Immunoglobulin A antibodies and methods of making and using

Abstract

The present invention relates to immunoglobulin A antibodies and methods of making and using the same. In particular, the invention provides antibodies, such as IgA antibodies and IgG-IgA fusion molecules, compositions comprising such antibodies, and methods of making and using such antibodies and compositions.

Description

[0001] This application is a divisional application of the invention application with the application date of February 22, 2010, Chinese application number 2020800097585, and invention name “Immunoglobulin A antibodies and preparation and use methods”.

[0002] Cross-references to related patent applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 795,367, filed on January 22, 2019, and U.S. Provisional Application No. 62 / 838,071, filed on April 24, 2019, each of which is hereby incorporated by reference in its entirety and claims the benefit of priority. Technical Field

[0004] The present disclosure relates to antibodies, such as IgA antibodies and IgG-IgA fusion molecules, and compositions comprising such antibodies, as well as methods of making and using such antibodies and compositions. Background Art

[0005] Immunoglobulin A (IgA) is a major class of antibodies present in most mammalian mucosal secretions and is the first line of defense against invasion of inhaled and ingested pathogens on vulnerable mucosal surfaces. In humans, there are two IgA isotypes, IgA1 and IgA2, the difference being that the 13-residue extension in the hinge region of the IgA1 heavy chain (HC) does not exist in the IgA2 molecule (Leusen, Mol. Immunol. 68: 35-9 (2015)). Both isotypes are abundant in all organs and tissues, except in the intestine where IgA2 is dominant and in serum where almost only IgA1 monomers are found (Kerr, Biochem. J. 271: 285-96 (1990)). IgA2 has three allotypes: m1 (Tsuzukida et al., Proc Natl Acad Sci USA 76: 1104-8 (1979)), m2 ( et al., Proc Natl Acad Sci USA 75:966–9 (1978)) and mn (Chintalacharuvu et al., J Immunol 152:5299-304 (1994)). The m2 and mn allotypes form a typical light chain (LC)-HC disulfide bond, while the presence of proline 221 at position HC in IgA2m1 results in LC-LC disulfide bond formation (Chintalacharuvu et al., J Immunol 157:3443-9 (1996)). The mutation of proline 221 to arginine (P221R) in the IgA2m1 allotype (which is found in m2 and mn allotypes) restores the typical LC-HC bond (Lohse et al., Cancer Res 76:403-17 (2016) and Chintalacharuvu et al. (1996)). The sequence identity between IgA1 and IgA2 isotypes is quite high, approximately 90%, and is even higher within the IgA2 allotype, with only six residues differing between m1 and m2 and two residues differing between m1 or m2 and mn (Chintalacharuvu et al. (1994)).

[0006] In contrast to other human immunoglobulin classes, IgA has the unique ability to exist naturally as both a monomeric soluble species and a polymeric soluble species, and only polymeric IgA (pIgA) can bind to pIgR for subsequent transcytosis (Yoo et al., Clin. Immunol. 116:3-10 (2005)). Oligomerization of IgA is promoted by an 18-residue extension at the C-terminus of the HC (called the tail) and a 137-amino acid connecting chain (JC). The penultimate residue Cys471 of the IgA tail of the first IgA monomer mediates a disulfide bond with Cys15 of JC, while Cys471 of the second IgA monomer mediates a disulfide bond with Cys69 of JC to form a covalent IgA dimer held together by a single JC (Zikan et al., Mol Immunol 23:541-4 (1986) and Halpern et al., J Immunol 111:1653-60 (1973)). Since each IgA monomer is composed of two HCs, each with a tail, the IgA dimer has two unpaired Cys471 residues through which additional IgA monomers can be attached. Higher order IgA oligomers, such as trimers, tetramers and pentamers, have been reported (Suzuki et al., Proc Natl Acad Sci USA 112: 7809-14 (2015)). Serum IgA is mainly monomeric, while polymeric IgA is produced by plasma cells in the lamina propria. The presence of JC in polymeric IgA is necessary for binding to pIgR on the basolateral side of the epithelium and active transport to the apical side of the mucosal tissue (Wu et al., Clin Dev Immunol 11: 205-13 (2004)). Upon transcytosis, the extracellular domain of pIgR is proteolytically cleaved to generate the so-called secretory component (SC), which remains covalently attached to the polymeric IgA heavy chains via a disulfide bond between Cys467 in pIgR and Cys311 in one HC (Fallgreen-Gebauer et al., Biol Chem Hoppe-Seyler 374:1023–8 (1993) and Bastian et al., Adv Exp Med Biol 371A:581-3 (1995)). This complex is considered to be secretory IgA (sIgA), the major determinant of mucosal immunity (Mantis et al., Mucosal Immunol 4:603-11 (2011) and Johansen et al. Mucosal Immunol 4:598-602 (2011)).

[0007] Compared with traditional IgG-based therapies, immunoglobulin A (IgA) research has highlighted the multiple potential therapeutic applications and unique mechanisms of action of monomeric and polymeric immunoglobulin A (IgA) antibodies (Yoo et al. (2005), Bakema et al., MAbs 3:352-61 (2011) and Leusen (2015)). In oncology, monomeric and polymeric anti-EGFR and anti-CD20 IgAs have shown superior tumor cell killing to IgG, driven by FcαRI-mediated cytotoxicity or more efficient receptor binding and downregulation (Pascal et al., Haematologica 97:1686-94 (2012), Boross et al., EMBOMolecular Medicine 5:1213-26 (2013) and Lohse et al. (2016)). The cytotoxic activity of IgA can be further enhanced by the dual binding of IgG / A fusions or hybrid molecules to FcγR and FcαRI (Li et al., Oncotarget (2017) and Kelton et al., Chem Biol 21: 1603-9 (2015)). For infectious diseases, IgA multivalent target binding achieved excellent antigen binding and neutralization in influenza infection models (Suzuki et al. (2015)). In addition, in a mouse model of polycystic kidney disease, human IgA dimers (dIgA) can be effectively delivered to the renal cavity by binding to polymeric immunoglobulin receptors (pIgR), while IgG molecules cannot (Olsan et al., Journal of Biological Chemistry 290: 15679-86 (2015)). Exploiting the specific transcytosis activity of IgA may allow access to therapeutic targets within the luminal side of mucosal tissues that cannot be effectively targeted by current IgG therapies (Bakema et al. (2011), Olsan et al. (2015) and Borrok et al., JCI Insight 3 (2018)).

[0008] The preparation of recombinant monomeric IgA is more challenging than that of mature IgG molecules. IgA antibodies often have poor expression and heterologous glycosylation. Human IgG1 usually has only two N-linked glycosylation sites, one for each C HThe 2 domains of IgA contain one, while human IgA contains multiple glycosylation sites that may be sensitive to glycan heterogeneity (Leusen (2015)). IgA1 has multiple O-linked glycosylation sites in the hinge region and also two N-linked glycosylation sites in the HC constant domain. Although IgA2 molecules are not modified by O-linked glycans, they do contain four (IgA2m1) or five (IgA2m2 and IgA2mn) N-linked glycosylation sites (Yoo et al. (2005) and Bakema et al. (2011)). JC also contains one N-linked glycosylation site. The assembly of three polypeptide chains (LC, HC and JC) leads to multiple oligomeric states and further contributes to the overall complexity of recombinant polymeric IgA (Rouwendal et al., MAbs 8:74-86 (2016) and Brunke et al., MAbs 5:936-45 (2013)). As the size of IgA oligomers increases, not only does the number of glycosylation sites increase, but more glycan heterogeneity may also occur.

[0009] IgA has previously been shown to have a short circulating half-life (<1 day to about 4 days) in multiple species (Challacombe et al., Immunology 36:331-8 (1979) and Leusen (2015)). Unlike IgG, IgA does not bind to the neonatal receptor FcRn and therefore cannot undergo endosomal recycling and escape lysosomal degradation (Roopenian et al., Nat Rev Immunol 7:715-25 (2007)). In addition to lack of FcRn binding, immature N-linked glycans can also shorten the serum half-life of recombinant IgA by making it a susceptible target for carbohydrate-specific endocytic receptors such as the asialoglycoprotein receptor (ASGPR) (Boross et al. (2013) and (Rifai et al., J Exp Med 191:2171-82 (2000)) and the mannose receptor (Lee et al., Science 295:1898-901 (2002) and Heystek et al., J Immunol 168:102-7 (2002)). These clearance receptors, which are highly concentrated in the liver, recognize glycoproteins with incompletely sialylated N-linked glycans and clear them from the circulation (Tomana et al., Gastroenterology 94:762-70 (1988) and Daniels et al., Hepatology 9:229-34(1989)).

[0010] Therefore, there is a need in the art for IgA antibodies with longer half-lives and methods for their preparation that improve expression levels and production of polymeric IgA. Summary of the invention

[0011] The present disclosure relates to IgA antibodies and compositions comprising such antibodies, as well as methods of making and using such antibodies and compositions.

[0012] In certain embodiments, the present disclosure relates to an isolated IgA antibody. For example, but not limited to, an isolated IgA antibody or fragment thereof of the present disclosure comprises a substitution at amino acid V458. In certain embodiments, amino acid V458 is substituted with isoleucine (i.e., V458I). In certain embodiments, the isolated IgA antibody is an IgA1, IgA2mn, or IgA2m1 antibody.

[0013] In certain embodiments, the isolated IgA antibody or fragment thereof of the present disclosure comprises a substitution at amino acid 1458. In certain embodiments, amino acid 1458 is substituted with valine (ie, 1458V). In certain embodiments, the isolated IgA antibody is an IgA2m2 antibody.

[0014] The present disclosure further provides an isolated IgA antibody comprising a substitution at amino acid N459 and / or S461. In certain embodiments, amino acid N459 is substituted with glutamine (i.e., N459Q). In certain embodiments, amino acid S461 is substituted with alanine (i.e., S461A). In certain embodiments, the IgA antibody is an IgA1 or IgA2m1 antibody.

[0015] The present disclosure also provides an isolated IgA antibody comprising one or more substitutions selected from the group consisting of N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459, S461, and combinations thereof. In certain embodiments, the IgA antibody has a substitution at amino acid N459 and is an IgA1, IgA2m1, or IgA2m2 antibody. In certain embodiments, the IgA antibody has a substitution at amino acid N166 and is an IgA2m1 or IgA2m2 antibody. In certain embodiments, the IgA antibody has a substitution at amino acid S212 and is an IgA2m2 antibody. In certain embodiments, the IgA antibody has a substitution at amino acid N263 and is an IgA1, IgA2m1, or IgA2m2 antibody. In certain embodiments, the IgA antibody has a substitution at amino acids N337, I338, T339 and is an IgA2m1 or IgA2m2 antibody. In certain embodiments, the IgA antibody has a substitution at amino acids N337, I338, T339 and has one or more substitutions at T168, N211, S212, S213, N263, T265, N459, S461, and combinations thereof. In certain embodiments, the IgA antibody is an IgA2m2 antibody and comprises substitutions at amino acids N166, S212, N263, N337, I338, T339, and N459. For example, but not limited to, substitutions at amino acids N166, S212, N263, N337, I338, T339 and N459 can be N166A, S212P, N263Q, N337T, I338L, T339S and N459Q.

[0016] The present disclosure further provides an isolated IgG-IgA fusion molecule. In certain embodiments, the isolated IgG-IgA fusion molecule may include a full-length IgG antibody, which is fused to the Fc region of an IgA antibody at its C-terminus, wherein the Fc region of the IgA antibody includes a sequence including P221 or R221 to the C-terminus of the heavy chain of the IgA antibody, wherein the IgG antibody further includes the deletion of amino acid K447. The present disclosure provides an isolated IgG-IgA fusion molecule that may include a full-length IgG antibody, which is fused to the Fc region of an IgA antibody at its C-terminus, wherein the Fc region of the IgA antibody includes a sequence including C242 to the C-terminus of the heavy chain of the IgA antibody. In certain embodiments, the IgG antibody includes the deletion of amino acid K447. In certain embodiments, the IgG antibody is selected from the group consisting of IgG1 antibody, IgG2 antibody, IgG3 antibody and IgG4 antibody. For example, but not limited to, the IgG antibody can be an IgG1 antibody. In certain embodiments, the IgA antibody is selected from the group consisting of an IgA1 antibody, an IgA2m1 antibody, an IgA2m2 antibody, and an IgA2mn antibody. For example, but not limited to, the IgA antibody is an IgA2m1 antibody.

[0017] The present disclosure further provides nucleic acids encoding the separation of IgA antibodies or IgG-IgA fusion molecules disclosed herein and host cells comprising such nucleic acids. The present disclosure further provides a method for producing antibodies, including culturing host cells disclosed herein, thereby producing IgA antibodies or IgG-IgA fusion molecules. The method may further include recovering IgA antibodies or IgG-IgA fusion molecules from host cells.

[0018] The present disclosure provides a pharmaceutical composition comprising an IgA antibody or IgG-IgA fusion molecule disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition may further include an additional therapeutic agent.

[0019] The present disclosure further provides methods of treating an individual suffering from a disease, wherein the method comprises administering to the individual an effective amount of an IgA antibody or IgG-IgA fusion molecule disclosed herein. In certain embodiments, the disease is an inflammatory disease, an autoimmune disease, or cancer.

[0020] The present disclosure provides a method for enhancing the expression of IgA dimers. In certain embodiments, the method includes increasing the amount of DNA encoding a joining chain (JC) introduced into a first cell relative to the amount of DNA encoding a light chain (LC) and a heavy chain (HC), wherein the enhanced expression is relative to the amount of IgA dimers produced in a second cell into which equal amounts of JC, LC, and HC DNA are introduced. For example, but not limited to, the ratio of the amount of DNA encoding HC to the amount of DNA encoding LC to the amount of DNA encoding JC introduced into the first cell (HC:LC:JC) is approximately from about 1:1:2 to about 1:1:5.

[0021] In certain embodiments, the present disclosure provides a method for enhancing the expression of IgA dimers, trimers or tetramers. In certain embodiments, the method includes reducing the amount of DNA encoding a joining chain (JC) introduced into a first cell relative to the amount of DNA encoding a light chain (LC) and a heavy chain (HC), wherein the enhanced expression is relative to the following: the amount of IgA trimers or tetramers produced in a second cell into which a greater amount of HC and LC DNA relative to the amount of JC DNA is introduced. In certain embodiments, the ratio of the amount of DNA encoding HC to the amount of DNA encoding LC to the amount of DNA encoding JC introduced into the first cell (HC:LC:JC) is from about 1:1:0.25 to about 1:1:0.5.

[0022] The present disclosure provides a method for increasing the production of IgA1 or IgA2m1 polymers. In certain embodiments, the method includes expressing an IgA1 or IgA2m1 antibody having a substitution (e.g., V458I) at amino acid V458 in a first cell, wherein the increased production is relative to the amount of IgA1 or IgA2m1 polymers produced in a second cell expressing an IgA1 or IgA2m1 antibody without substitution at amino acid V458. The present disclosure further provides a method for increasing the production of IgA2m2 dimers, comprising expressing an IgA2m2 antibody having a substitution (e.g., I458V) at amino acid I458 in a first cell, wherein the increased production is relative to the amount of IgA2m2 dimers produced in a second cell expressing an IgA2m2 antibody without substitution at amino acid I458. In certain embodiments, methods for increasing the production of IgA1 or IgA2m1 polymers include expressing in a first cell an IgA1 or IgA2m1 antibody having a substitution at amino acid N459 and / or S461 (e.g., N459Q and / or S461A), wherein the increased production is relative to the amount of IgA1 or IgA2m1 polymers produced in a second cell expressing an IgA1 or IgA2m1 antibody without a substitution at amino acid N459 or S461. In certain embodiments, methods for decreasing the production of IgA2m2 polymers include expressing in a first cell an IgA2m2 antibody having a substitution at amino acid C471 (e.g., C471S), wherein the decreased production is relative to the amount of IgA2m2 polymers produced in a second cell expressing an IgA2m2 antibody without a substitution at amino acid C471. In certain embodiments, an IgA antibody comprising a substitution at amino acid C471 (eg, C471S) may further comprise a substitution at P221 (eg, P221R),

[0023] The present disclosure provides a method of enhancing transient expression of an IgA2m2 antibody, comprising expressing an IgA2m2 antibody comprising a substitution at an amino acid selected from the group consisting of N166, S212, N263, N337, I338, T339, N459, and a combination thereof in a first cell, wherein the enhanced transient expression is relative to the transient expression amount produced in a second cell expressing an IgA2m2 antibody without a substitution at an amino acid selected from the group consisting of N166, S212, N263, N337, I338, T339, N459, and a combination thereof.

[0024] The present disclosure further provides a method for expressing a dimer of an IgG-IgA fusion molecule, including expressing an IgG-IgA fusion molecule comprising a full-length IgG antibody, wherein the full-length IgG antibody is fused to the Fc region of an IgA antibody at its C-terminus. In certain embodiments, the Fc region of an IgA antibody comprises a sequence comprising P221 or R221 to the C-terminus of an IgA antibody heavy chain, wherein the IgG antibody comprises the deletion of amino acid K447. In certain embodiments, the present disclosure provides a method for expressing a dimer, trimer and tetramer of an IgG-IgA fusion molecule, including expressing an IgG-IgA fusion molecule comprising a full-length IgG antibody, wherein the full-length IgG antibody is fused to the Fc region of an IgA antibody at its C-terminus, wherein the Fc region of an IgA antibody comprises a sequence comprising C242 to the C-terminus of an IgA antibody heavy chain. In certain embodiments, an IgG antibody comprises the deletion of amino acid K447.

[0025] The present disclosure provides methods for purifying the IgA and IgG-IgA fusion molecules disclosed herein and / or for purifying specific oligomeric states, such as dimers, trimers or tetramers, of the IgA and IgG-IgA fusion molecules disclosed herein. In certain embodiments, the method for purifying the IgA antibody from a mixture comprising the IgA antibody and at least one host cell protein comprises: applying the mixture to a column comprising protein L to bind the IgA antibody, washing the protein L column with a wash buffer comprising PBS, and eluting the IgA antibody from the protein L column by an elution buffer comprising phosphoric acid. In certain embodiments, a method for purifying an IgA antibody or IgG-IgA fusion molecule in an oligomeric state from a mixture comprising an IgA antibody or IgG-IgA fusion molecule and at least one host cell protein may include: applying the mixture to an affinity purification column comprising protein L or protein A to bind the IgA antibody or IgG-IgA fusion molecule, washing the affinity purification column with a wash buffer, eluting the IgA antibody or IgG-IgA fusion molecule from the affinity purification column with an elution buffer to form a first eluate, applying the first eluate to a size exclusion chromatography column to separate different IgA oligomeric states, and obtaining an effluent comprising the IgA antibody or IgG-IgA fusion molecule in an oligomeric state. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A -B and Figure 1C-Figure 1D . Protein sequences of human IgA heavy chain constant domain and J chain. Figure 1A Panel A of -B: Human heavy chain constant domains of IgA1, IgA2m1, and IgA2m2 C H 1,C H 2,C H3. Hinge (Brezski et al. CurrOpin Immunol 40:62–9 (2016)) and tail ( et al. 75:966–9 (1978). Mismatches relative to the IgA1 sequence are highlighted in gray, N-linked glycosylation motifs are boxed, and asterisks indicate amino acid differences between IgA2m2 and IgA1 and IgA2m1 in the tail. Figure 1A Panel B of -B: Protein sequence of human J chain, with the N-linked glycosylation motif indicated by a box. Figure 1C : Human heavy constant chain domain C of IgA2mn H 1,C H 2,C H 3. Protein sequences of the hinge and tail. N-linked glycosylation sites are indicated by boxes. Figure 1D : Schematic representation of the oligomeric states of IgA with light chain (LC), heavy chain (HC) and joining chain (JC). IgA polymers represent trimer, tetramer and pentamer species.

[0027] Figures 2A-2F. The oligomeric state of recombinantly produced IgA is affected by the amount of J chain DNA used in transfection and the sequence of the heavy chain tail. Figures 2A-2C: Overlays of normalized analytical size exclusion chromatograms of affinity purified IgA from small-scale transient transfections of the following isotypes / allotypes using different ratios of light chain (LC), heavy chain (HC) and connecting chain (JC) DNA. Figure 2A: IgA1, Figure 2B: IgA2m1 or Figure 2C: IgA2m2. Monomer (M), dimer (D) and polymer (P) peaks are indicated. Values ​​are normalized based on the highest signal in each chromatogram. Figures 2D-2F are the relative amounts of monomer, dimer and trimer / tetramer species of variants produced by IgA, quantified by analytical SEC. Figure 2D: Effect of mutations at positions 458 and 467 in the IgA tail of IgA1, IgA2m1 and IgA2m2 on trimer / tetramer formation. Effect of removal of mutations at positions 458 and 467 in the IgA tail of IgA1, IgA2m1 and IgA2m2 on trimer / tetramer formation.

[0028] Figures 3A-3D .Biophysical and structural characterization of recombinant IgA oligomers. Figures 3A-3D Panel A: Overlay of analytical size exclusion chromatograms of purified IgA1, IgA2m1, IgA2m1 P221R and IgA2m2 monomers, dimers and tetramers. Figures 3A-3DPanel B: SDS-PAGE analysis of non-reduced (DTT) and reduced (+DTT) IgA1, IgA2m1, IgA2m1 P221R and IgA2m2 monomers (M), dimers (D) and tetramers (T). The heavy chain (HC), light chain (LC) and connecting chain (*) are indicated in the reduced sample, and the LC-LC dimer of IgA2m1 is indicated by an arrow. Figures 3A-3D Panels CD, top: reference-free 2D classes from negative-stain electron microscopy, used for Figures 3A-3D Panel C: IgA2m2 dimer or Figures 3A-3D Panel D: IgA2m2 tetramer. Figures 3A-3D Panels CD, bottom: original image particles compared to their assigned 2D class, shown next to an IgA model superimposed on the 2D class with the Fc domain and Fab fragment highlighted.

[0029] Figures 4A-4B .Recombinantly produced IgA oligomers are stable and functional in vitro. Figures 4A-4B Panel A: In vitro transcytosis of anti-mIL-13 hIgA monomers, dimers, and tetramers in MDCK cells transfected with human pIgR. IgA polymers are transcytosed, but monomers are not. Figures 4A-4B Panel B: Thermal stability of anti-mIL-13 IgA, IgG1 and IgG1 Fab fragments measured by differential scanning fluorimetry (DSF). Only one melting transition was observed for all samples.

[0030] Figure 5A-5B and Figure 5C .Recombinant IgA oligomers show rapid serum clearance in vivo. Figure 5A: Serum time concentration curves of IgA or IgG in mice. Total serum exposure of Balb / c mice receiving a single 5 mg / kg intravenous (IV) dose of IgA or IgG molecules at 5 minutes, 15 minutes, 30 minutes, 1 hour, 1 day, 3 days, 7 days and 14 days after administration. All mice were bled retro-orbitally under isoflurane to assess serum concentration curves. Human serum IgA monomers were administered at 10 mg / kg and are shown as dashed lines. Figure 5B- Figure 5C : Tissue distribution of IgA or IgG in mice 1 hour after injection. All figures are mean ± SEM of n = 4 per group. FIG5B : Concentration difference of intact antibody in each tissue (except blood) normalized to blood minus, expressed as 125 I (% ID / g tissue). (C) 111 In (% ID / g tissue) minus 125 I (% ID / g tissue) was used to determine catabolic antibody concentration values.

[0031] Figure 6A-6B and Figure 6C .Incomplete glycosylation of recombinant IgA molecules. Figure 6A: Schematic diagram of N-linked glycan processing. Figure 6B: Global N-linked glycan analysis of recombinant IgA and IgA purified from human serum. Glycan analysis by mass spectrometry after antibody deglycosylation and subsequent glycan enrichment. While human serum IgA has a sialylation rate of over 90%, all recombinantly expressed IgA molecules have a sialylation rate of less than 60%. Figure 6C : Site-specific N-linked glycan analysis of IgA2m1 dimers revealed heterogeneous glycan compositions between different N-linked glycosylation sites on the IgA2m1 heavy chain (HC) and joining chain (JC).

[0032] Figures 7A-7C and Figure 7D-7E .IgG1-IgA2m1 Fc fusions and aglycosylated IgA2m2 show increased serum exposure in vivo compared to wild-type IgA and demonstrate the ability to transcytose in vitro. Figure 7A: Schematic representation of an IgA2m2 tetramer with a light chain (LC, black), a heavy chain (HC, white), and a linker chain with 41 N-linked glycosylation sites (diamonds) (left) or without glycosylation (right). Figure 7B: Schematic representation of IgG1, IgA2m1 dimer, or IgG1-IgA2m1 Fc dimer formats, including LC (black), IgG1 HC (dotted line), IgA2m1 HC (white), JC, and N-linked glycosylation (diamonds). Figure 7C: Analytical SEC of iodinated IgG1-IgA2m1 Fc dimer or tetramer after incubation in mouse plasma for 0 h (black), 24 h (orange), or 96 h (blue). The original IgG1-L-P221R IgA2m1 Fc tetramer and dimer showed degradation similar to the anti-HER2 IgG1 (trastuzumab) peak, while the re-engineered IgG1ΔK-P221 IgA2m1 Fc or IgG1ΔK-C242IgA2m1 Fc dimer was stable. Figure 7D: Serum time concentration curves of IgA or IgG in mice. Total serum exposure of Balb / c mice administered a single 30 mg / kg IV dose of IgA molecules. Internal concentration data of a typical human IgG1 (anti-gD) previously administered as a single intravenous (IV) injection of 30 mg / kg are shown as a dotted line. All mice were bled retro-orbitally under isoflurane or by cardiac puncture to assess serum concentration curves. Figure 7E: In vitro transcytosis of hIgA in MDCK cells transfected with human pIgR.

[0033] Figures 8A-8B . Original negative stain EM images of IgA2m2 dimer and tetramer purification. Figures 8A-8BPanel A: Negative stain electron microscopy (EM) raw image of purified IgA2m2 dimer showing well-dispersed particles. Figures 8A-8B Panel B: Negative stain EM raw image of purified IgA2m2 tetramer showing well-dispersed radial particles.

[0034] Fig. 9 . Distribution of intact antibodies normalized to plasma concentrations. Tissue distribution of IgA or IgG in mice 1 hour after injection. All values ​​represent %ID / g of tissue corrected for blood, normalized to %ID / mL of plasma. All graphs are mean ± SD for n = 4 per group.

[0035] Fig.10 .Tissue distribution of intact antibody after one day. The tissue concentration of intact antibody for each tissue was normalized by subtracting blood and expressed as 125 I (%ID / g tissue) was calculated one day after injection. All graphs are mean ± SEM of n=4 per group.

[0036] Fig.11 .Tissue distribution of degraded antibodies after one day. 111 In (% ID / g tissue) minus 125 Catabolism antibody values ​​were determined by % ID / g tissue and calculated 1 day after injection. All graphs are mean ± SEM of n=4 per group.

[0037] Fig.12 Schematic diagram of IgG1-IgA2m1 Fc fusion oligomers. The IgG1-L-P221R IgA2m1 Fc fusion (Borrok et al. MAbs 7:743–51 (2015)) was made as dimers and tetramers but had poor stability in mouse plasma (Fig. 7C). To eliminate potential furin cleavage sites, the C-terminal lysine (K) and intervening leucine residues (L) of IgG1 were deleted. In addition, the IgA2m1 wild-type (WT) sequence was restored with proline at position 221 to make the IgG1ΔK-P221IgA2m1 Fc fusion. The IgG1ΔK-C242 IgA2m1 Fc fusion was designed similarly, but the IgA2m1 Fc begins at residue C242, thereby deleting the IgA2m1 hinge (Δhinge).

[0038] Fig.13Global glycan analysis of engineered IgA oligomers. Global N-linked glycan analysis of CHO recombinantly produced dimers of anti-mIL-13 IgG1ΔK fused to P221 or C242 IgA2m1 Fc and aglycosylated anti-HER2 IgA2m2 tetramers. Both dimers of anti-mIL-13 IgG1ΔK fused to P221 or C242 IgA2m1 Fc had approximately 20% sialylation, and as expected, no glycosylation was detected for the aglycosylated anti-HER2 IgA2m2 tetramers.

[0039] Fig.14 Protein sequences of IgA heavy chain constant domains from humans and other species. Human heavy chain constant domain C H 1. C H 2. C H 3. Hinge (Brezski et al. (2016)) and tail ( et al. (1978). Conservation of protein sequences between species is highlighted in grey, while N-linked glycosylation motifs are indicated by boxes.

[0040] Figure 15A-Figure 15C . Fig.15A : Analytical size exclusion chromatogram of affinity purified xmuIL13.huIgA1 from small-scale transient transfections performed in Expi293 cells with ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA ranging from 1:1:0.25 to 1:1:2. Fig. 15B : Analytical size exclusion chromatograms of affinity purified xmuIL13.huIgA1 from small-scale transient transfections performed in Expi293 cells with ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA ranging from 1:1:1 to 1:1:5. Fig. 15C : The number of dimer and tetramer species of IgA antibodies produced.

[0041] Figure 16A-16B . Fig.16A : Analytical size exclusion chromatograms of affinity purified xmuIL13.IgA2m1 from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA. Fig. 16B : The number of dimer and tetramer species of IgA antibodies produced.

[0042] Figure 17A-17B . Fig.17A: Analytical size exclusion chromatograms of affinity purified xmuIL13.IgA2m1.P221R from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA. Fig. 17B : The number of dimer and tetramer species of IgA antibodies produced.

[0043] Figure 18A-18E . Fig.18A : Analytical size exclusion chromatograms of affinity purified xmuIL13.huIgA2m2 from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA. Fig.18B : Analytical size exclusion chromatogram of affinity purified xmuIL13.huIgA2m2 from small-scale transient transfection in Expi293 cells with ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA ranging from 1:1:1 to 1:1:5. Fig. 18C : The number of dimer and tetramer species of IgA antibodies produced. Fig.18D : Analytical size exclusion chromatograms of affinity purified xmuIL13.huIgA2m2 from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), joining chain (JC), and secretory component (SC) DNA. Fig.18E : The heavy chain, light chain and J chain of xmuIL13.huIgA2m2 were confirmed by mass spectrometry.

[0044] Fig.19 Depicted are Biacore analyses of the following anti-IL-13 antibodies of the following isotypes / allotypes: IgA1 dimer, IgA2m1 dimer, IgA2m2 dimer, and IgA2m2 tetramer.

[0045] Figure 20A-20B . Fig. 20A : Analytical size exclusion chromatograms of affinity purified xmuGP120.3E5.huIgA1 from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA. Fig. 20B : Analytical size exclusion chromatograms of affinity purified xmuGP120.3E5.IgA2m1.P221R from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA.

[0046] Fig.21Depicted are analytical size exclusion chromatograms of affinity purified xmuGP120.3E5.huIgA2m2 from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA.

[0047] Fig. 22 Depicted are analytical size exclusion chromatograms of affinity purified xmuGP120.3E5.huIgA2m2 from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA.

[0048] Fig.23 Depicted are analytical size exclusion chromatograms of affinity purified mouse xgD.5B6.hIgA2m2 from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC), and joining chain (JC) DNA.

[0049] Figure 24A-24C Modifications of glycosylation sites of IgA2m2 and J chain are depicted. Fig.24A : Summary of modifications of IgA2m2 heavy and J chains and their in vitro expression compared with wild type. Fig. 24B : Summary of transient expression of monoglycosylated variants of IgA2m2. Fig.24C : Summary of transient expression of IgA2m2 glycosylation variants with multiple mutations.

[0050] Fig.25 Depicted are analyses of the receptor binding properties of IgA monomers, wild-type IgA2m2 tetramers, and IgA2m2 tetramers (aglycosylated) and J chain (glycosylated) from human serum.

[0051] Fig.26 An analysis of the glycan characteristics of each IgA molecule is described.

[0052] Fig. 27 .Concentration-time curve of IgA molecules after a single 10 mg / kg IV injection in female Balb / C mice.

[0053] Figure 28A-28B . Fig.28A : Analysis of cysteine ​​mutations to prevent disulfide bond formation with secretory component or J chain. Fig.28B : When a linker is added to the light and heavy chains, the formation of IgA2m2 dimers and higher-order oligomers requires C471 but not C311.

[0054] Fig.29 Co-transfection analysis of secretory component, joining chain, light chain and heavy chain is described.

[0055] Figure 30A-Figure 30E . Fig. 30A : Expression levels of the xmuIL13.IgA2m2 variant generated to abrogate pIgR binding. Fig. 30B : Analytical size exclusion chromatogram of xmuIL13.IgA2m2 variants from small-scale transient transfections performed in Expi293 cells. Fig. 30C : Biacore analysis of the xmuIL13.IgA2m2 variant binding to mouse pIgR. Fig.30D : Biacore analysis of xmuIL13.IgA2m2 variants binding to human pIgR. Fig.30E : Biacore analysis of xmuIL13.IgA2m2 variants binding to human FcαRI.

[0056] Figure 31A-Figure 31B Analysis of cell culture conditions to increase sialylation of anti-Jag1 IgA2m2 is described. Fig.31A : Matrix of cell culture conditions for the xJAG1.2B3.hIgA2m2 stable cell line. Fig.31B : Analysis of the effects of cell culture conditions on the glycosylation of xJAG1.2B3.hIgA2m2.

[0057] Fig.32 The stability of the IgA variants was profiled by differential scanning fluorimetry (DSF).

[0058] Figure 33A-Figure 33D Characterization and engineering of a full-length anti-murine IL-13 IgG1.Leu-P221R.IgA2m1 Fc fusion molecule to enhance oligomer stability are described. Fig.33A : Analytical size exclusion chromatograms of affinity purified glycosylated full-length anti-murine IL-13 IgG1.Leu-P221R.IgA2m1 Fc fusion molecules from small-scale transient transfections performed in Expi293 cells with varying ratios of light chain (LC), heavy chain (HC) and joining chain (JC) DNA. Fig.33B : Biacore analysis of IgA oligomers binding to mouse pIgR. Fig.33C : Summary of IgA oligomer binding to mouse and human pIgR. Fig.33D : Stability of IgA oligomers determined by DSF.

[0059] Figure 34A-Figure 34C . Fig.34A : IgG1 full-length-IgA Fc construct design to eliminate furin sites and instability. Fig.34B : Full-length anti-murine IL-13 IgG1-IgA Fc transient expression data of engineered constructs. Fig.34C: Mouse plasma stability data of engineered anti-murine IL-13 IgA molecules.

[0060] Figure 35A-Figure 35B . Fig.35A : Wasatch analysis of IgA oligomer binding to human FcαRI. Fig.35B : Summary of the binding of IgA oligomers to FcαRI determined by Wasatch surface plasmon resonance (SPR).

[0061] Figure 36A-Figure 36D . Fig.36A : Wasatch analysis of binding of IgA2m2 dimers and tetramers produced by transient expression in CHO cells and Expi293 cells to mouse and human pIgR. Fig.36B : Wasatch analysis of binding of IgA2m2 glycosylation variants to mouse pIgR. Fig.36C : Wasatch analysis of binding of IgA2m2 glycosylation variants to human pIgR. Fig.36D : Summary of IgA oligomer binding to mouse and human pIgR determined by Wasatch SPR.

[0062] Figure 37A-Figure 37C . Fig.37A : Expression profile of hIgG1-hIgA1 fusion molecules. Fig.37B : Analytical size exclusion chromatogram of hIgG1-hIgA1 fusion molecule. Fig.37C : Biacore analysis of binding of hIgG1-hIgA1 fusion molecules to mouse and human pIgR and human FcαRI.

[0063] Fig.38 Depicted is the analysis of N-linked glycosylation removal of various IgA1 antibodies.

[0064] Fig.39 . Recombinantly expressed human anti-mIL-13IgA2m2 was affinity purified on a Capto L column. Then, a 3.5μm, 7.8mm x 300mm Water's XBridge Protein BEH SEC The Capto L eluate was analyzed by size exclusion chromatography (SEC) on HPLC. Three major peaks were observed in the analytical SEC elution profile, corresponding to higher order aggregates (peak 1, including trimers, tetramers, and pentamers), dimers (peak 2), and monomers (peak 3) as determined by multi-angle light scattering (MALS) and negative stain electron microscopy.

[0065] Fig.40.Separation of the mixture of recombinant human anti-mIL-13IgA2m2 oligomeric species seen in the eluate from the Capto L affinity column was attempted by size exclusion chromatography (SEC) using a HiLoad 16 / 600 Superose 6 preparative grade (pg) column. Four major peaks were observed in the Superose 6 elution profile, corresponding to high molecular weight aggregates (peak 1, eluting in the void volume of the column), higher order aggregates (peak 2, likely including trimers, tetramers, and pentamers), dimers (peak 3), and monomers (peak 4) as determined by multi-angle light scattering (MALS) coupled analytical SEC and negative stain electron microscopy. The molar mass (MW) and polydispersity index (PDI) of the protein measured from the fractions of peaks 2 and 3 are shown.

[0066] Fig.41 .Use 3.5μm, 7.8mm x 300mm Water's XBridge Protein BEH SEC The column separated recombinant human anti-mIL-13IgA2m2 dimers from higher order aggregates by size exclusion chromatography (SEC) on HPLC. Three major peaks were observed in the analytical SEC elution profile, corresponding to higher order aggregates (peak 1, including trimers, tetramers and pentamers), dimers (peak 2) and monomers (peak 3) as determined by multi-angle light scattering (MALS) coupled analytical SEC and negative stain electron microscopy.

[0067] Figures 42A-42D . Figures 42A-42D Panel A: Using 3.5μm, 7.8mm x 300mm Water's XBridgeProtein BEH SEC The elution of the Capto L affinity column of human anti-mIL-13IgA2m2 was analyzed by size exclusion chromatography (SEC) on HPLC. Three major peaks were observed corresponding to higher order aggregates (peak 1, including trimers, tetramers and pentamers), dimers (peak 2) and monomers (peak 3), as determined by multi-angle light scattering (MALS) coupled to analytical SEC and negative stain electron microscopy. Figures 42A-42D Panel B: Water's XBridgeProtein BEH SEC at 3.5μm, 7.8mm x 300mm The product was purified on a chromatographic column and peak 1 was separated from Figure (A). Fig.41 Shown here is a 3.5μm, 7.8mm x 300mm Water's XBridge Protein BEH SEC coupled to a MALS detector. Post-column analysis of peak 1. The molar mass (MW) and polydispersity index (PDI) determined by MALS were consistent with the expected mass for predominantly tetrameric IgA2m2. Figures 42A-42D Panel C: Water's XBridge Protein BEH SEC at 3.5 μm, 7.8 mm x 300 mm The product was purified on a chromatographic column and peak 2 was separated from Figure (A). Fig.41 Shown here is Water's XBridgeProtein BEH SEC on a 3.5μm, 7.8mm x 300mm instrument coupled to a MALS detector. Post-cleanup analysis of Peak 2 on column. MW and PDI determined by MALS were consistent with the expected mass for predominantly dimeric IgA2m2. Figures 42A-42D Panel D: SDS-PAGE analysis of the Figures 42A-42D The purified proteins of peaks 1 and 2 in panels B and C of FIG. The reduced sample bands were observed to migrate with the expected masses for the heavy chain (HC), light chain (LC), and J chain (JC).

[0068] Figures 43A-43B Panel A: Representative raw image of negative stain electron microscopy (EM) of human anti-mIL-13IgA2m2 particles from peak 1 in Figure 42B. Panel B: Reference-free 2D class of negative stain EM of particles from peak 1 in Figure 42B, indicating that the sample is predominantly tetramers.

[0069] Figures 44A-44B . Figures 44A-44B Panel A: Representative raw negative stain electron microscopy (EM) images of human anti-mIL-13IgA2m2 particles from peak 2 in Figure 42C. Figures 44A-44B Panel B: Reference-free 2D cluster of negative stain EM of particles from peak 2 in Figure 42C, indicating that the sample is predominantly dimers.

[0070] Fig.45 .from Figures 42A-42D Mass spectrometry analysis of human anti-mIL-13 IgA2m2 dimer purified from peak 2 in panel C. Mass spectrometry analysis after heat denaturation, reduction with dithiothreitol, and deglycosylation with PNGaseF confirmed the presence of the correct joining chain (JC), light chain (LC), and heavy chain (HC).

[0071] Figures 46A-46C . Figures 46A-46C Panel A: Using 3.5μm, 7.8mm x 300mm Water's XBridgeProtein BEH SEC The elution of human anti-mIL-13IgA1 from a Capto L affinity column was analyzed by size exclusion chromatography (SEC) on HPLC. Before separation of oligomers, three major peaks were observed corresponding to higher order aggregates (peak 1, including trimers, tetramers and pentamers), dimers (peak 2) and monomers (peak 3). Figures 46A-46C Panel B: 3.5μm, 7.8mm x 300mm Water's XBridge Protein BEH SEC The chromatographic column was used to purify and separate peak 2 in Figure (A), such as Fig.41 Shown here is a 3.5μm, 7.8mm x 300mm Water'sXBridge Protein BEH SEC coupled to a multi-angle light scattering (MALS) detector. Analysis of peak 2 after purification on column. The molar mass (MW) and polydispersity index (PDI) determined by MALS were consistent with the expected mass for predominantly dimeric IgA1. Figures 46A-46C C: Purified proteins from peak 2 of panel (B) were analyzed by SDS-PAGE under non-reducing (-DTT) or reducing (+DTT) conditions. The reduced sample bands were observed to migrate with the expected masses of heavy chain (HC), light chain (LC) and J chain (JC).

[0072] Figures 47A-47C . Figures 47A-47C Panel A: Using 3.5μm, 7.8mm x 300mm Water's XBridgeProtein BEH SEC The elution of human anti-mIL-13IgA2m1 from a Capto L affinity column was analyzed by size exclusion chromatography (SEC) on HPLC. Before separation of oligomers, three major peaks were observed corresponding to higher order aggregates (peak 1, including trimers, tetramers and pentamers), dimers (peak 2) and monomers (peak 3). Figures 47A-47C Panel B: 3.5μm, 7.8mm x 300mm Water's XBridge Protein BEH SEC The chromatographic column was used to purify and separate peak 2 in Figure (A), such as Fig.41 Shown here is a 3.5μm, 7.8mm x 300mm Water'sXBridge Protein BEH SEC coupled to a multi-angle light scattering (MALS) detector. Post-column analysis of peak 2. The molar mass (MW) and polydispersity index (PDI) determined by MALS were consistent with the expected mass for predominantly dimeric IgA2m1. Figures 47A-47C C: Purified proteins from peak 2 of panel (B) were analyzed by SDS-PAGE under non-reducing (-DTT) or reducing (+DTT) conditions. The reduced sample bands were observed to migrate with the expected masses of heavy chain (HC), light chain (LC) and J chain (JC).

[0073] Figures 48A-48C . Figures 48A-48C Panel A: Using 3.5μm, 7.8mm x 300mm Water's XBridgeProtein BEH SEC The elution of the Capto L affinity column of human anti-mIL-13IgA2m1 containing the P221R mutation in the heavy chain was analyzed by size exclusion chromatography (SEC) on HPLC. Before separation of oligomers, three major peaks were observed corresponding to higher order aggregates (peak 1, including trimers, tetramers and pentamers), dimers (peak 2) and monomers (peak 3). Figures 48A-48C Panel B: 3.5μm, 7.8mm x 300mm Water's XBridge Protein BEH SEC The chromatographic column was used to purify and separate peak 2 in Figure (A), such as Fig.41 Shown. Shown is the 3.5μm, 7.8mm x 300mm Water'sXBridge Protein BEH SEC Peak 2 was analyzed after purification on a chromatographic column. Figures 48A-48C Panel C: SDS-PAGE analysis of the Figures 48A-48C The purified protein of peak 2 of panel B. The reduced sample bands were observed to migrate with the expected masses of heavy chain (HC), light chain (LC) and J chain (JC).

[0074] Fig.49 The ability of monomeric and polymeric anti-HER2 IgA antibodies to cause death of HER2+ breast cancer cell lines KPL-4, BT474-M1, and SKBR3 is depicted.

[0075] Fig.50 Depicted is the ability of monomeric and polymeric anti-HER2 IgA antibodies to cause cell death of SKBR3 breast cancer cells in the presence of neutrophils from different donors.

[0076] Fig.51The ability of glycosylated and nonglycosylated IgA polymers and monomers to cause cell death in SKBR3 breast cancer cells is described.

[0077] Figure 52A-Figure 52B . Fig.52A : Biacore analysis of IgA oligomers and tetramers binding to human FcαRI. Fig.52B : Summary of the binding of IgA oligomers and tetramers to FcαRI determined by Biacore SPR. DETAILED DESCRIPTION

[0078] For clarity and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0079] I. Definitions;

[0080] Ⅱ. Antibodies;

[0081] III. Methods for antibody preparation and purification;

[0082] IV. Treatment methods;

[0083] V. Pharmaceutical compositions;

[0084] VI. Articles of manufacture; and

[0085] VII. Exemplary Embodiments.

[0086] I. Definition

[0087] As used herein, 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, in accordance with practice in the art, "about" can mean within 3 or more than 3 standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within a certain order of magnitude of a certain value, preferably within 5 times, more preferably within 2 times.

[0088] Unless the context clearly dictates otherwise, the terms "a", "an", and "the" include plural references. The terms "one / a kind" and "one / more" and "at least one" are used interchangeably herein. In addition, "and / or" as used herein will be considered a specific disclosure of each of two specific features or components, with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B, A or B, A (alone) and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0089] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies, antibody fragments, and antibody fusion molecules, so long as they exhibit the desired antigen-binding activity.

[0090] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0091] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0092] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0093] The term "IgA antibody" refers to an antibody of the IgA class, including the IgA isotypes IgA1 and IgA2, and the three allotypes of IgA2, ml, m2 and mn.

[0094] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer drugs disclosed below.

[0095] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0096] An "effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount that is effective to achieve the desired therapeutic or preventive outcome at the necessary dosage and time period. For example, but not by way of limitation, an "effective amount" may refer to an amount of an antibody disclosed herein that is capable of alleviating, minimizing, and / or preventing the symptoms of a disease and / or disorder, prolonging survival, and / or prolonging the time until a disease and / or disorder recurs.

[0097] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) of the Fc region may or may not be present. In certain embodiments, the human IgA heavy chain Fc region extends from Pro221 (P221), Arg221 (R221), Val222 (V222), Pro223 (P223), or from Cys242 (C242) to the carboxyl terminus of the heavy chain (see Figure 1AUnless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0098] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Fc receptors include, but are not limited to, FcαRI (recognizes the Fc region of IgA antibodies) and FcγRII (recognizes the Fc region of IgG antibodies). FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both of which have similar amino acid sequences, the main difference being their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Annu. Rev. Immunol. 15: 203-234 (1997). FcRs are reviewed in, e.g., Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). The term "FcR" herein encompasses other FcRs, including those to be identified in the future.

[0099] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.). In vivo binding to human FcRn and serum half-life of human FcRn high affinity binding polypeptides can be determined, e.g., in transgenic mice or transfected human cell lines expressing human FcRn or in primates administered polypeptides having variant Fc regions. WO 2000 / 042072 (Presta) describes antibody variants with improved or reduced binding to FcRs. See also, e.g., Shields et al. al., J. Biol. Chem. 9(2):6591-6604(2001).

[0100] "Framework" or "FR" refers to the variable domain residues excluding the complementarity determining regions (CDRs). The FR of a variable domain is usually composed of the following four FR domains: FR1, FR2, FR3, and FR4. Therefore, CDR and FR sequences usually appear in the following sequence in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0101] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0102] The terms "host cell," "host cell line," and "host cell culture," as used interchangeably herein, refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the primary transformed cell, without regard to the number of passages. Progeny may not be identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein.

[0103] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0104] "Human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. In general, the subgroup of sequences is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In certain embodiments, for VL, the subgroup is subgroup κI as in Kabat et al., supra. In certain embodiments, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0105] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will substantially comprise at least one, usually two, variable domains, wherein all or substantially all CDRs correspond to HVRs of non-human antibodies, and all or substantially all FRs correspond to FRs of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in "humanized form," such as a non-human antibody, refers to an antibody that has been humanized.

[0106] As used herein, the term "hypervariable region" or "HVR" refers to each of the following: regions of an antibody variable domain that are hypervariable in sequence (also referred to herein as "complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., supra. Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3), and three in VL (L1, L2, L3).

[0107] "Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0108] As used interchangeably herein, "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0109] An "isolated" antibody is one that has been separated from a component of its natural environment. In certain embodiments, the antibody is purified to a purity greater than 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0110] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0111] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or different vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0112] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by base sequences, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically expressed as from 5' to 3'. In this article, the term nucleic acid molecule encompasses synthetic forms of deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes sense strands and antisense strands, as well as single-stranded and double-stranded forms. In addition, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for direct expression in vitro and / or in vivo (e.g., in a host or patient) of the antibodies of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the coding molecule so that mRNA may be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi: 10.1038 / nm.4356 or EP 2101823 B1).

[0113] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the preparation of monoclonal antibody preparations, such variants are usually present in small amounts), each antibody comprising the population is identical and / or binds to the same epitope. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies used according to the currently disclosed subject matter can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0114] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical compositions.

[0115] "Natural antibodies" refer to natural immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also referred to as a variable heavy chain domain or a heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, and the two types are called kappa (κ) and lambda (λ).

[0116] As used herein, the term "package insert" refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0117] " Amino acid sequence identity percentage " about reference polypeptide sequence is defined as the percentage of amino acid residues in candidate sequence identical with the amino acid residues in reference polypeptide sequence after comparing sequences and introducing room (if necessary) to realize maximum sequence identity percentage, without considering any conservative substitution as a part of sequence identity for the purpose of comparison. Comparison for determining amino acid sequence identity percentage can be realized by various means within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ClustalW, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for realizing maximum comparison on the total length of the compared sequence. Alternatively, sequence comparison computer program ALIGN-2 can be used to generate identity percentage values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code has been filed with user documentation in the US Copyright Office, Washington D.C., 20559, where it is registered under US Copyright Registration No. TXU510087 and as described in WO 2001 / 007611.

[0118] Unless otherwise stated, for the purposes of this article, the BLOSUM50 comparison matrix is ​​used to generate the values ​​of amino acid sequence identity percentage using the ggsearch program of the FASTA package version 36.3.8c or higher. The FASTA package is by W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W. R. Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al., (1997) Genomics 46:24-36 creation, and can be obtained from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure that a global rather than a local alignment is performed. The amino acid identity percentages are given in the alignment header of the output.

[0119] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredients contained in the preparation to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.

[0120] As used herein, "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0121] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual to be treated, and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In certain embodiments, the antibodies of the present disclosure can be used to delay the development of a disease or slow the progression of a disease.

[0122] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using the VH or VL domains from antibodies that bind to the antigen, respectively, to screen for libraries of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0123] The term "vector" as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0124] II. Antibodies

[0125] In certain embodiments, the present disclosure is based in part on methods of engineering antibodies, such as IgA antibodies and IgG-IgA fusion molecules, to exhibit improved serum retention and increased polymeric antibody production. In certain embodiments, the antibodies of the present disclosure exhibit binding to FcRn. In certain embodiments, the antibodies of the present disclosure exhibit increased IgR-mediated transcytosis. In certain embodiments, the antibodies of the present disclosure exhibit reduced binding and / or no binding to FcαRI. In certain embodiments, the antibodies of the present disclosure can provide excellent safety in a therapeutic setting by minimizing proinflammatory responses after administration.

[0126] In certain embodiments, the present disclosure provides antibodies, such as IgA antibodies and IgG-IgA fusion molecules, that exhibit improved serum retention. For example, but not by way of limitation, antibodies, such as IgA antibodies and IgG-IgA Fc fusion molecules, of the present disclosure are stable in plasma for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, or up to about 5 days. In certain embodiments, antibodies, such as IgA antibodies and IgG-IgA fusion molecules of the present disclosure are stable in plasma for up to about 4 days.

[0127] In certain embodiments, the disclosure provides antibodies with reduced glycosylation or no glycosylation, such as IgA antibodies and IgG-IgA fusion molecules. For example, but not by way of limitation, compared to unmodified IgA or unmodified IgG-IgA fusion molecules, the antibodies of the disclosure exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% glycosylation reduction. In certain embodiments, the antibodies of the disclosure are less than about 0.5%, less than about 1%, less than about 2%, less than about 5% glycosylation, less than about 10% glycosylation, less than about 20% glycosylation, less than about 30% glycosylation, or less than about 40% glycosylation. In certain embodiments, the antibodies of the disclosure have 0% glycosylation, i.e., no glycosylation.

[0128] A. Exemplary Antibodies

[0129] 1. IgA antibody variants

[0130] The present disclosure provides IgA antibodies, such as IgA1, IgA2m1, IgA2m2, and IgA2mn antibodies, which have been modified to reduce the extent to which the antibody is glycosylated. Deletion of antibody glycosylation sites can be achieved by changing the amino acid sequence of the antibody to remove one or more glycosylation sites. In certain embodiments, the antibodies of the present disclosure can be modified to remove one or more, two or more, three or more, four or more, five or more, or six or more glycosylation sites, such as N-linked glycosylation sites and / or O-linked glycosylation sites.

[0131] In certain embodiments, the antibodies of the present disclosure may be modified to remove one or more N-linked glycosylation motifs NXS / T, wherein X is any amino acid. In certain embodiments, removal of an N-linked glycosylation site may include modifying, e.g., mutating, one or more amino acids present in the glycosylation site motif. For example, but not by way of limitation, the N, X, and / or S / T amino acids may be modified, e.g., mutated, in the motif of the glycosylation site. In certain embodiments, all three amino acids of the motif may be mutated.

[0132] In certain embodiments, the antibodies of the present disclosure may be modified to remove one or more, two or more, three or more, four or more, or five or more glycosylation sites from the heavy chain constant domain. For example, but not by way of limitation, the antibodies of the present disclosure may be modified to remove one or more, two or more, three or more, or all four N-linked glycosylation sites at amino acids 166, 211, 263, and / or 337 of the heavy chain constant domain. In certain embodiments, the antibodies of the present disclosure may be modified to remove one or more glycosylation sites in the heavy chain tail (see Figure 1A ). For example, but not by way of limitation, an antibody of the present disclosure may be modified to remove an N-linked glycosylation site at amino acid 459 at the tail end of the heavy chain. In certain embodiments, an IgA1 antibody of the present disclosure may be modified to remove one or more N-linked glycosylation sites at amino acids 263 and / or 449. In certain embodiments, an IgA2m1 antibody of the present disclosure may be modified to remove one or more N-linked glycosylation sites at amino acids 166, 263, 337, and / or 449. In certain embodiments, an IgA2m2 or IgA2mn antibody of the present disclosure may be modified to remove one or more N-linked glycosylation sites at amino acids 166, 211, 263, 337, and / or 449. In certain embodiments, an antibody may be modified to remove all N-linked glycosylation sites from the heavy chain of the antibody, including the heavy chain constant domain and the tail end.

[0133] In certain embodiments, the antibodies of the present disclosure may be aglycosylated. For example, but not limited to, the aglycosylated antibodies of the present disclosure are antibodies that are not glycosylated on the heavy chain of the antibody including the heavy chain constant region and the tail. In certain embodiments, the aglycosylated antibodies of the present disclosure are antibodies that are not glycosylated on the heavy chain (including the heavy chain constant region and the tail) and are not glycosylated on the J chain.

[0134] In certain embodiments, the present disclosure provides an IgA antibody having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve modifications, e.g., substitutions, at amino acids 166, 168, 211, 212, 213, 263, 265, 337, 338, 339, 459, and / or 461 to reduce glycosylation of the IgA antibody. For example, but not by way of limitation, the present disclosure provides an IgA antibody having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve modifications, e.g., substitutions, at amino acids N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459, and / or S461 to reduce glycosylation of the IgA antibody.

[0135] In certain embodiments, the IgA1 antibodies of the present disclosure have one or more, two or more, three or more, or four modifications at amino acids 263, 265, 459, and / or 461, such as at amino acids N263, T265, N459, and / or S461. In certain embodiments, the IgA2m1 antibodies of the present disclosure have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight modifications at amino acids 166, 168, 263, 265, 337, 338, 339, 459, and / or 461, such as at amino acids N166, T168, N263, T265, N337, I338, T339, N459, and / or S461. In certain embodiments, the IgA2m2 or IgA2mn antibodies of the present disclosure have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve modifications at amino acids 166, 168, 211, 212, 213, 263, 265, 337, 338, 339, 459 and / or 461, e.g., at amino acids N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459 and / or S461. In certain embodiments, an IgA2m1, IgA2m2, or IgA2mn antibody of the disclosure is modified at all three amino acids 337, 338, and 339, such as at amino acids N337, I338, and T339.

[0136] In certain embodiments, the IgA antibodies of the present disclosure, such as IgA2m1, IgA2m2, or IgA2mn antibodies, have a modification at amino acid N166. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA2m1, IgA2m2, or IgA2mn antibodies, have a modification at amino acid N168. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA2m2, or IgA2mn antibodies, have a modification at amino acid S211. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA2m2, or IgA2mn antibodies, have a modification at amino acid S212. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA2m2, or IgA2mn antibodies, have a modification at amino acid S213. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA1, IgA2m1, IgA2m2 or IgA2mn antibodies, have modifications at amino acid N263. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA1, IgA2m1, IgA2m2 or IgA2mn antibodies, have modifications at amino acid N265. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA2m1, IgA2m2 or IgA2mn antibodies, have modifications at three amino acids N337, I338 and T339. In certain embodiments, the IgA antibodies of the present disclosure, such as IgA1, IgA2m1, IgA2m2 or IgA2mn antibodies, have modifications at amino acid N459. In certain embodiments, an IgA antibody of the disclosure, such as an IgA1, IgA2m1, IgA2m2, or IgA2mn antibody, has a modification at amino acid S461.

[0137] In certain embodiments, the amino acid N may mutate to an A, G, or Q amino acid. In certain embodiments, the amino acid S may mutate to an A amino acid. In certain embodiments, the amino acid T may mutate to an A amino acid. In certain embodiments, the IgA antibodies of the present disclosure, such as the IgA2m2 or IgA2mn antibodies of the present disclosure, may be modified to include one or more, two or more, three or more, four or more, five or more, six or more, or all seven of the following mutations N166A, S212P, N263Q, N337T, I338L, T339S, and N459Q. For example, but not limited to, the IgA1 antibodies of the present disclosure may be modified to include one or more or all two of the following mutations N263Q and N459Q. In certain embodiments, the IgA2m1 antibody of the present disclosure may be modified to include one or more, two or more, three or more, four or more, five or more, or all six of the following mutations N166A, N263Q, N337T, I338L, T339S, and N459Q. In certain embodiments, the IgA2m2 or IgA2mn antibody of the present disclosure may be modified to include one or more, two or more, three or more, four or more, five or more, six or more, or all seven of the following mutations N166A, S212P, N263Q, N337T, I338L, T339S, and N459Q.

[0138] In certain embodiments, the J chain of the antibody of the present disclosure may be modified to remove one or more glycosylation sites. In certain embodiments, the antibody of the present disclosure may be modified to remove the N-linked glycosylation site at amino acid 49 of the J chain, for example, by modifying one or more amino acids of the glycosylation site motif comprising amino acids 49, 50 and 51. For example, but not limited to, the amino acid N49 and / or the amino acid S51 of the J chain may be modified. In certain embodiments, the amino acid N may mutate to an A, G or Q amino acid and / or the amino acid S may mutate to an A amino acid. For example, but not limited to, the J chain of the antibody of the present disclosure may include an N49A / G / Q mutation and / or an S51A mutation. In certain embodiments, the J chain of the antibody of the present disclosure may include an N49Q mutation.

[0139] In certain embodiments, the antibodies of the present disclosure may be modified to remove one or more, two or more, three or more, four or more, or five or more glycosylation sites from the heavy chain, and modified to remove one glycosylation site from the J chain. In certain embodiments, the antibodies of the present disclosure have one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve modifications, e.g., substitutions, at amino acids N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459, and / or S461 of the heavy chain, and one or two modifications, e.g., substitutions, at amino acids N49 and / or S51 of the J chain. In certain embodiments, the antibodies of the present disclosure have one or more, two or more, three or more, four or more, five or more, six or more, or all seven modifications, e.g., substitutions, at amino acids N166, S212, N263, N337, I338, T339, and / or N459 of the heavy chain, and one or two modifications, e.g., substitutions, at amino acids N49 and / or S51 of the J chain.

[0140] In certain embodiments, the IgA antibodies of the present disclosure, such as IgA1, IgA2m1, IgA2m2 or IgA2mn antibodies, may have one or more modifications at amino acid N459 or S461 to reduce glycosylation of the IgA antibody. In certain embodiments, the modification of amino acid N459 and / or S461 results in an enhanced ability of the antibody to produce polymers such as dimers, trimers, tetramers and pentamers.

[0141] In certain embodiments, antibodies of the present disclosure, such as IgA antibodies, may have a modification, such as a substitution, at amino acid 458. In certain embodiments, the present disclosure provides IgA1, IgA2m1, and IgA2mn antibodies having a substitution at amino acid V458. In certain embodiments, amino acid V458 may be mutated to isoleucine (i.e., V458I). In certain embodiments, the present disclosure provides IgA2m2 antibodies having a substitution at amino acid I458. In certain embodiments, amino acid I458 may be mutated to valine (i.e., I458V). In certain embodiments, as described herein, one or more of these modifications may be present in antibodies with reduced or no glycosylation.

[0142] In certain embodiments, antibodies of the present disclosure, such as IgA antibodies, may have modifications, such as substitutions, at amino acid C471 and / or C311. In certain embodiments, the IgA antibody may have a mutation at amino acid C471, such as C471S. In certain embodiments, the IgA antibody may have a mutation at amino acid C311, such as C311S.

[0143] In some embodiments, the antibodies of the present disclosure may be modified to produce antibody variants with certain improved properties. For example, but not by way of limitation, antibodies of the present disclosure with reduced glycosylation may exhibit improved serum retention. In certain embodiments, antibodies of the present disclosure with reduced glycosylation may have an enhanced ability to produce polymers such as dimers, trimers, tetramers, and pentamers. In certain embodiments, antibodies of the present disclosure with reduced glycosylation may exhibit reduced binding to the IgA-specific hFc receptor FcαRI, such as no binding to FcαRI. In certain embodiments, antibodies of the present disclosure with modifications enhance the ability to produce polymers (e.g., dimers, trimers, tetramers, and pentamers) compared to antibodies without one of the above modifications at amino acids 458, 459, and / or S461. In certain embodiments, antibodies disclosed herein with modifications at amino acid C471 have a reduced ability to produce polymers such as dimers, trimers, tetramers, and pentamers.

[0144] 2. IgG-IgA fusion molecules

[0145] The disclosure further provides an antibody comprising at least a portion of an IgG antibody and at least a portion of an IgA antibody, referred to herein as an IgG-IgA fusion molecule. In certain embodiments, the IgG-IgA fusion molecule of the present disclosure has enhanced resistance to proteases (e.g., furin), and increased activity and / or serum half-life (see Table 9). In certain embodiments, the IgG-IgA fusion molecule of the present disclosure binds to FcRn.

[0146] In certain embodiments, the IgG antibody of the IgG-IgA fusion molecule of the present disclosure can be a full-length IgG antibody. In certain embodiments, the IgG antibody can be any IgG antibody that binds to a neonatal Fc receptor (FcRn). For example, but not limited to, the IgG antibody can be IgG1, IgG2, IgG3 or IgG4. In certain embodiments, the IgG antibody is an anti-IgG1 antibody. In certain embodiments, the IgG antibody is an anti-IgG2 antibody. In certain embodiments, the IgG antibody is an anti-IgG3 antibody. In certain embodiments, the IgG antibody is an anti-IgG4 antibody.

[0147] In certain embodiments, the IgG-IgA fusion molecules of the present disclosure may include an IgG antibody fused to a fragment of an IgA antibody. In certain embodiments, the IgA antibody may be an IgA1 antibody, an IgA2m1 antibody, an IgA2mn antibody, or an IgA2m2 antibody. In certain embodiments, the IgA fragment may be about 300 amino acids in length, about 250 amino acids in length, about 200 amino acids in length, about 150 amino acids in length, about 100 amino acids in length, about 80 amino acids in length, about 60 amino acids in length, about 40 amino acids in length, or about 20 amino acids in length. In certain embodiments, the length of the IgA fragment is about 250 amino acids. In certain embodiments, the length of the IgA fragment is about 20 amino acids, such as about 18 amino acids. For example, but not limited to, the IgA fragment may include the Fc region of the IgA antibody. In certain embodiments, the IgA fragment may include the CH2 and CH3 domains of the IgA antibody. In certain embodiments, the IgA fragment may further include the hinge region of the IgA antibody. In certain embodiments, the IgA fragment may further include the tail end of the IgA antibody.

[0148] In certain embodiments, the IgG-IgA fusion molecules of the present disclosure may include an IgG antibody and an Fc region of an IgA antibody. In certain embodiments, the IgG-IgA fusion molecules may include an IgG antibody fused to the Fc region of an IgA antibody disclosed herein at the C-terminus of the IgG antibody. For example, but not limited to, the IgG-IgA fusion molecules may include a full-length IgG heavy chain sequence fused to the Fc region of an IgA heavy chain at the C-terminus of the IgG heavy chain sequence (see Figures 7B, 12 and 34A).

[0149] In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may include a sequence from P221 to the C-terminus of the heavy chain. For example, but not limited to, the IgA antibody portion may include amino acids P221-Y472 of the IgA antibody. In certain embodiments, the Fc region of an IgA antibody, such as an IgA1 or IgA2m1 antibody, may include a sequence from P221 to the C-terminus of the heavy chain. In certain embodiments, the P221 amino acid may be mutated to arginine (R), i.e., P221R. In certain embodiments, the Fc region of an IgA antibody, such as an IgA2m2 or IgA2mn antibody, may include a sequence from R221 to the C-terminus of the heavy chain, such as amino acids R221-Y472 of the IgA antibody. Alternatively, the Fc region of an IgA antibody may include a sequence from C242 to the C-terminus of the heavy chain, which deletes the hinge region of the IgA antibody. For example, but not by way of limitation, the IgA portion of the fusion molecule may include amino acids C242-Y472 of an IgA antibody. In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may include a sequence from V222 to the C-terminus of the heavy chain. For example, but not by way of limitation, the IgA antibody portion may include amino acids V222-Y472 of an IgA antibody, such as an IgA1, IgA2m1, IgA2mn, or IgA2m2 antibody. In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may include a sequence from P223 to the C-terminus of the heavy chain. For example, but not by way of limitation, the IgA antibody portion may include amino acids P223-Y472 of an IgA antibody, such as an IgA1, IgA2m1, IgA2mn, or IgA2m2 antibody. In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may include a sequence from C241 to the C-terminus of the heavy chain. For example, but not limited to, the IgA antibody portion may include amino acids C241-Y472 of an IgA antibody such as IgA1, IgA2m1, IgA2m2, or IgA2mn antibody.

[0150] In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may comprise a sequence from P237 to the C-terminus of the heavy chain. For example, but not limited to, the IgA antibody portion may include amino acids P237-Y472 of an IgA antibody, such as IgA1, IgA2m1, IgA2mn, or IgA2m2 antibody. In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may comprise a sequence from P238 to the C-terminus of the heavy chain. For example, but not limited to, the IgA antibody portion may include amino acids P238-Y472 of an IgA antibody, such as IgA2m1, IgA2m2, or IgA2mn antibody. In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may comprise a sequence from S238 to the C-terminus of the heavy chain. For example, but not by way of limitation, the IgA antibody portion may include amino acids S238-Y472 of an IgA antibody, such as an IgA1 antibody. In certain embodiments, the IgA portion of the fusion molecule, such as an Fc region of an IgA antibody, may include a sequence from P239 to the C-terminus of the heavy chain. For example, but not by way of limitation, the IgA antibody portion may include amino acids P239-Y472 of an IgA antibody, such as an IgA1, IgA2m1, IgA2m2, or IgA2mn antibody. In certain embodiments, the IgA portion of the fusion molecule, such as an Fc region of an IgA antibody, may include a sequence from P240 to the C-terminus of the heavy chain. For example, but not by way of limitation, the IgA antibody portion may include amino acids P240-Y472 of an IgA antibody, such as an IgA2m1, IgA2m2, or IgA2mn antibody. In certain embodiments, the IgA portion of the fusion molecule, such as the Fc region of an IgA antibody, may include a sequence from S240 to the C-terminus of the heavy chain. For example, but not limited to, the IgA antibody portion may include amino acid S240 of an IgA antibody, such as an IgA1 antibody. In certain embodiments, the IgA portion of the fusion molecule does not include the tail end of the IgA antibody, such as amino acids 454-472.

[0151] In certain embodiments, the IgG-IgA fusion molecules of the present disclosure may include an Fc region from one IgA isotype and a hinge region from a second isotype. For example, but not by way of limitation, the IgG-IgA fusion molecules of the present disclosure may include a hinge region from an IgA2, such as an IgA2m1, IgA2m2, or IgA2mn antibody, and include an Fc region from an IgA1 antibody.

[0152] In certain embodiments, the heavy chain of an IgG antibody has been modified to remove a C-terminal lysine, such as amino acid K447 of an IgG antibody (e.g., IgG1, IgG2, IgG3, and IgG4). For example, but not limitation, the present disclosure provides an IgG-IgA fusion molecule comprising an IgG antibody lacking amino acid K447 and an IgA portion comprising amino acids P221-Y472 or R221-Y472 of an IgA antibody.

[0153] In certain embodiments, the connection between the Fc region of an IgG antibody and an IgA antibody may comprise the amino acid sequence TQKSLSLSPGPVPPPPPCC (SEQ ID NO: 1) or a fragment thereof or a conservative substitution thereof. In certain embodiments, the connection between the Fc region of an IgG antibody and an IgA antibody may comprise the amino acid sequence TQKSLSLSPGC (SEQ ID NO: 2) or a fragment thereof or a conservative substitution thereof. Non-limiting examples of conservative substitutions are provided in Table 1. In certain embodiments, the connection between the Fc region of an IgG antibody and an IgA antibody may comprise the amino acid sequence TQKSLSLSPGPVPPPPPCC (SEQ ID NO: 1) or a fragment thereof or a conservative substitution thereof. Fig.34A The amino acid sequence disclosed in .

[0154] In certain embodiments, the IgG-IgA Fc fusions of the present disclosure are stable in plasma for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, or up to about 5 days. For example, but not by way of limitation, the IgG1-IgA Fc fusions of the present disclosure are stable in plasma for up to about 4 days.

[0155] In certain embodiments, the IgG-IgA fusion molecules of the present disclosure may further include one or more amino acid substitutions as described above to reduce glycosylation. For example, but not by way of limitation, the IgG-IgA fusion molecules of the present disclosure may be modified to remove glycosylation of the heavy chain and / or J chain of the IgA antibody of the IgG-IgA fusion molecule. In certain embodiments, the IgA antibody of the IgG-IgA fusion molecule is aglycosylated. In certain embodiments, the IgG-IgA fusion molecules of the present disclosure bind to FcRn but not to FcαRI.

[0156] In certain embodiments, the IgG-IgA fusion molecules of the present disclosure may further include substitutions to one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329. For example, but not by way of limitation, the IgG-IgA fusion molecules of the present disclosure may further include a substitution at amino acid 297, such as N297G.

[0157] In certain embodiments, the IgG-IgA fusion molecules of the present disclosure may further include substitutions at amino acids C471 and / or C311. In certain embodiments, the IgG-IgA fusion molecules of the present disclosure may have mutations at amino acids C471, such as C471S. In certain embodiments, the IgG-IgA Fc fusion molecules of the present disclosure may have mutations at amino acids C311, such as C311S.

[0158] B. Chimeric and Humanized Antibodies

[0159] In certain embodiments, the antibodies of the present disclosure are chimeric antibodies. Certain chimeric antibodies are described in, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In certain embodiments, the chimeric antibodies of the present disclosure comprise non-human variable regions (e.g., variable regions derived from mice, rats, hamsters, rabbits, or non-human primates (such as monkeys)) and human constant regions. In another example, a chimeric antibody may be a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0160] In certain embodiments, chimeric antibodies of the present disclosure may be humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibodies. Typically, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or portions thereof) are derived from non-human antibodies, and FR (or portions thereof) are derived from human antibody sequences. Humanized antibodies may also optionally include at least a portion of a human constant region. In certain embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), such as to restore or improve antibody specificity or affinity.

[0161] Humanized antibodies and methods for their preparation are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method for FR shuffling).

[0162] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the “best fit” method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)). al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0163] C. Human Antibodies

[0164] In certain embodiments, the antibodies of the present disclosure may be human antibodies. Human antibodies may be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0165] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or exists outside the chromosome of the animal or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, description of XENOMOUSE TM US Patent Nos. 6,075,181 and 6,150,584 for the technology; description US Patent No. 5,770,429 for technology; describes KM US Patent No. 7,041,870, and describes Human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with different human constant regions.

[0166] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185-91 (2005).

[0167] Human antibodies can also be produced by isolating the Fv clone variable domain sequence selected from the human phage display library. Such variable domain sequences can then be combined with the expected human constant domains. The technology of selecting human antibodies from the antibody library is described below.

[0168] D. Antibody variants

[0169] The current disclosed subject further provides amino acid sequence variants of the disclosed antibodies. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include but are not limited to the deletion and / or insertion and / or substitution of residues in the antibody amino acid sequence. Any combination of deletion, insertion and substitution can be performed to achieve the final construct, provided that the final antibody (i.e. modified) has the desired characteristics, such as antigen binding.

[0170] 1. Substitution, insertion and deletion variants

[0171] In certain embodiments, antibody variants may have one or more amino acid substitutions. The target site for substitution mutations includes HVR and FR. Non-limiting examples of conservative substitutions are shown under the heading "preferred substitutions" in Table 1. Non-limiting examples of more substantial changes are provided under the heading "exemplary substitutions" in Table 1, as further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activity (e.g., retained / improved antigen binding, weakened immunogenicity, or improved complement dependent cytotoxicity (CDC) or antibody dependent cell-mediated cytotoxicity (ADCC)).

[0172] Table 1.

[0173]

[0174] Amino acids can be grouped according to common side chain properties:

[0175] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0176] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0177] (3) Acidic: Asp, Glu;

[0178] (4) Basic: His, Lys, Arg;

[0179] (5) Residues that affect chain orientation: Gly, Pro;

[0180] (6) Aromatic: Trp, Tyr, Phe.

[0181] In certain embodiments, non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0182] In certain embodiments, a type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, relative to the parent antibody, one or more resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., but not limited to, affinity enhancement, immunogenicity reduction) and / or will substantially retain certain biological properties of the parent antibody. A non-limiting example of a substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0183] In certain embodiments, for example, HVRs may be altered (e.g., substituted) to improve antibody affinity. Such changes may occur in HVR "hot spots", i.e., residues encoded by codons that undergo high frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and / or residues in contact with the antigen (binding affinity of the resulting variant VH or VL is detected). Affinity maturation achieved by construction and reselection from a secondary library has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., Human Press, Totowa, NJ, (2001)). In certain embodiments of affinity maturation, diversity may be introduced into variable genes selected for maturation purposes by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0184] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the antigen binding ability of the antibody. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be outside the antigen contact residues of HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged, or comprises no more than one, two or three amino acid substitutions.

[0185] The method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and the antigen are identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues that are candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they have the desired properties.

[0186] Amino acid sequence insertion includes amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertion variants of antibody molecules include fusions of the N-terminal or C-terminal of the antibody with an enzyme (e.g., for antibody directed enzyme prodrug therapy (ADEPT)) or a polypeptide that increases the serum half-life of the antibody.

[0187] 2. Fc region variants

[0188] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant may comprise a human Fc region sequence (e.g., a human IgA Fc region or a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0189] In certain embodiments, the present disclosure provides antibody variants with some but not all effector functions, which make them desirable candidates for applications where the half-life of the antibody in vivo is important and certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the attenuation / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks IgA-specific hFc receptor (i.e., FcαRI) binding, but retains FcRn binding ability. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). For example, primary cells, NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Other non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA; and CYTOTOX Nonradioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)). In certain embodiments, alterations can be made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0190] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0191] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem 9(2):6591-6604 (2001)).

[0192] In certain embodiments, the antibody variants of the disclosure comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0193] In certain embodiments, changes made in the Fc region of an antibody disclosed herein, e.g., a bispecific antibody, can produce variant antibodies with extended half-life and improved neonatal Fc receptor (FcRn) binding, antibodies responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)) are described in US 2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as a substitution at Fc region residue 434 (U.S. Pat. No. 7,371,826).

[0194] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0195] 3. Cysteine-engineered antibody variants

[0196] In certain embodiments, it may be desirable to produce cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced by cysteine ​​residues. In particular embodiments, the substituted residues are present at accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody, and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered antibodies may be generated as described, for example, in U.S. Patent No. 7,521,541.

[0197] 4. Antibody derivatives

[0198] In certain embodiments, the antibodies of the present disclosure may be further modified to contain other non-proteinaceous parts known in the art and easily available. Suitable parts for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in manufacturing. Polymers may have any molecular weight and may or may not have side chains. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used for therapy under defined conditions, etc.

[0199] In certain embodiments, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). In certain embodiments, the radiation can have any wavelength, and includes but is not limited to wavelengths that are not harmful to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed.

[0200] 5. Immunoconjugates

[0201] The disclosed subject matter also provides immunoconjugates comprising an antibody described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof), or radioactive isotopes. For example, an antibody of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.

[0202] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody of the present disclosure is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunorubicin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vinblastine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and octataxel; trichothecenes and CC1065.

[0203] In certain embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthus protein, pokeweed antiviral proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcumin, crotonin, saponin inhibitor, gelatin, mitocin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0204] In certain embodiments, the immunoconjugate includes an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes can be used to prepare radioconjugates. Non-limiting examples include At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example, tc99m or I 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0205] Conjugates of antibody fragments and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020). Non-limiting examples of linkers are disclosed above.

[0206] The immunoconjugates specifically disclosed herein contemplate, but are not limited to, such conjugates prepared with cross-linking agents including, but not limited to, commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinyl sulfone)benzoate).

[0207] III. Antibody Preparation and Purification Methods

[0208] A. Antibody Preparation Methods

[0209] The antibodies disclosed herein can be prepared using any available or known technology in the art. For example, but not limited to, antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. Detailed procedures for producing antibodies of the present disclosure, such as IgA antibodies and IgG-IgA fusion molecules, are described in the following examples.

[0210] The subject matter of the present disclosure further provides an isolated nucleic acid encoding an antibody disclosed herein. For example, an isolated nucleic acid can encode an amino acid sequence encoding an aglycosylated antibody of the present disclosure. In certain embodiments, an isolated nucleic acid of the present disclosure can encode an amino acid sequence encoding an IgA antibody that has been modified to remove one or more, two or more, three or more, four or more, five or more, or six or more glycosylation sites, such as N-linked glycosylation sites and / or O-linked glycosylation sites. In certain embodiments, the isolated nucleic acid of the present disclosure can encode an amino acid sequence, the IgA antibody encoded by the amino acid sequence has one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more or twelve modifications, such as substitutions, at amino acid N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459 and / or S461. In certain embodiments, the isolated nucleic acid of the present disclosure can encode an amino acid sequence, the amino acid sequence encoding an IgG-IgA fusion molecule, such as an IgG-IgA Fc fusion molecule disclosed herein.

[0211] In certain embodiments, nucleic acid can be present in one or more vectors (e.g., expression vectors). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in host cells (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins) in which they are introduced. After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors (expression vectors) can guide the expression of nucleic acids operably connected thereto. Typically, expression vectors that are useful in recombinant DNA technology are typically in the form of plasmids (vectors). However, the disclosed subject matter is intended to include these other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) with equivalent functions.

[0212] In certain embodiments, nucleic acids encoding antibodies of the present disclosure and / or one or more vectors comprising the nucleic acids can be introduced into host cells. In certain embodiments, nucleic acids can be introduced into cells by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a virus or phage vector containing a nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In certain embodiments, a host cell may include, for example, a cell that has been transformed with: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody light chain, an amino acid sequence comprising an antibody heavy chain, and an amino acid sequence comprising an antibody J chain; (2) (a) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody light chain and an amino acid sequence comprising an antibody heavy chain, and (b) a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody J chain; or (3) (a) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody light chain, (b) a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody heavy chain, and (c) a third vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody J chain. In certain embodiments, a host cell can include, for example, a cell that has been transformed with: (a) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody light chain, (b) a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody heavy chain, (c) a third vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody J chain, and (d) a fourth vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody secretory component.

[0213] In certain embodiments, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell. In certain embodiments, the host cell is a 293 cell, for example, an Expi293 cell.

[0214] In certain embodiments, the method for preparing an antibody of the present disclosure comprises culturing a host cell (in which one or more nucleic acids encoding the antibody have been introduced) under conditions suitable for expressing the antibody, and optionally recovering the antibody from the host cell and / or the host cell culture medium. In certain embodiments, the antibody is recovered from the host cell by chromatography techniques.

[0215] Regarding the recombinant production of antibodies of the present disclosure, nucleic acids encoding antibodies such as those described above can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding heavy and light chains of antibodies).

[0216] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli.) Antibodies can be separated from bacterial cell paste in a soluble fraction after expression, and can be further purified.

[0217] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used with insect cells have been identified, particularly for transfecting Spodoptera frugiperda cells.

[0218] Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0219] In certain embodiments, plant cell cultures can be used as host cells. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0220] In certain embodiments, vertebrate cells may also be used as hosts. For example, but not by way of limitation, mammalian cell lines adapted to growth in suspension may be useful. Non-limiting examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described, e.g., in Mather et al., Annals of NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, which include DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0221] In certain embodiments, animal systems can be used to produce antibodies of the present disclosure. One animal system for preparing hybridomas is the mouse system. Producing hybridomas in mice is a well-established process. Immunization protocols and techniques for isolating immune spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York).

[0222] 1. Preparation of polymerized IgA

[0223] The present disclosure provides a method for preparing polymerized IgA. In certain embodiments, the method of the present disclosure can be used to prepare an IgA polymer containing two or more IgA monomers, such as about two to about five IgA monomers. For example, but not limited to, the method of the present disclosure can be used to produce IgA dimers, trimers, tetramers and / or pentamers. In certain embodiments, such methods include changing the amount of DNA encoding J chains introduced (such as transfected) into cells and the ratio of the amount of DNA encoding light chains (LC) and / or heavy chains (HC). In certain embodiments, such methods include changing the amount of DNA encoding J chains introduced (such as transfected) into cells and the ratio of the amount of DNA encoding LC, HC and secretory component (SC).

[0224] The present disclosure provides a method for increasing the production of IgA dimers. In certain embodiments, the method for increasing the production of IgA dimers includes increasing the amount of DNA encoding the J chain introduced, for example, transfected into the cell, relative to the amount of DNA encoding the light chain and the heavy chain. In certain embodiments, the enhanced expression is relative to the following: the amount of IgA dimers produced in cells that introduce, for example, transfected with equal amounts of J chain, heavy chain, and light chain DNA. For example, but not limited to, the method can be used to produce IgA1, IgA2m1, IgA2m1.P221R dimers, IgA2m2, and IgA2mn dimers. In certain embodiments, the method may include introducing, for example, transfecting host cells with a ratio of the amount of DNA encoding the heavy chain to the amount of DNA encoding the light chain to the amount of DNA encoding the J chain (HC:LC:JC), i.e., about 1:1:2, about 1:1:3, about 1:1:4, or about 1:1:5 to increase the production of IgA dimers, for example, a ratio of about 1:1:2 to about 1:1:5. In certain embodiments, the method may include transfecting cells with about 2 times, about 3 times, about 4 times, or about 5 times the amount of DNA encoding the light chain and / or the amount of DNA encoding the heavy chain.

[0225] The present disclosure provides a method for increasing the output of IgA polymers. For example, but not limited to, the present disclosure provides a method for increasing the output of IgA dimers, trimers, tetramers and / or pentamers. In certain embodiments, the method for increasing the output of IgA polymers (such as dimers, trimers, tetramers and / or pentamers) includes reducing the amount of DNA encoding J chains introduced into cells relative to the amount of DNA encoding light and heavy chains. In certain embodiments, the increased output is relative to the amount of IgA polymers (such as dimers, trimers, tetramers and / or pentamers) prepared in cells, and the cell introduces, for example, transfected with an equivalent amount of heavy and light chain DNA relative to the amount of J chain DNA. For example, but not limited to, the method can be used to prepare IgA1, IgA2m1, IgA2m1P221R, IgA2m2 or IgA2mn polymers, such as dimers, trimers, tetramers and / or pentamers. In certain embodiments, the method may include the ratio (HC:LC:JC) of the amount of the DNA encoding the heavy chain and the amount of the DNA encoding the light chain and the amount of the DNA encoding the J chain, i.e., about 1:1:0.25 or about 1:1:0.5, for example, about 1:1:0.25 to about 1:1:0.5 ratio transfection host cells, to increase the production of IgA trimers, tetramers and / or pentamers. In certain embodiments, the amount of the DNA encoding the J chain can be the amount of the DNA encoding the light chain and / or the amount of the DNA encoding the heavy chain less than about 3 times, less than about 2 times or less than about 1 times. In certain embodiments, the amount of the DNA encoding the J chain can be the amount of the DNA encoding the light chain and / or the amount of the DNA encoding the heavy chain less than about 0.5 times or less than about 0.25 times.

[0226] In certain embodiments, the method for increasing the production of IgA1, IgA2m1 and / or IgA2mn trimers, tetramers and pentamers may include expressing an IgA1 antibody, an IgA2m1 antibody and / or an IgA2mn antibody having a substitution at amino acid V458 in a cell. In certain embodiments, amino acid V458 may be mutated to isoleucine (i.e., V458I). In certain embodiments, the increase in the production of IgA1, IgA2m1 and / or IgA2mn trimers, tetramers and pentamers is relative to the production of IgA1, IgA2m1 and / or IgA2mn trimers, tetramers and pentamers obtained by expressing IgA1 antibody, IgA2m1 antibody and / or IgA2mn antibody in a cell without a substitution at amino acid V458.

[0227] In certain embodiments, a method of increasing the production of IgA2m2 dimers may include expressing an IgA2m2 antibody having a substitution at amino acid 1458. In certain embodiments, amino acid 1458 may be mutated to valine (i.e., I458V). In certain embodiments, the increase in the production of IgA2m2 dimers is relative to the production of IgA2m2 dimers obtained by expressing an IgA2m2 antibody without a substitution at amino acid 1458.

[0228] In certain embodiments, methods for increasing the production of IgA polymers may include removing one or more glycosylation sites from an IgA antibody, such as by amino acid substitution (as described above), the increase in production being, for example, relative to the production of IgA polymers produced by an antibody IgA that has not been modified to remove glycosylation sites. In certain embodiments, methods for increasing the production of IgA polymers may include one or more substitutions at amino acids N459 and / or S461. For example, but not by way of limitation, an IgA antibody may have a substitution at amino acid N459. In certain embodiments, an IgA antibody may have a substitution at amino acid S461. In certain embodiments, an IgA antibody may have a substitution at amino acid N459 and / or S461. Non-limiting examples of such substitutions include mutating N459 to A, G, or Q. In certain embodiments, amino acid S461 may mutate to A. In certain embodiments, methods for increasing the production of IgA1 polymers include expressing an IgA1 antibody having a substitution at amino acid N459 and / or S461, such as a substitution at amino acid N459 and S461, for example, wherein the enhanced expression is relative to the amount of IgA1 polymer produced by expressing an IgA1 antibody without a substitution at amino acid N459 and / or S461. In certain embodiments, methods for increasing the production of IgA2 polymers (e.g., IgA2m1, IgA2m2, and IgA2mn polymers) include expressing an IgA2 antibody having a substitution at amino acid N459 and / or S461, such as a substitution at amino acid N459 and S461. In certain embodiments, the increase in the production of IgA2 polymers is relative to the production of IgA2 polymers obtained by expressing an IgA2 antibody without a substitution at amino acid N459 and / or S461.

[0229] In certain embodiments, a method for reducing the production of IgA polymers (e.g., increasing the production of IgA monomers) comprises expressing an IgA antibody, such as an IgA1, IgA2m1, IgA2m2, or IgA2mn antibody, having a substitution at amino acid C471 (e.g., a C471S mutation). For example, but not by way of limitation, a method for reducing the production of IgA2m2 polymers comprises expressing an IgA2m2 antibody having a substitution at amino acid C471 (e.g., a C471S mutation). In certain embodiments, the reduction in the production of IgA polymers (e.g., IgA2m2 polymers) is relative to the production of IgA polymers (e.g., IgA2m2 polymers) obtained by expressing an IgA antibody (e.g., IgA2m2 antibody) without a substitution at amino acid C471.

[0230] 2. Preparation of polymerized IgG-IgA fusion molecules

[0231] The present disclosure further provides methods for producing IgG-IgA fusion molecules of the present disclosure. In certain embodiments, the present disclosure provides methods for producing dimers of IgG-IgA fusion molecules disclosed herein. In certain embodiments, the present disclosure provides methods for producing polymers (e.g., dimers, trimers and / or tetramers) of IgG-IgA fusion molecules disclosed herein.

[0232] In certain embodiments, the method involves producing a dimer of an IgG-IgA fusion molecule. For example, but not by way of limitation, a method for expressing a dimer of an IgG-IgA fusion molecule may include expressing an IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence from P221 or R221 to the C-terminus of the IgA antibody heavy chain, and the IgG antibody comprises a deletion of amino acid K447.

[0233] In certain embodiments, the method relates to the preparation of polymers of IgG-IgA fusion molecules disclosed herein. For example, but not limited to, the method for expressing polymers of IgG-IgA fusion molecules includes expressing IgG-IgA fusion molecules comprising full-length IgG antibodies, wherein the full-length IgG antibodies are fused to the Fc region of IgA antibodies at their C-terminal ends, wherein the Fc region of IgA antibodies includes sequences from C242 to the C-terminal ends of the heavy chains of IgA antibodies. In certain embodiments, the IgG antibodies include the deletion of amino acid K447. In certain embodiments, the polymers of IgG-IgA fusion molecules produced by the method include dimers, trimers and / or tetramers of IgG-IgA fusion molecules.

[0234] B. Antibody Purification Methods

[0235] The present disclosure further provides methods for purifying the antibodies disclosed herein.For example, but not by way of limitation, the present disclosure provides methods for separating the oligomeric states of the antibodies disclosed herein, for example, separating the dimerization state from the tetramerization state of the antibodies.

[0236] In certain embodiments, the method for purifying the antibodies of the present disclosure may include purifying the antibodies using a protein affinity column. In certain embodiments, the method may also include performing size exclusion chromatography (SEC). For example, but not limited to, SEC may be performed to purify and / or separate antibodies of a specific oligomeric state disclosed herein, such as IgA antibodies and / or IgG-IgA fusion molecules. In certain embodiments, SEC may be performed to purify and / or separate an oligomeric state of antibodies disclosed herein, such as a dimeric state, a trimeric state, a tetrameric state, and / or a pentameric state.

[0237] In certain embodiments, the protein affinity column can be a MabSelect Sure (GE Healthcare) column. In certain embodiments, antibodies of the present disclosure, such as IgA samples containing primarily one oligomeric state, can be affinity purified using MabSelectSure (GE Healthcare) followed by SEC using a HiLoad Superdex column (GE Healthcare).

[0238] In certain embodiments, antibodies of the present disclosure, such as IgA antibodies disclosed herein, can be purified with Protein L (GE Healthcare) and then with SEC. In certain embodiments, the protein L column can be washed with a first wash buffer comprising Tris buffer (25mM Tris, pH 7.5, 150mM NaCl, 5mM EDTA, 2mM NaN3). In certain embodiments, the protein L column can be further washed with a second wash buffer comprising Triton X-114 buffer (25mM Tris, pH 7.5, 150mM NaCl, 5mM EDTA, 0.1% Triton X-114, 2mM NaN3) to remove endotoxin. In certain embodiments, the protein L column can be washed with a third wash buffer comprising Tris buffer, with a fourth wash buffer comprising KP buffer (0.4M potassium phosphate, pH 7.0, 5mM EDTA, 0.02% Tween20, 2mM NaN3) and / or with a fifth wash buffer comprising Tris buffer. Alternatively or additionally, the Protein L column may be washed one or more times with a wash buffer comprising PBS.

[0239] In certain embodiments, the antibody can be eluted from the protein L column using a buffer comprising 150 mM acetic acid, pH 2.7, which can be neutralized with 1 M arginine, 0.4 M succinate, pH 9.0. Alternatively or additionally, the antibody can be eluted from the protein L column using a buffer comprising 50 mM phosphoric acid, pH 3.0. In certain embodiments, the eluted antibody can be neutralized with 20X PBS, pH 11.

[0240] In certain embodiments, where the IgA sample contains complex oligomers, the Protein L eluate can be analyzed using a 3.5 μm, 7.8 mm x 300 mm XBridge Protein BEH SEC column (Waters) is further purified, for example, to separate antibodies of specific oligomeric states (e.g., dimer, trimer and / or tetramer states). In certain embodiments, an injection volume of no more than 100 μL (wherein the total protein is less than 1 mg) is run at 1 mL / min, using Agilent 1260 Infinity HPLC, with 0.2 M arginine, 0.137 M succinate, pH 5.0 as mobile phase, 200 μL fractions are collected. In certain embodiments, fractions from SEC can be selectively collected to mainly separate a kind of oligomeric state. One or more injections can be performed, and fractions of a given oligomer from each injection can be pooled together.

[0241] IV. Treatment Methods

[0242] The disclosed subject matter further provides methods of using the disclosed antibodies, such as IgA and IgG-IgA fusion molecules. In certain embodiments, the methods are directed to therapeutic uses of the disclosed antibodies.

[0243] In certain embodiments, one or more antibodies of the disclosed subject matter can be used to treat a disease and / or condition in a subject. For example, but not by way of limitation, the antibodies of the disclosed subject matter can be used to treat inflammatory diseases, autoimmune diseases, and cancer. In certain embodiments, the antibodies of the disclosed subject matter can be used to treat cancer. In certain embodiments, antibodies of the disclosed subject matter that lack binding to FcαRI and cannot activate FcαRI can be used to treat inflammatory diseases, autoimmune diseases, and cancer. In certain embodiments, antibodies of the disclosed subject matter can be used to treat diseases and / or conditions that require antibody transcytosis to achieve a therapeutic effect and / or access to a therapeutic target. For example, but not by way of limitation, antibodies of the disclosed subject matter can be used to treat diseases and / or conditions that require antibody transcytosis across mucosal membranes.

[0244] In certain embodiments, the present disclosure provides antibodies in methods for treating individuals with specific diseases and / or illnesses, the method comprising administering an effective amount of an antibody or a composition comprising the antibody to the individual. In certain embodiments, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In certain embodiments, the present disclosure provides antibodies for inhibiting specific molecular pathways and / or mechanisms. In certain embodiments, the present disclosure provides antibodies in methods for inhibiting specific molecular pathways and / or mechanisms in individuals, the method comprising administering effective antibodies to individuals to inhibit specific molecular pathways and / or mechanisms. In certain embodiments, the present disclosure provides antibodies for activating specific molecular pathways and / or mechanisms. In certain embodiments, the present disclosure provides antibodies in methods for inhibiting specific molecular pathways and / or mechanisms in activated individuals, the method comprising administering effective antibodies to individuals to inhibit specific molecular pathways and / or mechanisms.

[0245] As used interchangeably herein, "individual," "patient," or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0246] The present disclosure further provides the use of antibodies in the manufacture or preparation of drugs for treating diseases and / or disorders of subjects. In certain embodiments, the drugs are used to treat specific diseases and / or disorders. In certain embodiments, the drugs are used in methods for treating specific diseases and / or disorders, the method comprising administering an effective amount of the drugs to an individual with the disease. In certain embodiments, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In certain embodiments, the drugs are used to inhibit or activate specific molecular pathways and / or mechanisms. In certain embodiments, the methods for inhibiting or activating specific molecular pathways and / or mechanisms in individuals, comprising administering an effective amount of drugs to individuals to inhibit specific molecular pathways and / or mechanisms.

[0247] In certain embodiments, the antibodies used in the disclosed methods of treatment may be present in a pharmaceutical composition, as described herein. In certain embodiments, the pharmaceutical composition may include a pharmaceutically acceptable carrier, as described herein. In certain embodiments, the pharmaceutical composition may include one or more antibodies of the present disclosure.

[0248] Additionally or alternatively, the pharmaceutical composition may include a second therapeutic agent. When one or more disclosed antibodies are administered together with another therapeutic agent, one or more antibodies and another therapeutic agent may be administered sequentially or simultaneously. Such combination therapies described above encompass combined administration (wherein two or more therapeutic agents are included in the same or separate preparation) and separate administration, where separate administration, the administration of antibodies disclosed herein may be performed before, simultaneously and / or after the administration of another therapeutic agent or medicament. In certain embodiments, the administration of antibodies disclosed herein and the administration of other therapeutic agents are performed within about one month from each other, or within about one week, two weeks or three weeks, or within about one day, two days, three days, four days, five days or six days.

[0249] Antibodies of the present disclosure (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations, bolus administrations, and pulse infusions at various time points.

[0250] The antibodies disclosed herein will be formulated, dosed, and applied in a manner consistent with good medical practice. Factors to be considered in this case include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the time schedule of administration, and other factors known to practitioners. The antibodies are not required, but are optionally co-formulated with one or more preparations currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of the antibody present in the formulation used, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration as described herein, or in about 1% to 99% of the dosages described herein, or in any dosage and any route determined to be suitable empirically / clinically.

[0251] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. In certain embodiments, the antibodies of the present disclosure may be administered as needed. In certain embodiments, the antibodies may be administered to the patient at one time or in a series of treatments. For example, but not as a limitation, the antibodies and / or pharmaceutical compositions comprising the antibodies, as disclosed herein, may be administered twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, or once a year.

[0252] In certain embodiments, depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be, for example, an initial candidate dose administered to a patient by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose may range from about 1 μg / kg to 100 mg / kg or more. In certain embodiments, the daily dose may be greater than about 100 mg / kg. In certain embodiments, the dosage may be adjusted to achieve a plasma antibody concentration of 1-1000 μg / ml, and in some methods 25-300 μg / ml.

[0253] For repeated administration for several days or longer, depending on the disease, treatment will generally continue until the desired disease symptom suppression occurs. An exemplary dosage of an antibody ranges from about 0.05 mg / kg to about 10 mg / kg. In certain embodiments, one or more dosages of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Alternatively, the antibody may be administered as a sustained release formulation, in which case less frequency of administration is required. Dosage and frequency may vary according to the half-life of the antibody in the patient. In certain embodiments, such dosages may be administered intermittently, such as weekly or every three weeks (e.g., so that the patient receives about twice to about twenty times, or, for example, about six doses of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses.

[0254] In certain embodiments, the method may further include monitoring the subject and determining the effectiveness of the treatment. For example, the progress of the therapy can be easily monitored by conventional techniques and assays.

[0255] V. Pharmaceutical Compositions

[0256] The subject matter of the present disclosure further provides pharmaceutical compositions containing one or more antibodies of the present disclosure, such as IgA and IgG-IgA Fc fusion proteins, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition can include a combination of multiple (e.g., two or more) antibodies and / or antigen-binding portions thereof of the subject matter of the present disclosure.

[0257] In certain embodiments, the disclosed pharmaceutical compositions and formulations can be prepared in the form of a lyophilized formulation or an aqueous solution by combining an antibody having the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). For example, but not by way of limitation, lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. In certain embodiments, aqueous antibody formulations may include those described in U.S. Pat. No. 6,171,586 and WO2006 / 044908, the formulations in the latter comprising a histidine-acetate buffer. In certain embodiments, the purity of the antibody may be greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9%.

[0258] Pharmaceutical carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polyols. peptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0259] The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the antibody may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0260] The pharmaceutical composition of the present disclosure can also be administered in combination therapy, i.e., in combination with other agents. In certain embodiments, the pharmaceutical composition disclosed herein may also contain more than one active ingredient necessary for the specific indication treated, such as an active ingredient with complementary activity that does not adversely affect each other. In certain embodiments, the pharmaceutical composition may include a second active ingredient for treating the same disease treated by a first therapeutic agent. Such active ingredients are suitably present in an amount combination effective for the intended purpose. For example, but not limited to, the preparation of the present disclosure may also contain more than one active ingredient necessary for the specific indication treated, preferably an active ingredient with complementary activity that does not adversely affect each other. For example, it may be necessary to further provide a second therapeutic agent for treating the same disease. Such active ingredients are suitably present in an amount combination effective for the intended purpose.

[0261] The preparations disclosed herein can be administered by a variety of methods known in the art. The route of administration and / or mode of administration depends on the desired result. The active compound can be prepared together with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such preparations are described in, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978. In certain embodiments, the pharmaceutical composition is manufactured under the conditions of the Good Manufacturing Practice (GMP) of the U.S. Food and Drug Administration.

[0262] Sustained-release preparations containing the disclosed antibodies can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrix being in the form of shaped articles such as films or microcapsules. In certain embodiments, the active ingredient can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0263] In order to administer the antibodies of the present disclosure by certain routes of administration, it may be necessary to coat the compound with a material or co-administer the compound with a material to prevent its inactivation. For example, the compound can be administered to a subject in a suitable carrier such as a liposome or a diluent. Pharmaceutical diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al., J. Neuroimmunol. 7: 27 (1984)).

[0264] Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except in the case where any conventional media or agents are incompatible with the active compound, they are contemplated for use in the pharmaceutical compositions of the present disclosure. Supplementary active compounds may also be incorporated into the composition.

[0265] The therapeutic composition must be sterile, substantially isotonic, and stable under manufacturing and storage conditions. The composition can be formulated into a solution, microemulsion, liposome or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by maintaining the required particle size and by using a surfactant in the case of dispersion. In many cases, it is preferred to include an isotonic agent in the composition, such as sugar, polyols such as mannitol, sorbitol or sodium chloride. The extended absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, such as monostearate and gelatin.

[0266] Sterile injectable solutions can be prepared by mixing the desired amount of one or more disclosed antibodies with one or more of the above-listed ingredients in a suitable solvent as needed, followed by sterilization microfiltration, such as filtration through a sterile filtration membrane. Typically, dispersions are prepared by mixing the active compound into a sterile carrier that contains a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional required ingredients from its previously sterile filtered solution.

[0267] The therapeutic compositions can also be administered with medical devices known in the art. For example, the therapeutic compositions of the present disclosure can be administered with a needle-free subcutaneous injection device, such as those disclosed in, for example, U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules that can be used in the present disclosure include: U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known.

[0268] For therapeutic compositions, formulations disclosed herein include those suitable for oral, nasal, topical (including oral and sublingual), rectal, vaginal and / or parenteral administration. The formulations can be conveniently presented in unit dosage form and can be prepared by any known method in the field of pharmaceutical technology. The amount of the antibody that can be combined with a carrier material to produce a single dosage form varies according to the subject being treated and the specific mode of administration. The amount of the antibody that can be combined with a carrier material to produce a single dosage form is generally the amount of the composition that produces the therapeutic effect. Typically, in percentage, the amount ranges from about 0.01% to about 99% active ingredient, about 0.1% to about 70% or about 1% to about 30%.

[0269] Dosage forms for topical or transdermal administration of the compositions of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutical carrier and any preservatives, buffers or propellants that may be required.

[0270] The phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, which are usually administered by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0271] These pharmaceutical compositions may also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can be sterilized by the above-mentioned sterilization procedures and by adding various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.) to ensure that there is no microorganism. It may also be desirable to include isotonic agents, such as sugar, sodium chloride, etc. in the composition. In addition, the extended absorption of injectable drug forms can be achieved by comprising agents (e.g., aluminum monostearate and gelatin) that delay absorption.

[0272] In certain embodiments, when the antibodies of the present disclosure are administered to humans and animals as a medicament, they can be administered alone or as a pharmaceutical composition comprising, for example, from about 0.01% to about 99.5% (or about 0.1 to about 90%) of an antibody described herein, with a pharmaceutically acceptable carrier.

[0273] Ⅵ. Products

[0274] The disclosed subject matter further relates to an article of manufacture, e.g., containing one or more of the disclosed antibodies, for use in the treatment and / or prevention of the above-mentioned diseases and / or disorders.

[0275] In certain embodiments, the article comprises a container and a label or package insert on or associated with the container. Non-limiting examples of suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed of a variety of materials such as glass or plastic. The container can hold a composition that is effective, by itself or in combination with another composition, for treating, preventing, and / or diagnosing a condition, and the container can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0276] In certain embodiments, at least one active agent in the composition is an antibody of the presently disclosed subject matter.The label or package insert may indicate that the composition is used to treat the condition of choice.

[0277] In certain embodiments, the article of manufacture may include (a) a first container containing a composition comprising an antibody of the present disclosure; and (b) a second container containing a composition comprising an additional cytotoxic agent or other therapeutic agent. In certain embodiments, the article of manufacture may further include a package insert indicating that the composition can be used to treat a specific condition.

[0278] Alternatively or additionally, the article of manufacture may further include an additional container, such as a second or third container, comprising a pharmaceutically acceptable buffer, such as, but not limited to, bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0279] VII. Exemplary Embodiments

[0280] A. In certain non-limiting embodiments, the presently disclosed subject matter provides an isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises a substitution at amino acids V458, N459, and / or S461.

[0281] A1. The aforementioned isolated IgA antibody according to A, wherein amino acid V458 is substituted by isoleucine (V458I), amino acid N459 is substituted by glutamine (N459Q), glycine (N459G) or alanine (N459A), and / or amino acid S461 is substituted by alanine (S461A).

[0282] B. In certain non-limiting embodiments, the presently disclosed subject matter provides an isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises a substitution at amino acid 1458.

[0283] B1. The aforementioned isolated IgA antibody according to B, wherein amino acid I458 is substituted by valine (I458V).

[0284] C. In certain non-limiting embodiments, the presently disclosed subject matter provides an isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises one or more substitutions at an amino acid selected from the group consisting of N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459, S461, and combinations thereof.

[0285] C1. The aforementioned isolated IgA antibody according to C, wherein the substitutions at amino acids N166, S212, N263, N337, I338, T339 and N459 are N166A, S212P, N263Q, N337T, I338L, T339S and N459Q.

[0286] C2. The aforementioned isolated IgA antibody according to any one of A-C1, wherein the IgA antibody is an IgA1, IgA2m1, IgA2m2 or IgA2mn antibody.

[0287] C3. The aforementioned isolated IgA antibody according to any one of C and C1, wherein the IgA antibody has substitutions at amino acids N337, I338 and T339, and one or more substitutions at T168, N211, S212, S213, N263, T265, N459, S461 and combinations thereof.

[0288] C4. The aforementioned isolated IgA antibody according to C3, wherein the IgA antibody is an IgA2m1, IgA2m2 or IgA2mn antibody.

[0289] C5. The aforementioned isolated IgA antibody according to any one of A-C4, wherein the IgA antibody is a humanized, chimeric antibody or a human antibody.

[0290] D. In certain non-limiting embodiments, the presently disclosed subject matter provides an isolated IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence including P221 or R221 to the C-terminus of the heavy chain of the IgA antibody, wherein the IgG antibody further comprises a deletion of amino acid K447.

[0291] E. In certain non-limiting embodiments, the presently disclosed subject matter provides an isolated IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence including C242 to the C-terminus of the IgA antibody heavy chain.

[0292] E1. The aforementioned isolated IgG-IgA fusion molecule according to E, wherein the IgG antibody further comprises a deletion of amino acid K447.

[0293] E2. The aforementioned isolated IgG-IgA fusion molecule according to any one of D-E1, wherein the IgG antibody is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody and an IgG4 antibody.

[0294] E3. The aforementioned isolated IgG-IgA fusion molecule according to any one of D-E2, wherein the IgG antibody is an IgG1 antibody.

[0295] E4. The aforementioned isolated IgG-IgA fusion molecule according to any one of D-E3, wherein the IgA antibody is selected from the group consisting of IgA1 antibody, IgA2m1 antibody, IgA2m2 antibody and IgA2mn antibody.

[0296] E5. The aforementioned isolated IgG-IgA fusion molecule according to any one of D-E4, wherein the IgA antibody is an IgA2m1 antibody.

[0297] F. In certain non-limiting embodiments, the presently disclosed subject matter provides an isolated nucleic acid encoding the IgA antibody of any one of A-C4 or the IgG-IgA fusion molecule of any one of D-E5.

[0298] G. In certain non-limiting embodiments, the presently disclosed subject matter provides a host cell comprising a nucleic acid of F.

[0299] H. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for producing an IgA antibody or IgG-IgA, comprising culturing a G host cell to produce an IgA antibody or an IgG-IgA fusion molecule.

[0300] H1. According to the aforementioned method of H, it further comprises recovering the IgA antibody or IgG-IgA fusion molecule from the host cell.

[0301] H2. The aforementioned IgA antibody or IgG-IgA fusion molecule is prepared from H or recovered from H1.

[0302] I. In certain non-limiting embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising one or more IgA antibodies of any one of A-C4 and H2, or one or more IgG-IgA fusion molecules of any one of D-E5 and H2, and a pharmaceutically acceptable carrier.

[0303] I1. The aforementioned pharmaceutical composition according to I, further comprising an additional therapeutic agent.

[0304] J. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating an individual suffering from a disease, wherein the method comprises administering to the individual an effective amount of one or more IgA antibodies of any one of A-C4 and H2, or one or more IgG-IgA fusion molecules of any one of D-E5 and H2.

[0305] J1. The aforementioned method according to J, wherein the disease is an inflammatory disease, an autoimmune disease or cancer.

[0306] K. The aforementioned IgA antibody according to any one of A-C4 and H2 or the aforementioned IgG-IgA fusion molecule according to any one of D-E5 and H2, for use as a medicament.

[0307] L. The aforementioned IgA antibody according to any one of A-C4 and H2 or the aforementioned IgG-IgA fusion molecule according to any one of D-E5 and H2, for use in treating a disease.

[0308] M. The aforementioned IgA antibody or IgG-IgA fusion molecule according to L, wherein the disease is an inflammatory disease, an autoimmune disease or cancer.

[0309] N. In certain non-limiting embodiments, the presently disclosed subject matter provides use of an IgA antibody of any one of A-C4 and H2 or an IgG-IgA fusion molecule of any one of D-E5 and H2 in the manufacture of a medicament for treating a disease.

[0310] N1. The aforementioned use according to N, wherein the disease is an inflammatory disease, an autoimmune disease or cancer.

[0311] O. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for enhancing expression of IgA dimers, comprising increasing the amount of DNA encoding a joining chain (JC) introduced into a first cell relative to the amount of DNA encoding a light chain (LC) and a heavy chain (HC), wherein the enhanced expression is relative to the amount of IgA dimers produced in a second cell into which equal amounts of JC, LC, and HC DNA are introduced.

[0312] O1. According to the aforementioned method of O, the ratio of the amount of DNA encoding the HC to the amount of DNA encoding the LC to the amount of DNA encoding the JC (HC:LC:JC) introduced into the first cell is from about 1:1:2 to about 1:1:5.

[0313] P. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of enhancing expression of an IgA dimer, trimer, or tetramer, comprising reducing the amount of DNA encoding a joining chain (JC) introduced into a first cell relative to the amount of DNA encoding a light chain (LC) and a heavy chain (HC), wherein the enhanced expression is relative to the amount of IgA trimer or tetramer produced in a second cell into which a greater amount of HC and LC DNA relative to the amount of JC DNA is introduced.

[0314] P1. According to the aforementioned method of P, wherein the ratio of the amount of DNA encoding the HC to the amount of DNA encoding the LC to the amount of DNA encoding the JC introduced into the first cell (HC:LC:JC) is from about 1:1:0.25 to about 1:1:0.5.

[0315] Q. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of increasing production of an IgA1 or IgA2m1 polymer, comprising expressing in a first cell an IgA1 or IgA2m1 antibody having a substitution at amino acid V458, wherein the increased production is relative to the amount of IgA1 or IgA2m1 polymer produced in a second cell expressing the IgA1 or IgA2m1 antibody without a substitution at amino acid V458.

[0316] Q1. The aforementioned method according to Q, wherein the amino acid is substituted by isoleucine (V458I).

[0317] R. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for increasing the production of IgA2m2 dimers, comprising expressing in a first cell an IgA2m2 antibody having a substitution at amino acid 1458, wherein the increased production is relative to the amount of IgA2m2 dimers produced in a second cell expressing the IgA2m2 antibody without a substitution at amino acid 1458.

[0318] R1. The aforementioned method according to R, wherein the amino acid is substituted by valine (I458V).

[0319] S. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of increasing production of IgA1 or IgA2m1 polymers, comprising expressing in a first cell an IgA1 or IgA2m1 antibody having a substitution at amino acid N459 or S461, wherein the increased production is relative to the amount of IgA1 or IgA2m1 polymers produced in a second cell expressing the IgA1 or IgA2m1 antibody without a substitution at amino acid N459 or S461.

[0320] S1. The aforementioned method according to S, wherein amino acid N459 is substituted by N459Q, N459G or N459A mutation and / or amino acid S461 is substituted by S461A mutation.

[0321] T. A method for reducing the production of IgA2m2 polymers, comprising expressing in a first cell an IgA2m2 antibody having a substitution at amino acid C471, wherein the reduced production is relative to the amount of IgA2m2 polymers produced in a second cell expressing the IgA2m2 antibody without the substitution at amino acid C471.

[0322] T1. The aforementioned method according to T, wherein amino acid C471 is substituted by a C471S mutation.

[0323] U. In certain non-limiting embodiments, the present disclosure provides a method of enhancing transient expression of an IgA2m2 antibody, comprising expressing an IgA2m2 antibody comprising a substitution at an amino acid selected from the group consisting of N166, S212, N263, N337, I338, T339, N459, and a combination thereof in a first cell, wherein the enhanced transient expression is relative to the transient expression amount produced in a second cell expressing an IgA2m2 antibody without a substitution at an amino acid selected from the group consisting of N166, S212, N263, N337, I338, T339, N459, and a combination thereof.

[0324] V. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of expressing a dimer of an IgG-IgA fusion molecule, comprising expressing an IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence comprising P221 or R221 to the C-terminus of the heavy chain of the IgA antibody, and the IgG antibody comprises a deletion of amino acid K447.

[0325] W. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for expressing dimers, trimers, and tetramers of IgG-IgA fusion molecules, comprising expressing an IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence including C242 to the C-terminus of the IgA antibody heavy chain.

[0326] W1. The aforementioned method according to W, wherein the IgG antibody comprises a deletion of amino acid K447.

[0327] X. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for purifying an IgA antibody from a mixture comprising an IgA antibody and at least one host cell protein, comprising:

[0328] (a) applying the mixture to a column comprising protein L to bind the IgA antibody;

[0329] (b) washing the protein L column with a wash buffer comprising PBS; and

[0330] (c) eluting the IgA antibody from the Protein L column with an elution buffer containing phosphoric acid.

[0331] Y. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for purifying an IgA antibody or IgG-IgA fusion molecule in an oligomeric state from a mixture comprising an IgA antibody or IgG-IgA fusion molecule and at least one host cell protein, comprising:

[0332] (a) applying the mixture to an affinity purification column containing protein L or protein A to bind the IgA antibody or IgG-IgA fusion molecule;

[0333] (b) washing the affinity purification column with a washing buffer;

[0334] (c) eluting the IgA antibody or IgG-IgA fusion molecule from the affinity purification column with an elution buffer to form a first eluate; and

[0335] (d) applying the first eluate to a size exclusion chromatography column to separate IgA antibodies or IgG-IgA fusion molecules in different oligomeric states and obtaining an effluent containing the IgA antibodies or IgG-IgA fusion molecules in oligomeric states.

[0336] The following examples are merely illustrative of the subject matter of the present disclosure and should not be considered limiting in any way. Example 1: Evaluation of the effects of glycosylation and FcRn binding on pharmacokinetic parameters of polymeric IgA in mice

[0337] Compared with traditional IgG-based drugs, IgA antibodies have broad potential as a novel therapeutic platform due to their superior receptor-mediated cytotoxic activity, potent pathogen neutralization, and transcytotic transport across mucosal barriers mediated by the polymeric immunoglobulin receptor (pIgR). However, the transition of IgA to clinical development has been challenged by complex expression and characterization as well as rapid serum clearance, which is thought to be mediated by glycan receptor clearance of recombinantly produced IgA monomers with incompletely sialylated N-linked glycans. In this example, a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimer, and polymeric human IgA is provided. In addition, two strategies to overcome the rapid serum clearance of polymeric IgA are identified: (1) removal of N-linked glycosylation sites to produce aglycosylated or partially aglycosylated polymeric IgA, and (2) engineering polymeric IgG-IgA Fc fusions to bind to FcRn.

[0338] method:

[0339] Plasmid cloning and sequence alignment.The antibody variable domain sequences used included humanized anti-human HER2 antibody (Carter et al., Proc Natl Acad Sci USA 89:4285–9 (1992)) and mouse anti-mouse IL-13 antibody (Genentech). Protein sequences of human IgA constant heavy chains IgA1, IgA2m1 and IgA2m2, other IgA species and human J chain were obtained from Uniprot (www.uniprot.org) or NCBI (www.ncbi.nlm.nih.gov / protein). Other species obtained included a mutation in IgA2m1, namely P221R, which stabilizes the light chain-heavy chain disulfide as previously reported (Chintalacharuvu et al., J Immunol 157:3443-9 (1996)). Genes encoding fusions of antibody variable domains with human light chain and human IgA1, IgA2m1 and IgA2m2 heavy chain constant domains were synthesized and cloned into mammalian pRK vector (Eaton et al., Biochemistry 25:8343–7 (1986)). Site-directed mutagenesis was used to introduce point mutations. All plasmids were sequence verified. Sequence alignments were performed using GSeqWeb (Genentech) and Excel (Microsoft).

[0340] Small-scale antibody expression and purification. Expi293T TM Cells were transiently transfected at 30 mL scale with 15 μg of DNA of LC and HC (for IgA monomers) or with a total of 30 μg of DNA of LC, HC and JC in different ratios (for IgA oligomers) (Bos et al., Journal of Biotechnology 180: 10–6 (2014) and Bos et al., Biotechnol Bioeng 112: 1832–42 (2015)). IgA was affinity purified in batches with Protein L (GE Healthcare) because all antibodies contain a kappa light chain. Protein L eluates were characterized by analytical SEC-HPLC (Tosoh Bioscience LLC TSKgelSuperSW3000 column, Thermo Scientific Dionex UltiMate 3000HPLC). Constant volumes were loaded onto the column and the area under each curve was quantified using Chromeleon Chromatography Data System software (Thermo Scientific).

[0341] Large-scale antibody expression and purification.IgA, IgG, and IgG-IgA Fc fusions were transiently expressed in CHO DP12 cells as previously described (Wong et al., Biotechnol Bioeng 106:751–63 (2010)). For low expressing clones, TI stable cell lines were generated. IgG and IgG-IgA Fc fusions were affinity purified using MabSelect Sure (GE Healthcare) followed by size exclusion chromatography (SEC) using a HiLoad Superdex 200pg column (GE Healthcare). IgA was affinity purified using Capto L (GE Healthcare) followed by SEC. For IgA samples where the DNA ratio successfully biased expression toward predominantly one oligomeric state, a HiLoad Superdex 200pg column (GE Healthcare) was used for SEC. For IgA samples containing a complex mixture of oligomers, a 3.5 μm, 7.8 mm x 300 mm Xbridge Protein BEH The SEC column (Waters) better separated the dimer and tetramer peaks.

[0342] SEC-MALS. Polymeric IgA at 3.5μm, 7.8mm x 300mm Xbridge Protein BEH The samples were run on a SEC column (Waters) and injected directly into a DAWN HELEOS / Optilab T-rEX II (Wyatt) multi-angle light scattering detector for molar mass determination and polydispersity measurements.

[0343] Differential Scanning Fluorescence (DSF). DSF was performed as previously described (Lombana et al., Sci Rep 5:17488 (2015)).

[0344] In vitro transcytosis assay.Madin-Darby canine kidney (MDCKII) cells (European Collection of Authenticated Cell Cultures, Salisbury, UK) were transduced with retrovirus containing cDNA encoding the human pIgR gene (Retro-X, Takara Bio; OriGene, Rockville, MD). The expression of the pIgR gene was confirmed by qRT-PCR and Western blotting. MDCKII cells expressing pIgR were maintained in DMEM supplemented with 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin (Thermo Fisher, Carlsbad, CA) and 2 μg / ml puromycin (Takara Bio, Mountain View, CA). For transcytosis assays, cells were seeded on 0.4 μm Millicell 24-well cell inserts (Millipore, Burlington, MA) and cultured for 4 days. On the day of the experiment, cells were washed twice with FluoroBrite DMEM (Thermo Fisher) and 6 μg of IgA molecules were added to the basolateral compartment. After 24 hours of incubation, the culture medium from the apical and basolateral compartments was collected for ELISA analysis.

[0345] Electron microscope. Purified anti-IL-13 IgA2m2 dimer and tetramer samples were first cross-linked by incubation in 0.015% glutaraldehyde (Polysciences, Inc.) at room temperature for 10 minutes. After fixation, the samples were diluted with TBS buffer to reach a concentration of 10 ng / μL. Then 4 μl of each sample was incubated on a freshly glow-discharged 400-mesh copper grid covered with a thin layer of continuous carbon before being treated with 2% (w / v) uranyl acetate negative stain (Electron MicroscopySciences). IgA dimers and tetramers were then detected using a Tecnai Spirit T12 (Thermo Fisher) at 120 keV at 25,000x Imaging was performed at a magnification of 1000 x 1000. Images were recorded using a Gatan 4096 x 4096 pixel CCD camera under low-dose conditions. Approximately 5000 particles of IgA dimers and tetramers were then selected and extracted using the e2boxer.py software in the EMAN2 package (Tang et al., J Struct Biol 157:38–46 (2007)) using a particle box size of 128 pixels. The reference-free 2D class in the RELION imaging software package (Scheres J Struct Biol 180:519–30 (2012)) was used to generate an average image of the two samples.

[0346] Global N-linked glycan composition analysis (LC-MS analysis). 10 μg of each IgA sample was denatured with 8M guanidine hydrochloride at a volume ratio of 1:1 and reduced with 100 mM dithiothreitol (DTT) at 95°C for 10 minutes. The samples were diluted with 100 mM Tris HCl, pH 7.5 to a final concentration of 2 M guanidine hydrochloride and then N-linked deglycosylation was performed with 2 μl of P0705S PNGase F (New England BioLabs) overnight at 37°C. After deglycosylation, 150 ng of each sample was injected into an Agilent 1260 Infinity LC chromatography system and eluted by the following: an isocratic gradient of 2% to 32% solvent B (solvent A: 99.88% water, containing 0.1% formic acid and 0.02% trifluoroacetic acid; solvent B: 90% acetonitrile, containing 9.88% water plus 0.1% formic acid and 0.02% trifluoroacetic acid). The HPLC system was coupled to a G6520B Q-TOF mass spectrometer via an Agilent G4240A Chip Cube MS system. The samples were enriched and separated for glycans using a porous graphitized carbon column constructed within a G4240-64025 mAb-Glyco chip in the Chip Cube MS system. Data acquisition: 1.9 kV spray voltage; 325 °C gas temperature; 5 l / min drying gas flow; 160 V fragmentor voltage; 65 V skimmer voltage; 750 V oct 1RF Vpp voltage; 400 to 3000 m / z scan range; positive polarity; MS1 centroid data acquisition using extended dynamic range (2 GHz) instrument mode; 3 spectra / s; 333.3 ms / spectrum; 3243 transients / spectrum; CE set to 0. The acquired mass spectral data were searched against a glycan library in the Agilent MassHunter Qualitative Analysis software using a combination of accurate mass and 10 ppm mass tolerance and expected retention times for glycan identification. N-linked glycans were quantified label-free relative to all identified N-linked glycans within each sample based on the AUC in the extracted compound chromatograms for each glycan.

[0347] N-linked glycopeptide site mapping analysis (LC-MS / MS analysis).10 μg IgA was reduced with 10 mM DTT at 37°C for 1 hr, alkylated with 10 mM iodoacetamide at room temperature for 20 minutes, digested with 0.2 μg trypsin (Promega) and 0.2 μg chymotrypsin (Thermo Fisher Scientific) at 37°C overnight, quenched with 0.1% trifluoroacetic acid (TFA), and cleaned with C18 (3M Empore C18 extraction disk) grade pipette tips (50% acetonitrile, 49.9% water, 0.1% TFA). 200 fmol of sample was injected into the Waters NanoAcquity UPLC system by an autosampler and separated on a Waters Acquity M-Class BEH C18 column (0.1 mm×100 mm, 1.7 μm resin) at 45°C. Elution was performed using a gradient of 2% to 40% solvent B (solvent A: 99.9% water, 0.1% formic acid; solvent B 99.9% acetonitrile, 0.1% formic acid).

[0348] The separated peptides were analyzed online by nanospray ionization into an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) using the following data acquisition parameters: 60,000 resolution; 375-1600 m / z scan range; positive polarity; centroid mode; 1 m / z isolation width, 0.25 activation Q and 10 ms activation time; CID activation; CE set to 35. Data were collected in data-dependent mode, parent ions were analyzed in FTMS, and the top 15 most abundant ions were selected for fragmentation and analysis in ITMS. The acquired mass spectrometric data were searched against protein sequences using Protein Metrics Byonic software and analyzed in Protein Metrics Byologic software. Peptide identification for each glycosylation site was manually verified based on a combination of MS2 fragmentation spectra, extracted ion chromatograms (XICs), and retention times. N-linked glycopeptides were label-free quantified relative to their unmodified peptides by integration of the AUC of the XIC.

[0349] Mouse studies.Female Balb / C mice (6-8 weeks old) were obtained from Charles River Laboratories. Upon arrival, all mice were housed in a pathogen-free animal facility at room temperature at 21°C under a standard 12h light / 12h dark cycle with free access to food and water. All mice received a single intravenous (IV) injection of the corresponding antibody (IgG or IgA). At various times after injection, blood samples (150-200 μL) were collected by retro-orbital sinus or cardiac puncture under isoflurane anesthesia. The samples were collected into serum separator tubes. The blood was allowed to clot at room temperature for at least 20 minutes. The clotted samples were kept at room temperature until centrifugation, starting within 1 hour of the collection time. Each sample was centrifuged at 2-8°C for 5 minutes at a relative centrifugal force of 1500-2000 x g. Serum was separated from the blood sample within 20 minutes after centrifugation and transferred to a labeled 2.0-mL polypropylene conical-bottom microcentrifuge tube.

[0350] Only animals that appeared healthy and had no obvious abnormalities were used in research. All animal work performed was reviewed and approved by Genentech's Institutional Animal Care and Use Committee (IACUC).

[0351] IgA ELISA was used for transcytosis and pharmacokinetic studies. IgA antibody levels were measured by sandwich ELISA. The wells of a 384-microtiter plate were coated overnight at 4°C with 25 μl of coating buffer (0.05 M sodium carbonate, pH 9.6) containing 2 μg / ml goat anti-human κ antibody (Southern Biotech, Cat#2060-01), and then blocked for 2 hours at 37°C with 50 μl of PBS containing 0.5% BSA. Samples (25 μl) diluted in sample buffer (1x PBS, pH 7.4, 0.5% BSA, 0.35 M NaCl, 0.05% Tween 20, 0.25% CHAPS, 5 mM EDTA) were then added to the blocked plate and incubated for 2 hours at room temperature. After incubation, 25 μl of horseradish peroxidase-conjugated goat anti-human IgA (Southern Biotech, Cat#2053-05) was added and incubated for 1 hour at room temperature. The plate was then incubated with 25 μl TMB (Moss, Cat# TMBE-1000) for 15 min and the reaction was terminated with 25 μl 1M H3PO4. The absorbance was measured at 450 nm using a disk analyzer, with a decrease at 630 nm. Between steps, the plate was washed six times with 200 μl wash buffer (PBS containing 0.05% Tween-20). As a quantitative reference, a standard curve was established for each IgA molecule using serially diluted stock material (20 ng / ml-0.15 ng / ml). The IgA ELISA can tolerate biological matrices up to 10% mouse serum and 10% tissue lysate.

[0352] Radiochemistry. Iodine-125 125 I] was obtained from Perkin Elmer (Boston, MA) as 0.1 N sodium hydroxide containing sodium iodide. 1 mCi 125 I (~3 μL) was used for the indirect iodine method (Pierce Chemical Co., Rockford, IL) at a specific activity of ~10 μCi / μg. 125 I was randomly labeled with tyrosine residues. 111 In and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated (randomly via lysine) mAbs were achieved in 0.3 M ammonium acetate pH 7 for 1 h 111 Radiosynthesis of In-labeled antibodies (~8 μCi / μg). Purification of all radioimmunoconjugates was achieved using a NAP5 column equilibrated in PBS and confirmed by size exclusion chromatography.

[0353] The antibodies were radioiodinated using the indirect iodine addition method (Chizzonite et al., J Immunol; 147:1548–56 (1991)). TM The protein of radiolabeling is purified by column (GE Healthcare Life Sciences, cat.17-0853-01).After radioiodination, the labeled antibody is characterized by SEC-HPLC to compare with unlabeled antibody.The sample is injected into Agilent 1100 series HPLC (Agilent Technology, Santa Clara, CA) and Yarra SEC-3000, 3 μM 300mm x 7.8mm (Phenomenex, Torrance, CA, cat.00H-4513-K0) size exclusion column in series, and eluted with phosphate buffered saline (PBS pH 7.0) at a flow rate of 0.8mL / min for 20 minutes.The elution is monitored by the absorbance at 280nm and by measuring the radioactivity of the eluted fraction in an online Gabiγ counter (Elysia-Raytest, Germany).

[0354] Tissue distribution study design and analysis. The protocol, housing, and anesthesia were approved by the Institutional Animal Care and Use Committee of Genentech Laboratory Animal Resources and complied with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care.

[0355] Female BALB-c mice weighing 20-30 g and 6-7 weeks old were obtained from Jackson / West (CA). Six groups of 12 mice were used in this study. 125 I isolation, 100 μL of 30 mg / mL sodium iodide was intraperitoneally administered 1 hour and 24 hours before administration. All mice received 125 I-labeled antibodies and 111 A single IV injection of a mixture of In-labeled antibodies (5 μCi each) plus the corresponding unmodified antibodies for a total dose of 5 mg / kg. Groups of 4 mice were bled retro-orbitally under isoflurane (inhalation) at 5min, 15min, 30min, 1hr, 4hr, 12hrs, 1 day, 2 days and 3 days after injection. At 1 hour, 1 day and 3 days; 4 animals were euthanized by thoracotomy under ketamine (75-80mg / kg) / xylene (7.5-15mg / kg) anesthesia. The following tissues were collected, rinsed in cold PBS, blotted, weighed and frozen: brain, liver, lung, kidney, spleen, heart, stomach, small intestine, muscle, skin, fat, large intestine. Use 1480WIZARD TM The gamma counter measures the radioactivity of the sample in the energy window. 111 In(245keV; attenuation t 1 / 2 =2.8 days) and 125 I(35keV; attenuation t 1 / 2 =59.4 days) counts with automatic background and decay correction. Data were analyzed and plotted using GraphPad Prism (version Windows 7.00, GraphPad Software, San Diego California USA, www.graphpad.com).

[0356] Mouse plasma stability. Mouse plasma (containing the anticoagulant lithium heparin) was obtained from BioIVT (Westbury, NY), and a buffer control was prepared by mixing bovine serum albumin (Sigma-Aldrich; St. Louis, MO, cat. A2058) with PBS (PBS + 0.5% BSA). Radiolabeled antibodies were mixed into mouse plasma or buffer control at 5 μCi of radiolabeled tracer and then incubated in an incubator set at 37°C and 5% CO2. At set time points of 0, 24, and 96 hours of incubation, samples were removed from the incubator and stored in a -80°C freezer until analysis.

[0357] Samples were analyzed by the SEC-HPLC method described above and samples were diluted 1:1 in PBS. Resulting chromatograms were compared between time points to monitor changes from the parent peak at time zero.

[0358] Antibody kinetics as determined by Wasatch. A 96×96 array-based SPR imaging system (Carterra USA) was used to analyze the kinetics of purified IgA, IgG-IgA Fc fusions or IgG at 25°C. Antibodies were diluted at 10 μg / ml in 10 mM sodium acetate buffer pH 4.5 and directly immobilized on an SPR sensing prism CMD200M chip (XanTec Bioanalytics, Germany) using amine coupling using a continuous flow microspotter. Antigens diluted in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20, 1 mM EDTA) were injected at different concentrations for 3 minutes and allowed to dissociate for 10 minutes, with regeneration using 10 mM glycine pH 2.5 between cycles. Antigens were from R&D Systems (mIL-13, 413-ML-025 / CF; mpIgR, 2800-PG-050; hpIgR, 2717-PG-050; hFcαRI, 3939-FA-050), SinoBiologicals (hHER2, 10004-H08H) or Genentech, co-expressed with species-specific βeta-2 microglobulin (m / hFcRn). Data were processed using the Wasatch kinetics software tool.

[0359] Antibody kinetics measured by Biacore. The binding kinetics of anti-IL-13 or anti-HER2 IgA2m2 antibodies were measured using surface plasmon resonance on a Biacore T200 instrument (GE Healthcare). All kinetic experiments were performed at a flow rate of 30 μL / min at 25 ° C, with a running buffer of 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween 20, and 1 mM EDTA. Fab binders from human Fab capture kits (GE Healthcare) were immobilized on CM5 sensor chips by amine-based coupling. IgA antibodies at a concentration of 50-100 ug / mL were captured at a speed of 5 uL / min for 210 seconds. Recombinant human FcαRI antigen (R&D Systems, 3939-FA-050) bound to antibodies was measured using concentrations of 1000 nM, 333 nM, and 111 nM. Sensorgrams for binding were recorded using a 90 s injection time followed by a 120 s dissociation time and surface regeneration with two 60 s injections of glycine pH 2.1. A 1:1 Langmuir binding model was used to calculate kinetics and binding constants.

[0360] result:

[0361] Factors affecting the formation of IgA oligomers. The recombinant production of monomeric IgA is well known and can be achieved by co-expression of light chains (LC) and heavy chains (HC), similar to the production of IgG. In contrast, the assembly of polymeric IgA requires the co-expression of LC, HC, and joining chain (JC), and the resulting oligomeric state of IgA is less well characterized. To better understand the assembly process of IgA oligomers, the expression of various human IgA isotypes and allotypes was characterized, including IgA1, IgA2m1, IgA2m1.P221R (disulfide-stabilized LC-HC pairing), and IgA2m2 ( Figure 1A -B (A). The murine variable domains of an anti-mouse interleukin 13 (mIL-13) antibody were cloned as chimeras with human κ LC and IgA HC constant domains. The chimeric LC and HC were then co-expressed in the presence and absence of human JC ( Figure 1A -B (panel B). After affinity purification with protein L, IgA prepared in 30 mL Expi293T transient expression without co-transfected JC yielded relatively pure monomers. In these experiments, cells were transfected with equal amounts of LC and HC DNA. In contrast, transfection of equal mass amounts of LC, HC, and JC DNA produced a variety of oligomeric species, corresponding to IgA monomers, dimers, and aggregates containing three to five IgA monomers ( Figure 1D 2A-2C). It was found that IgA1, IgA2m1 and IgA2m1.P221R produced mainly dimeric IgA (Fig. 2A-2B), while IgA2m2 produced approximately equal amounts of dimers and aggregates (Fig. 2C). Similar oligomer distributions were observed in CHO transient expression scaled up to the "up" level.

[0362] Separation of IgA dimers from aggregates by secondary purification proved challenging on a larger scale. To bias the assembly toward dimer formation, the amount of JC DNA was increased relative to the amount of LC and HC DNA to promote enhanced JC expression levels. This resulted in an increase in the relative percentage of dimer species and a decrease in the relative percentage of aggregate species (Figure 2A-2C; see also Figure 15A-Figure 15C , Figure 16A-16B , Figure 17A-17B and Figure 20A-20B In contrast, decreasing the amount of JC DNA relative to the amount of LC and HC DNA resulted in an increase in the percentage of higher order aggregates (trimers / tetramers / pentamers) ( Figure 18A-18E and Figure 21-23 For the IgA2m2 species, the ability to influence oligomeric species based on the amount of JC DNA was most pronounced. In addition, co-transfection of the secretory component, connecting chain, light chain, and heavy chain produced higher order oligomers compared to co-transfection of the connecting chain, light chain, and heavy chain without the secretory component ( Fig.29 ).

[0363] To understand why IgA2m2 tends to form larger oligomers than IgA1 or IgA2m1, the amino acid sequences of the HC tails of different isotypes / allotypes were compared. Although the sequences of the IgA1 and IgA2m1 tails are identical, IgA2m2 differs in two residues. Residues 458 and 467 are both valine in IgA1 and IgA2m1, while IgA2m2 has isoleucine and alanine, respectively, at these positions ( Figure 1A -B, panel A, asterisks). We therefore investigated whether these two amino acid differences could explain the unique propensity of recombinant IgA2m2 to form larger oligomers. Indeed, when valine at position 458 in IgA1 or IgA2m1 was replaced by isoleucine, more polymers were produced, independent of whether alanine or valine was present at residue 467 (Fig. 2D). In contrast, when position 458 in IgA2m2 was changed from isoleucine to valine, the content of polymer species decreased in favor of an increase in the content of dimers.

[0364] Mutations of certain cysteine ​​residues in the heavy chain of the IgA2m2 antibody were generated to prevent disulfide bond formation with the secretory component or the linker chain, and analyses were performed to determine the effect of such mutations on oligomer formation. Mutation of Cys311 to serine prevented disulfide bonding with the secretory component, while mutation of Cys471 to serine prevented disulfide bonding with the linker chain. Fig.28B As shown, when a linker chain was added to the light and heavy chains, the formation of IgA2m2 dimers and higher-order oligomers required mutation of C471 but not C311.

[0365] Glycosylation is known to play a role in IgA oligomerization (Chuang et al., J Immunol 158:724–32 (1997)). Therefore, mutations were made to remove each N-linked glycosylation site in IgA1 and IgA2m2. Four separate mutations (N459A / G / Q or S461A) that removed N-linked glycosylation sites from the tail of IgA1 or IgA2m2 also increased the amount of polymers produced, whereas mutations that removed glycosylation sites outside the tail did not alter oligomer formation (see Figures 2E and 2F, respectively; see also Fig.38 ). Therefore, in addition to adjusting the DNA ratio in transfection, the formation of IgA polymers can be increased by adding isoleucine at the amino residue 458 of the tail or by preventing N-linked glycosylation of the IgA tail segment.

[0366] Large-scale purification and biophysical characterization of IgA monomers and oligomers.Using the insights gained from small-scale expression into IgA oligomer formation, monomeric, dimeric, and tetrameric IgA were scaled up using CHO transient expression. Monomers and dimers of IgA1, IgA2m1, IgA2m1.P221R, and IgA2m2 were isolated, as well as tetramers of IgA2m2 ( Figures 3A-3D Non-reducing SDS-PAGE analysis of these samples showed major molecular weight bands consistent with the expected masses of ∼150 kDa, ∼310 kDa, and ∼610 kDa for IgA monomer, dimer, and tetramer, respectively ( Figures 3A-3D (B). These expected masses are based on the amino acid sequence without glycosylation and assume the incorporation of one JC per oligomer. The molar masses of the purified oligomeric species were also measured by SEC-MALS and found to be consistent with the expected masses for dimeric and tetrameric IgA (Table 2). Reducing SDS-PAGE analysis of the purified IgA samples confirmed the presence of monomeric LC and HC bands at ∼25 kDa and ∼55 kDa, respectively, while a band for JC could also be detected in the oligomeric samples just below 25 kDa ( Figures 3A-3D The identities of LC, HC and JC were additionally confirmed by mass spectrometry after reduction and enzymatic deglycosylation ( Fig.18E Negative stain electron microscopy (EM) was also used to further verify the oligomeric state of the isolated species. IgA2m2 dimer ( Figures 3A-3D Panel C) and tetramer ( Figures 3A-3D Negative stain images of (D) and (E) confirm the presence of two or mainly four IgA molecules, respectively. In the dimer, two IgA molecules are linked tail-to-tail by their Fc domains into an elongated particle, while in the tetramer, the interaction between the four Fc domains produces a compact complex of four IgA molecules. The original images of both samples show the presence of well-behaved monodisperse particles ( Figures 8A-8B ).

[0367] Table 2. Molar mass of recombinant IgA measured by SEC-MALS

[0368]

[0369]

[0370] Injection of protein into Waters Xbridge Protein BEH Molar mass and polydispersity were measured on an analytical size exclusion chromatography (SEC) column coupled to a Wyatt DAWN HELEOS II / Optilab T-rEX multi-angle light scattering (MALS) detector. The predicted molecular weight (MW) of IgA molecules is based solely on the amino acid composition, assuming that one JC is incorporated per dimer or tetramer, and does not take into account any potential N-linked or O-linked glycans.

[0371] Anti-mIL-13 IgA monomers, dimers, and tetramers bound murine IL-13 with similar affinity as anti-mIL-13 IgG1 (Tables 3 and Fig.19 ), indicating that the Fab region is correctly folded and functional in recombinant IgA. As expected, mouse and human pIgR binding was only seen in IgA oligomers, while both monomeric and oligomeric anti-mIL-13 IgA bound human FcαRI with similar affinity (Table 3). In addition, IgA2m2 purified from transient expression in CHO cells or Expi293 cells showed similar binding to mouse and human pIgR ( Fig.36A Due to the pIgR binding capacity, all IgA oligomers (but not monomers) were able to undergo transcytosis in vitro using an MDCK cell line ectopically expressing human pIgR ( Figures 4A-4B In addition, IgA monomers and oligomers showed higher stability than anti-mIL-13 human IgG1 and similar or higher stability than IgG1 Fab fragments, as measured by differential scanning fluorimetry (DSF). Figures 4A-4B Figure B).

[0372] Table 3. Binding affinities of anti-mouse IL-13 IgA and IgG molecules to antigen and receptor.

[0373]

[0374]

[0375] NB: No binding. All experiments were performed at least n=3 times.

[0376] Pharmacokinetic profile and biodistribution of recombinant IgA.The serum concentration-time curves of the disclosed recombinant IgA oligomers were analyzed and those data comparable to the data reported previously with recombinant monomers were identified (Figure 5A and Table 4) (Boross et al. (2013), Rouwendal et al. (2016) and Lohse et al., Br J Haematol 112:4170 (2017)). After single administration of the recombinant IgA oligomers, a very rapid serum clearance rate (>200 mL / day / kg) was observed. Serum-purified human IgA monomers exhibited a slower overall clearance rate and the serum PK curves were generally consistent with those of highly sialylated IgA monomers reported previously (Rouwendal et al. (2016)). In addition to characterizing the serum concentration-time curves, radiolabeled biodistribution studies were also performed on mice with dual I-125 and In-111 labeled antibodies. The dual-tracer method provided the ability to distinguish intact antibodies (I-125) before lysosomal degradation and internalized / catabolized antibodies (In-111 minus I-125) (Figure 5B and Figure 5C )(Boswell et al., Bioconjugate Chem 21:2153–63 (2010), Mandikian et al., Mol Cancer Ther 17:776–85 (2018) and Rajan et al., MAbs 9:1379–88 (2017)). Briefly, iodine diffused rapidly out of the cells and was cleared after the iodinated antibodies underwent lysosomal degradation, while the In-111-labeled antibodies showed intercellular accumulation of In-111-adducts after lysosomal degradation. Since the clearance rate of IgA antibodies is so rapid compared to IgG1, it is difficult to directly compare the tissue distribution data because the raw tissue values represent interstitial and vascular concentrations. Therefore, the intact antibody distribution data were blood-corrected as previously described to represent only the interstitial concentration in the tissues (Boswell et al., Mol Pharmaceutics 11:1591–8 (2014)). Compared to IgG1, a slightly higher enrichment of intact IgA oligomers was observed in the liver, stomach, small intestine, large intestine, and skin 1 hour later (all pIgR-expressing tissues (Asano et al., Scand J Immunol 60:267–72 (2004) and Wang et al., Scand J Immunol 83:235–43 (2016)), although at lower levels (Figure 5B). High levels of IgA degradation were also found in the antibody forms studied 1 hour after administration in the liver and small intestine ( Figure 5C ). To account for the differences in total blood concentrations observed between the antibody forms, the ratio of individual tissue to plasma concentration was also described ( Fig. 9After 1 day, almost no intact IgA antibodies remain in the tissue ( Fig.10 ) and the greatest catabolism was detected in the liver ( Fig.11 ), although catabolism in the small intestine may not be detected because In-111 is not well accumulated in enterocytes at later time points (Boswell et al., British Journal of Pharmacology 168:445–57 (2013)). Without being bound by a particular theory, it is hypothesized that reduced degradation and final clearance mechanisms of IgA compared to IgG can further enhance the absorption of IgA molecules by mucosal tissues.

[0377] It has been reported that the sialylation content on the N-linked glycans of monomeric IgA molecules is negatively correlated with the antibody clearance rate through specific glycan receptors (Rouwendal et al. (2016)). Therefore, the published IgA molecules were analyzed to determine their overall sialylation content. Glycans were classified according to the level of processing, with complexes and sialylation being the most required for IgA molecules (Figure 6A). Recombinantly produced IgA1, IgA2m1, IgA2m1.P221R, dimers of IgA2m2, and IgA2m2 monomers and tetramers were approximately 20-50% sialylated (Figure 6B, Fig.25 and Fig.26 and Table 5). This suggests that IgA molecules contain incompletely processed glycans that can be recognized by glycan receptors. In addition, the sialylation content of each site on the IgA2m1 dimer was examined and it was found that all sites, including the site on JC, contained incompletely processed glycans, indicating that incomplete glycan processing does not occur only at one specific site ( Figure 6C and Table 6). In contrast to published recombinant IgA molecules, IgA purified from human serum had 95% sialylation content (Figure 6B and Table 5) and was monomeric as determined by SEC-MALS. Since serum IgA is known to be primarily monomeric (Kerr (1990)), it is likely enriched in highly sialylated molecules, as sialylation content positively correlates with systemic exposure of antibodies. Without being bound by a particular theory, it was thought that this increased level of sialylation in human purified IgA monomers compared to recombinant IgA monomers would correlate with reduced serum clearance of this molecule in mice. In fact, this proved to be true, suggesting that binding to specific glycan receptors in the liver may be an important clearance mechanism for IgA monomers (Figure 5A, Fig.25 and Fig. 27 ).

[0378] Table 4. Estimated pharmacokinetic parameters following IV bolus injection of 5 mg / kg IgA monomer / oligomers in Balb / C mice (mean values)

[0379]

[0380]

[0381] AUC 最终 = area under the concentration-time curve, final measurable concentration; CL = clearance; observed C max = maximum concentration; IV = injection; # Balb / c mice were given 10 mg / kg intravenous bolus. Note: Since sparse PK analysis was performed on all mouse PK data, the data of individual mice in each group were pooled and SD is not reported.

[0382] Generation of IgA variants with reduced pIgR binding. IgA2m2 variants were generated to determine the effects of mutations on pIgR binding. IgA2m2 variants were generated with amino acids Y411, V413 and T414 mutated to alanine (referred to herein as "411-414AAA"), P440R mutation, C311S mutation, or a combination thereof. Fig. 30A The expression levels of such variants are provided in . Fig. 30B SEC characterization of small scale purified anti-IL-13 IgA2m2 variants is provided. Figure 30C-Figure 30D As shown, IgA2m2 variants with Y411, V413 and T414 amino acid mutations did not bind to mouse pIgR or human pIgR, while the P440R variant resulted in a 10-fold decrease in affinity for mouse pIgR and a significant loss of binding ability to human pIgR. In addition, IgA2m2 variants with amino acid 411, 413 and 414 mutations also did not bind to FcαRI ( Fig.30E ).

[0383] Table 5. Global N-linked glycan analysis of monomeric and polymeric IgA molecules.

[0384]

[0385]

[0386]

[0387] Cell culture conditions were modified to increase the sialylation content of IgA antibodies. The culture conditions of cells expressing IgA antibodies were modified to increase the sialylation content of the antibodies. The cell culture conditions tested were Fig.31A As provided in Fig.31B As shown, in the 7-day harvest, sialylation of IgA2m2 antibody was enhanced after addition of sialyltransferase (ST) and galactosyltransferase (GT) in the presence of galactose and N-acetylmannosamine (ManNac).

[0388] Table 6. Site-specific N-linked glycan analysis of IgA2m1 dimers.

[0389]

[0390]

[0391]

[0392] Improving the pharmacokinetic profile of recombinant IgA by glycosylation site and FcRn engineering. Two parallel approaches were taken to reduce the clearance of recombinant polymeric IgA in mice. First, all five N-linked glycosylation motifs in IgA2m2 and a single site in JC were removed by mutagenesis to generate a molecule without glycans (aglycosylation) (Figure 7A and Fig.25 ). Aglycosylated IgA polymers are not recognized by glycan receptors and allow for the study of pharmacokinetics independent of glycan receptor-mediated clearance mechanisms. Figure 36B-Figure 36D As shown, each IgA2m2 glycosylation variant has similar binding to mouse pIgR and human pIgR. To remove the N-linked glycosylation motif NXS / T in IgA2m2, there are three cases, N is mutated to A / G / Q or S / T is mutated to A or the motif is restored to the aglycosylated IgA1 sequence ( Figure 1A -B, panel A). JC residue N49 was mutated to A / G / Q or S51 was mutated to A. Individual IgA2m2 mutations N166A, S212P, N263Q, N337T.I338L.T339S and N459Q were found, and the N49QJC mutation gave the highest transient expression level, however, the combination of mutations that removed all five glycosylation sites in IgA2m2 resulted in poor expression, while further addition of the J chain N49Q mutation completely abolished it. See also Fig.24A -C. Chimeric immunoglobulins are generally expressed at lower levels in mammalian cells compared to immunoglobulins from a single species. Therefore, the murine anti-mIL13 variable domain was switched to humanized anti-HER2 to generate humanized IgA, but this did not improve transient expression levels. Therefore, a CHO targeted integration (TI) stable cell line was generated to increase the expression level of aglycosylated human anti-HER2 IgA2m2 polymer, which was purified as a mixture of oligomeric species.

[0393] Next, two IgG1-IgA Fc fusions were designed to exploit FcRn binding as a means of reducing lysosomal degradation (Figure 7B). Previous studies have shown that this approach rescues IgA monomer serum clearance to levels comparable to IgG1 (Li et al. (2017) and Borrok et al. (2015)). Initially, dimeric and tetrameric versions of the previously reported IgG1-IgA2m1P221R Fc fusion (Borrok et al. (2015)) were prepared, but these were observed to exhibit instability in mouse plasma at 4 days (Figure 7C). The major truncated product eluted at a similar time in analytical SEC as the full-length anti-HER2 IgG1 (trastuzumab), suggesting that this instability was caused by endoprotease cleavage at the IgG1-IgA2m1 P221R Fc junction. The amino acid sequence of the junction was examined, and a stretch of positively charged residues similar to a furin-like cleavage site was identified ( Fig.12 To mitigate proteolytic cleavage, these positively charged residues at the junction were eliminated by removing the C-terminal K447 of the IgG1 heavy chain and starting IgA2m1Fc with P221-native IgA2m1 residues (rather than the P221R mutation) or starting IgA2m1 Fc with C242 (which deletes the IgA2m1 hinge) ( Fig.12 and Fig.34A The C242 Fc start site was also included because it is the first residue in the IgA1 Fc crystal structure construct and is therefore assumed to be a stable truncated protein (Herr et al., Nature 423:614–20 (2003)). When the re-engineered IgG1ΔK-P221IgA2m1 Fc and IgG1ΔK-C242 IgA2m1 Fc fusions were produced as dimers, both were found to be stable in mouse plasma for up to 4 days (Figure 7C). Fig.34B Transient expression data of full-length anti-IL-13 IgG1-IgA Fc fusions are provided. Some engineered fusion molecules showed improved expression compared to IgG1 and the original construct ( Fig.34B and Fig.37A ). In addition, if Fig.33A As shown, increasing the amount of JC DNA compared to the amounts of LC and HC DNA resulted in the production of more dimer species than higher order oligomer species.

[0394] IgG1-IgA1 fusions were also generated by fusing IgG1 at the lower hinge residues E233 or L234 to the Fc of IgA1 at C241 or C242. Fig.37BAs shown, the IgG1-IgA1 fusion was expressed mainly as a dimer, similar to IgA1. In addition, the IgG1-IgA1 fusion bound to human and mouse pIgR and human FcαRI in a manner similar to IgA1 ( Fig.37C ).

[0395] The stability of the engineered IgA antibodies and IgG1-IgA fusion molecules was analyzed by differential scanning fluorimetry (DSF), confirming that there was no loss in stability compared to the IgA1 dimer ( Fig.32 ).

[0396] The engineered IgA antibodies and IgG1-IgA fusion molecules were further characterized for global glycan content, antigen binding, and receptor binding. The aglycosylated anti-HER2 IgA2m2 polymers were indeed devoid of glycosylation, whereas the anti-mIL-13 IgG1ΔK-P221IgA2m1 Fc and IgG1ΔK-C242 IgA2m1 Fc fusions contained only ~20% of the complexed sialylated glycans ( Fig.13 and Table 8). It was found that the aglycosylated anti-HER2 IgA2m2 polymer had similar binding affinity to human (h)HER2, murine (m)pIgR, and hpIgR as the glycosylated IgA2m2 tetramer, but it did not bind to the IgA-specific hFc receptor hFcαRI as determined by the Wasatch SPR assay (Table 7; see also Figure 33B-Figure 33C Interestingly, IgA2m2 tetramers lacking glycosylation on the IgA2m2 HC but retaining glycosylation on the J chain also failed to bind hFcαRI ( Figure 35A-Figure 35B ), as determined by the Wasatch SPR assay, indicating that glycosylation of IgA HCs is required for receptor binding. However, as shown using the Biacore SPR system (a commonly used SPR system in the pharmaceutical industry), the glycosylation state of IgA polymers did not affect binding to hFcαRI ( Figure 52A-Figure 52B ).like Figure 52A-Figure 52B As shown, the aglycosylated anti-HER2 IgA2m2 polymer (referred to as "xHER2 4D5.IgA2m2 tetramer N168A.S214P.N252Q.N326T.I327L.T328S.N461Q,J-N71Q") and the partially aglycosylated anti-IL-13 IgA2m2 oligomer retained hFcαRI binding as determined by Biacore SPR assay. Without being bound by a particular theory, the difference in results obtained from the two SPR systems (i.e., Wasatch and Biacore systems) may be due in part to the different strategies used to immobilize antibodies to the chip used in the SPR system disclosed in the present method.

[0397] Furthermore, anti-mIL-13IgG1ΔK-P221 IgA2m1 Fc and IgG1K-C242 IgA2m1 Fc dimers had similar binding affinities to mIL-13, mFcRn, and hFcRn as anti-mIL-13IgG1 (Table 7), and similar binding affinities to mpIgR, hpIgR, and hFcαRI as IgA2m1 dimers (Tables 3 and 7), as determined by Wasatch SPR assays. Thus, the IgG1-Ig2m1A Fc fusion retains the desired properties of IgG and polymeric IgA.

[0398] Table 7. Binding affinities of IgG1-IgA2m1 Fc fusion dimers and non-glycosylated IgA2m2 tetramers to antigen and receptor using the Wasatch binding assay.

[0399]

[0400]

[0401] NB: No combination. *K D All experiments were performed at least 3 times.

[0402] Table 8. Global N-linked glycan analysis of IgG1-IgA2m1 Fc fusion oligomers and aglycosylated IgA2m2 tetramers.

[0403]

[0404]

[0405] In vitro pIgR-mediated transcytosis and serum concentration-time profiles of both newly generated antibody forms were measured in mice. The IgG1-IgA2m1 Fc fusion showed the most significant improvement in overall IgA serum exposure in mice (Figure 7D, Fig.34C and Table 9 ), but had the lowest level of in vitro transcytosis compared with aglycosylated IgA2m2 polymers, which showed the highest level of in vitro transcytosis ( FIG. 7E ).

[0406] Table 9. Estimated pharmacokinetic parameters (mean values) following IV bolus injection of 30 mg / kg IgA oligomers to Balb / C mice.

[0407]

[0408] AUC 最终 = area under the concentration-time curve, final measurable concentration; CL = clearance; observed C max= maximum concentration; IV = intravenous injection. Note: Since sparse PK analysis was performed on all mouse PK data, data from individual mice in each group were pooled and SD is not reported.

[0409] discuss:

[0410] IgA has the potential to expand the therapeutic scope of monoclonal antibodies beyond the current capabilities provided by IgG. This is due in part to the versatility of IgA to form both monomeric and polymeric species. Significant progress has been made in the recombinant preparation of monomeric IgA over the past few years (Leusen (2015), Dicker et al., Bioengineered (2016), Vasilev et al., Biotechnol Adv (2015), and Virdi et al., Cell Mol Life Sci (2015)), providing a robust route to isolate well-characterized material with increased sialylation content of N-linked glycans, resulting in improved serum clearance (Rouwendal et al. (2016)). While the potent cytotoxic properties of monomeric IgA are an attractive feature for oncology indications, polymeric IgA requires pIgR-mediated transcytosis to reach targets beyond the epithelial barrier. Prior to the work described herein, only recombinantly prepared mixtures of monomers and oligomers were used for in vivo experiments studying transcytosis (Olsan et al. (2015) and Rifai et al. (2000)). The experiments described in this example established a robust expression and purification pathway that allowed enrichment of dimeric and tetrameric IgA. In particular, modulation of the amount of JC DNA used for transfection or the glycosylation state of the IgA tail region can affect the distribution of oligomeric species. Interestingly, the N-linked glycosylation site of the IgA tail is the only site that is extremely conserved between species ( Fig.14 ), providing a method to control the formation of higher-order IgA oligomers in vivo. For the purification of recombinant IgA, dimers were easily separated from tetramers using protein L affinity chromatography followed by HPLC-SEC.

[0411] It has been previously demonstrated that enhancing the sialylation level of recombinantly expressed IgA monomers reduces serum clearance by overcoming glycan receptor-mediated catabolism (Rouwendal et al. (2016)). As an alternative strategy, the contribution of glycans to oligomeric IgA serum clearance was eliminated and completely aglycosylated IgA2m2 polymers were generated. The lack of N-linked glycans did not affect binding to pIgR as assessed by surface plasmon resonance. Surprisingly, in an in vitro MDCK transcytosis assay, aglycosylated species were observed to significantly improve transcytosis compared to glycosylated tetramers or dimers. This improvement was not observed in previous studies of human IgA1 dimers, which had N-linked glycans removed from the antibody Fc region but still had carbohydrates present on the murine J chain (Chuang et al., (1997)). Without being bound by a particular theory, one explanation is that the lack of glycans eliminates binding to glycan receptors, thereby providing undisturbed and more efficient transcytosis via pIgR binding. Interestingly, binding of tetrameric IgA2m2 to FcαRI was dependent on glycosylation, in contrast to the lack of effect observed on binding of monomeric IgA2m1 engineered to contain a reduced number of glycosylation sites. Although the cytotoxic effects of polymeric IgA were not analyzed, IgA therapeutics that can transcytose without activating FcαRI would be desirable for inflammatory diseases as this would prevent the proinflammatory response caused by neutrophil migration (Aleyd et al., Immunol Rev 268:123–38 (2015)).

[0412] In the experiments disclosed herein, recombinantly produced glycosylated IgA oligomers and monomers were rapidly cleared from serum, similar to what has been observed previously for monomeric and polymeric IgA (Rouwendal et al. (2016) and Chuang et al., (1997)). The rapid clearance was attributed to potential binding to glycan receptors and subsequent degradation in lysosomes. Biodistribution studies of glycosylated IgA support this conclusion, indicating that catabolism is greatest in the liver and small intestine. To better understand the role of having glycans, IgA polymers without glycans (aglycosylation) were generated and their in vivo PK profiles were directly compared to the glycosylated versions. There was no significant difference (<2-fold) in overall mouse serum exposure for aglycosylated IgA2m2 polymers compared to glycosylated polymers. This suggests that N-linked glycans play a minimal role in the clearance of mouse IgA polymers. Although further studies are needed to understand why aglycosylated IgA oligomers fail to enhance serum clearance, it appears that pIgR-mediated transcytosis and / or clearance may play an important role in determining the overall serum concentration and fate of polymeric IgA in mice. Indeed, the equilibrium binding affinity of tetrameric IgA for pIgR is at least in the picomolar range, allowing efficient binding to the abundant pIgR receptors followed by transcytosis. However, detailed biodistribution studies with tissue distribution profiles are needed to better interpret the serum concentration-time profiles of the molecules and the distribution of aglycosylated polymeric IgA. An important caveat in studying the pharmacokinetic properties and biodistribution of polymeric IgA molecules in rodents is that the expression pattern of pIgR differs between rodents and humans, which may confound the ultimate clinical interpretation. In particular, high expression of pIgR in rodent and rabbit hepatocytes has been implicated in the biliary clearance of polymerized IgA (Daniels et al. (1989)), which is not thought to occur in humans, where pIgR expression is instead found in cholangiocytes (Tomana et al. (1988)). Therefore, the exact role of pIgR in the biodistribution and clearance of polymerized IgA molecules in mice needs to be evaluated separately.

[0413] An alternative strategy to avoid accelerated serum clearance and improve exposure has been through engineering of an IgG1-IgA2m1Fc fusion. The ability to bind FcRn allows recycling in endosomes, thereby avoiding sorting for lysosomal degradation. Previous reports on monomeric IgG-IgA Fc fusions reported serum clearance comparable to that of IgG (Li et al. Oncotarget (2017) and Borrok et al. (2015)). In addition, improved pharmacokinetics were observed for monomeric IgA fused to an albumin-binding peptide (Meyer et al., MAbs 8:87–98 (2016)). Since no large effect was observed with removal of glycans from IgA polymers, glycosylated fusion proteins were prepared. Although the dimeric IgG1-IgA2 Fc fusion showed improved overall serum exposure, it was not comparable to that of IgG, as was observed for the monomeric IgG-IgA Fc fusion (Li et al. Oncotarget (2017) and Borrok et al. (2015)). By surface plasmon resonance, we demonstrated that the dimeric IgG1-IgA2m1 Fc fusion can bind to both FcRn and pIgR, although transcytosis in vitro was attenuated in the MDCK model. Although binding to FcRn prolongs the terminal half-life, the interaction of the IgG1-IgA2m1 Fc dimer with pIgR may provide a clearance mechanism, especially in the early stages, which leads to pIgR-mediated transcytosis and / or clearance, resulting in reduced serum concentrations compared with IgG.

[0414] IgA in serum is mainly composed of IgA1 monomers secreted by bone marrow cells, while polymeric IgA2 is secreted by plasma cells in the lamina propria, the site of transcytosis (Yoo et al. 116: 3-10 (2005)). The high affinity between polymeric IgA and pIgR naturally leads to rapid clearance of polymeric IgA from the circulation, provided that harmful antigens in the circulation as IgA-antigen complexes are effectively cleared (Shroff et al., Infect Immun 63: 3904–13 (1995)) and can be used in therapeutic settings to limit drug activity to limited tissues and for a short period of time, which may be particularly beneficial when stimulating cytokine receptors. The approximately 20-fold increase in IgA concentration in the serum of pIgR-deficient NOD mice further supports the rapid clearance of polymeric IgA from mouse serum through pIgR (Simpfendorfer et al., PLoS ONE 10: e0121979 (2015)). Example 2: Purification of recombinant IgA antibodies

[0415] Recombinant IgA containing kappa light chain is expressed as secretory protein in CHO cells and affinity captured from cell culture supernatant using Capto L (GE Healthcare) column. After capture, the column is washed with 5 column volumes (CV) of Tris buffer (25mM Tris, pH 7.5, 150mM NaCl, 5mM EDTA, 2mM NaN3), 20CV of Triton X-114 buffer (25mMTris, pH7.5, 150mM NaCl, 5mM EDTA, 0.1% Triton X-114, 2mM NaN3) (to remove endotoxin), 5CV of Tris buffer, 5CV of KP buffer (0.4M potassium phosphate, pH7.0, 5mM EDTA, 0.02% Tween20, 2mM NaN3) and 10CV of Tris buffer. IgA was eluted with 150 mM acetic acid (pH 2.7) and immediately neutralized with 1 / 5 volume of 1 M arginine, 0.4 M succinate (pH 9.0).

[0416] After affinity purification, recombinant IgA was purified using size exclusion chromatography (SEC). For recombinant IgA samples that were primarily present in one oligomeric state (≥90% of a single type of oligomer), SEC was performed using a HiLoad Superdex 200 pg column (GE Healthcare) followed by peak clipping to avoid contaminants of the deleterious oligomeric state. For IgA samples containing a complex mixture of nearly equal amounts of oligomers (e.g., ~40% dimers and ~60% higher order aggregates), several purification methods were tested. Fig.39 The human anti-mIL-13 IgA2m2 oligomer mixture shown was used to test different types of purification as described below.

[0417] SEC using a HiLoad Superose 6 16 / 600 pg column (GE Healthcare) was not able to adequately separate IgA dimers from higher order oligomers in the higher molecular weight range, e.g. Fig.40As shown. On the Superose 6 elution profile, Peak 1 elutes close to ~35-39 mL, which corresponds to the void volume of the column and is therefore likely an aggregated protein. Peaks 2 and 3 overlap significantly, so Peak 3 appears as a shoulder on the trailing edge of Peak 2. As described above, analysis of fractions near the leading edge of Peak 2 and the trailing edge of Peak 3 by SEC-MALS yielded molar masses of 735,000 g / mol and 375,300 g / mol, respectively. The expected molecular weights of human anti-mIL-13IgA2m2 monomers are ~148 kDa, dimers ~312 kDa, trimers ~460 kDa, tetramers ~608 kDa, and pentamers ~756 kDa. This suggests that Peaks 2 and 3 may contain a mixture of pentamers, tetramers, trimers, and dimers. Peak 4 eluted later at around ~60 mL and may correspond to monomeric IgA.

[0418] SEC using a HiLoad Superdex 200 pg column (GE Healthcare) or a HiLoad Sephacryl 400 pg column (GE Healthcare) also failed to adequately separate IgA dimers from higher order oligomers. Attempts to separate the oligomers by cation exchange chromatography using an SPHP column (GE Healthcare), anion exchange chromatography using a Q FF column (GE Healthcare), and hydrophobic interaction chromatography (HIC) using a 5 μm, 7.8 x 75 mm ProPac HIC-10 column (Dionex) were also unsuccessful.

[0419] In contrast, for human anti-mIL-13IgA2m2, a 3.5 μm, 7.8 mm x 300 mm XBridgeProtein BEH Small-scale purification using SEC columns (Waters) allows for optimal separation of IgA dimers from higher order oligomers, such as Fig.41 As shown. To maximize the separation, an injection volume of no more than 100 μL (wherein the total protein is less than 1 mg) was run at 1 mL / min using an Agilent 1260 Infinity HPLC with 0.2 M arginine, 0.137 M succinate, pH 5.0 as the mobile phase, and 200 μL fractions were collected. The fractions were then selectively pooled to separate primarily one oligomeric state. Multiple runs were performed, and the pooled fractions of a given oligomer from each run were combined.

[0420] The identity, purity and oligomeric state of IgA found in the pooled fractions were characterized by SEC-MALS using a 3.5 μm, 7.8 mm x 300 mm XBridge Protein BEH SEC column(Waters)( Figures 42A-42D ), SDS-PAGE is as follows ( Figures 42A-42D ), negative staining electron microscopy is as follows ( Figures 43A-43B and Figures 44A-44B ) and mass spectra are as follows ( Fig.45 ). Recombinant IgA was injected into a 3.5 μm, 7.8 mm x 300 mm Waters XBridge Protein BEH column at a flow rate of 1 mL / min using an Agilent 1260 Infinity HPLC with 0.2 M arginine, 0.137 M succinate, pH 5.0 as the mobile phase. SEC-MALS was performed on a size exclusion chromatography (SEC) column. Fig.39 Shown is an example analytical SEC profile of recombinant anti-mIL-13 IgA2m2 from Capto L affinity purification. Protein eluted from the analytical SEC column was directly injected into a Wyatt DAWN HELEOS II / Optilab T-rEX multi-angle light scattering (MALS) detector to measure the molar mass and polydispersity of the various IgA oligomeric states present in a given sample.

[0421] For anti-mIL-13IgA2m2 antibody, Waters XBridge Protein BEH Three main peaks were identified on analytical SEC: Figures 42A-42D Panel A). The expected molecular weight of human anti-mIL-13IgA2m2 monomer is ~148 kDa, dimer ~312 kDa, trimer ~460 kDa, tetramer ~608 kDa, and pentamer ~756 kDa. All expected molecular weights are based on amino acid sequence composition and do not take into account potential N-linked or O-linked glycans, as the sugar composition is often heterogeneous and variable. After separation on the SEC column, MALS determined the molar mass of Peak 1 to be 658,000 g / mol + / - 0.510% ( Figures 42A-42D This indicates that peak 1 is primarily tetrameric IgA2m2. The molar mass of peak 2 was determined by MALS to be 343,700 g / mol + / - 0.646% ( Figures 42A-42D This indicates that peak 2 is mainly dimeric IgA2m2. Peak 3, which elutes later than the dimer, is likely monomeric IgA ( Figures 42A-42D Figure A).

[0422] From Figures 42A-42DPanels BC SDS-PAGE analysis of unreduced purified peaks 1 and 2 showed major bands migrating around the expected molecular weights for IgA tetramers and dimers, respectively ( Figures 42A-42D This is consistent with the molar mass identified by MALS ( Figures 42A-42D After reduction with DTT, three bands were observed on the gel ( Figures 42A-42D Panel D). The expected molecular weights are 50.2 kDa for the heavy chain (HC), 23.8 kDa for the light chain (LC), and 15.6 kDa for the joining chain (JC). All expected molecular weights are based on the amino acid sequence composition and do not take into account potential N-linked glycans or O-linked glycans, as the sugar composition is often heterogeneous and variable. The three bands run approximately at the predicted molecular weights for all three chains, with HC and JC running slightly larger. HC has five predicted N-linked glycosylation sites, while JC has one predicted N-linked glycosylation site, which, if occupied, would increase the molecular weight and reduce migration on the gel. SDS-PAGE was performed by mixing recombinant IgA proteins with LDS sample buffer (ThermoFisher Scientific) with or without the addition of 10 mM dithiothreitol (DTT) and heating at 70°C for 10 min. Samples were then run on 4-12% Bolt Bis-Tris Plus gels (Thermo Fisher Scientific) in MES buffer (Thermo Fisher Scientific) and stained with ClearPAGE Instant Blue stain (Expedeon).

[0423] Negative staining electron microscopy (EM) was used to analyze the Figures 42A-42D Panels BC of human anti-mIL-13 IgA2m2 purification peaks 1 and 2. Purified IgA2m2 samples were first cross-linked by incubation in 0.015% glutaraldehyde (Polysciences, Inc.) at room temperature for 10 minutes. After fixation, samples were diluted using TBS buffer to reach a concentration of 10 ng / μL. 4 μL of each sample was then incubated on a freshly glow-discharged 400-mesh copper grid covered with a thin layer of continuous carbon before treatment with 2% (w / v) uranyl acetate negative stain (Electron Microscopy Sciences). IgA was then ionized using a Tecnai Spirit T12 (Thermo Fisher) at 120 keV at 25,000x The images were imaged at magnifications of 1000 x 1000. Images were recorded using a Gatan 4096 x 4096 pixel CCD camera under low-dose conditions. Approximately 5000 particles per IgA sample were then selected and extracted using the e2boxer.py software within the EMAN2 package, using a particle box size of 128 pixels. The reference-free 2D classifier within the RELION imaging software package was used to generate an average image for both samples. The raw image files and reference-free 2D classifiers for IgA from purified peaks 1 and 2 are shown ( Figures 43A-43B and Figures 44A-44B Peak 1 is mainly tetrameric IgA2m2, with some pentamers, trimers and dimers ( Figures 43A-43B Peak 2 is dimeric IgA2m2 ( Figures 44A-44B ).

[0424] Mass spectrometry confirmed the presence of JC, LC and HC within 5Da of the expected molecular weight, wherein the amino terminal residues of JC and HC form pyroglutamic acid. Mass spectrometry was performed by heating 0.5mg / mL of IgA at 97°C for 30 minutes in the presence of 5mM DTT to reduce and denature the protein. The sample was then cooled on ice and then deglycosylated overnight with 1,000 units of PNGaseF (NEB) at 37°C. Agilent 1290 Infinity UHPLC was then used to inject reduced, denatured and deglycosylated IgA into a 3 μm, 4.6x 50mm reverse phase chromatography PLRP-S column (Agilent) at a speed of 1mL / min. A 5%-60% buffer B gradient was performed in 6 minutes using an aqueous solution (buffer A) containing 0.05% trifluoroacetic acid (TFA) and an acetonitrile solution (buffer B) containing 0.05% TFA. The proteins eluted from the reverse phase column were directly injected into an Agilent 6230 electrospray ionization time-of-flight mass spectrometer (ESI-TOF) for intact mass measurement.

[0425] Apart from Fig.41 , Figures 42A-42D , Figures 43A-43B , Figures 44A-44B and Fig.45 In addition to the success with IgA2m2 described in , this separation technique is also applicable to all other isotypes and allotypes tested, including IgA1 ( Figures 46A-46C )、IgA2m1 wild type ( Figures 47A-47C ) and IgA2m1 containing the P221R mutation to restore the disulfide bond between the light and heavy chains ( Figures 48A-48C Example 3: In vitro analysis of recombinant IgA antibodies and IgG-IgA fusion molecules

[0426] The ability of IgA antibodies and IgG-IgA fusion molecules to trigger cancer cell death was analyzed in vitro in the HER2+ breast cancer cell lines KPL-4, BT474-M1, and SKBR3 using the CellTiter-Glo luminescent cell viability assay. The assay was repeated as follows. Peripheral blood was collected from healthy donors using EDTA as an anticoagulant. The EasySep TM Human neutrophils used as effector cells were isolated from peripheral blood using the Direct Human Neutrophil Isolation Kit (STEMCELL Technologies). Neutrophils and HER2-amplified target cells (SK-BR3; density of 10,000 cells per well) were incubated in a black, clear-bottom 96-well plate (Corning) at a ratio of 20:1 for 48 hours in the presence of the detection reagent. Target cell viability was measured by luminescent relative light units (RLU) using the Cell Titer-Glow luminescent cell viability reagent (Promega cat#G7570). Target cell killing activity was calculated as follows: ((RLU untreated – RLU treated) / RLU untreated) × 100%.

[0427] like Fig.49 As shown, SKBR3 and BT474-M1 cell lines are resistant to HER IgA2m1 monomer (in Fig.49 In particular, the anti-HERIgA2m1 monomer caused significant killing of SKBR3 cells compared to the effect on BT474-M1 cell viability. However, the KPL-4 cell line was insensitive to anti-IgA antibodies. To further analyze whether the ability of IgA antibodies to cause cancer cell death is specific to the donor of neutrophils, neutrophils from two different donors were used in the cell viability assay. Fig.50 As shown in Figure 2, neutrophils from two different donors were able to mediate the death of SKBR3 cells in the presence of monomeric anti-HER2 IgA antibodies and monomeric IgG-IgA fusion molecules, indicating that the efficacy of the antibodies and fusion molecules is not donor-specific. Compared with monomeric anti-HER2 IgA antibodies, polymeric anti-HER2 IgA antibodies resulted in less cell death ( Fig.50 ).

[0428] Additional experiments were performed to determine whether the glycosylation state of the antibody affects its ability to cause cancer cell death. Glycosylated monomeric and tetrameric anti-HER IgA antibodies resulted in significant killing of SKBR3 cells ( Fig.51). However, the aglycosylated tetrameric anti-HER IgA antibody did not cause cell death of the targeted SKBR3 cells. Without being bound by a particular theory, these results suggest that glycosylation can affect the effectiveness of IgA antibodies.

[0429] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. For purposes of illustration and description, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0430] It is obvious to those skilled in the art that various modifications and changes can be made to the composition and method of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Therefore, it is intended that the disclosed subject matter includes modifications and changes within the scope of the attached claims and their equivalents.

[0431] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entireties.

[0432] The present disclosure relates to the following embodiments:

[0433] 1. An isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises a substitution at amino acid V458, N459 and / or S461.

[0434] 2. An isolated IgA antibody according to claim 1, wherein amino acid V458 is substituted by isoleucine (V458I), amino acid N459 is substituted by glutamine (N459Q), glycine (N459G) or alanine (N459A), and / or amino acid S461 is substituted by alanine (S461A).

[0435] 3. An isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises a substitution at amino acid 1458.

[0436] 4. The isolated IgA antibody according to item 3, wherein amino acid I458 is substituted by valine (I458V).

[0437] 5. An isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises one or more substitutions at an amino acid selected from the group consisting of N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459, S461, and combinations thereof.

[0438] 6. An isolated IgA antibody according to item 5, wherein the substitutions at amino acids N166, S212, N263, N337, I338, T339 and N459 are N166A, S212P, N263Q, N337T, I338L, T339S and N459Q.

[0439] 7. The isolated IgA antibody according to items 1-6, wherein the IgA antibody is an IgA1, IgA2m1, IgA2m2 or IgA2mn antibody.

[0440] 8. An isolated IgA antibody according to item 5 or 6, wherein the IgA antibody has substitutions at amino acids N337, I338 and T339, and one or more substitutions at T168, N211, S212, S213, N263, T265, N459, S461 and combinations thereof.

[0441] 9. The isolated IgA antibody according to item 8, wherein the IgA antibody is an IgA2m1, IgA2m2 or IgA2mn antibody.

[0442] 10. The isolated IgA antibody according to any one of items 1 to 9, wherein the IgA antibody is a humanized antibody, a chimeric antibody or a human antibody.

[0443] 11. An isolated IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence including P221 or R221 to the C-terminus of the heavy chain of the IgA antibody, and wherein the IgG antibody further comprises a deletion of amino acid K447.

[0444] 12. An isolated IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence including C242 to the C-terminus of the heavy chain of the IgA antibody.

[0445] 13. An isolated IgG-IgA fusion molecule according to claim 12, wherein the IgG antibody further comprises a deletion of amino acid K447.

[0446] 14. The isolated IgG-IgA fusion molecule according to any one of items 11 to 13, wherein the IgG antibody is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody and an IgG4 antibody.

[0447] 15. An isolated IgG-IgA fusion molecule according to any one of items 11-14, wherein the IgG antibody is an IgG1 antibody.

[0448] 16. The isolated IgG-IgA fusion molecule according to any one of items 11 to 15, wherein the IgA antibody is selected from the group consisting of an IgA1 antibody, an IgA2m1 antibody, an IgA2m2 antibody and an IgA2mn antibody.

[0449] 17. The isolated IgG-IgA fusion molecule according to any one of items 11 to 16, wherein the IgA antibody is an IgA2m1 antibody.

[0450] 18. An isolated nucleic acid encoding the antibody according to any one of items 1 to 10 or the IgG-IgA fusion molecule according to any one of items 11 to 17.

[0451] 19. A host cell comprising the nucleic acid according to item 18.

[0452] 20. A method for preparing an IgA antibody or IgG-IgA, comprising culturing the host cell according to item 19 to prepare the IgA antibody or IgG-IgA fusion molecule.

[0453] 21. The method according to item 20, further comprising recovering the IgA antibody or IgG-IgA fusion molecule from the host cell.

[0454] 22. The IgA antibody or IgG-IgA fusion molecule prepared according to item 20 or recovered according to item 21.

[0455] 23. A pharmaceutical composition comprising one or more IgA antibodies according to any one of items 1 to 10 or item 22 or one or more IgG-IgA fusion molecules according to any one of items 11 to 17 or item 22 and a pharmaceutically acceptable carrier.

[0456] 24. A pharmaceutical composition according to item 23, further comprising an additional therapeutic agent.

[0457] 25. A method of treating an individual suffering from a disease, wherein the method comprises administering to the individual an effective amount of one or more IgA antibodies according to any one of items 1-10 or item 22 or one or more IgG-IgA fusion molecules according to any one of items 11-17 or item 22.

[0458] 26. The method of claim 25, wherein the disease is an inflammatory disease, an autoimmune disease or cancer.

[0459] 27. An IgA antibody according to any one of items 1 to 10 or item 22 or an IgG-IgA fusion molecule according to any one of items 11 to 17 or item 22 for use as a medicament.

[0460] 28. An IgA antibody according to any one of items 1 to 10 or 22, or an IgG-IgA fusion molecule according to any one of items 11 to 17 or 22, for use in treating a disease.

[0461] 29. The IgA antibody or IgG-IgA fusion molecule according to item 28, wherein the disease is an inflammatory disease, an autoimmune disease or cancer.

[0462] 30. Use of the IgA antibody according to any one of items 1 to 10 or item 22 or the IgG-IgA fusion molecule according to any one of items 11 to 17 or item 22 for preparing a medicament for treating a disease.

[0463] 31. The use according to item 30, wherein the disease is an inflammatory disease, an autoimmune disease or cancer.

[0464] 32. A method for enhancing expression of IgA dimers, comprising increasing the amount of DNA encoding a joining chain (JC) introduced into a first cell relative to the amount of DNA encoding a light chain (LC) and a heavy chain (HC), wherein the enhanced expression is relative to the amount of IgA dimers produced in a second cell into which equal amounts of JC, LC and HC DNA are introduced.

[0465] 33. A method according to claim 32, wherein the ratio of the amount of DNA encoding the HC to the amount of DNA encoding the LC to the amount of DNA encoding the JC (HC:LC:JC) introduced into the first cell is from about 1:1:2 to about 1:1:5.

[0466] 34. A method for enhancing expression of an IgA dimer, trimer or tetramer comprising reducing the amount of DNA encoding a joining chain (JC) introduced into a first cell relative to the amount of DNA encoding a light chain (LC) and a heavy chain (HC), wherein the enhanced expression is relative to the amount of IgA trimer or tetramer produced in a second cell into which a greater amount of HC and LC DNA relative to the amount of JC DNA is introduced.

[0467] 35. A method according to claim 34, wherein the ratio of the amount of DNA encoding the HC to the amount of DNA encoding the LC to the amount of DNA encoding the JC (HC:LC:JC) introduced into the first cell is from about 1:1:0.25 to about 1:1:0.5.

[0468] 36. A method for increasing production of IgA1 or IgA2m1 polymers, comprising expressing in a first cell an IgA1 or IgA2m1 antibody having a substitution at amino acid V458, wherein the increased production is relative to the amount of IgA1 or IgA2m1 polymers produced in a second cell, the second cell expressing the IgA1 or IgA2m1 antibody without a substitution at amino acid V458.

[0469] 37. A method according to claim 36, wherein the amino acid is substituted by isoleucine (V458I).

[0470] 38. A method for increasing the production of IgA2m2 dimers, comprising expressing an IgA2m2 antibody having a substitution at amino acid 1458 in a first cell, wherein the increased production is relative to the amount of IgA2m2 dimers produced in a second cell, the second cell expressing an IgA2m2 antibody without a substitution at amino acid 1458.

[0471] 39. A method according to claim 38, wherein the amino acid is substituted by valine (I458V).

[0472] 40. A method for increasing production of IgA1 or IgA2m1 polymers, comprising expressing in a first cell an IgA1 or IgA2m1 antibody having a substitution at amino acid N459 or S461, wherein the increased production is relative to the amount of IgA1 or IgA2m1 polymers produced in a second cell, the second cell expressing an IgA1 or IgA2m1 antibody without a substitution at amino acid N459 or S461.

[0473] 41. A method according to claim 40, wherein amino acid N459 is replaced by a N459Q, N459G or N459A mutation and / or amino acid S461 is replaced by a S461A mutation.

[0474] 42. A method for reducing the production of IgA2m2 polymers, comprising expressing in a first cell an IgA2m2 antibody having a substitution at amino acid C471, wherein the reduced production is relative to the amount of IgA2m2 polymers produced in a second cell, the second cell expressing the IgA2m2 antibody without a substitution at amino acid C471.

[0475] 43. A method according to claim 42, wherein amino acid C471 is replaced by a C471S mutation.

[0476] 44. A method of enhancing transient expression of an IgA2m2 antibody, comprising expressing an IgA2m2 antibody comprising a substitution at an amino acid selected from the group consisting of N166, S212, N263, N337, 1338, T339, N459, and combinations thereof in a first cell, wherein the enhanced transient expression is relative to the amount of transient expression produced in a second cell, the second cell expressing the IgA2m2 antibody without a substitution at an amino acid selected from the group consisting of N166, S212, N263, N337, 1338, T339, N459, and combinations thereof.

[0477] 45. A method for expressing a dimer of an IgG-IgA fusion molecule, comprising expressing an IgG-IgA fusion molecule, wherein the IgG-IgA fusion molecule comprises a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence including P221 or R221 to the C-terminus of the heavy chain of the IgA antibody, and wherein the IgG antibody comprises a deletion of amino acid K447.

[0478] 46. ​​A method for expressing a dimer, trimer or tetramer of an IgG-IgA fusion molecule, comprising expressing an IgG-IgA fusion molecule comprising a full-length IgG antibody fused at its C-terminus to the Fc region of an IgA antibody, wherein the Fc region of the IgA antibody comprises a sequence including C242 to the C-terminus of the heavy chain of the IgA antibody.

[0479] 47. A method according to claim 46, wherein the IgG antibody comprises a deletion of amino acid K447.

[0480] 48. A method for purifying an IgA antibody from a mixture comprising the IgA antibody and at least one host cell protein, comprising:

[0481] (a) applying the mixture to a column comprising protein L to bind the IgA antibody;

[0482] (b) washing the protein L column with a wash buffer comprising PBS; and

[0483] (c) eluting the IgA antibody from the Protein L column with an elution buffer containing phosphoric acid.

[0484] 49. A method for purifying an IgA antibody or IgG-IgA fusion molecule in an oligomeric state from a mixture comprising an IgA antibody or IgG-IgA fusion molecule and at least one host cell protein, comprising:

[0485] (a) applying the mixture to an affinity purification column containing protein L or protein A to bind the IgA antibody or IgG-IgA fusion molecule;

[0486] (b) washing the affinity purification column with a washing buffer;

[0487] (c) eluting the IgA antibody or IgG-IgA fusion molecule from the affinity purification column with an elution buffer to form a first eluate; and

[0488] (d) applying the first eluate to a size exclusion chromatography column to separate the IgA antibodies or IgG-IgA fusion molecules in different oligomeric states and obtaining an effluent containing the IgA antibodies or IgG-IgA fusion molecules in oligomeric states.

Claims

1. An isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises a substitution at amino acid V458, N459 and / or S461.

2. The isolated IgA antibody of claim 1, wherein amino acid V458 is substituted by isoleucine (V458I), amino acid N459 is substituted by glutamine (N459Q), glycine (N459G) or alanine (N459A), and / or amino acid S461 is substituted by alanine (S461A).

3. An isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises a substitution at amino acid 1458.

4. The isolated IgA antibody according to claim 3, wherein amino acid I458 is substituted with valine (I458V).

5. An isolated IgA antibody or fragment thereof, wherein the IgA antibody comprises one or more substitutions at an amino acid selected from the group consisting of N166, T168, N211, S212, S213, N263, T265, N337, I338, T339, N459, S461, and combinations thereof.

6. The isolated IgA antibody of claim 5, wherein the substitutions at amino acids N166, S212, N263, N337, I338, T339 and N459 are N166A, S212P, N263Q, N337T, I338L, T339S and N459Q.

7. The isolated IgA antibody of claims 1-6, wherein the IgA antibody is an IgA1, IgA2m1, IgA2m2 or IgA2mn antibody.

8. The isolated IgA antibody of claim 5 or 6, wherein the IgA antibody has substitutions at amino acids N337, I338, and T339, and one or more substitutions at T168, N211, S212, S213, N263, T265, N459, S461, and combinations thereof.

9. The isolated IgA antibody according to claim 8, wherein the IgA antibody is an IgA2m1, IgA2m2 or IgA2mn antibody.

10. The isolated IgA antibody according to any one of claims 1 to 9, wherein the IgA antibody is a humanized antibody, a chimeric antibody or a human antibody.

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