heavy chain constant region comprises an N135Q amino acid substitution compared to the corresponding WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0025] In some embodiments, the heavy chain constant region further comprises an N45.2G amino acid substitution, an N114T amino acid substitution, an I115L amino acid substitution, a T116S amino acid substitution, and an amino acid substitution selected from the group consisting of N15.2G, N15.2Q, and N15.2T, relative to a WT IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the heavy chain constant region further comprises a C86S amino acid substitution, a P124R amino acid substitution, a deletion of C147, a deletion of Y148, a L15.3I amino acid substitution, a T16S amino acid substitution, or a combination thereof, relative to a WT IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0026] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH2 region and an IgA2 CH3 region, wherein the IgA2 heavy chain constant region comprises a C86S amino acid substitution, an N114T amino acid substitution, an I115L amino acid substitution, a T116S amino acid substitution, and an N135Q amino acid substitution compared to a WT IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0027] Provided herein is an antibody or functional fragment thereof comprising an antigen-binding domain and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA CH3 region, wherein the IgA2 heavy chain constant region comprises an N45.2G amino acid substitution, a C86S amino acid substitution, an N114T amino acid substitution, an I115L amino acid substitution, a T116S amino acid substitution, an N135Q amino acid substitution, a deletion of C147, and a deletion of Y148 compared to a wild-type IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA2 heavy chain constant region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 16.

[0028] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA2 CH3 region, wherein the IgA2 heavy chain constant region comprises an N45.2G amino acid substitution, a C86S amino acid substitution, an N114T amino acid substitution, an I115L amino acid substitution, a T116S amino acid substitution, and a deletion of a C-terminal tail piece, compared to a WT IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0029] In some embodiments, the C-terminal tailpiece deletion comprises a deletion of C-terminal amino acid residues P131 to Y148 relative to the WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0030] In some embodiments, the IgA2 heavy chain constant region of an antibody described herein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO:17.

[0031] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA2 CH3 region, wherein the IgA2 heavy chain constant region comprises an N45.2G amino acid substitution, an N114T amino acid substitution, an I115L amino acid substitution, a T116S amino acid substitution, an N135Q amino acid substitution, an amino acid substitution selected from the group consisting of N15.2G, N15.2Q, and N15.2T, an L15.3I amino acid substitution, and a T16S amino acid substitution, relative to a WT IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0032] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA2 CH3 region, wherein the IgA2 heavy chain constant region comprises, relative to a WT IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme, an N45.2G amino acid substitution, an N114T amino acid substitution, an I115L amino acid substitution, a T116S amino acid substitution, an amino acid substitution selected from the group consisting of N15.2G, N15.2Q, and N15.2T, an L15.3I amino acid substitution, a T16S amino acid substitution, and a deletion of C-terminal amino acids P131 to Y148.

[0033] In some embodiments, an antibody or functional fragment thereof disclosed herein comprises a C86S amino acid substitution, a P124R amino acid substitution, a deletion of C147, a deletion of Y148, or a combination thereof, relative to a wild-type IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme. In some embodiments, the IgA2 heavy chain constant region comprises an amino acid sequence at least 95% identical to a sequence selected from any one of SEQ ID NOs: 18-21. In some embodiments, the antibody or functional fragment thereof is aglycosylated. In some embodiments, the antibody or functional fragment thereof has an extended circulating half-life relative to a corresponding IgA comprising one or more of wild-type amino acid residues N45.2, N114, I115, T116, N15.2, L15.3, T16, or the C-terminal amino acid residues P131-Y148, numbered according to the IMGT scheme.

[0034] In some embodiments, the antibodies or functional fragments thereof described herein induce increased antibody-dependent cellular cytotoxicity (ADCC) compared to a corresponding antibody comprising an IgG heavy chain constant domain. In some embodiments, the antibodies or functional fragments thereof exhibit increased thermal stability compared to a corresponding IgA comprising one or more of wild-type amino acid residues N45.2, N114, I115, T116, N15.2, L15.3, and T16, or C-terminal amino acid residues P131-Y148, numbered according to the IMGT scheme. In some embodiments, the antibodies or functional fragments thereof exhibit reduced glycosylation compared to a corresponding IgA comprising one or more of wild-type amino acid residues N45.2, N114, I115, T116, N15.2, L15.3, and T16, or C-terminal amino acid residues P131-Y148, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region exhibits increased affinity binding to an FcαR expressed on an immune effector cell compared to a corresponding IgA comprising one or more of wild-type amino acid residues N45.2, N114, I115, T116, N15.2, L15.3, and T16, or C-terminal amino acid residues P131 to Y148, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region further comprises a hinge region. In some embodiments, the hinge region comprises an IgA hinge amino acid sequence or a variant or fragment thereof. In some embodiments, the hinge region comprises a human IgA hinge amino acid sequence or a variant or fragment thereof. In some embodiments, the hinge is an IgA1 hinge or an IgA2 hinge, or a variant or fragment thereof. In some embodiments, the constant domain further comprises a light chain constant region. In some embodiments, the light chain constant region is a kappa light chain constant region, and the kappa light chain constant region comprises the sequence of SEQ ID NO: 31. In some embodiments, the IgA heavy chain constant region further comprises one or more albumin binding domains. In some embodiments, one or more albumin binding domains are fused to the C-terminus of the CH3 region. In some embodiments, the constant region comprises a light chain constant region, and one or more albumin binding domains are fused to the light chain constant region.In some embodiments, the antibody or functional fragment thereof has a longer circulating half-life compared to a corresponding IgA antibody that does not contain one or more albumin-binding domains, hi some embodiments, the antibody or functional fragment thereof exhibits reduced complement-dependent cytotoxicity (CDC) compared to a corresponding antibody that contains an IgG heavy chain constant domain, as measured in an appropriate in vitro CDC assay.

[0035] In some embodiments, the antibodies or functional fragments thereof described herein exhibit increased antibody-dependent cell-mediated cytotoxicity (ADCC) compared to a corresponding wild-type IgA antibody, as measured in an appropriate in vitro ADCC assay. In some embodiments, the antigen-binding domain comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region of the antibodies described herein comprises at least one of the following complementarity-determining regions (CDRs): HC-CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 33-40; HC-CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 41-48; and HC-CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 49-56.

[0036] In some embodiments, the light chain variable region of an antibody described herein comprises at least one of the following complementarity determining regions (CDRs): LC-CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 57-64; LC-CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 65-72; and LC-CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 73-80.

[0037] In some embodiments, the heavy chain variable region of the described antibody comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity to an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 81-86. In some embodiments, the light chain variable region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identity to an amino acid sequence selected from any one of SEQ ID NOs: 5, 8, 95-100. In some embodiments, the heavy chain CDRs and light chain CDRs are each derived from an IgG antibody. In some embodiments, the heavy chain variable region further comprises four framework regions (FW): HC-FW1, HC-FW2, HC-FW3, and HC-FW4. In some embodiments, the light chain variable region further comprises four framework regions (FW): LC-FW1, LC-FW2, LC-FW3, and LC-FW4.

[0038] In some embodiments, the heavy chain FW region and the light chain FW region are each derived from an IgG antibody, hi some embodiments, the heavy chain FW region and the light chain FW region are each derived from an IgA antibody.

[0039] In some embodiments, the antibody or functional fragment thereof described herein is a chimeric antibody, a heavy chain antibody, a single chain antibody, a humanized antibody, a human antibody, a monoclonal antibody, a deimmunized antibody, a bispecific antibody, a multispecific antibody, a multivalent antibody, or a combination thereof.

[0040] In some embodiments, antigen-binding fragments of antibodies described herein include Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody, linear antibody, single domain antibody (sdAb), VHH domain, or multispecific antibody formed from antibody fragments. In some embodiments, the variable domain specifically binds to GD2, CD20, CD47, CD38, CD19, EGFR, HER2, PD-L1, or CD25. In some embodiments, the antibody or functional fragment thereof further comprises an enzyme, substrate, cofactor, fluorescent marker, chemiluminescent marker, peptide tag, magnetic particle, drug, toxin, radionuclide, binding site for a secondary antibody, metal-binding domain, or a combination thereof.

[0041] Provided herein is a pharmaceutical composition comprising the antibody or functional fragment thereof of any one of the above aspects and a pharmaceutically acceptable carrier.

[0042] Provided herein is a method of treating a subject in need thereof or a composition for use in treating a subject in need thereof, the method or composition comprising administering to the subject a therapeutic dose of an antibody or functional fragment thereof of any one of the above embodiments, or a pharmaceutical composition disclosed above.

[0043] In some embodiments, the antibody or functional fragment thereof, or pharmaceutical composition has cytolytic activity against target cells. In some embodiments, the target cells are cancer cells. In some embodiments, the antibody or functional fragment thereof, or pharmaceutical composition inhibits tumor growth. In some embodiments, the antibody or functional fragment thereof, or pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. In some embodiments, the antibody or functional fragment thereof, or pharmaceutical composition is administered to a subject in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an anti-cancer agent, a chemotherapeutic agent, radiation therapy, a cytotoxic agent, a nutritional supplement such as an NSAID, a corticosteroid, an antioxidant, or a combination thereof. In some embodiments, the second therapeutic agent is administered before, simultaneously with, or after administration of the antibody or functional fragment thereof, or pharmaceutical composition.

[0044] Provided herein is an isolated nucleic acid encoding the antibody or functional fragment thereof of any one of the above embodiments.

[0045] Provided herein is an isolated nucleic acid molecule encoding a heavy chain polypeptide, the isolated nucleic acid molecule comprising a first nucleic acid sequence encoding an IgA heavy chain constant region, wherein the first nucleic acid sequence is selected from any one of SEQ ID NOs: 25 to 32. In some embodiments, the isolated nucleic acid molecule of the above aspect further comprises a second nucleic acid sequence encoding a variable heavy chain region, wherein the second nucleic acid sequence is selected from any one of SEQ ID NOs: 87 to 84.

[0046] Provided herein is a vector comprising the isolated nucleic acid molecule of any one of the above aspects.

[0047] Provided herein is a host cell comprising the isolated nucleic acid molecule of any one of the above aspects. In some embodiments, the host cell of any one of the above aspects further comprises an isolated nucleic acid molecule encoding a light chain polypeptide, wherein the isolated nucleic acid molecule encoding the light chain polypeptide comprises a nucleic acid sequence encoding a variable light chain region, wherein the nucleic acid sequence is selected from any one of SEQ ID NOs: 101-108. In some embodiments, the isolated nucleic acid molecule encoding the light chain polypeptide further comprises a nucleic acid sequence encoding a kappa light chain constant region, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO: 32.

[0048] Provided herein is a host cell that expresses the antibody or functional fragment thereof according to any one of the above aspects. In some embodiments, the host cell is a bacterial cell or a mammalian cell. In some embodiments, the host cell is a CHO cell or a HEK293 cell.

[0049] Provided herein are methods for producing an antibody or functional fragment thereof, the method comprising: (a) culturing a host cell of any one of the above aspects in a culture medium under conditions that allow expression of the polypeptide encoded by the isolated nucleic acid molecule and assembly of the antibody or functional fragment thereof; and (b) purifying the antibody or functional fragment thereof from the cultured host cell or the host cell culture medium. In some embodiments, the purifying step is by size exclusion chromatography.

[0050] Provided herein is an antibody or functional fragment thereof that selectively binds to a CD20 polypeptide or a variant thereof, the antibody or functional fragment thereof comprising: (a) an IgA heavy chain constant region comprising an amino acid sequence selected from any one of SEQ ID NOs: 16-21; and (b) a variable heavy chain region comprising at least one of a complementarity-determining region heavy chain 1 (HC-CDR1) of SEQ ID NO: 33, a HC-CDR2 of SEQ ID NO: 41, and a HC-CDR3 of SEQ ID NO: 49; (c) a variable light chain region comprising at least one of a complementarity-determining region light chain 1 (LC-CDR1) of SEQ ID NO: 57, a LC-CDR2 of SEQ ID NO: 65, and a LC-CDR3 of SEQ ID NO: 73; or (d) the variable heavy chain of (b) and the variable light chain of (c). In some embodiments, the variable heavy chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 81, and the variable light chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0051] Provided herein is an antibody or functional fragment thereof that selectively binds to a GD2 protein or a variant thereof, the antibody or functional fragment thereof comprising: (a) an IgA heavy chain constant region comprising an amino acid sequence selected from any one of SEQ ID NOs: 16-21; and (b) a variable heavy chain region comprising at least one of a complementarity-determining region heavy chain 1 (HC-CDR1) of SEQ ID NO: 34, a HC-CDR2 of SEQ ID NO: 42, and a HC-CDR3 of SEQ ID NO: 50; (c) a variable light chain region comprising at least one of a complementarity-determining region light chain 1 (LC-CDR1) of SEQ ID NO: 58, a LC-CDR2 of SEQ ID NO: 66, and a LC-CDR3 of SEQ ID NO: 74; or (d) the variable heavy chain of (b) and the variable light chain of (c). In some embodiments, the variable heavy chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the variable light chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0052] Provided herein is an antibody or functional fragment thereof that selectively binds to a Her2 protein or a variant thereof, the antibody or functional fragment thereof comprising: (a) an IgA heavy chain constant region comprising an amino acid sequence selected from SEQ ID NOs: 16-21; and (b) a variable heavy chain region comprising at least one of a complementarity-determining region heavy chain 1 (HC-CDR1) of SEQ ID NO: 35, a HC-CDR2 of SEQ ID NO: 43, and a HC-CDR3 of SEQ ID NO: 51; (c) a variable light chain region comprising at least one of a complementarity-determining region light chain 1 (LC-CDR1) of SEQ ID NO: 59, a LC-CDR2 of SEQ ID NO: 67, and a LC-CDR3 of SEQ ID NO: 75; or (d) the variable heavy chain of (b) and the variable light chain of (c). In some embodiments, the variable heavy chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 82, and the variable light chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 96.

[0053] Provided herein is an antibody or functional fragment thereof that selectively binds to a gp75 protein or a variant thereof, the antibody or functional fragment thereof comprising: (a) an IgA heavy chain constant region comprising an amino acid sequence selected from SEQ ID NOs: 16-21; and (b) a variable heavy chain region comprising at least one of a complementarity-determining region heavy chain 1 (HC-CDR1) of SEQ ID NO: 36, a HC-CDR2 of SEQ ID NO: 44, and a HC-CDR3 of SEQ ID NO: 52; (c) a variable light chain region comprising at least one of a complementarity-determining region light chain 1 (LC-CDR1) of SEQ ID NO: 60, a LC-CDR2 of SEQ ID NO: 68, and a LC-CDR3 of SEQ ID NO: 76; or (d) the variable heavy chain of (b) and the variable light chain of (c). In some embodiments, the variable heavy chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 83, and the variable light chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 97.

[0054] Provided herein is an antibody or functional fragment thereof that selectively binds to a CTLA4 protein or a variant thereof, the antibody or functional fragment thereof comprising: (a) an IgA heavy chain constant region comprising an amino acid sequence selected from SEQ ID NOs: 16-21; and (b) a variable heavy chain region comprising at least one of a complementarity-determining region heavy chain 1 (HC-CDR1) of SEQ ID NO: 37, a HC-CDR2 of SEQ ID NO: 45, and a HC-CDR3 of SEQ ID NO: 53; (c) a variable light chain region comprising at least one of a complementarity-determining region light chain 1 (LC-CDR1) of SEQ ID NO: 61, a LC-CDR2 of SEQ ID NO: 69, and a LC-CDR3 of SEQ ID NO: 77; or (d) the variable heavy chain of (b) and the variable light chain of (c). In some embodiments, the variable heavy chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 84, and the variable light chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 98.

[0055] Provided herein is an antibody or functional fragment thereof that selectively binds to a CD47 protein or a variant thereof, the antibody or functional fragment thereof comprising: (a) an IgA heavy chain constant region comprising an amino acid sequence selected from SEQ ID NOs: 16-21; and (b) a variable heavy chain region comprising at least one of a complementarity-determining region heavy chain 1 (HC-CDR1) of SEQ ID NO: 38, a HC-CDR2 of SEQ ID NO: 46, and a HC-CDR3 of SEQ ID NO: 54; (c) a variable light chain region comprising at least one of a complementarity-determining region light chain 1 (LC-CDR1) of SEQ ID NO: 62, a LC-CDR2 of SEQ ID NO: 70, and a LC-CDR3 of SEQ ID NO: 78; or (d) the variable heavy chain of (b) and the variable light chain of (c). In some embodiments, the variable heavy chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 85, and the variable light chain comprises a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 99.

[0056] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA2 CH3 region, wherein the IgA2 heavy chain constant region comprises an N45.2G amino acid substitution, an N135Q amino acid substitution, a deletion of C147, and a deletion of Y148 compared to a WT IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0057] Provided herein is an antibody or functional fragment thereof comprising: an antigen-binding domain; and a constant domain comprising an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA2 CH3 region, wherein the IgA2 heavy chain constant region comprises an amino acid substitution selected from the group consisting of an N45.2G amino acid substitution, an N114T amino acid substitution, an I115L amino acid substitution, a T116S amino acid substitution, an N15.2G, an N15.2Q, and an N15.2T, and a deletion of C-terminal amino acids P131 to Y148, relative to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0058] In some embodiments, the antibody or functional fragment thereof of the above aspect further comprises a C86S amino acid substitution, a P124R amino acid substitution, a deletion of C147, a deletion of Y148, a L15.3I amino acid substitution, a T16S amino acid substitution, or a combination thereof, relative to a WT IgA heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 1, numbered according to the IMGT scheme.

[0059] In some embodiments, the IgA2 heavy chain constant region comprises an amino acid sequence at least 95% identical to a sequence selected from any one of SEQ ID NOs: 18 to 21. In some embodiments, the antibody or functional fragment thereof is aglycosylated.

[0060] In some embodiments, the antibody or functional fragment thereof has a circulating half-life that is increased relative to a corresponding IgA that includes one or more of wild-type amino acid residues N45.2, N114, I115, T116, N15.2, or the C-terminal amino acid residues P131 to Y148, numbered according to the IMGT scheme.

[0061] In some embodiments, the antibody or functional fragment thereof induces increased antibody-dependent cellular cytotoxicity (ADCC) compared to a corresponding antibody comprising an IgG heavy chain constant domain. In some embodiments, the antibody or functional fragment thereof exhibits increased thermal stability compared to a corresponding IgA comprising one or more of wild-type amino acid residues N45.2, N114, I115, T116, N15.2, or C-terminal amino acid residues P131-Y148, numbered according to the IMGT scheme. In some embodiments, the antibody or functional fragment thereof exhibits reduced glycosylation compared to a corresponding IgA comprising one or more of wild-type amino acid residues N45.2, N114, I115, T116, N15.2, or C-terminal amino acid residues P131-Y148, numbered according to the IMGT scheme. Incorporation by Reference

[0062] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0063] The features of the present disclosure are set forth with particularity 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 and accompanying drawings that set forth illustrative embodiments, in which the principles of the disclosure are utilized. [Brief explanation of the drawings]

[0064] [Figure 1]1 depicts the amino acid sequence (SEQ ID NO: 1) of the IgA2 heavy chain (UniProt Reference A0A0G2JMB2). Highlighted amino acids indicate amino acids that are subject to substitution in some embodiments described herein. Underlined sequences indicate tail pieces, all or part of which may be deleted in some embodiments described herein.

[0065] [Figure 2] 2 depicts the amino acid sequence (SEQ ID NO: 2) of the IgA2 heavy chain (UniProt Reference No. P01877). Highlighted amino acids indicate amino acids that are subject to substitution in some embodiments described herein. Underlined sequences indicate tail pieces, all or part of which may be deleted in some embodiments described herein.

[0066] [Figure 3] 3 depicts the amino acid sequence (SEQ ID NO: 3) of a representative IgA2 heavy chain (UniProt reference number A0A286YEY5). Highlighted amino acids indicate amino acids that are subject to substitution or deletion in some embodiments described herein. In some further embodiments, the corresponding amino acids may be deleted in an equivalent IgA antibody or an equivalent chimeric antibody having an IgA2 constant region.

[0067] [Figure 4] Figures 4A-4D are schematic diagrams of engineered IgA variants compared to wild-type IgA (IgA2(m1)). Figure 4A shows a depiction of the wild-type (WT) IgA;IgA2m1 antibody. Wild-type (WT) IgA;IgA2m1 contains three N-glycosylation sites in the CH domain and one glycosylation site in the tailpiece.

[0068] Figure 4B shows a depiction of the engineered IgA3.0+ (plus) variant. The engineered IgA3.0+ variant was generated by engineering the IgA2m1 antibody to contain a stabilized heavy-to-light chain linkage via the CH1-P124R mutation, the removal of two free cysteines: one mutated to serine (CH2-C86S), and the second cysteine ​​removed by deletion of the last two amino acids of the tailpiece (CH3-CHS-C147del). Additionally, three N-linked glycosylation sites were removed by substitution of critical amino acids within the three N-glycosylation motifs. The mutations in the three N-glycosylation motifs include CH1-N45.2G; CH2-N114T-I115L-T116S; and CH3-CHS-N135Q.

[0069] Figure 4C shows a depiction of an engineered IgA3.0- or IgA3.0min variant containing a stabilized heavy and light chain linkage (CH1-P124R mutation), a deletion of the entire tail piece (CH3-CHS-P131-Y148del), and the absence of two free cysteines, one of which is mutated to serine (CH2-C86S) and the second cysteine ​​in the tail piece (CH3-CHS-C147del). Furthermore, three N-linked glycosylation sites have been eliminated by substitution of crucial amino acids within two N-glycosylation motifs, namely CH1-N45.2G and CH2-N114T-I115L-T116S, and deletion of the tailpiece CH3-CHS-N135Qdel.

[0070] Figure 4D shows a depiction of an engineered IgA4.0 variant that contains all of the features of IgA3.0min and additionally contains a mutation in the final N-linked glycosylation motif, CH2-N15.2. Thus, the IgA4.0 variant contains a stabilized heavy-to-light chain linkage (CH1-P124R mutation), a deletion of the entire tailpiece (CH3-CHS-P131-Y148del), and the absence of two free cysteines, one of which is mutated to serine (CH2-C86S) and the second cysteine ​​in the tailpiece (CH3-CHS-C147del). Additionally, four N-linked glycosylation sites have been eliminated by substitution of critical amino acids within four N-glycosylation motifs (CH1-N45.2G; CH2-N114T-I115L-T116S; CH3-CHS-N135Q; and one of CH2-N15.2G; CH2-N15.2Q; CH2-N15.2T; or CH2-N15.2T-L15.3I-T16S). The IgA4.0 variant is an unglycosylated IgA.

[0071] [Figure 5] 5A and 5B are graphs demonstrating the yield of IgA3.0min antibody variants.

[0072] FIG. 5A is a graph showing the concentration of anti-Her2 IgA antibodies, ie, IgA2-Her2, determined by ELISA on supernatants of HEK293F cells transfected with various heavy chain (HC):light chain (LC):pAdvantage ratios.

[0073] Figure 5B is a graph showing the concentration of anti-CD47 IgA3.0min antibody, i.e., IgA3.0min antibody containing the anti-CD47 variable domain (IgA3.0min-C47A8-CQ), as determined by ELISA on supernatants of HEK293F cells transfected with various heavy chain (HC):light chain (LC):pAdvantage ratios. The variable domain was obtained from the C47A8-CQ antibody.

[0074] [Figure 6] 6A to 6C are graphs showing a comparison of the yield of antibody IgA3.0min antibody variants in HEK293F cells and ExpiCHO-S cells, as determined by ELISA.

[0075] Figure 6A is a graph showing the production rate of anti-CD20 IgA3.0min-Obi antibody in HEK293F cells compared to the production rate of anti-CD20 IgA3.0min-Obi antibody in ExpiCHO cells. The IgA3.0min-Obi antibody contains IgA3.0min with the obinutuzumab (Obi) variable domain.

[0076] Figure 6B is a graph showing a comparison of the production rates of anti-Her2 IgA3.0min-Her2 antibodies in HEK293F cells and ExpiCHO cells. The IgA3.0min-Her2 antibodies contain IgA3.0min with an anti-Her2 variable domain.

[0077] Figure 6C is a graph showing the comparison of the production rates of anti-mCTLA4 IgA3.0min-mCTLA4 antibodies in HEK293F cells and ExpiCHO cells. IgA3.0min-mCTLA4 contains IgA3.0min with the anti-mCTLA4 variable domain.

[0078] [Figure 7] 7A-7D are graphs demonstrating the concentration of IgA3.0min antibody variants in ExpiCHO-S cells transfected with various heavy chain (HC):light chain (LC):pAdvantage ratios.

[0079] 7A is a graph showing the concentration of anti-GD2 IgA3.0min-ch14.18 antibody, which contains IgA3.0min with the ch14.18 variable domain.

[0080] Figure 7B is a graph showing the concentration of anti-gp75 IgA3.0min-TA99 antibody, which contains IgA3.0min with the TA99 variable domain.

[0081] Figure 7C is a graph showing the concentration of anti-Her2 IgA3.0min-Her2 antibody, which contains IgA3.0min with an anti-Her2 variable domain (the anti-Her2 variable domain was derived from the anti-Her2 antibody trastuzumab).

[0082] Figure 7D is a graph showing the concentration of anti-CD20 IgA3.0min-Obi antibody, which contains IgA3.0min with the obinutuzumab (Obi) variable domain.

[0083] 8A-8D are graphs showing the production rate of IgA4.0 antibody variants in ExpiCHO-S cells transfected with various heavy chain (HC):light chain (LC):pAdvantage ratios.

[0084] [Figure 8-1] 8A is a graph showing the production of the anti-CD20 IgA4.0_NG-Obi variant, which contains variable domains from IgA4.0 and Obi with the CH2-N15.2G mutation at the glycosylation site.

[0085] [Figure 8-2] Figure 8B is a graph showing the production of the anti-CD20 IgA4.0_NT-Obi variant, which contains variable domains from IgA4.0 and Obi with the CH2-N15.2T mutation at the glycosylation site.

[0086] [Figure 8-3]Figure 8C is a graph showing the production of the anti-CD20 IgA4.0_NQ-Obi variant, which contains variable domains from IgA4.0 and Obi with the CH2-N15.2Q mutation at the glycosylation site.

[0087] [Figure 8-4] Figure 8D is a graph showing the production of the anti-CD20 IgA4.0_NLT-TIS-Obi variant, which contains variable domains from IgA4.0 and Obi with N15.2T-L15.3I-T16S mutations at glycosylation sites.

[0088] [Figure 9-1] 9A-9B are graphs showing the elution profiles of IgA3.0min-Obi from supernatants derived from the producer cell lines ExpiCHO-S (FIG. 9A) and HEK293F (FIG. 9B) using a KappaSelect column (GE Healthcare).

[0089] [Figure 9-2] 9C-9D are graphs showing the SEC separation profiles of IgA3.0min-Obi from the producing cell lines ExpiCHO-S (FIG. 9C) and HEK293F (FIG. 9D). No aggregates are observed in the SEC separation.

[0090] [Figure 10-1] 10A-10B are graphs showing the elution profiles of IgA3.0min-Her2 from supernatants derived from the producer cell lines ExpiCHO-S (FIG. 10A) and HEK293F (FIG. 10B) using a KappaSelect column (GE Healthcare).

[0091] [Figure 10-2]10C-10D are graphs showing the SEC elution profiles of IgA3.0min-Her2 from the producing cell lines ExpiCHO-S (FIG. 10C) and HEK293F (FIG. 10D). No aggregates are observed by SEC.

[0092] [Figure 11-1] FIG. 11A is a graph showing a typical elution profile of IgA4.0-Obi from supernatant derived from the producer cell line ExpiCHO-S using a KappaSelect column (GE Healthcare).

[0093] [Figure 11-2] Figure 11B is a graph showing the SEC elution profile of IgA4.0-Obi from the producer cell line ExpiCHO-S. No aggregates are observed by SEC.

[0094] 12A to 12E are graphs showing the binding of IgA3.0+-Obi and IgA3.0min-Obi to CD20-positive Daudi cells by flow cytometry.

[0095] [Figure 12-1] FIG. 12A is a graph showing an unstained CD20+ Daudi cell control.

[0096] [Figure 12-2] FIG. 12B is a graph showing Daudi cells stained with a negative control of secondary antibody only.

[0097] [Figure 12-3] Figure 12C is a graph showing staining with anti-CD20 IgA Obi (5 μg / mL) positive control.

[0098] [Figure 12-4]Figure 12D is a graph showing Daudi cells stained with IgA3.0+-Obi (supernatant). Figure 12E is a graph showing Daudi cells stained with IgA3.0min-Obi (supernatant). Figures 12D-12E are graphs showing Daudi cells stained with supernatant from HEK293F cells transfected with IgA3.0min-Obi or IgA3.0+-Obi. Both variants, IgA3.0+-Obi (Figure 12D) and IgA3.0min-Obi (Figure 12E), bind to CD20-positive Daudi to the same extent as IgA Obi (Figure 12C). [Figure 12-5] Same as above.

[0099] [Figure 13] Figure 13 is a graph showing the binding of the Fc portion of IgA3.0min-Obi to PMNs, as assessed by a PMN binding assay. PMNs were added to ELISA plate wells coated with various concentrations of antibody. A series of washes determines the binding strength of the Fc portion of IgA3.0min-Obi and IgA2 to PMNs. Binding is plotted after the sixth wash. IgA3.0min-Obi shows equal to better binding to PMNs than wild-type IgA2.

[0100] [Figure 14] FIG. 14 shows equivalent coating concentrations of IgA3.0 min compared to wild-type IgA2.

[0101] [Figure 15]Figure 15 shows binding analysis of IgA4.0 variants. Supernatants from ExpiCHO-S cells transfected with IgA4.0 variants IgA4.0_NT-Obi, IgA4.0_NQ-Obi, IgA4.0_NG-Obi, and IgA4.0_NLT-TIS-Obi were evaluated for binding to CD20-expressing SKBR3 cells. All IgA4.0-Obi variants bind to SKBR3-CD20 to the same extent as supernatants from ExpiCHO-S cells transfected with IgA3.0min-Obi or purified IgA3.0min-Obi.

[0102] 16A-16D are graphs showing that IgA variants induce PMN-mediated ADCC against target cells.

[0103] [Figure 16-1] FIG. 16A is a graph showing that the IgA3.0min-Her2 antibody induces ADCC to a similar or better extent than IgA2-Her2 in SKBR3 cells, as determined by a chromium release assay.

[0104] [Figure 16-2] Figures 16B-16C are graphs showing that IgA3.0min-Obi antibodies produced by HEK293F cells or ExpiCHO-S cells induce similar levels of ADCC against Ramos cells (Figure 16B) and Daudi cells (Figure 16C), as assessed by a chromium release assay.

[0105] [Figure 16-3]Figure 16D is a graph showing induction of ADCC by IgA variants against Daudi cells. Purified IgA3.0min-Obi, supernatants from cells transfected with IgA3.0min-Obi, IgA4.0_NG-Obi, IgA4.0_NQ-Obi, IgA4.0_NT-Obi, and IgA4.0_NLT-TIS-Obi show increased ADCC compared to purified IgG1-Obi. The IgA4.0-obi variants, IgA4.0_NG-Obi, IgA4.0_NQ-Obi, IgA4.0_NT-Obi, and IgA4.0_NLT-TIS-Obi, show similar levels of ADCC induction compared to purified IgA3.0min-Obi and supernatants from cells transfected with IgA-3.0min-Obi.

[0106] Figures 17A-17C are graphs showing the thermal stability of IgA variants. The thermal stability of IgA3.0min and IgA4.0 was determined using a SYPRO Orange thermal shift assay. Figure 17A is a graph demonstrating that the engineered IgA3.0min variants, IgA3.0min with the Obi variable domain and IgA3.0min with the 2.3D11 variable domain, exhibit increased thermal stability compared to wild-type IgA2 (m1), i.e., IgA2 with the ch14.18 variable domain and IgA2 with the Her2 variable domain.

[0107] [Figure 17-1]Figure 17A also demonstrates that the engineered IgA4.0min variants, IgA4.0 NQ, IgA4.0 NT, IgA4.0 NLT-TIS, and IgA4.0 NG, all with the Obi variable domain, exhibit increased thermal stability compared to wild-type IgA2(m1), i.e., IgA2 with the ch14.18 variable domain and IgA2 with the Her2 variable domain. The increased thermal stability of the engineered IgA3.0min variants and the engineered IgA4.0 variants compared to wild-type IgA2(m1) is indicated by a shift to higher temperatures.

[0108] [Figure 17-2] Figure 17B is a graph showing a plot of the mean Tm values ​​of the analyzed engineered IgA3.0min and engineered IgA4.0 variants relative to wild-type IgA2 (m1). The plot shows that the engineered IgA3.0min and engineered IgA4.0 variants are more thermostable compared to wild-type IgA2.

[0109] [Figure 17-3] Figure 17C is a graph showing antibodies exposed to increasing temperatures tested for functionality in PMN-ADCC against CD20-expressing Raji cells. Wild-type IgA2(m1) shows a gradual decrease in efficacy from 47°C onwards, while both IgA3.0min-Obi and IgA4.0_NT-Obi remain effective. At 71°C, both IgA3.0min-Obi and IgA4.0_NT-Obi show greater than 50% killing efficacy, while IgA2(m1) does not function at this temperature.

[0110] [Figure 18]Figure 18 shows the effect of PNGase F treatment on deglycosylation of engineered IgA3.0min and engineered IgA4.0 variants compared to wild-type IgA2. Wild-type IgA2 (IgA2(m1)-UMAB10) shows the largest shift, while engineered IgA3.0min variant IgA3.0min-Obi shows a minor shift, indicating reduced glycosylation compared to wild-type IgA2. Engineered IgA4.0 variant IgA4.0_NG-Obi shows no shift, indicating reduced glycosylation compared to engineered IgA3.0min and wild-type IgA2.

[0111] 19A-19G show the gross glycosylation profiles of wild-type IgA2 and engineered IgA variants as determined using MALDI-TOF-MS.

[0112] [Figure 19-1] FIG. 19A is a graph showing the gross glycosylation profile of wild-type IgA2; IgA2-Her2 produced by HEK293 cells.

[0113] [Figure 19-2] Figure 19B is a graph showing the total glycosylation profile of the engineered IgA3.0min variant, IgA3.0min-Her2, produced by HEK293 cells, which shows all signals from the single glycosylation site present in the engineered IgA3.0min variant.

[0114] [Figure 19-3]Figure 19C is a graph showing glycosylation site-specific analysis of the engineered IgA3.0min variant, IgA3.0min-Obi, produced in HEK293F and ExpiCHO-S. IgA3.0min-Obi produced in ExpiCHO-S exhibited less free galactose, indicating that it was less susceptible to ASGPR-dependent clearance in the liver.

[0115] [Figure 19-4] Figures 19D-19G show native MS analyses of four IgA4.0 variants: IgA4.0_NG-Obi (Figure 19D), IgA4.0_NQ-Obi (Figure 19E), IgA4.0_NT-Obi (Figure 19F), and IgA4.0_NLT-TIS-Obi (Figure 19G). The large plot shows the broad mass range of the antibodies, and the inset is a zoom-in of the highest peak. The observed masses are virtually equal to the theoretical masses excluding the presence of bulky N-glycans, indicating the absence of glycosylation in the IgA4.0 variants.

[0116] 20A-20B are graphs showing pharmacokinetic and drug distribution analysis of engineered IgA variants. 100 μg amounts of wild-type IgG1-dinutuximab, wild-type IgA2-dinutuximab, and IgA3.0-dinutuximab, IgA3.0-obinutuzumab, and IgA4.0-obinutuzumab were injected into BALB / c mice, and blood was analyzed by ELISA at the indicated time points.

[0117] [Figure 20-1] FIG. 20A is a graph showing that the IgA3.0min variant; IgA3.0min-dinutuximab exhibited an extended half-life compared to its wild-type IgA2 counterpart.

[0118] [Figure 20-2]Figure 20B is a graph showing a comparison of IgA3.0min and IgA4.0 in the obinutuzumab format. The engineered IgA4.0 variant, IgA4.0-obinutuzumab, exhibited a better half-life profile than the engineered IgA3.0min variant, IgA3.0min-obinutuzumab. Obinutuzumab IgA4.0 was still detectable after 120 hours.

[0119] [Figure 21] Figures 21A-21B are photographs showing the biodistribution of IgG1 dinutuximab and an engineered IgA3.0min variant, IgA3.0min dinutuximab, containing the variable domains from dinutuximab. Indium-111 radiolabeled IgG1 dinutuximab and an engineered IgA3.0min variant, IgA3.0min dinutuximab, were injected intravenously into mice for biodistribution analysis. Mice were monitored to track antibody distribution after 24 hours (Figure 21A) and 48 hours (Figure 21B). Clear infiltration of both the IgG1 and the engineered IgA3.0min variant into the tumor is observed.

[0120] [Figure 22] Figure 22 is a graph showing quantification of the distribution of radiolabeled IgG1 dinutuximab and the engineered IgA3.0min variant, IgA3.0min dinutuximab, in the mice from Figures 21A-21B. The ratio of tumor signal to liver signal was determined and adjusted for background signal. The plot shows that the engineered IgA3.0min variant, IgA3.0min dinutuximab, showed an increased tumor / liver ratio compared to IgG1 dinutuximab.

[0121] [Figure 23]Figure 23 is a graph showing tumor growth inhibition in an established tumor model following administration of an engineered IgA3.0min variant, IgA3.0min-Her2. A431-Luc2-Her2 cells were intraperitoneally injected into hCD89 transgenic or non-transgenic SCID mice on day 0, followed by subcutaneous injection of PEG-CSF on day 6. Bioluminescence signals were measured from day 6 onward, on which day mice were randomly assigned to different treatment groups and significant signals were measured. Treatment began on day 7, with daily intraperitoneal injection of 10 μg of IgA3.0min-Her2 for 10 days. Bioluminescence signals were measured at the indicated time points. DETAILED DESCRIPTION OF THE INVENTION

[0122] The following description and examples illustrate in detail the embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will appreciate that there are numerous variations and modifications of the present disclosure, which are encompassed within the scope of the present disclosure.

[0123] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0124] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0125] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may for clarity be described herein in the context of separate embodiments, the present disclosure may also be practiced in a single embodiment. definition

[0126] The following definitions are for the assistance of those skilled in the art, and are intended for the present application, and are not to be construed as being related or unrelated, for example, to any co-owned patent or application. Although any method and material similar to or equivalent to the methods and materials described herein can be used to carry out the testing of the present disclosure, preferred materials and methods are described herein.Therefore, the terminology used herein is merely intended to describe specific embodiments and is not limiting.

[0127] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0128] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments, but not necessarily in all embodiments of the present disclosure.

[0129] As used in the specification and claims, the word "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), the word "having" (and any form of having, e.g., "have" and "has"), the word "including" (and any form of including, e.g., "includes" and "include"), or the word "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is intended that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to practice the methods of the disclosure.

[0130] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, per practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes 10 and any amount from 9 to 11.

[0131] In yet another example, the term "about," when used in relation to a reference numerical value, can also include a range of values ​​of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is recited in this application and in the claims, unless otherwise specified, the term "about" is assumed to mean within an acceptable range of error for the particular value.

[0132] As used herein, the term "antibody" refers to an immunoglobulin (Ig), whether natural or partially or wholly synthetically produced. The term also encompasses any polypeptide or protein having a binding domain that is, or is homologous to, an antigen-binding domain. The term further includes "antigen-binding fragment" or "functional fragment thereof," or other interchangeable terms for similar binding fragments, such as "fragment of an antibody," "antibody fragment," "functional fragment of an antibody," and others described below.

[0133] Antibodies include, for example, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, chemically engineered antibodies, deimmunized antibodies, affinity matured antibodies, multispecific antibodies (e.g., bispecific and multireactive antibodies), heteroconjugate antibodies, antibody fragments, and combinations thereof (e.g., monoclonal antibodies that are also deimmunized, humanized antibodies that are also deimmunized, etc.).

[0134] The antibody can be, for example, murine, chimeric, humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody. The antigen-binding fragment can include, for example, a Fab', F(ab')2, Fab, FV, rlgG, scFV, hcAb (heavy chain antibody), single domain antibody, VHH, VNAR, sdAb, or nanobody.

[0135] The term "monoclonal antibody," as used herein, refers to an antibody produced by a single clone of B cells and binding to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells and binding to different epitopes of the same antigen. A whole antibody generally consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, while each light chain contains an N-terminal variable (VL) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. The VH and VL regions have a similar general structure, each containing four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody that is responsible for antigen binding.

[0136] As used herein, a "chimeric antibody" refers to an antibody that contains amino acid sequences derived from two different species or two different sources, including synthetic molecules. Non-limiting examples include antibodies containing non-human CDRs and human variable region frameworks or constant or Fc regions, antibodies with binding domains derived from two different monoclonal antibodies, or antibodies containing mutations of one or more amino acid residues to enhance or reduce the biological activity or binding of a portion of the antibody. In certain embodiments, recombinant antibodies are produced from recombinant DNA molecules or synthesized. In certain embodiments, the antibodies described herein are polypeptides encoded by one or more polynucleotides.

[0137] As used herein, "recognize" refers to the association or binding of an antigen with an antigen-binding domain. As used herein, "antigen" refers to an antigenic substance that can induce an immune response in a host. The antigenic substance can be a molecule, such as a costimulatory molecule, that can induce an immune response in a host.

[0138] As used herein, "antibody construct" refers to a construct containing an antigen-binding domain and an Fc domain.

[0139] As used herein, a "binding domain" refers to an antibody or non-antibody domain.

[0140] As used herein, "antigen-binding domain" refers to an antibody- or non-antibody-derived binding domain capable of binding to an antigen. The antigen-binding domain may be a tumor antigen-binding domain or a binding domain capable of binding to an antigen (e.g., a molecule, etc.) on an antigen-presenting cell. When more than one antigen-binding domain is present in a given conjugate or antibody construct, the antigen-binding domains may be numbered (e.g., the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, etc.). Different antigen-binding domains within the same conjugate or construct may target the same or different antigens (e.g., a first antigen-binding domain capable of binding to a tumor antigen, a second antigen-binding domain capable of binding to a molecule on an antigen-presenting cell (APC antigen), and a third antigen-binding domain capable of binding to an APC antigen). The term "antigen-binding domain" refers to a fragment of an antibody that contains a region that specifically binds to an epitope and is complementary to part or all of an antigen. The antigen-binding domain may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0141] As used herein, the term "antigen" refers to a molecule or a portion of a molecule that can react with a recognition site on an antibody. The term "antigen" also includes a molecule or a portion of a molecule (also called an "immunogen") that can elicit an immune response, either by itself or in conjunction with an adjuvant or carrier. The term "antigen" as used herein includes a molecule or a portion of a molecule (epitope) that can elicit the production of an antibody or that can bind to an antibody. The term encompasses materials that react strongly with an antibody and have high specificity for the antibody, as well as materials that react weakly with an antibody and / or have low affinity for the antibody.

[0142] The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen-binding site, known as a paratope, within the variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes arise from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes formed by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on an antigen. Various techniques known to those skilled in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in Antibodies. Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248: 443-463), and peptide cleavage analysis.In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9: 487-496).Another method that can be used to identify the amino acid in a polypeptide that interacts with the antigen-binding domain of an antibody is hydrogen / deuterium exchange, which is detected by mass spectrometry.Generally speaking, hydrogen / deuterium exchange involves deuterium-labeling a protein of interest, and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium labeled).After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids that interact with the antibody.See, for example, Ehring (1999) Analytical Biochemistry 267 (2): 252-259;Engen and Smith (2001) Anal.Chem.73: 256A-265A.X-ray crystallography of antigen / antibody complexes can also be used for epitope mapping.

[0143] As used herein, an "antibody antigen-binding domain" refers to a binding domain derived from an antibody that is capable of binding to an antigen.

[0144] As used herein, "Fc domain" refers to an antibody- or non-antibody-derived Fc domain capable of binding to an Fc receptor. As used herein, "Fc domain" and "Fc-containing domain" may be used interchangeably.

[0145] As used herein, a "target binding domain" refers to a construct containing an antibody- or non-antibody-derived antigen-binding domain capable of binding to an antigen.

[0146] As used herein, the abbreviations for natural l-enantiomeric amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine ​​(C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X can represent any amino acid. In some embodiments, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).

[0147] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of good medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0148] The phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

[0149] The terms "cancer," "tumor," "proliferative disease," "malignant tumor," or "malignant disease" refer to a physiological condition in mammals characterized by deregulated cell growth. Cancer is a class of diseases in which groups of cells exhibit uncontrolled or unwanted growth. Cancer cells can spread to other locations, which can lead to the formation of metastases. The spread of cancer cells within the body can occur, for example, via lymph or blood. Uncontrolled growth, invasion, and metastasis formation are also referred to as malignant characteristics of cancer. These malignant characteristics distinguish cancer from benign tumors, which generally do not invade or metastasize.

[0150] An "antigen recognition moiety" or "antibody recognition domain" refers to a molecule or portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, antibody-like molecule, or fragment thereof, and the antigen is a tumor antigen or an infectious disease antigen.

[0151] The terms "antibody fragment," "antibody fragment," "functional fragment of an antibody," "antigen-binding portion," or their grammatical equivalents are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23 (9): 1126-1129 (2005)). The antibody fragment desirably contains, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the stalk region; (iii) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778). (1998)) and (v) diabodies that are dimers of polypeptide chains, where each polypeptide chain is too short to allow pairing of the VH and VL on the same polypeptide chain, and where the VH and VL are connected by a peptide linker that drives pairing between complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule with a bifunctional antigen-binding site. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent No. 8,603,950. Other antibody fragments may include the variable fragment of a heavy chain antibody (VHH).

[0152] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A common way to define common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, G.E. and Schirmer, R.H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, it is possible to define groups of amino acids in which the amino acids within the group are preferentially exchanged with each other and thus have approximately the same effect on the overall protein structure (Schulz, G.E. and Schirmer, R.H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, such as substitution of arginine with lysine, or vice versa, which can maintain a positive charge; substitution of aspartic acid with glutamic acid, or vice versa, which can maintain a negative charge; substitution of threonine with serine, which can maintain a free -OH; and substitution of asparagine with glutamine, which can maintain a free -NH2. Alternatively or additionally, a therapeutic IgA antibody may comprise the amino acid sequence of a reference protein with at least one non-conservative amino acid substitution.

[0153] The term "non-conservative mutation" or "non-conservative amino acid substitution" involves the substitution of amino acids between different groups, such as the substitution of tryptophan with lysine or serine with phenylalanine, where the non-conservative amino acid substitution is preferably one that does not interfere with or inhibit the biological activity of the therapeutic IgA antibody. Non-conservative amino acid substitutions can enhance the biological activity of the therapeutic IgA antibody, thus increasing the biological activity of the therapeutic IgA antibody compared to the wild-type therapeutic IgA antibody.

[0154] As used herein, "humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are antibodies in which residues from a complementarity-determining region (CDR) of a human immunoglobulin (recipient antibody) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to all or substantially all of a non-human immunoglobulin and all or substantially all of the FR regions corresponding to all or substantially all of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0155] As used herein, an "isolated antibody" is an antibody that has been separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with the antibody's diagnostic or therapeutic use, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous components. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0156] "Substantially purified" means that the antibody or functional fragment thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The term includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations in which the antibody has less than about 30%, less than about 20%, less than about 10%, or less than about 5% (by dry weight) of contaminating proteins and culture medium. In some embodiments, the antibody can be purified by chromatography, e.g., size exclusion chromatography or ion exchange chromatography.

[0157] As used herein, the term "complementarity-determining region" (CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is necessary for antigen binding. Generally, each variable domain has three CDR regions, identified as CDR1, CDR2, and CDR3. The CDRs of a variable heavy chain can be CDR-H1, CDR-H2, and CDR-H3. The CDRs of a variable light chain can be CDR-L1, CDR-L2, and CDR-L3. Exemplary hypervariable loops are located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are: L1 at amino acid residues 24-34, L2 at amino acid residues 50-56, L3 at amino acid residues 89-97, H1 at amino acid residues 31-35B, H2 at amino acid residues 50-65, and H3 at amino acid residues 95-102 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991)). Thus, unless otherwise specified, HVs may be contained within the corresponding CDRs and are referred to herein as "hypervariable loops" of VHs, and VL domains should be construed to also encompass the corresponding CDRs, and vice versa. The more highly conserved regions of variable domains are referred to as framework regions (FRs), as defined below. Native heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), which are largely in a beta-sheet configuration and connected by three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, together with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies.Structural analysis of antibodies has revealed the relationship between the sequences and the shape of the binding site formed by the complementarity-determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215: 175-182 (1990)). Despite the large degree of sequence variability, five of the six loops adopt only a small repertoire of main-chain conformations, referred to as "canonical structures." These conformations are determined primarily by the length of the loop and, secondarily, by the presence of key residues at certain positions within the loops and framework regions that determine conformation through packing, hydrogen bonding, or the ability to adopt unusual main-chain conformations.

[0158] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs within each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) a method based on sequence variability across species (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, Md.)); and (2) a method based on crystallographic studies of antigen-antibody complexes (Allazikani et al. (1997) J. Molec. Biol. 273: 927-948)). CDRs may refer to CDRs defined by either method or a combination of both methods.

[0159] The "constant region" of an antibody refers to the constant region of the antibody light chain, i.e., the light chain constant region, or the constant region of the antibody heavy chain, i.e., the heavy chain constant region, either alone or in combination. The constant region does not vary with antigen specificity.

[0160] As used herein, the term "heavy chain region" includes an amino acid sequence derived from the constant domain of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain region includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. In certain embodiments, an antibody or antigen-binding fragment thereof may include an Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, an antibody or antigen-binding fragment thereof lacks at least a region of the constant domain (e.g., all or part of the CH2 domain). In certain embodiments, at least one, preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain region includes a fully human hinge domain. In other preferred embodiments, the heavy chain region includes a fully human Fc region (e.g., hinge, CH2, and CH3 domain sequences derived from a human immunoglobulin). In certain embodiments, the constant domains comprising the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of the polypeptide may comprise a domain derived from an IgA molecule and a hinge region derived from an IgA1 or IgA2 molecule. In other embodiments, the constant domain is a chimeric domain comprising regions from different immunoglobulin molecules. For example, the hinge may comprise a first region derived from an IgA1 molecule and a second region derived from an IgA2 molecule. As noted above, those skilled in the art will understand that the constant domain of the heavy chain region may be modified, and thus the amino acid sequence of the constant domain of the heavy chain region may vary from that of a naturally occurring (wild-type) immunoglobulin molecule. That is, the polypeptides of the invention disclosed herein may comprise one or more alterations or modifications of the heavy chain constant domain (CH1, hinge, CH2, or CH3) and / or the light chain constant domain (CL). Exemplary modifications include the addition, deletion, or substitution of one or more amino acids within one or more domains. In some embodiments, the modification is selected from the modifications in Table 2.

[0161] Antibodies or antigen-binding fragments thereof of the present disclosure may comprise a CDR3 region that is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Antibodies or antigen-binding fragments thereof of the present disclosure may comprise a CDR3 region that is at least about 18 amino acids in length.

[0162] As used herein, the term "hinge region" includes the region of a heavy chain molecule that joins the CH1 and CH2 domains. The hinge region may contain approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998 161: 4083).

[0163] As used herein, the term "Fv" refers to the minimum antibody fragment containing a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.

[0164] The folding of these two domains gives rise to six hypervariable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv, containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, although with a lower affinity than the entire binding site.

[0165] "Heavy chain variable region" or "VH", in the context of an antibody, refers to the fragment of a heavy chain containing three CDRs, intervening sequences known as framework regions, which are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0166] "Light chain variable region" or "VL", in the context of an antibody, refers to the fragment of a light chain containing three CDRs, intervening sequences known as framework regions, which are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0167] The six hypervariable loops (three loops each from the heavy and light chains) contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody, but even a single variable domain (or half of an Fv, containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, although with a lower affinity than the entire binding site.

[0168] "Framework" or FR residues are those variable domain residues other than the hypervariable region residues.

[0169] An antibody is understood in the art to be a glycoprotein or antigen-binding portion thereof having at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). Both the heavy and light chain variable regions contain framework regions (FR or FWR) and hypervariable regions (HVR). HVRs are the amino acid residues of an antibody involved in antigen binding. Hypervariable regions generally contain amino acid residues from complementarity-determining regions (CDRs) and have the highest sequence variability and / or are involved in antigen recognition. With the exception of CDR1 of VH, CDRs generally contain amino acid residues that form hypervariable loops. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs referred to as abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) include L1 at amino acid residues 31-34, L2 at amino acid residues 50-55, L3 at amino acid residues 89-96, H1 at amino acid residues 31-35B, H2 at amino acid residues 50-58, and H3 at amino acid residues 95-102 (see, e.g., Fransson, Front. Biosci. 13: 1619-1633 (2008)).

[0170] Unless otherwise specified, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. The variable region is the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p.91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain derived from an antibody that binds to the antigen (see, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991)). The four FWR regions are generally more conserved, while the CDR regions (CDR1, CDR2, and CDR3) represent hypervariable regions and are arranged from the NH2-terminus to the COOH-terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens, while the constant regions, depending on the isotype, may mediate the binding of the immunoglobulin to host tissues or factors. Antibodies also include chimeric antibodies, humanized antibodies, and recombinant antibodies, human antibodies generated from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to those skilled in the art.

[0171] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which usually determines the class to which the antibody belongs.

[0172] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa ("κ") and lambda ("λ") light chains refer to the two major antibody light chain isotypes.

[0173] An antibody or antigen-binding fragment thereof "specifically binds" or "preferentially binds" to a target if it binds to the target with greater affinity and / or avidity than it binds to an epitope on an unrelated polypeptide. The specificity of an antibody or antigen-binding fragment, or portion thereof, can be determined based on affinity and / or avidity. Methods for determining such specific binding are well known in the art. According to certain embodiments of the present disclosure, an antibody or antigen-binding fragment thereof may bind to a human cancer antigen but not to cancer antigens from other species. Alternatively, in certain embodiments, an antibody or antigen-binding fragment thereof may bind to a human cancer antigen and also to cancer antigens from one or more non-human species. For example, an antibody or antigen-binding fragment thereof may bind to a human cancer antigen and, optionally, may also bind to, or not bind to, one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee cancer antigens.

[0174] Dissociation of antigen and antigen-binding protein (K D Affinity, represented by the equilibrium constant for ), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein: K D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can be expressed as 1 / K D The affinity constant (K A) As will be clear to those skilled in the art, affinity can be determined in a manner known per se depending on the particular antigen of interest. Thus, an antibody or antigen-binding fragment thereof as defined herein binds to a first antigen with an affinity (as described above, e.g., K ) that is at least 50 times, e.g., at least 100 times, preferably at least 1000 times, up to 10,000 times or more greater than the affinity with which said amino acid sequence or polypeptide binds to another target or polypeptide. D An antibody or antigen-binding fragment thereof is said to be "specific" for a first target or antigen relative to a second target or antigen if it binds at a specific binding affinity (appropriately expressed as a specificity value). When an antibody or antigen-binding fragment thereof is "specific" for a target or antigen relative to another target or antigen, the antibody or antigen-binding fragment thereof may bind to the target or antigen, but preferably not to other targets or antigens. However, as will be understood by those skilled in the art, in some embodiments, an antibody or antigen-binding fragment thereof may specifically bind to a target, such as a cancer-associated antigen, and have a functional effect, for example, inhibiting / preventing tumor progression, when the binding site on the target is shared or partially shared by multiple different ligands.

[0175] In some embodiments, the dissociation constant (K D ) is about 1 μM, 100 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 Up to M, e.g. 10 -9 M to 10 -13 In another aspect of the invention, antibodies or antigen-binding fragments thereof are provided that have increased affinity for their target, e.g., affinity matured antibodies. Affinity matured antibodies are antibodies with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, which result in improved affinity of the antibody for its antigen. These antibodies bind to antigens with an affinity of about 5×10 -9 M, 2 x 10-9 M, 1 x 10 -9 M, 5 x 10 -10 M, 2 x 10 -10 M, 1 x 10 -10 M, 5 x 10 -11 M, 1 x 10 -11 M, 5 x 10 -12 M, 1 x 10 -12 M or less K D In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that have at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or greater increase in affinity compared to a wild-type IgA antibody or a wild-type IgG antibody, or both, containing the heavy and light chain sequences. In other embodiments, the antibodies or functional fragments thereof provided herein compete for binding to the same epitope as the corresponding antibody from which the variable domain is derived. In some embodiments, antibodies or antigen-binding fragments thereof that bind to the same epitope as and / or compete for binding to the same epitope as an antibody exhibit effector function activity, such as, for example, Fc-mediated cellular cytotoxicity, including ADCC activity.

[0176] K D can be measured by any suitable assay. For example, K D can be measured by radiolabeled antigen binding assay (RIA) (see, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999); Presta et al., Cancer Res. 57: 4593-4599 (1997)). For example, KD can be measured using a surface plasmon resonance assay (e.g., using a BIACORE®-2000 or BIACORE®-3000). For example, K D can be measured using competitive ELISA.

[0177] Avidity is a measure of the strength of binding between an antigen-binding molecule and an associated antigen. Avidity is related to both the affinity of the antigenic determinant to its antigen-binding site on the antigen-binding molecule and the number of associated binding sites present on the antigen-binding molecule. Generally, antigen-binding proteins bind to their cognate or specific antigens with a binding activity of 10 or more. -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, more preferably 10 -8 ~10 -12 Dissociation constant in moles / liter (K D ) (i.e., 10 5 ~10 12 Liters / mole or greater, preferably 10 7 ~10 12 liters / mole or greater, or even more preferably 10 8 ~10 12 Association constant (K) in liters / mole A )) to join. 10 -4 K greater than moles / liter D The value is either (or 10 4 M -1 Less than K A Any K value is generally considered to be indicative of nonspecific binding. D Generally, 10 -10 M(0.1nM)~10 -5 The stronger the interaction, the greater the K DThe binding site on the anti-LAP antibody or antigen-binding fragment thereof described herein preferably binds with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, e.g., less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and various modifications thereof known per se in the art; as well as other techniques mentioned herein.

[0178] The term "k on ", as used herein, is intended to refer to the rate constant for the association of an antibody or antigen-binding fragment thereof with an antigen.

[0179] The term "Koff," as used herein, is intended to refer to the rate constant for dissociation of an antibody or antigen-binding fragment thereof from the antibody / antigen complex.

[0180] The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below), (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from the immunoglobulin sequences disclosed herein. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human immunoglobulin VH and VL sequences, but are sequences that do not naturally occur within the human antibody germline repertoire in vivo.

[0181] With respect to antibodies or antigen-binding fragments thereof, the term "specificity" or "specific" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or antigen-binding fragment thereof can bind. The specificity of an antibody or antigen-binding fragment or portion thereof can be determined based on affinity and / or avidity. Affinity is determined by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (K D ) and is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein: K D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can be expressed as 1 / K D The affinity constant (K A) As will be clear to those skilled in the art, affinity can be determined in a manner known per se depending on the particular antigen of interest. Thus, an antibody or antigen-binding fragment thereof as defined herein binds to a first antigen with an affinity (as described above, e.g., K) that is at least 50 times, e.g., at least 100 times, preferably at least 1000 times, up to 10,000 times or more greater than the affinity with which said amino acid sequence or polypeptide binds to another target or polypeptide. D An antibody or antigen-binding fragment thereof is said to be "specific" for a first target or antigen relative to a second target or antigen if it binds with a specific binding affinity (appropriately expressed as a specific binding affinity value). When an antibody or antigen-binding fragment thereof is "specific" for a target or antigen relative to another target or antigen, the antibody or antigen-binding fragment thereof may bind to the target or antigen, but preferably does not bind to other targets or antigens.

[0182] However, as will be appreciated by those skilled in the art, in some embodiments, when the binding site on the target is shared or partially shared by multiple different ligands, the antibody or antigen-binding fragment thereof will specifically bind to the target antigen and have a functional effect, for example, inhibiting / preventing tumor progression.

[0183] Avidity is a measure of the strength of binding between an antigen-binding molecule and an associated antigen. Avidity is related to both the affinity of an antigenic determinant to its antigen-binding site on the antigen-binding molecule and the number of associated binding sites present on the antigen-binding molecule. Generally, antigen-binding proteins bind to their cognate or specific antigens in a range of 10 to 120%. -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, more preferably 10 -8 ~10 -12 Dissociation constant in moles / liter (K D ) (i.e., 10 5 ~10 12 Liters / mole or greater, preferably 10 7 ~1012 liters / mole or greater, or even more preferably 10 8 ~10 12 Association constant (K) in liters / mole A )) to join. 10 -4 K greater than moles / liter D The value is either (or 10 4 M -1 Less than K A Any K value is generally considered to be indicative of nonspecific binding. D Generally, 10 -10 M(0.1nM)~10 -5 The stronger the interaction, the greater the K D The binding site on the anti-LAP antibody or antigen-binding fragment thereof described herein preferably binds with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, e.g., less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and various modifications thereof known per se in the art; as well as other techniques mentioned herein.

[0184] The term "fusion protein," as used herein, refers to a polypeptide that comprises the amino acid sequence of an antibody or fragment thereof and the amino acid sequence of a heterologous polypeptide (ie, an unrelated polypeptide).

[0185] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are single domain antibodies. A "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to a single variable region (VH or ') that can confer antibody-antigen binding. In other words, a single variable domain does not need to recognize a target antigen by interacting with another variable region. A single domain antibody monomer is a single arm that allows antigen binding by each antibody variable region (VH * Examples of single domain antibodies include antibodies derived from camelids (camels and llamas) and cartilaginous fish (e.g., nurse sharks) and antibodies derived from these (Ward et al., Nature (1989) 341: 544-546; Dooley and Flajnik, Dev Comp Immunol (2006) 30: 43-56; Muyldermans et al., Trend Biochem Sci (2001) 26: 230-235; Holt et al., Trends Biotechnol (2003):21: 484-490; WO2005 / 035572; TO03 / 035694; Davies and Riechmann, Febs Lett (1994) 339: 285-290; WO2005 / 035572; TO03 / 035694; Davies and Riechmann, Febs Lett (1994) 339: 285-290; WO2005 / 035572; TO03 / 035694). 004; WO02 / 051870), in which a single domain antibody variable region or a single variable region of the antibody other than the variable domain is present in the antigen-binding arm (e.g., homomultimer or heteromultimer).

[0186] As used herein, the term "modification" refers to an amino acid substitution or deletion of one or more amino acid residues in the heavy chain constant region of an antibody compared to the WT heavy chain constant region of a WT antibody. In some embodiments, the modification is in an amino acid residue in the IgA CH1 region of the heavy chain constant region. In some embodiments, the modification is in an amino acid residue in the IgA CH2 region of the heavy chain constant region. In some embodiments, the modification is in an amino acid residue in the IgA CH3 region of the heavy chain constant region. In some embodiments, the modification is selected from Table 2. In some embodiments, one or more modifications disclosed herein result in improved properties compared to the corresponding WT antibody containing one or more modifications.

[0187] The term "improved properties" refers to characteristics associated with an antibody comprising one or more modifications disclosed herein that are improved compared to a parent WT antibody that does not contain one or more modifications. Such improved properties include, but are not limited to, increased thermal stability, extended circulating half-life, increased ADCC, reduced aggregation, reduced aggregation with serum proteins, increased tumor targeting, increased stability, reduced glycosylation, and increased binding to FcαR expressed on immune cells. In some embodiments, the improved property is an increase in one or more effector functions of the antibody or functional fragment thereof compared to the corresponding WT IgA. Effector functions are biological activities attributable to the Fc region of an antibody that vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); and phagocytosis.

[0188] In some embodiments, an antibody or functional fragment thereof disclosed herein (e.g., comprising one or more modifications disclosed herein in an IgA heavy chain constant region) has one or more improved properties of at least 2%, 3%, 4%, 5%, 7%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 100% compared to a corresponding WT IgA antibody or a corresponding WT IgG antibody.

[0189] As used herein, the term "corresponding" unmodified antibody refers to a wild-type antibody that is identical in sequence to an antibody that comprises one or more selected modifications disclosed herein, particularly in the heavy chain constant region, but without the one or more selected modifications described herein. In some embodiments, the corresponding antibody can be a WT IgA antibody comprising a WT IgA heavy chain constant region. In some embodiments, the corresponding antibody can be a WT IgG antibody comprising a WT IgG heavy chain constant region. In some embodiments, the corresponding WT IgA antibody is a WT IgA1 antibody. In some embodiments, the corresponding WT IgA antibody is a WT IgA2 antibody. In some embodiments, the corresponding WT IgA antibody comprises a wild-type IgA heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the corresponding WT IgA2 antibody comprises a wild-type IgA2 heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the corresponding WT IgA antibody comprises a wild-type IgA heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the corresponding WT IgA antibody comprises a wild-type IgA heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the WT IgA heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the WT IgA2 heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid modifications disclosed herein are relative to the amino acid residue at a selected position in a WT IgA heavy chain constant region (e.g., a WT IgA2 heavy chain constant region) comprising the amino acid sequence set forth in SEQ ID NO: 1, numbered according to the IMGT scheme.

[0190] In some embodiments, the WT IgA heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the WT IgA2 heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the amino acid modifications disclosed herein are relative to the amino acid residue at a selected position in a WT IgA heavy chain constant region (e.g., a WT IgA2 heavy chain constant region) comprising the amino acid sequence set forth in SEQ ID NO: 2, numbered according to the IMGT scheme.

[0191] In some embodiments, the WT IgA heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the WT IgA2 heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the amino acid modifications disclosed herein are relative to the amino acid residue at a selected position in a WT IgA heavy chain constant region (e.g., a WT IgA2 heavy chain constant region) comprising the amino acid sequence set forth in SEQ ID NO: 3, numbered according to the IMGT scheme.

[0192] The term "Fab," as used herein, refers to a region of an antibody that consists of one constant domain and one variable domain of each of the heavy and light chains (monovalent antigen-binding fragment), but where the heavy chain has been truncated to lack the CH2 and CH3 domains (i.e., VH, CH1, VL, and CL), and may also lack part or all of the hinge region. Fab can be produced by digesting whole antibody with the enzyme papain. Fab can refer to this region in isolation or in the context of a full-length antibody, immunoglobulin construct, or Fab fusion protein.

[0193] The term Fab', as used herein, can be obtained by treating whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain containing the VH and a single constant domain. Two Fab' fragments can be obtained from such treated antibody.

[0194] "scFv" refers to an antibody fragment comprising the VF1 and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. See, e.g., U.S. Patent Nos. 4,946,778, 5,260,203, 5,455,030, and 5,856,456. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315. The complex between the VF1 and VL domains of an Fv fragment can also be stabilized by disulfide bonds (U.S. Patent No. 5,747,654).

[0195] The term "in vivo half-life" or "circulating half-life," as used herein, refers to the circulation of an antibody or functional fragment thereof in a given animal and is represented by the time required for half of the amount administered to the animal to be cleared from the circulation.

[0196] The term "extended circulating half-life", as used herein, means that an antibody comprising one or more modifications relative to a WT IgA antibody provided in accordance with the invention persists in serum or plasma for a longer period of time and / or has a longer half-life of the maximum measured serum or plasma concentration compared to the same antibody not containing the same modifications, i.e., a WT antibody, for example a WT IgA antibody.

[0197] The term "increased thermostability" refers to a greater retention of biological activity, e.g., ADCC, antigen binding, FcαR binding, of an antibody or functional fragment disclosed herein compared to a corresponding WT IgA antibody after an incubation period at a certain temperature. Increased thermostability can be assessed, for example, under one or more (e.g., several) temperature conditions. For example, the one or more (e.g., several) temperatures can be any temperature within the range of 45°C to 95°C, e.g., 45, 50, 55, 60, 65, 70, 75, 80, 85, or 95°C (or intermediate, e.g., 62°C, 68°C, 72°C, etc.), for a suitable incubation period (hours), e.g., 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, or 60 minutes (or intermediate, e.g., 23 minutes, 37 minutes, etc.), at one or more (e.g., several) pHs within the range of 3 to 9, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 (or intermediate), such that the variant retains residual activity. However, longer incubation periods can also be used. The term "increased thermal stability" may be used interchangeably with "improved thermal stability."

[0198] As used herein, the term "naturally occurring," when referring to a glycosylation site or amino acid residue within an IgA heavy chain constant region, refers to the fact that the glycosylation site or amino acid residue can be found in the IgA heavy chain constant region in nature, e.g., can be isolated from a natural source, and has not been intentionally modified by humans (including viruses) in the laboratory. A polypeptide or polynucleotide sequence that is present in an organism is a naturally occurring sequence.

[0199] The terms "disease," "disorder," or "condition" are used interchangeably herein and refer to any alteration in some state of the body or organs, an interruption or disturbance in the performance of function, and / or causing symptoms such as discomfort, impairment, distress, or even death to the afflicted person or those in contact with them. A disease or disorder can also relate to an ailment, a chronic condition of illness, a chronic minor illness, a chronic disease, a disorder, a pathology, a sickness, a complaint, or a pretense.

[0200] The term "in need thereof," in reference to therapeutic or prophylactic treatment, means having the disease, being diagnosed with the disease, or needing to prevent the disease, for example, for someone at risk of developing the disease. Thus, a subject in need thereof can be a subject in need of treating or preventing the disease.

[0201] As used herein, the term "administering" refers to placing a compound (e.g., an antibody or antigen-binding fragment thereof disclosed herein) into a subject by a method or route that results in at least partial delivery of the agent to a desired site. Pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof disclosed herein can be administered by any reasonable route that results in effective treatment in a subject, including, but not limited to, intravenous, intraarterial, direct injection or infusion into tissue parenchyma, etc. If necessary or desired, administration can include, for example, intracerebroventricular ("icv") administration, intranasal administration, intracranial administration, intracavitary administration, intracerebellar administration, or intrathecal administration.

[0202] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of ectopic cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0203] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be demonstrated by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors in the first place.

[0204] As used herein, "subject," "patient," "individual," and similar terms are used interchangeably and refer to vertebrates, mammals, primates, or humans. Mammals include, but are not limited to, humans, primates, rodents, feral animals, farm animals, sport animals, and pet animals, including wild or domestic animals. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, for example, cattle, horses, pigs, deer, bison, water buffalo, feline species, such as domestic cats, and canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. The terms "individual," "patient," and "subject" are used interchangeably herein. The subject may be male or female.

[0205] In some embodiments, the subject is a mammal. The mammal may be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Non-human mammals can be advantageously used as subjects representing animal models of conditions or disorders of uncontrolled cell growth (e.g., cancer). Non-limiting examples include mouse tumor models. Furthermore, the compositions and methods described herein can be used to treat domestic animals and / or pet animals. The subject may be a subject previously diagnosed or identified as suffering from cancer. The subject may be a subject who has been diagnosed and is currently undergoing treatment, or who is seeking treatment, monitoring, adjustment or modification of an existing therapeutic treatment, or who is at risk of developing a given disorder (e.g., cancer).

[0206] A "cytotoxic agent" refers to an agent that has a cytotoxic and / or cytostatic effect on a cell. A "cytotoxic effect" refers to the depletion, elimination, and / or death of a target cell. A "cytostatic effect" refers to the inhibition of cell proliferation.

[0207] As used herein, the terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to a series of amino acid residues connected to one another by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein," "peptide," and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein," "peptide," and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. These terms encompass, for example, native and artificial proteins, protein fragments and polypeptide analogs of protein sequences (e.g., muteins, variants, and fusion proteins), as well as proteins that are post-translationally or otherwise covalently or non-covalently modified. A peptide, polypeptide, or protein can be a monomer or a polymer. A polypeptide can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptides can be isolated from nature or produced by recombinant or synthetic means. In some embodiments, the polypeptide is a "variant." "Variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not counting any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity.In some embodiments, variants have at least about 90% amino acid sequence identity. In some embodiments, variants have at least about 95% amino acid sequence identity to the native sequence polypeptide. A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation of another chemical moiety (e.g., polyethylene glycol or albumin, such as human serum albumin), phosphorylation, and glycosylation.

[0208] The terms "increased / prolonged / elevated," "increase / prolong / increase," or "enhance" are all used herein to generally mean an increase by a statistically significant amount. For the avoidance of doubt, the terms "increased / prolonged / elevated", "increase / prolong / elevate", or "enhance" mean an increase of at least 10% compared to a reference level, for example at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase of 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or greater compared to a reference level.

[0209] The term "fusion protein," as used herein, refers to a polypeptide that comprises the amino acid sequence of an antibody or fragment thereof and the amino acid sequence of a heterologous polypeptide (ie, an unrelated polypeptide).

[0210] The terms "decrease", "reduce", "reduce", "reduction", "lower" or "lowering" or "inhibit" are all used herein to generally mean a decrease / reduction / decrease / reduction by a statistically significant amount. For example, the terms "decrease," "reduce," "reduce," "reduction," or "inhibit" refer to a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease (e.g., tumor size after treatment compared to the reference level before treatment), or any decrease between 10 and 100% compared to a reference level. With respect to a marker or symptom, these terms refer to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, preferably to an acceptable level that falls within the normal range for individuals without a given disease. Reduce / reduce / shrink or inhibit can refer, for example, to the symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or size of dormant tumors.

[0211] The terms "synthetic polynucleotide," "synthetic gene," or "synthetic polypeptide," as used herein, mean that the corresponding polynucleotide sequence or portion thereof, or amino acid sequence or portion thereof, is derived from a novel or modified, designed, or synthetic sequence compared to the equivalent naturally occurring sequence. Synthetic polynucleotides (antibodies or antigen-binding fragments) or synthetic genes can be prepared by methods known in the art, including, but not limited to, chemical synthesis of nucleic acid or amino acid sequences. Synthetic genes generally differ from naturally occurring genes at the amino acid level or polynucleotide level (or both) and are generally located in the context of synthetic expression control sequences. Synthetic gene polynucleotide sequences may not necessarily encode proteins with different amino acids compared to naturally occurring genes; for example, they may encompass synthetic polynucleotide sequences that incorporate different codons but encode the same amino acids (i.e., the nucleotide change is a silent mutation at the amino acid level). IgA antibody

[0212] IgA has two subclasses (IgA1 and IgA2) and can be produced in a monomeric form as well as a dimeric and secreted form. In some embodiments, an IgA antibody can be monomeric. In some embodiments, an IgA antibody can comprise one or more IgA1 amino acid sequences. In some embodiments, an IgA antibody can comprise one or more IgA2 amino acid sequences. In some embodiments, an IgA antibody can comprise one or more IgA1 amino acid sequences and one or more IgA2 amino acid sequences.

[0213] In some embodiments, the IgA antibody is an IgA2 antibody of the allotypes IgA2m(1), IgA2(m)2, or IgA2n. In some embodiments, the IgA2m(1) antibody is a Caucasian IgA2m(1) antibody. In some embodiments, the IgA2m(2) antibody is an African IgA2m(2) antibody or an Asian IgA2m(2) antibody.

[0214] In some embodiments, an IgA antibody comprises a heavy chain constant region comprising at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% IgA amino acids, hi some embodiments, an IgA antibody comprises a light chain constant region comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% IgA amino acids.

[0215] In some embodiments, the IgA antibody comprises a heavy chain constant region comprising one or more of the IgA CH3 region, the IgA CH2, or the IgA CH1 region, or any combination thereof. In some embodiments, the IgA antibody comprises a light chain region comprising the IgA CH1 region. In some embodiments, the IgA antibody comprises a light chain region comprising a kappa light chain constant region. In some embodiments, the IgA antibody comprises a heavy chain constant region comprising one or more amino acids of the IgG CH3 region, the IgG CH2, or the IgG CH1 region, or any combination thereof. In some embodiments, the IgA antibody comprises a heavy chain constant region comprising the IgA CH3 region, the IgA CH2, and the IgA CH1 region, or any combination thereof. In some embodiments, the IgA antibody comprises a heavy chain constant region comprising the IgA CH3 region, the IgA CH2, and the IgG CH1 region, or any combination thereof. In some embodiments, the IgA2 antibody comprises a heavy chain constant region comprising one or more of the IgA2 CH3 region, the IgA2 CH2, or the IgA2 CH1 region, or any combination thereof.

[0216] In some embodiments, the IgA antibody comprises an IgG light chain variable region. In some embodiments, the IgA antibody comprises an IgG heavy chain variable region. In some embodiments, the IgA antibody comprises an IgG light chain variable region and an IgG heavy chain variable region.

[0217] In some embodiments, the IgA antibody may be a humanized antibody. In some embodiments, the IgA antibody may be a chimeric antibody. In some embodiments, the IgA antibody may be a human antibody.

[0218] In some embodiments, the IgA antibody may be a monospecific antibody. In some embodiments, the IgA antibody may be a bispecific antibody. In some embodiments, the IgA antibody may be a trispecific antibody. In some embodiments, the IgA antibody may be a multispecific antibody.

[0219] In some embodiments, the IgA antibody may be a bispecific antibody. In some embodiments, the IgA antibody simultaneously associates with two antigens on the cell surface. In some examples, the IgA antibody binds to two different antigens sequentially. For example, the IgA antibody binds to the first antigen first, thereby limiting the space explored by the second antibody arm. As a result, the local concentration of the second antigen may be significantly increased, thereby facilitating the binding of the second antibody arm.

[0220] In some embodiments, the IgA antibody may comprise at least a portion of an Fc domain. In some embodiments, the IgA antibody comprises a heavy chain constant region comprising a CH3 domain, a CH2 domain, and a CH1 domain. In some embodiments, the IgA antibody comprises a light chain constant region comprising a CH1 domain.

[0221] In some embodiments, the IgA antibody induces complement-dependent cytotoxicity (CDC). In some embodiments, the IgA antibody induces polymorphonuclear neutrophil (PMN)-mediated tumor cell lysis. In some embodiments, the IgA antibody induces programmed cell death (PCD) via a caspase-independent pathway. In some embodiments, the IgA antibody induces antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the IgA antibody induces neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC).

[0222] In some embodiments, IgA antibodies have a superior ability to recruit neutrophils for antibody-dependent cellular cytotoxicity (ADCC) compared to corresponding IgG antibodies. In some embodiments, IgA antibodies may require a lower effector:target (E:T) ratio. In some embodiments, IgA antibodies may require a lower tumor-opsonizing antibody concentration compared to other types of antibodies (e.g., IgG). In some embodiments, IgA antibodies may induce neutrophil-mediated phagocytosis or trogocytosis of tumor cells after IgA antibody-neutrophil association. In some embodiments, IgA antibodies may induce trogoptosis of tumor cells after IgA antibody-neutrophil association. This tumor cell killing mechanism is primarily mediated by interaction with the Fc receptor (FcαRI; CD89), the best-characterized IgA receptor for IgA. FcαRI is expressed on monocytes, macrophages, granulocytes, a subset of dendritic cells, and Kupffer cells and binds to both monomeric and dimeric IgA isoforms with intermediate affinity. IgA binding to FcαRI mediates effector functions such as phagocytosis, oxidative burst, cytokine release, antigen presentation, and ADCC. Two IgA isotypes, IgA1 and IgA2, and three allotypes, IgA2m(1), IgA2m(2), and IgA2n, have been identified in humans. In some embodiments, IgA antibodies induce polymorphonuclear cell (PMN)-mediated ADCC more efficiently than IgG antibodies.

[0223] In some embodiments, IgA does not bind to B cells, T cells, platelets, and / or red blood cells. For example, in some embodiments, IgA antibodies may have low immunogenicity.

[0224] In some embodiments, the IgA antibody is a therapeutic antibody. In some embodiments, the IgA antibody can be a recombinant antibody. In some embodiments, the IgA antibody is made in a cell line. In some embodiments, the cell line is CHO. In some embodiments, the cell line is SP20. In some embodiments, the cell line is a HEK239 cell line. In some embodiments, the HEK293 cell line is HEK293F.

[0225] In some embodiments, the antibodies or functional fragments thereof provided herein comprise an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibodies or functional fragments thereof provided herein comprise a variable heavy chain domain derived from an IgG antibody. In some embodiments, the antibodies or functional fragments thereof provided herein comprise a variable light chain domain derived from an IgG antibody. The variable domains can be derived from, for example, dinutuximab, obinutuzumab, Unituxin, TA99, 2.3D11, C47A8-CQ, UMAB10, and trastuzumab and Rituxan (rituximab); other antibodies with therapeutic activity within the scope of the invention include, but are not limited to, Avastin, Herceptin, 3F8, 8H9, abagovomab, abciximab, actoxumab, , adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, aracizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anifrolumab, anrukinzumab, apolizumab, arcitumomab, acelizumab, atinumab, atlizumab, atolizumab, bapineuzumab, basiliximab, bavituximab Mab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab vedotin, caplacizumab, capromab pendetide, carlumab, catumaxomab , cBR96-doxorubicin immunoconjugate, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, concizumab, CR6261, crenezumab, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab,dorlimomab alitox, dorozitumab, durigotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efangumab, eldelumab, elotuzumab, ersilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, facilicumab, Numab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, IMAB362, imicilomab, imgatuzumab, in Clacumab, indatuximab ravtansine, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, roderucizumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatumumab, marjetuximab, maslimomab, matuzumab, mabryri Mumab, mepolizumab, metelitumumab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimu- mab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narunatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentane, occaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumumab, olokizumab, omalizumab, onartuzumab,Ontuximab, Oportuzumab monatox, Oregovomab, Olticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozolarizumab, Pagibaximab, Palivizumab, Panitumumab, Pancomab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placuramab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, P RO140, kiruzumab, racotumomab, radletumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, cetoxaximab, sevilumab, SGN-CD19A, SGN-CD33A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirumab, sora Nezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab, tigatuzumab, tildrakizumab, TNX-650, tocilizumab, toralizumab, tositumomab, tobetumab, tralokinumab, trastuzumab, TRBS07, tregalizumab, treme Limulumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vanticutumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, hu14.18K322A, zolimomab alitox, rituximab (Rituxan®, CD20), trastuzumab (Herceptin®),Alemtuzumab (Campath®, CD52), Ibritumomab tiuxetan (Zevalin®, CD20), Tositumomab-I-131 (Bexxar®: CD20), Cetuximab (Erbitux®), Bevacizumab (VEGF), Panitumumab (Vectibix®, EGFR), Ofatumumab (Arzerra®, CD20), Ipilimumab (Y pervoy®, CTLA-4), brentuximab (brentiuximab), vedotin (Adectris®, CD30), pertuzumab (Perjecta®, HER2), adotrastuzumab, emtansine (Kadcyla®, HER2), obinutuzumab (Gazyva®, CD20), nivolumab, and pembrolizumab (anti-PD-1s).

[0226] In one aspect, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4, 7, 81-86. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody comprising that sequence retains the ability to bind to the same antigen as a wild-type antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within the amino acid sequence of any one of SEQ ID NOs: 4, 7, 81-86. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody comprises a VH sequence of the amino acid sequence of SEQ ID NO: 4, 7, 81-86, including one or more post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 33-40, (b) an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 41-48, and (c) an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 49-56.

[0227] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5, 8, 95-100. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to the same antigen as a wild-type antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted from any one of the amino acid sequences of any one of SEQ ID NOs: 5, 8, 95-100. In some embodiments, the substitutions, insertions, or deletions are present in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VL sequence of any one of SEQ ID NOs: 5, 8, 95-100, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from: (a) an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 57-64; (b) an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 65-72; and (c) an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 73-80.

[0228] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises the amino acid sequence of any one of SEQ ID NOs: 4, 7, 81-86, and the VL comprises the amino acid sequence of any one of SEQ ID NOs: 5, 8, 95-100, optionally including post-translational modifications of these sequences.

[0229] In one embodiment, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH selected from any VH in Table 9. In one embodiment, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VL selected from any VL in Table 9. In one embodiment, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH selected from any VH in Table 9 and a VL selected from any VL in Table 9. In one embodiment, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH selected from any VH in Table 9 and a VL selected from any VL in Table 9, wherein the selected VH and VL are paired according to Table 9. In one embodiment, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises an HC-CDR3 selected from any HC-CDR3 in Table 5 and an LC-CDR3 selected from any LC-CDR3 in Table 6, and the selected HC-CDR3 and LC-CDR3 are paired according to Table 9. In one embodiment, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises an HC-CDR2 selected from any CDR-H2 in Table 5 and an LC-CDR2 selected from any CDR-L2 in Table 6, and the selected HC-CDR2 and LC-CDR2 are paired according to Table 9. In one embodiment, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises an HC-CDR1 selected from any HC-CDR1 in Table 5 and an LC-CDR1 selected from any LC-CDR1 in Table 6, and the selected HC-CDR1 and LC-CDR1 are paired according to Table 9.In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises an HC-CDR1, HC-CDR2, and HC-CDR3 selected from any of the HC-CDR1, HC-CDR2, and HC-CDR3 in Table 5 and an LC-CDR1, LC-CDR2, and LC-CDR3 selected from any of the LC-CDR1, LC-CDR2, and LC-CDR3 in Table 6, wherein the selected HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 are paired according to Table 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises any one of the HC-CDR1, HC-CDR2, and HC-CDR3 selected from any of the HC-CDR1, HC-CDR2, and HC-CDR3 in Table 5, and any IgA heavy chain constant region selected from SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises any one of LC-CDR1, LC-CDR2, and LC-CDR3 selected from any of LC-CDR1, LC-CDR2, and LC-CDR3 in Table 6, and any IgA heavy chain constant region selected from SEQ ID NOs: 16-21.

[0230] In some embodiments, the antibody or antigen-binding fragment thereof comprises any one of HC-CDR1, HC-CDR2, and HC-CDR3 selected from any of HC-CDR1, HC-CDR2, and HC-CDR3 in Table 5, and any one of LC-CDR1, LC-CDR2, and LC-CDR3 selected from any of LC-CDR1, LC-CDR2, and LC-CDR3 in Table 6, and any IgA heavy chain constant region selected from SEQ ID NOs: 16-21, wherein the selected HC-CDR1, HC-CDR2, HC-CDR3, selected LC-CDR1, LC-CDR2, LC-CDR3, and selected IgA heavy chain constant region are paired according to Table 9. IgA antibody modification

[0231] Described herein are IgA antibodies that contain one or more amino acid substitutions and / or one or more amino acid deletions.

[0232] In some embodiments, the amino acid numbering of the IgA antibodies described herein is shown according to the IMGT unique numbering for C and C-like domains (as 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 numbered according to the IMGT scheme and compared to amino acid residues at selected positions in a WT IgA heavy chain constant region (e.g., a WT IgA2 heavy chain constant region) comprising the amino acid sequence set forth in SEQ ID NO: 1. Note that US 62 / 824,864, the entire contents of which are incorporated herein, also includes antibodies and antibody constructs comprising modified IgA heavy chain constant regions numbered according to the IMGT numbering scheme. Some amino acids therein are inadvertently labeled differently, and the specific amino acid positions described in US 62 / 824,864 correspond to the specific amino acid positions presented herein below. Positions C92, N120, I121, and T122 referred to in US 62 / 824,864 correspond to amino acid residues C86, N114, I115, and T116 of the antibodies described herein, labeled exactly according to the IMGT numbering scheme (the IMGT numbering is shown in Table 11 for reference to position numbering only). The reference wild-type heavy chain constant region sequences of these antibodies disclosed in US 62 / 824,864 and the reference wild-type heavy chain constant region sequence of the present application are the same, i.e., SEQ ID NO: 1, as shown in Figure 1. As such, the inadvertent misrepresentation of residues C86, N114, I115, and T116 in US 62 / 824,864 as C92, N120, I121, and T122 in the IMGT nomenclature scheme will be apparent to one skilled in the art.

[0233] In some embodiments, an IgA antibody disclosed herein comprises a deletion of at least four glycosylation sites in the constant region. In some embodiments, an IgA antibody disclosed herein comprises a deletion of at least three N-linked glycosylation sites in the constant region of the antibody. In some embodiments, an IgA antibody comprises a deletion of at least three N-linked glycosylation sites in the constant region of the antibody and at least one O-linked glycosylation site in the constant region of the antibody. In some embodiments, an IgA antibody or functional fragment disclosed herein comprises one or more modifications disclosed herein (e.g., in Table 2) in the IgA heavy chain constant region. In some embodiments, an IgA antibody or functional fragment disclosed herein comprises one or more modifications disclosed herein (e.g., in Table 2) in the IgA1 heavy chain constant region. In some embodiments, an IgA antibody or functional fragment disclosed herein comprises one or more modifications disclosed herein (e.g., in Table 2) in the IgA2 heavy chain constant region. In some embodiments, the one or more modifications are in amino acid residues within the CH1, CH2 and / or CH3 regions of the IgA1 heavy chain constant region.

[0234] In some embodiments, the IgA antibody comprises a deleted tail piece. In some embodiments, the one or more modifications are in amino acid residues within the CH1, CH2, and / or CH3 regions of the IgA2 heavy chain constant region. In some embodiments, the IgA CH1 region comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the IgA CH2 region comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the IgA CH2 region comprises an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 111. In some embodiments, an IgA antibody or functional fragment thereof disclosed herein comprises a deletion 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, or 3 to 4 C-terminal amino acids. In some embodiments, an IgA2 antibody comprises a deletion 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, or 3 to 4 C-terminal amino acids. In some embodiments, the C-terminal amino acids are from amino acids 131-148 of an IgA2 antibody, numbered according to the IMGT scheme.

[0235] In some embodiments, the IgA2 antibody comprises a deletion of amino acids 131-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 147-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 146-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 145-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 144-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 143-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 142-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 141-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 140-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 139-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 138-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 137-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 136-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 135-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 134-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 133-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 132-148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acids 131-148, numbered according to the IMGT scheme.In some embodiments, the IgA2 antibody comprises a deletion of amino acids P131 to Y148, numbered according to the IMGT scheme.

[0236] In some embodiments, the IgA antibody comprises a mutation at the C-terminal asparagine (N) amino acid. In some embodiments, the mutation is a non-conservative amino acid substitution. In some embodiments, the mutation deletes the glycosylation site at the C-terminal asparagine (N) amino acid of IgA. In some embodiments, the IgA2 antibody comprises a mutation at N135, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at N135, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a N135Q mutation, numbered according to the IMGT scheme.

[0237] In some embodiments, the IgA antibodies or functional fragments thereof disclosed herein comprise modifications at least two naturally occurring glycosylation sites in the IgA heavy chain constant region. In some embodiments, the IgA antibodies or functional fragments thereof disclosed herein comprise modifications at least three naturally occurring glycosylation sites in the IgA heavy chain constant region. In some embodiments, the IgA antibodies or functional fragments thereof disclosed herein comprise modifications at least four naturally occurring glycosylation sites. In some embodiments, the glycosylation sites comprise N-linked glycosylation sites. In some embodiments, the glycosylation sites comprise naturally occurring asparagine residues. In some embodiments, the glycosylation sites are within the CH2 region, the CH3 region, and / or the CH1 region. In some embodiments, the IgA heavy chain constant region comprises modifications at N45.2G, N15.2, L15.3, T16, N114, I115, T116, N135, or a combination thereof, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises an amino acid substitution that is N45.2G, N45.2A, N15.2G, N15.2Q, N15.2T, L15.3I, T16S, N114T, I115L, T116S, N135Q, or a combination thereof, numbered according to the IMGT scheme. In some embodiments, an unglycosylated antibody or functional fragment thereof is provided herein. In some embodiments, the unglycosylated antibody comprises modifications at all four naturally occurring glycosylation sites in the IgA2 heavy chain constant region. In some embodiments, the unglycosylated antibody provided herein comprises modifications at residues N45.2, N15.2, L15.3, T16, N114, I115, T116, and N135, numbered according to the IMGT scheme. In some embodiments, the aglycosylated antibodies provided herein comprise modifications at residues N45.2, N15.2, N114, I115, T116, and N135, numbered according to the IMGT scheme.

[0238] In some embodiments, the non-glycosylated antibodies provided herein comprise modifications at residues N45.2, N15.2, L15.3, T16, N114, I115, T116, and deletion of C-terminal tailpiece residues P131-Y148, numbered according to the IMGT scheme. In some embodiments, the non-glycosylated antibodies provided herein comprise modifications at residues N45.2, N15.2, N114, I115, T116, and P131-Y148, numbered according to the IMGT scheme.

[0239] In some embodiments, provided herein are antibodies or functional fragments thereof comprising modifications at residues N45.2, N15.2, L15.3, T16, N114, I115, T116, and N135, numbered according to the IMGT scheme. In some embodiments, provided herein are antibodies or functional fragments thereof comprising modifications at residues N45.2, N15.2, N114, I115, T116, and N135, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions that are N45.2G, N45.2A, N15.2G, N114T, I115L, T116S, and N135Q, numbered according to the IMGT scheme. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions that are N45.2G, N45.2A, N15.2Q, L15.3I, T16S, N114T, I115L, T116S, and N135Q. In some embodiments, the IgA heavy chain constant region comprises amino acid substitutions that are N45.2G, N45.2A, N15.2G, N15.2T, N114T, I115L, T116S, and N135Q, numbered according to the IMGT scheme. In some embodiments, an antibody that comprises a combination of amino acid substitutions in the IgA heavy chain constant region that are N45.2G, N45.2A, N15.2G, N114T, I115L, T116S, and N135Q, numbered according to the IMGT scheme, is an aglycosylated antibody. In some embodiments, the aglycosylated antibody comprises the following combination of amino acid substitutions: N45.2G, N45.2A, N15.2G, T16S, N114T, I115L, T116S, and a deletion of the C-terminal tailpiece.

[0240] In some embodiments, an antibody comprising the combination of amino acid substitutions N45.2G, N45.2A, N15.2Q, N114T, I115L, T116S, and N135Q in the IgA heavy chain constant region, numbered according to the IMGT scheme, is an aglycosylated antibody. In some embodiments, an aglycosylated antibody comprises the combination of amino acid substitutions N45.2G, N45.2A, N15.2Q, T16S, N114T, I115L, T116S, and a deletion of the C-terminal tailpiece. In some embodiments, an antibody comprising the combination of amino acid substitutions N45.2G, N45.2A, N15.2T, N114T, I115L, T116S, and N135Q in the IgA heavy chain constant region, numbered according to the IMGT scheme, is an aglycosylated antibody. In some embodiments, the non-glycosylated antibody comprises the following combination of amino acid substitutions: N45.2G, N45.2A, N15.2T, T16S, N114T, I115L, T116S, and a deletion of the C-terminal tailpiece. In some embodiments, an antibody comprising the following combination of amino acid substitutions in the IgA heavy chain constant region, numbered according to the IMGT scheme, is a non-glycosylated antibody. In some embodiments, the aglycosylated antibody comprises the following combination of amino acid substitutions: N45.2G, N45.2A, N15.2T, L15.3I, T16S, T16S, N114T, I115L, T116S, and a deletion of the C-terminal tailpiece.

[0241] In some embodiments, the aglycosylated antibody exhibits an increased circulating half-life compared to a corresponding antibody comprising at least one glycosylation site (e.g., WT residues N45.2, N114, N15.2, and N135, numbered according to the IMGT scheme) or a corresponding WT IgA antibody. In some embodiments, the aglycosylated antibody exhibits reduced aggregation compared to a corresponding antibody comprising at least one glycosylation site (e.g., WT residues N45.2, N114, N15.2, and N135, numbered according to the IMGT scheme) or a corresponding WT IgA antibody. In some embodiments, the aglycosylated antibody exhibits reduced aggregation with serum proteins compared to a corresponding antibody comprising at least one glycosylation site (e.g., WT residues N45.2, N114, N15.2, and N135, numbered according to the IMGT scheme) or a corresponding WT IgA antibody. In some embodiments, the aglycosylated antibody exhibits reduced aggregation compared to a corresponding antibody comprising at least one glycosylation site (e.g., WT residues N45.2, N114, N15.2, and N135, numbered according to the IMGT scheme) or a corresponding WT IgA antibody. In some embodiments, the aglycosylated antibody exhibits increased thermal stability compared to a corresponding antibody comprising at least one glycosylation site (e.g., WT residues N45.2, N114, N15.2, and N135, numbered according to the IMGT scheme) or a corresponding WT IgA antibody. In some embodiments, the aglycosylated antibody exhibits increased binding affinity to an Fc receptor compared to a corresponding WT IgA. In some embodiments, the aglycosylated antibody induces ADCC of target cells. In some embodiments, the aglycosylated antibody specifically binds to a target antigen. In some embodiments, the aglycosylated antibody specifically binds to a target cell.

[0242] In some embodiments, the antibody comprises a mutation at N45.2, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at N45.2, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises N45.2G, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the N45.2 amino acid.

[0243] In some embodiments, the antibody comprises a mutation at P124, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at P124, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises P124R, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended 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.

[0244] In some embodiments, the antibodies described herein comprise a mutation at C86 (inadvertently referred to as C92 in US Pat. No. 6,224,864, the entire contents of which are incorporated herein, as noted above) as numbered according to the IMGT scheme. In some embodiments, the antibodies comprise a non-conservative mutation at C86 as numbered according to the IMGT scheme. In some embodiments, the antibodies comprise C86S as numbered according to the IMGT scheme. In some embodiments, the antibodies have reduced aggregation compared to antibodies without a mutation at the C86 amino acid. In some embodiments, the antibodies have reduced aggregation with serum proteins compared to antibodies without a mutation at the C86 amino acid. In some embodiments, the antibodies have reduced in vitro or in vivo aggregation compared to antibodies without a mutation at the C86 amino acid.

[0245] In some embodiments, the IgA2 antibody comprises a mutation at C86 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at C86 according to the numbering scheme of the IMGT scheme. In some embodiments, the IgA2 antibody comprises C86S according to the numbering scheme of the IMGT scheme. In some embodiments, the IgA2 antibody has reduced aggregation compared to an IgA2 antibody without a mutation at the C86 amino acid. In some embodiments, the IgA2 antibody has reduced aggregation with serum proteins compared to an IgA2 antibody without a mutation at the C86 amino acid. In some embodiments, the IgA2 antibody has reduced in vitro or in vivo aggregation compared to an IgA2 antibody without a mutation at the C86 amino acid.

[0246] In some embodiments, the antibody comprises a mutation at N114 (inadvertently referred to as N120 in US 62 / 824,864, which is incorporated herein in its entirety, as noted above) numbered according to the IMGT scheme. In some embodiments, the antibody comprises a non-conservative mutation at N114 numbered according to the IMGT scheme. In some embodiments, the antibody comprises N114T numbered according to the IMGT scheme. In some embodiments, the antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the N114 amino acid.

[0247] In some embodiments, the antibody comprises a mutation at I115 (inadvertently referred to as I121 in US Pat. No. 6,222,824,864, incorporated herein in its entirety, as noted above), numbered according to the IMGT scheme. In some embodiments, the antibody comprises a non-conservative mutation at I115, numbered according to the IMGT scheme. In some embodiments, the antibody comprises I115L, numbered according to the IMGT scheme. In some embodiments, the antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the I115 amino acid.

[0248] In some embodiments, the antibody comprises a mutation at T116 (inadvertently referred to as T122 in US Pat. No. 6,224,864, incorporated herein in its entirety, as noted above) numbered according to the IMGT scheme. In some embodiments, the antibody comprises a non-conservative mutation at T116 numbered according to the IMGT scheme. In some embodiments, the antibody comprises T116S numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the T116 amino acid.

[0249] In some embodiments, the IgA2 antibody comprises a mutation at N114, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at N114, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises an N14T, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the N114 amino acid.

[0250] In some embodiments, the IgA2 antibody comprises a mutation at I115 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at I115 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises I115L according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the I115 amino acid.

[0251] In some embodiments, the IgA2 antibody comprises a mutation at T116, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at T116, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises T116S, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the T116 amino acid.

[0252] In some embodiments, the IgA2 antibody comprises a mutation at N15.2 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at N15.2 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises N15.2G, N15.2Q, or N15.2T according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the N15.2 amino acid.

[0253] In some embodiments, the IgA2 antibody comprises a mutation at L15.3 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at L15.3 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises L15.3I according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the L15.3 amino acid.

[0254] In some embodiments, the IgA2 antibody comprises a mutation at T16 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at T16 according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a T16S numbering according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody without a mutation at the T16S amino acid.

[0255] In some embodiments, the IgA2 antibody comprises a mutation at C147, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at C147, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises 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 without a mutation at the C147 amino acid.

[0256] In some embodiments, the IgA2 antibody comprises a mutation at Y148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a non-conservative mutation at Y148, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody comprises a deletion of amino acid Y148, numbered according to the IMGT scheme.

[0257] In some embodiments, the IgA antibody comprises one or more albumin-binding domains. In some embodiments, the one or more albumin-binding domains are fused to the light or heavy chain of an IgA constant region. In some embodiments, the one or more albumin-binding domains are fused to the heavy chain of an IgA constant region. In some embodiments, the one or more albumin-binding domains are fused to the C-terminal region of the CH3 region of the heavy chain of the IgA constant region. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody that does not comprise one or more albumin-binding domains. In some embodiments, the IgA2 antibody comprises one or more albumin-binding domains and has a circulating half-life within 1%, 5%, or 10% of the circulating half-life of the corresponding IgG antibody. In some embodiments, the IgA2 antibody comprises one or more albumin-binding domains and has a circulating half-life longer than the circulating half-life of the corresponding IgG antibody.

[0258] In some embodiments, the IgA antibody comprises one or more mutations described in Lohse S. et al. Cancer Res. 2015; 76 (2): 403-17; Meyer S. et al. mAbs. 2016; 8 (1): 87-98; or Leusen J. et al. Molecular Immunology. 2015; 68: 35-39.

[0259] In some embodiments, one or more mutations or deletions result in an extension or shortening of the circulating half-life of the IgA antibody. In some embodiments, one or more mutations or deletions result in an extension of the circulating half-life of the IgA antibody. For example, one or more mutations may extend the serum half-life of an IgA antibody in humans to 21 days or longer. Furthermore, one or more mutations may extend the serum half-life of an IgA antibody in mice to 9 days or longer. In some embodiments, one or more mutations may extend the serum half-life of an IgA antibody to a level equivalent to the serum half-life of an immunoglobulin G (IgG) molecule. In some embodiments, one or more mutations or deletions result in a shortening of the circulating half-life of an IgA antibody.

[0260] In some embodiments, one or more mutations may extend the serum half-life of an IgA antibody by at least about 7 days to about 30 days or more. In some embodiments, one or more mutations may extend the serum half-life of an IgA antibody by at least about 7 days. In some embodiments, one or more mutations may extend the serum half-life of an IgA antibody by up to about 30 days. In some embodiments, one or more mutations may extend the serum half-life of an IgA antibody by about 7 days to about 8 days, about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 15 days, about 7 days to about 20 days, about 7 days to about 25 days, about 7 days to about 30 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 15 days, about 8 days to about 20 days, about 8 days to about 25 days, about 8 days to about 30 days, The serum half-life of an IgA antibody may be extended by about 9 to about 10 days, about 9 to about 15 days, about 9 to about 20 days, about 9 to about 25 days, about 9 to about 30 days, about 10 to about 15 days, about 10 to about 20 days, about 10 to about 25 days, about 10 to about 30 days, about 15 to about 20 days, about 15 to about 25 days, about 15 to about 30 days, about 20 to about 25 days, about 20 to about 30 days, or about 25 to about 30 days. In some embodiments, one or more mutations may extend the serum half-life of an IgA antibody by about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days. Thus, in some embodiments, an antibody or functional fragment thereof disclosed herein exhibits a longer circulating half-life compared to a corresponding wild-type IgA antibody. In some embodiments, the antibodies exhibit a circulating 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 a corresponding WT IgA antibody.

[0261] In some embodiments, the IgA antibody exhibits increased stability. In some embodiments, the one or more mutations and / or one or more deletions result in increased stability of the IgA antibody compared to a corresponding IgA antibody that does not contain the one or more mutations and / or one or more deletions.

[0262] In some embodiments, the IgA antibody exhibits reduced aggregation. Antibody aggregation is a more common manifestation of physical instability. Protein aggregates generally have reduced activity due to epitope multiplicity and / or conformational changes and, more importantly, have greater immunogenic potential. Immunoglobulin aggregates are known to cause severe renal failure and anaphylactoid reactions, such as headache, fever, and chills. Therefore, reducing aggregation in antibody therapeutics is advantageous. Furthermore, 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 the IgA antibody compared to a corresponding IgA antibody that does not contain the one or more mutations and / or one or more deletions. In some embodiments, the antibodies or functional fragments disclosed herein exhibit reduced aggregation compared to a corresponding wild-type IgA antibody. In some embodiments, the antibodies exhibit 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% reduced aggregation compared to a corresponding WT IgA antibody. In some embodiments, the antibodies or functional fragments thereof disclosed herein exhibit reduced aggregation with serum proteins compared to a corresponding WT IgA antibody.In some embodiments, the antibodies exhibit 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% reduced aggregation compared to a corresponding WT IgA antibody.

[0263] In some embodiments, the IgA antibodies provided herein have aggregate levels ranging from at least about 0.1% to a maximum of about 5%. In some embodiments, the IgA antibodies provided herein have aggregate levels ranging from at least about 0.1%. In some embodiments, the IgA antibodies provided herein have aggregate levels ranging from a maximum of about 5%. In some embodiments, the IgA antibodies provided herein have aggregate levels ranging from about 0.1% to about 0.5%, about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 3%, about 0.1% to about 4%, about 0.1% to about 5%, about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 0.5% to about 5%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 3% to about 4%, about 3% to about 5%, or about 4% to about 5%. In some embodiments, the IgA antibodies provided herein have aggregate levels ranging from about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5%.

[0264] The therapeutic antibodies disclosed herein may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indomethacin, benzoylalanine ... phosphorus-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0265] Methods for substituting or deleting amino acids are known in the art. For example, amino acid substitution or deletion can be performed by site-directed mutagenesis (e.g., Zoller and Smith Nucl. Acids Res. 10: 6487 (1982)). Mutagenesis can be performed by synthesizing oligonucleotides with one or more modifications in the sequence of the constant domain of the antibody to be modified. Antibodies of the present disclosure (e.g., containing one or more modifications in the IgA heavy chain constant region) can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc. Site-directed mutagenesis allows for the generation of mutants by using specific oligonucleotide sequences encoding the DNA sequence of the desired mutation and a sufficient number of flanking oligonucleotides to provide primer sequences of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction to be traversed. Generally, primers of about 17 to about 75 nucleotides in length or longer, with about 10 to about 25 or more residues on either side of the junction of the sequence to be altered, are preferred. Several such primers, which introduce a variety of different mutations at one or more positions, can be used to generate a library of mutants.

[0266] The technique of site-directed mutagenesis is well known in the art (see, for example, Kunkel et al., Methods Enzymol, 154: 367-82, 1987). Generally, site-directed mutagenesis is performed by first obtaining a single-stranded vector containing a DNA sequence encoding the desired peptide, or by melting the two strands of a double-stranded vector apart. An oligonucleotide primer bearing the desired mutated sequence is generally prepared synthetically. This primer is then annealed with the single-stranded vector and subjected to a DNA polymerizing enzyme, such as T7 DNA polymerase, to completely synthesize a strand bearing the mutation. Thus, a heteroduplex is formed, in which one strand encodes the original, unmutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones containing recombinant vectors bearing the mutated sequence arrangement are selected. As will be appreciated, this technique generally uses phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors useful for site-directed mutagenesis include vectors such as M13 phage. These phages are readily commercially available, and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis, thereby eliminating the step of transferring the gene of interest from a plasmid to a phage. Site-directed mutagenesis has also been used to identify amino acid residues that affect the plasma clearance of mouse IgG1 hinge-Fc fragments, as described in Kim Jin-Kyoo et al., (1994) Eur. J. Immunol. 24: 542-548).

[0267] Alternatively, PCR can be used with commercially available thermostable enzymes such as Taq DNA polymerase to incorporate mutagenic oligonucleotide primers into the amplified DNA fragments, which can then be cloned into appropriate cloning or expression vectors.For PCR-mediated mutagenesis procedures, see, for example, Tomic et al., Nucleic Acids Res., 18(6): 1656, 1987, and Upender et al., Biotechniques, 18 (1): 29-30, 32, 1995.PCR using thermostable polymerase and thermostable ligase can also be used to incorporate phosphorylated mutagenic oligonucleotides into the amplified DNA fragments, which can then be cloned into appropriate cloning or expression vectors (see, for example, Michael, Biotechniques, 16 (3): 410-2, 1994).

[0268] Other methods known to those skilled in the art for generating sequence variants of an antibody Fc region or its FcR-binding domain can be used. For example, a recombinant vector encoding the amino acid sequence of an antibody constant domain or a fragment thereof can be treated with a mutagen such as hydroxylamine to obtain sequence variants. Mutants that confer desirable properties, such as increased ADCC, reduced aggregation, increased affinity for FcR, and / or increased in vivo half-life, can be screened using routine assays, such as those described below.

[0269] Synthetic gene construction requires the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest.Gene synthesis can be carried out using several techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar techniques for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.Single or multiple amino acid substitutions, deletions, and / or insertions can be made using known methods of mutagenesis, recombination, and / or shuffling, and then tested using related screening procedures, such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO95 / 17413; or WO95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0270] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0271] Semi-synthetic gene construction is realized by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling.Semi-synthetic construction is typically represented by the process of utilizing synthesized polynucleotide fragments in combination with PCR techniques.Thus, a defined region of a gene can be synthesized de novo, while other regions can be amplified using site-directed mutagenic primers, while still other regions can be subjected to error-prone PCR or non-error-prone PCR amplification.The polynucleotide subsequences can then be shuffled. Other covalent modifications

[0272] The covalent modification of antibody is also included in the scope of the present invention.The covalent modification of antibody can be carried out by chemical synthesis of antibody or by enzymatic or chemical cleavage, if applicable.Another type of covalent modification of antibody is introduced into molecule by reacting the targeted amino acid residue of antibody with an organic derivatizing agent that can react with selected side chain or N-terminal residue or C-terminal residue.

[0273] Cysteinyl residues most commonly are reacted with haloacetic acids (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, alpha-bromo-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0274] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino-terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.

[0275] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires the reaction to be performed under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with the lysine groups as well as the arginine epsilon-amino group.

[0276] Specific modifications of tyrosyl residues can be made, with particular interest in introducing spectroscopic labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125I or 131I to prepare labeled proteins for use in radioimmunoassay. Carboxyl side chains (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (RN-C-N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0277] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated forms of these residues are within the scope of the present invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0278] Another type of covalent modification involves chemically or enzymatically coupling glycosides to antibodies. These procedures are advantageous because they do not require production of the antibody in a host cell with glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling method used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO87105330, published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0279] Removal of any carbohydrate moieties present on an antibody can be achieved chemically or enzymatically. Chemical deglycosylation requires exposing the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Hakimuddin, et al. Arch. Biochem. Biophys. 259: 52 (1987) and Edge et al. Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved using various endo- and exoglycosidases, as described by Thotakura et al. Meth. Enzymol. 138: 350 (1987).

[0280] Another type of covalent modification of an antibody involves linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran. Such methods are known in the art; see, for example, U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337; 4,766,106; 4,179,337; 4,495,285; 4,609,546; or EP 315 456.

[0281] IgA antibody targets

[0282] The IgA antibodies described herein can be used to target antigens expressed on the surface of cells. In some embodiments, the IgA antibodies comprise an antigen-binding region that specifically binds to an antigen expressed on the surface of a target cell (e.g., a cancer cell). In some embodiments, the antigen is a human antigen. In some embodiments, the target cell is a human cell. In some embodiments, the IgA antibodies comprise an antigen-binding domain that specifically binds to an antigen that is one of the following proteins: CD20, GD2, CD47, CD38, EGFR, HER2, PD-L1, CD25, CD33, BCMA, CD44, CD21, CD64, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER3, folate-binding protein, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, EGFR, MUC-1, or MAGE-A1. , MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123, VEGF-R2, BCMA, CD19, CD22, CD30, CD33, CD123, CD38, CD44, CD70, CD274, CD45, CD123, CD1 38, CD171, ROR1, EGFR, EphA2, FBP, FAP, CEA, EGP2, EGP40, TAG72, PSMA, PSA, PAP, hsp70-2, M-CSF, LAGE-la, p53, NKG2D ligand, B7-H6, IL-13 R α 2, IL-11 R α, MUC1, MUC16, CA9, GD3, HMW-MAA, CD171, Lewis Y, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, PCTA-1, MAGE, ELF2M, IGF-I, IGF-II, IGF-I receptor, hTERT, WT1, MUC1, LMP2, HPV16, HPV18, RGL4, Me lanA, MART, ML-IAP, AFP, BCR, ABL, CYP1B1, PLAC1, BORIS, NY-BR-1, RGS5, SART3, EphA2, glypican-3, 5T4, 8H9, ανβ6B7-H3, B7-H6, CAIX, CA9, CSPG4, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FARFAP, FB P, KDR, MCSP, Mucl, Mucl6, NCAM, PRAME, ROR1, CD44v7 / 8, 8H9, NCAM, VEGF-R, TAG72, RAGE-1, MN-CA IX, RU1, RU2(AS), AchR, TEM1, TEM8, PAX5, OY-TES1, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, 2、PDGFR-β、カリクレイン4、PBF、PRAME、HSDL1、CA125、TADG-12、MUC16、マンナン-MIC-1、HERV-K-MEL、KK-LC-1、KM-HN-1、LAGE-1、MAGE-A4、SP17、S SX4、TAG1、TAG2、ENAH、マンマグロビン-A、NY-BR-1、BAGE-1、HERV-K-MEL、K K-LC-1, KM-KN-1, LAGE1, MAGE1A, MAGEA2, k, TRAG3, c-myc,B1, p62, DKK1, RU2AS, k-ras, ME1, NFYC, STEAP1, FGF5, RU2AS, hsp70-2, ARTC1, B-RAF, beta-catenin, CDC27, CDK4, CDK12, CDKN2A, CLPP, CSNK1A1, FN1, GAS7, GPNMB, HAUS3, LDLR-fucosyltransferase, MART2, MATN, MUM1, MUM2, MUM3, neo-PAP, myosin, PPP1R3B, PRDX5, PTPRK, RBAF600, SIRT2, SNRPD1, triosephosphate isomerase, OA1, RAB38, TRP1, TRP2, melanA, BAGE1, GAGE2, GAGE8, GAGE3, GAGE4, ​​G AGE5, GAGE6, GAGE7, GNTCF, LY6K, TRAG3, CASP8, SAGE, DEK-CAN, EFTUD2, FLT3-ITD, cyclin A1, FNDC3B, MAGEAG, G250, hepsin, intestinal carboxylesterase, PBF, CASP5, COA1, OGT, OS9, CALCA, MDM2, alpha actinin 4, elongation factor 2, fos-related antigen 1, legumain, sperm protein 17, carbonic anhydrase IX, folate receptor-α, neutrophil elastase, ephrin B2, glioma-associated antigen, beta-human chorionic gonadotropin, alpha-fetoprotein thyroglobulin, telomerase reverse transcriptase, intestinal carboxylesterase, prostein, or survivin. Ganglioside G2

[0283] In some embodiments, an IgA antibody or functional fragment thereof disclosed herein (i.e., an antibody comprising one or more modifications disclosed herein within the IgA heavy chain constant region) specifically binds to GD2. In some embodiments, the IgA antibody binds to O-acetylated GD2. In some embodiments, the IgA antibody binds to GD2 but not to O-acetylated GD2. In some embodiments, the IgA antibody binds to O-acetylated GD2 but not to GD2. In some embodiments, the antibody binds to both GD2 and O-acetylated GD2. In some embodiments, the antibody or functional fragment disclosed herein specifically binds to human GD2 polypeptide. Polypeptides and encoding nucleic acid sequences of GD2 of human origin and of several animals are publicly available, for example, from the NCBI website.

[0284] In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of antibody ch14.18. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of antibody ch14.18. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the light chain of antibody ch14.18. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of antibody ch14.18 and one, two, or three of CDR1, CDR2, or CDR3 of the light chain of antibody ch14.18. In some embodiments, the IgA antibody comprises CDR1, CDR2, or CDR3 of the heavy chain of antibody ch14.18 and CDR1, CDR2, or CDR3 of the light chain of antibody ch14.18. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of antibody ch14.18 and an IgA hinge. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of antibody ch14.18, an IgA hinge, and a CH1 IgA region. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of antibody ch14.18, an IgA hinge, a CH1 IgA region, and a CH2 IgA region. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of antibody ch14.18, an IgA hinge, a CH1 IgA region, a CH2 IgA region, and a CH3 IgA region. In some embodiments, the IgA antibody comprises a heavy chain variable region comprising the amino acid sequence: EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS [SEQ ID NO: 4]. In some embodiments, the IgA antibody comprises a light chain variable region comprising the amino acid sequence: EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPK LLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK [SEQ ID NO: 5].In some embodiments, the IgA antibody comprises a heavy chain variable region comprising the amino acid sequence: EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS [SEQ ID NO: 4], and a light chain variable region comprising the amino acid sequence: EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK [SEQ ID NO: 5].

[0285] In some embodiments, the IgA antibody comprises a variable heavy chain comprising one or more of the following: CDR1 comprising the amino acid sequence: EFTFTDYY [SEQ ID NO: 10]; CDR2 comprising the amino acid sequence: IRNRANGYTT [SEQ ID NO: 11]; and CDR3 comprising the amino acid sequence: ARVSNWAFDY [SEQ ID NO: 12]. In some embodiments, the IgA antibody comprises a variable light chain comprising one or more of the following: CDR1 comprising the amino acid sequence: QSLLKNNGNTFL [SEQ ID NO: 13]; CDR2 comprising the amino acid sequence: KVS [SEQ ID NO: 14]; and CDR3 comprising the amino acid sequence: SQSTHIPYT [SEQ ID NO: 15].

[0286] In some embodiments, the IgA antibody comprises a variable heavy chain comprising one or more of: a CDR1 comprising the amino acid sequence: EFTFTDYY [SEQ ID NO: 10]; a CDR2 comprising the amino acid sequence: IRNRANGYTT [SEQ ID NO: 11]; a CDR3 comprising the amino acid sequence: ARVSNWAFDY [SEQ ID NO: 12]; and a variable light chain comprising one or more of: a CDR1 comprising the amino acid sequence: QSLLKNNGNTFL [SEQ ID NO: 13]; a CDR2 comprising the amino acid sequence: KVS [SEQ ID NO: 14]; and a CDR3 comprising the amino acid sequence: SQSTHIPYT [SEQ ID NO: 15].

[0287] In some embodiments, the anti-GD2 IgA antibody comprises one or more (e.g., two, three, four, five, or six) of the HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 of the 3F8 antibody. In some embodiments, the anti-GD2 antibody comprises the variable heavy chain and / or variable light chain of the 3F8 antibody.

[0288] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 4; (b) a VL comprising the amino acid sequence of SEQ ID NO: 5; and (c) a combination thereof.

[0289] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0290] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 5.

[0291] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74; and a VH comprising the amino acid sequence of SEQ ID NO: 4.

[0292] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO:50; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:74.

[0293] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0294] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0295] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within the amino acid sequence of SEQ ID NO: 4. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 4, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 50.

[0296] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from any one of the amino acid sequences of SEQ ID NO: 5. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 5, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74.

[0297] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL sequence of SEQ ID NO: 5, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31. CD20

[0298] In some embodiments, the IgA antibody specifically binds to CD20. In some embodiments, the IgA antibody comprises an antigen-binding domain comprising a type II or type I / II CD20 binding region. In some embodiments, the antibodies or functional fragments disclosed herein specifically bind to a human CD20 polypeptide. Polypeptides and encoding nucleic acid sequences of CD20 of human origin and several animals are publicly available, for example, from the NCBI website. In some embodiments, the IgA antibody comprises an antigen-binding domain that specifically binds to a CD20 epitope, wherein the CD20 epitope is present within the following amino acid sequence: YNCEPANPSEKNSPSTQYCYS [SEQ ID NO: 6].

[0299] In some embodiments, the variable region of a CD20 antibody is described in PCTNL2017050581, which is incorporated by reference in its entirety. In some embodiments, the heavy chain variable region comprises one, two, or three of CDR1, CDR2, or CDR3 within the following amino acid sequence: QAYLQQSGAELVRPGASVKMSCKASGYTFT SYNLHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSRLTSED SAVYFCARSNSYGSTYWYFDVWGTGTTVTVSS [SEQ ID NO: 7]. In some embodiments, the light chain variable region comprises one, two, or three of CDR1, CDR2, or CDR3 within the following amino acid sequence: QIVLSQSPAVLFASPGEKVTMTCRARSSVSYMDWYQQKPRSSPKPWIYATSNLASGVPARFSGSG SGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGSGTKLEIKRADAAPTVSIFPPSS [SEQ ID NO: 8].

[0300] In some embodiments, the heavy chain variable region comprises the following amino acid sequence: QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNLHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSRLTSED SAVYFCARSNSYGSTYWYFDVWGTGTTVTVSS [SEQ ID NO: 7]. In some embodiments, the light chain variable region comprises the following amino acid sequence: QIVLSQSPAVLFASPGEKVTMTCRARSSVSYMDWYQQKPRSSPKPWIYATSNLASGVPARFSGSG SGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGSGTKLEIKRADAAPTVSIFPPSS [SEQ ID NO: 8].

[0301] In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of the antibody obinutuzumab. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of the antibody obinutuzumab. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the light chain of the antibody obinutuzumab. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of the antibody obinutuzumab and one, two, or three of CDR1, CDR2, or CDR3 of the light chain of the antibody obinutuzumab. In some embodiments, the IgA antibody comprises CDR1, CDR2, or CDR3 of the heavy chain of the antibody obinutuzumab, and CDR1, CDR2, or CDR3 of the light chain of the antibody obinutuzumab. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of the antibody obinutuzumab and an IgA hinge. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of the antibody obinutuzumab, an IgA hinge, and a CH1 IgA region. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of the antibody obinutuzumab, an IgA hinge, a CH1 IgA region, and a CH2 IgA region. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of the antibody obinutuzumab, an IgA hinge, a CH1 IgA region, a CH2 IgA region, and a CH3 IgA region.

[0302] In some embodiments, the IgA antibody specifically binds to the CD20 epitope EPANPSEK.

[0303] In some embodiments, the IgA antibody specifically binds to CD20 and has increased programmed cell death (PCD) functionality compared to rituximab having a constant region of the same isotype. In some embodiments, the IgA antibody specifically binds to CD20 and has increased antibody-dependent cellular cytotoxicity (ADCC) functionality compared to rituximab having a constant region of the same isotype. In some embodiments, the IgA antibody specifically binds to CD20 and has increased complement-dependent cytotoxicity (CDC) functionality compared to rituximab having a constant region of the same isotype.

[0304] In some embodiments, IgA antibodies have a shorter circulating half-life than the corresponding IgG antibody. In some embodiments, administration of anti-CD20 IgA antibodies is associated with fewer side effects due to B cell depletion than the corresponding IgG antibody. In some embodiments, administration of anti-CD20 antibodies is associated with more rapid B cell recruitment than the corresponding IgG antibody.

[0305] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 81; (b) a VL comprising the amino acid sequence of SEQ ID NO: 95; and (c) a combination thereof.

[0306] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 49; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 65; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 73.

[0307] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 49; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 95.

[0308] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 65; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 73; and a VH comprising the amino acid sequence of SEQ ID NO: 81.

[0309] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 49; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 73.

[0310] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 65; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 73. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0311] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 49; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 65; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 73.

[0312] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within the amino acid sequence of SEQ ID NO: 81. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 81, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 33, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0313] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from any one of the amino acid sequences of SEQ ID NO: 95. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 95, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 57; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 73.

[0314] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 81 and a VL sequence of SEQ ID NO: 95, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31.

[0315] In some embodiments, the present invention provides an antibody or functional fragment thereof that specifically binds to CD47. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of antibody UMAB10. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of antibody UMAB10. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the light chain of antibody UMAB10. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of antibody UMAB10 and one, two, or three of CDR1, CDR2, or CDR3 of the light chain of antibody UMAB10. In some embodiments, an IgA antibody comprises CDR1, CDR2, or CDR3 of the heavy chain of antibody UMAB10 and CDR1, CDR2, or CDR3 of the light chain of antibody UMAB10. In some embodiments, an IgA antibody comprises at least CDR3 of the heavy chain and an IgA hinge of antibody UMAB10. In some embodiments, an IgA antibody comprises at least CDR3, an IgA hinge, and a CH1 IgA region of the heavy chain of antibody UMAB10. In some embodiments, an IgA antibody comprises at least CDR3, an IgA hinge, a CH1 IgA region, and a CH2 IgA region of the heavy chain of antibody UMAB10. In some embodiments, an IgA antibody comprises at least CDR3, an IgA hinge, a CH1 IgA region, a CH2 IgA region, and a CH3 IgA region of the heavy chain of antibody UMAB10.

[0316] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 86; (b) a VL comprising the amino acid sequence of SEQ ID NO: 100; and (c) a combination thereof.

[0317] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 48; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0318] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 48; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0319] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 80; and a VH comprising the amino acid sequence of SEQ ID NO: 7.

[0320] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO:56; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:80.

[0321] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 80. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 48; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0322] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 48; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0323] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within the amino acid sequence of SEQ ID NO: 7. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 7, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0324] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from any one of the amino acid sequences of SEQ ID NO: 8. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 8, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 64; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 80.

[0325] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31. Her2

[0326] In some embodiments, provided herein is an antibody or functional fragment thereof that specifically binds to Her2. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of the antibody trastuzumab. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of the antibody trastuzumab. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the light chain of the antibody trastuzumab. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of the antibody trastuzumab and one, two, or three of CDR1, CDR2, or CDR3 of the light chain of the antibody trastuzumab. In some embodiments, the IgA antibody comprises CDR1, CDR2, or CDR3 of the heavy chain of the antibody trastuzumab, and CDR1, CDR2, or CDR3 of the light chain of the antibody trastuzumab. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of the antibody trastuzumab and an IgA hinge. In some embodiments, the IgA antibody comprises at least CDR3, an IgA hinge, and a CH1 IgA region of the heavy chain of the antibody trastuzumab. In some embodiments, the IgA antibody comprises at least CDR3, an IgA hinge, a CH1 IgA region, and a CH2 IgA region of the heavy chain of the antibody trastuzumab. In some embodiments, the IgA antibody comprises at least CDR3, an IgA hinge, a CH1 IgA region, a CH2 IgA region, and a CH3 IgA region of the heavy chain of the antibody trastuzumab.

[0327] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 82; (b) a VL comprising the amino acid sequence of SEQ ID NO: 96; and (c) a combination thereof.

[0328] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 43; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 51; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 75.

[0329] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 43; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 51; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 96.

[0330] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 75; and a VH comprising the amino acid sequence of SEQ ID NO: 82.

[0331] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 51; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 75.

[0332] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 75. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 43; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 51.

[0333] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 43; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 51; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 75.

[0334] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted within the amino acid sequence of SEQ ID NO: 82. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 82, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 35, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 51.

[0335] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from any one of the amino acid sequences of SEQ ID NO: 96. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 96, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 59; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 75.

[0336] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL sequence of SEQ ID NO: 96, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31. gp75

[0337] In some embodiments, provided herein are antibodies or functional fragments thereof that specifically bind to gp75 or tyrosine-associated protein 1. In some embodiments, the antibodies or functional fragments disclosed herein specifically bind to human gp75 polypeptide. Polypeptides and encoding nucleic acid sequences of gp75 of human origin and several animals are publicly available, for example, from the NCBI website. In some embodiments, an IgA antibody comprises at least CDR3 of the heavy chain of antibody TA99. In some embodiments, an IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of antibody TA99. In some embodiments, an IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the light chain of antibody TA99. In some embodiments, an IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 from the heavy chain of antibody TA99 and one, two, or three of CDR1, CDR2, or CDR3 from the light chain of antibody TA99. In some embodiments, an IgA antibody comprises CDR1, CDR2, or CDR3 from the heavy chain of antibody TA99 and CDR1, CDR2, or CDR3 from the light chain of antibody TA99. In some embodiments, an IgA antibody comprises at least CDR3 from the heavy chain and an IgA hinge of antibody TA99. In some embodiments, an IgA antibody comprises at least CDR3, an IgA hinge, and a CH1 IgA region from the heavy chain of antibody TA99. In some embodiments, an IgA antibody comprises at least CDR3, an IgA hinge, a CH1 IgA region, and a CH2 IgA region from the heavy chain of antibody TA99. In some embodiments, the IgA antibody comprises at least the CDR3, IgA hinge, CH1 IgA region, CH2 IgA region, and CH3 IgA region of the heavy chain of antibody TA99.

[0338] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 83; (b) a VL comprising the amino acid sequence of SEQ ID NO: 97; and (c) a combination thereof.

[0339] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 52; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 68; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0340] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 52; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 97.

[0341] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 68; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76; and a VH comprising the amino acid sequence of SEQ ID NO: 83.

[0342] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 52; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0343] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 68; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0344] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 52; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 68; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0345] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within the amino acid sequence of SEQ ID NO: 83. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 83, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0346] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from any one of the amino acid sequences of SEQ ID NO: 97. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 97, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 60; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 68; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0347] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL sequence of SEQ ID NO: 97, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31. CTLA4

[0348] In some embodiments, provided herein is an antibody or functional fragment thereof that specifically binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA4). In some embodiments, the antibody or functional fragment disclosed herein specifically binds to human CTLA4 polypeptide. In some embodiments, the antibody or functional fragment disclosed herein specifically binds to mouse CTLA4 polypeptide. Polypeptides and encoding nucleic acid sequences of CTLA4 of human origin and several animals are publicly available, for example, from the NCBI website.

[0349] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 84; (b) a VL comprising the amino acid sequence of SEQ ID NO: 98; and (c) a combination thereof.

[0350] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 77.

[0351] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 37; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 98.

[0352] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 77; and a VH comprising the amino acid sequence of SEQ ID NO: 84.

[0353] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 77.

[0354] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 77. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 37; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0355] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 77.

[0356] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within the amino acid sequence of SEQ ID NO: 84. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 84, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 37, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0357] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids of any one of the amino acid sequences of SEQ ID NO: 98 are substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 98, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 61; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 77.

[0358] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 84 and a VL sequence of SEQ ID NO: 98, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31. CD47

[0359] In some embodiments, the IgA antibody specifically binds to CD47. In some embodiments, the antibodies or functional fragments disclosed herein specifically bind to human CD47 polypeptide. CD47 polypeptides and encoding nucleic acid sequences of human origin and several animals are publicly available, for example, from the NCBI website. In some embodiments, the IgA antibody reduces the binding of SIRPα to CD47 expressed on the surface of cancer cells. In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) ranging from about 0.5 μM to about 999 μM compared to a corresponding wild-type antibody. In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) ranging from about 1 mM to about 1000 mM compared to a corresponding wild-type antibody. In some embodiments, the IgA antibody inhibits the interaction of human CD47 with signal regulatory protein α (SIRPα). In some embodiments, inhibition of the interaction of human CD47 with SIRPα increases the potency of the IgA antibody. In some embodiments, inhibiting the interaction between human CD47 and SIRPα increases phagocytosis and clearance of cancer cells at tumor sites. In some embodiments, the cancer cells are IgA-opsonized cancer cells. In some embodiments, the IgA antibody comprises an antigen-binding domain that binds to CD47 and an antigen-binding domain that specifically binds to a tumor-associated antigen (e.g., one described herein).

[0360] In some embodiments, the IgA antibody binds to CD47. In some embodiments, the IgA antibody reduces CD47 binding to cancer cells. For example, the IgA antibody can inhibit the interaction between human CD47 and signal-regulatory protein alpha (SIRPα). Furthermore, inhibiting the interaction between human CD47 and SIRPα can increase the potency of the IgA antibody. Inhibiting the interaction between human CD47 and SIRPα can increase phagocytosis and clearance of cancer cells at tumor sites. For example, the cancer cells can be IgA-opsonized cancer cells. In some embodiments, the IgA antibodies described herein have low affinity binding to CD47, thereby preventing the IgA antibody from binding to CD47 on cells other than cancer cells. In some embodiments, the low-affinity CD47 arm of the IgA antibodies described herein binds to tumor cells expressing CD47. In some examples, the low-affinity CD47 arm of the IgA antibodies described herein does not bind to cells expressing CD47 that are not tumor cells. In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of at least about 0.01 micromolar (μM) to about 999 μM or higher. In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of at least about 0.01 μM. In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of up to about 999 μM.In some embodiments, the IgA antibody binds to CD47 at about 0.01 μM to about 0.1 μM, about 0.01 μM to about 0.5 μM, about 0.01 μM to about 1 μM, about 0.01 μM to about 5 μM, about 0.01 μM to about 10 μM, about 0.01 μM to about 50 μM, about 0.01 μM to about 100 μM, about 0.01 μM to about 200 μM, about 0.01 μM to about 300 μM, about 0.01 μM to about 500 μM, about 0.01 μM to about 999 μM, about 0.1 μM to about 0.5 μM, about 0.1 μM to about 1 μM, about 0.1 μM to about 5 μM, about 0.1 μM to about 10μM, approximately 0.1μM to approximately 50μM, approximately 0.1μM to approximately 100μM, approximately 0.1μM to approximately 200μM, approximately 0.1μM to approximately 300μM, approximately 0.1μM to approximately 500μM, approximately 0.1μM to approximately 999μM, approximately 0.5μM to approximately 1μM, approximately 0.5μM to approximately 5μM, approximately 0.5μM to about 10μM, about 0.5μM to about 50μM, about 0.5μM to about 100μM, about 0.5μM to about 200μM, about 0.5μM to about 300μM, about 0.5μM to about 500μM, about 0.5μM to about 999μM, about 1μM to about 5μM, about 1μM to about 10μ M, approx. 1 μM to approx. 50 μM, approx. 1 μM to approx. 100 μM, approx. 200μM, about 5μM to about 300μM, about 5μM to about 500μM, about 5μM to about 999μM, about 10μM to about 50μM, about 10μM to about 100μM, about 10μM to about 200μM, about 10μM to about 300μM, about 10μM to about 500μM, about 10μM to about 9 It binds with a binding affinity (Kd) of 99 μM, about 50 μM to about 100 μM, about 50 μM to about 200 μM, about 50 μM to about 300 μM, about 50 μM to about 500 μM, about 50 μM to about 999 μM, about 100 μM to about 200 μM, about 100 μM to about 300 μM, about 100 μM to about 500 μM, about 100 μM to about 999 μM, about 200 μM to about 300 μM, about 200 μM to about 500 μM, about 200 μM to about 999 μM, about 300 μM to about 500 μM, about 300 μM to about 999 μM, or about 500 μM to about 999 μM.In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 50 μM, about 100 μM, about 200 μM, about 300 μM, about 500 μM, or about 999 μM.

[0361] In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of about 1 mM to about 1,000 millimolar (mM). In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of at least about 1 mM. In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of up to about 1,000 mM.In some embodiments, the IgA antibody binds to CD47 at about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 50 mM, about 1 mM to about 100 mM, about 1 mM to about 200 mM, about 1 mM to about 300 mM, about 1 mM to about 400 mM, about 1 mM to about 500 mM, about 1 mM to about 600 mM, about 1 mM to about 800 mM, about 1 mM to about 1,000 mM, about 5 mM to about 10 mM, about 5 mM to about 50 mM, about 5 mM to about 100 mM, about 5 mM to about 200 mM, about 5 mM to about 300 mM, about 5 mM to about 400 mM, about 5 mM to about 50 0 mM, about 5 mM to about 600 mM, about 5 mM to about 800 mM, about 5 mM to about 1,000 mM, about 10 mM to about 50 mM, about 10 mM to about 100 mM, about 10 mM to about 200 mM, about 10 mM to about 300 mM, about 10 mM to about 400 mM, about 10 mM to about 500 mM, about 10 mM to about 600 mM, about 10 mM to about 800 mM, about 10 mM to about 1,000 mM, about 50 mM to about 100 mM, about 50 mM to about 200 mM, about 50 mM to about 300 mM, about 50 mM to about 400 mM, about 50 mM to about 500 mM, about 50 mM to about 600 mM, about 50 mM to about 800 mM, about 50 mM to about 1,000 mM, about 100 mM to about 200 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 100 mM to about 500 mM, about 100 mM to about 600 mM, about 100 mM to about 800 mM, about 100 mM to about 1,000 mM, about 200 mM to about 300 mM, about 200 mM to about 400 mM, about 200 mM to about 500 mM, about 200 mM to about 600 mM, about 200 mM to about 800 mM, about 200 mM to about 1,000 mM, about 300 mM to about It binds with a binding affinity (Kd) of 400 mM, about 300 mM to about 500 mM, about 300 mM to about 600 mM, about 300 mM to about 800 mM, about 300 mM to about 1,000 mM, about 400 mM to about 500 mM, about 400 mM to about 600 mM, about 400 mM to about 800 mM, about 400 mM to about 1,000 mM, about 500 mM to about 600 mM, about 500 mM to about 800 mM, about 500 mM to about 1,000 mM, about 600 mM to about 800 mM, about 600 mM to about 1,000 mM, or about 800 mM to about 1,000 mM.In some embodiments, the IgA antibody binds to CD47 with a binding affinity (Kd) of about 1 mM, about 5 mM, about 10 mM, about 50 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 800 mM, or about 1,000 mM.

[0362] In some embodiments, provided herein is an antibody or functional fragment thereof that specifically binds to CD47. In some embodiments, the IgA antibody comprises at least CDR3 of the heavy chain of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the heavy chain of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises one, two, or three of CDR1, CDR2, or CDR3 of the light chain of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises one, two, or three of the CDR1, CDR2, or CDR3 of the heavy chain of antibody 2.3D11 or antibody C47A8-CQ and one, two, or three of the CDR1, CDR2, or CDR3 of the light chain of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises the CDR1, CDR2, or CDR3 of the heavy chain of antibody 2.3D11 or antibody C47A8-CQ and the CDR1, CDR2, or CDR3 of the light chain of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises at least the CDR3 of the heavy chain and an IgA hinge of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises at least the CDR3, IgA hinge, CH1 IgA region of the heavy chain of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises at least the CDR3, IgA hinge, CH1 IgA region, and CH2 IgA region of the heavy chain of antibody 2.3D11 or antibody C47A8-CQ. In some embodiments, the IgA antibody comprises at least the CDR3, IgA hinge, CH1 IgA region, CH2 IgA region, and CH3 IgA region of the heavy chain of antibody 2.3D11 or antibody C47A8-CQ.

[0363] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising the amino acid sequence of SEQ ID NO: 99; and (c) a combination thereof.

[0364] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 78.

[0365] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 38; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 99.

[0366] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 78; and a VH comprising the amino acid sequence of SEQ ID NO: 85.

[0367] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 78.

[0368] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 78. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 38; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54.

[0369] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 78.

[0370] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within the amino acid sequence of SEQ ID NO: 85. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 85, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 38, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54.

[0371] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids of any one of the amino acid sequences of SEQ ID NO: 99 have been substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 99, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 62; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 78.

[0372] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL sequence of SEQ ID NO: 99, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31.

[0373] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or more variable regions selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 86; (b) a VL comprising the amino acid sequence of SEQ ID NO: 100; and (c) a combination thereof.

[0374] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, two, three, four, five, or six CDRs selected from: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 47; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79.

[0375] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VH CDR sequences selected from: (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 47; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 55; and (d) a VL comprising the amino acid sequence of SEQ ID NO: 100.

[0376] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 71; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79; and a VH comprising the amino acid sequence of SEQ ID NO: 86.

[0377] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the CDRs: HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 55; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79.

[0378] In one aspect, the disclosure herein provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VL CDR sequences selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 71; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising at least one, at least two, or all three of the VH CDR sequences selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39; (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 47; and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 55.

[0379] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising CDRs: (a) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39; (b) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 47; (c) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 55; (d) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; (e) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 71; and (f) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79.

[0380] In one aspect, the antibody or antigen-binding fragment thereof comprises a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids within the amino acid sequence of SEQ ID NO: 86 have been substituted, inserted, and / or deleted. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., within the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 86, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from (a) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, (b) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and (c) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 55.

[0381] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising that sequence retains the ability to bind to an antigen. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from any one of the amino acid sequences of SEQ ID NO: 100. In some embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 100, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (a) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63; (b) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 71; and (c) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 79.

[0382] In one aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH similar to any of the embodiments presented above and a VL similar to any of the embodiments presented above. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL sequence of SEQ ID NO: 100, including post-translational modifications of these sequences. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgA heavy chain constant region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16-21. In some embodiments, the antibody or antigen-binding fragment thereof comprises a kappa light chain constant region of SEQ ID NO: 31. Improving IgA antibody properties and function

[0383] In some embodiments, the antibodies or functional fragments thereof disclosed herein exhibit improved stability compared to the corresponding wild-type IgA antibody. The term "increased stability," as used herein, includes increased thermal stability and / or reduced aggregation. Enhanced or improved stability can be determined, for example, by accelerated stability testing. Exemplary accelerated stability tests include, but are not limited to, tests characterized by elevated storage temperatures. Increased stability is indicated by a decrease in aggregate formation observed with the antibody compared to the corresponding wild-type IgA antibody. The stability of the antibodies and functional fragments thereof disclosed herein can be tested by measuring a change in the melting temperature transition of the antibody compared to the corresponding wild-type IgA antibody. In such embodiments, increased stability or increased thermal stability is evidenced by an increase in the melting temperature transition of the antibody or functional fragment thereof compared to the corresponding wild-type IgA or functional fragment thereof. In some embodiments, the antibody or functional fragment thereof has a higher melting temperature than the corresponding wild-type IgA antibody. In some embodiments, the melting temperature of an antibody or functional fragment thereof of the present disclosure is at least about 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.8-fold, 1.0-fold, or more higher than that of a corresponding wild-type IgA antibody. In some embodiments, the melting temperature of an antibody or functional fragment thereof of the present disclosure is at least about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 12°C, 15°C, or more higher than that of a corresponding wild-type IgA antibody. In some embodiments, the melting temperature of an antibody or functional fragment thereof of the present disclosure is at least about 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or more. In some embodiments, this increased stability is in the absence of additional disulfide bonds. Specifically, the increased stability is in the absence of additional disulfide bonds within the IgA heavy chain constant region. In one embodiment, the CH3 domain of the antibody disclosed herein does not contain an additional disulfide bond compared to the wild-type CH3 domain. In an alternative embodiment, the CH3 domain of the antibody disclosed herein contains at least one disulfide bond compared to the wild-type CH3 domain.

[0384] Additional methods for measuring protein aggregation are described in U.S. Patent Application No. 10 / 176,809, and US20030022243A1, the entire contents of which are incorporated herein by reference. Various analytical techniques for measuring protein stability are available in the art, such as those outlined below: Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, New York, Pubs., 1991; and Jones, A. Adv. Drug Delivery Rev. 10: 29-90, 1993. Stability can be measured at a selected temperature over a selected time period. Stability can be qualitatively and / or quantitatively determined in a variety of different ways, including determining the formation of aggregates (e.g., using size exclusion chromatography or by measuring turbidity and / or visual inspection). Methods include: assessment of charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; analysis of amino- or carboxy-terminal sequences; mass spectrometry; comparison of reduced or intact antibody SDS-PAGE analysis; peptide mapping (e.g., trypsin or LYS-C) analysis; and determination of antibody biological activity or antigen-binding function. Instability includes any one or more of the following: aggregation, oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, etc. The term "reduced aggregation" refers to a reduction in aggregation of an antibody of the present disclosure, or a functional fragment thereof, with other antibody molecules and / or other macromolecules, including serum proteins such as albumin, compared to the aggregation exhibited by a corresponding WT IgA antibody.

[0385] The increased thermal stability of an antibody compared to a corresponding WT IgA antibody or variant thereof can be determined by differential scanning calorimetry (DSC) using standard methods in the art (see, e.g., Sturtevant, 1987, Annual Review of Physical Chemistry 38: 463-488). The increased thermal stability of an antibody compared to a corresponding WT IgA antibody or variant thereof can also be determined using a protein thermal unfolding analysis. Alternatively, the increased thermal stability of an antibody compared to a corresponding WT IgA antibody or variant thereof can be determined using any applicable antibody assay that compares the performance of the antibody to the WT. For example, target cell ADCC, antigen binding, or binding to FcαR on immune cells. Immune effector functions of IgA antibodies

[0386] Provided herein are engineered IgA variants or antibodies that contain one or more modifications in the heavy chain constant region compared to a corresponding WT IgA antibody containing a WT heavy chain constant region. In some embodiments, the antibodies disclosed herein or functional fragments thereof (e.g., antibodies containing one or more modifications disclosed herein) exhibit improved properties.

[0387] In some embodiments, the antibodies or functional fragments thereof disclosed herein exhibit at least one increased effector function compared to a corresponding wild-type IgA antibody or functional fragment thereof. Effector functions are biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); and phagocytosis. For example, the antibodies or functional fragments thereof disclosed herein exhibit at least one increased effector function compared to a corresponding wild-type IgA antibody or functional fragment thereof or a corresponding wild-type IgG antibody. Antibody-dependent cellular cytotoxicity (ADCC) is the result of the formation of a complex between the IgA Fab portion of the antibody and an antigen on the cell surface and the binding of the Fc portion to an Fc receptor (FcαR) on an immune effector cell. For example, increased effector function can be increased binding affinity to Fc receptors (e.g., FcαR), increased ADCC; increased cellular immunity; increased binding to cytotoxic CD8 T cells; increased binding to NK cells; increased binding to macrophages; increased binding to polymorphonuclear cells; increased binding to monocytes; increased binding to macrophages; increased binding to large granular lymphocytes; increased binding to granulocytes; direct signal transduction to induce apoptosis; increased dendritic cell maturation; or increased T cell priming, opsonization, or opsonophagocytosis. In some embodiments, the antibody or its functional fragment induces lysis of cancer cells. Lysis can be induced by any mechanism, such as by mediating effector functions such as C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis, or direct induction of cell apoptosis. ADCC

[0388] "ADCC activity" refers to the ability of an antibody to elicit an ADCC response. ADCC is a cell-mediated response in which FcR-expressing antigen-nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to the surface of target cells and subsequently cause lysis (i.e., "killing") of the target cells (e.g., cancer cells). The primary mediator cells may be natural killer (NK) cells or neutrophils. ADCC activity can be assessed directly using in vitro assays, such as the 51Cr release assay using peripheral blood mononuclear cells (PBMCs) and / or NK effector cells described in the Examples and Shields et al. (2001) J. Biol. Chem., 276: 6591-6604, or another suitable method known in the art. ADCC activity can be expressed as the concentration of antibody that results in half-maximal target cell lysis. Thus, in some embodiments, the concentration of an antibody or antigen-binding fragment thereof of the present disclosure that results in the same level of lysis as the half-maximal lysis level achieved by the wild-type control is at least 2, 3, 5, 10, 20, 50, or 100 times lower than the concentration of the wild-type control itself.

[0389] Furthermore, in some embodiments, an antibody or functional fragment thereof of the present disclosure may exhibit increased maximal target cell lysis compared to a corresponding wild-type IgA. For example, the maximal target cell lysis of an antibody or functional fragment thereof of the present disclosure may be 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or more higher than that of a corresponding wild-type IgA antibody or a corresponding wild-type IgG antibody. In some embodiments, an antibody or functional fragment thereof disclosed herein induces increased ADCC compared to a corresponding wild-type IgG antibody comprising an IgG heavy chain constant domain. 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 a corresponding wild-type IgG antibody. CDC

[0390] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target (e.g., a cancer cell) in the presence of complement. 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) complexed with a cognate antigen. To assess complement activation, for example, a CDC assay as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed. Fc receptor binding

[0391] In some embodiments, the antibody or functional fragment thereof binds to an Fc receptor. In some embodiments, the Fc receptor is expressed on an immune effector cell. In some embodiments, the antibody or functional fragment thereof of the present disclosure binds to an Fc receptor on an immune effector cell and induces an effector function such as ADCC, CDC, and / or cytolysis of a target cell (e.g., a cancer cell). In some embodiments, the antibody or functional fragment thereof binds to an IgA receptor. In one embodiment, the IgA receptor is an Fc-alpha receptor (FcαR), such as FcαR for human IgA. In some embodiments, the FcαR is expressed on an immune effector cell. FcαR is present on immune effector cells, such as monocytes, macrophages, neutrophils, and other myeloid cells. FcαR is also found on metamyelocytes, myelocytes, promyelocytes, and some myeloblasts, such as myeloblasts derived from bone marrow. Such receptors are also found on myeloid cell lines, such as U937 cells, PLB985 cells, and HL60 cells. It has also been suggested that FcαR is present on lymphocytes. Expression of FcαR can be increased by activation of bone marrow cells. For example, stimulation of U937 and PLB985 cells with phorbol myristate acetate (PMA) increases the cell surface level of FcαR several-fold (Maliszewski, et al. (1990) J Exp. Med. 172: 1665). Other agents that can increase the surface level of FcαR include calcitriol, 1-25 dihydroxyvitamin D3, and interferon-γ (IFN-γ).

[0392] FcαR can interact with IgA1 and IgA2 in their monomeric, dimeric, and polymeric forms. Binding of the antibodi...

Claims

1. (a) an antigen-binding domain; (b) a constant domain; An antibody or a functional fragment thereof comprising: the constant domain comprises an IgA2 heavy chain constant region comprising an IgA2 CH1 region, an IgA2 CH2 region, and an IgA2 CH3 region, the IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO:1, each numbered according to Table 11, as compared to a WT IgA2 heavy chain constant region comprising: a. N45.2G amino acid substitution; b. P124R amino acid substitution; c. A C86S amino acid substitution; d. N114T amino acid substitution; e. I115L amino acid substitution; f. T116S amino acid substitution; g. N135Q amino acid substitution; h. Deletion of C147, and i. Deletion of Y148 wherein the functional fragment of the antibody retains the ability to specifically bind to the same antigen as that bound by the antibody. An antibody or a functional fragment thereof.

2. (a) an antigen-binding domain; (b) a constant domain; 1. An antibody or functional fragment thereof comprising: a. N45.2G amino acid substitution; b. P124R amino acid substitution; c. A C86S amino acid substitution; d. N114T amino acid substitution; e. I115L amino acid substitution; f. the T116S amino acid substitution, and g. Deletion of residues P131 to Y148 wherein the functional fragment of the antibody retains the ability to specifically bind to the same antigen as that bound by the antibody or a functional fragment thereof.

3. said IgA2 heavy chain constant region comprising the amino acid sequence of SEQ ID NO:1, as numbered according to Table 11, a. an amino acid substitution selected from the group consisting of N15.2G, N15.2Q, and N15.2T; b. L15.3I amino acid substitution, and c. T16S amino acid substitution; The antibody or functional fragment thereof of claim 2, further comprising:

4. An antibody or functional fragment thereof according to any one of claims 1 to 3, which is non-glycosylated.

5. The antibody or functional fragment thereof of any one of claims 1 to 4, wherein the antigen-binding domain binds to GD2, CD20, CD47, CD38, CD19, EGFR, HER2, PD-L1, or CD25.

6. A pharmaceutical composition comprising an antibody or functional fragment thereof according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier.

7. 7. A pharmaceutical composition according to claim 6 for use in treatment.

8. 6. An isolated nucleic acid encoding an antibody or functional fragment thereof according to any one of claims 1 to 5.

9. A vector comprising the isolated nucleic acid of claim 8.

10. A host cell comprising the isolated nucleic acid of claim 8.

11. The host cell of claim 10 which is a bacterial cell or a mammalian cell.

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