Glycoengineered recombinant IGA1 and cell lines

Abstract

Immunoglobulin A (IgA) nephropathy (IgAN) is a chronic kidney disease with an autoimmune mechanism wherein the kidneys are injured as innocent bystanders. Patients with IgAN develop characteristic glomerular IgA1-containing immunodeposits in the kidneys. These glomerular immunodeposits are enriched for IgA1 glycoforms with some hinge-region O-glycans galactose-deficient (Gd-IgA1) and IgG autoantibodies specific for Gd-IgA1. As disclosed herein, removal of circulatory IgG autoantibodies specific for Gd-IgA1 would represent a disease-specific treatment of IgAN and related diseases, such as IgAVN. Removal of such autoantibodies from the circulation may be accomplished by affinity methods using a Gd-IgA1 (autoantigen)-mimicking molecules. In addition, these Gd-IgA1-mimicking molecules can be used for targeting cells producing such autoantibodies. Therefore, disclosed herein are glycoforms and mutants of IgA1 and their uses for detection, epitope determination, and removal of pathogenic autoantibodies.

Description

TH Docket No.222120-2040 GLYCOENGINEERED RECOMBINANT IGA1 AND CELL LINES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No.63 / 584,016, filed September 20, 2023, and U.S. Provisional Application No.63 / 661,201, filed June 18, 2024, which are hereby incorporated herein by reference in their entireties. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government Support under Grant Nos. DK078244, DK082753, DK134489, and AI149431 awarded by the National Institutes of Health. The Government has certain rights in the invention. SEQUENCE LISTING

[0003] This application contains a sequence listing filed in ST.26 format entitled “222120-2040 Sequence Listing” created on September 16, 2024, and having 13,426 bytes. The content of the sequence listing is incorporated herein in its entirety. BACKGROUND OF THE INVENTION

[0004] IgA nephropathy (IgAN) is a chronic kidney disease with an autoimmune mechanism wherein the kidneys are injured as innocent bystanders. Patients with IgAN develop characteristic glomerular IgA1-containing immunodeposits in the kidneys. These glomerular immunodeposits are enriched for IgA1 glycoforms with some hinge-region O-glycans galactose- deficient (Gd-IgA1) and IgG autoantibodies specific for Gd-IgA1. Complement C3 is also present. It is thought that these immunodeposits originate from the circulating immune complexes (CICs). CICs of patients with IgAN contain Gd-IgA1 (autoantigen) bound by the corresponding IgG autoantibodies; other serum proteins may be also associated with these CICs (e.g., complement C3). Some of these CICs may deposit in the glomeruli to induce kidney injury. Compositions and methods are needed to model, diagnose, and treat IgAN. SUMMARY OF THE INVENTION

[0005] Gd-IgA1 in CICs is predominantly polymeric, with a high degree of galactose deficiency and minimal sialylation; these molecular glycoforms represent a very small proportion of total IgA1 in the circulation. In contrast, the predominant molecular form of circulatory IgA1 is monomeric and mostly fully glycosylated. Multiple lines of evidence suggest that elevated levels of circulatory Gd-IgA1 are not sufficient for development of IgAN and that IgG autoantibodies specific for Gd-IgA1 are necessary to form the pathogenic immune complexes. These IgG autoantibodies are thought to target glycoforms of Gd-IgA1 molecules that have some of the 3 to 6 O-glycans in the hinge region without galactose, i.e., having exposed residue(s) consistingTH Docket No.222120-2040 of N-acetylgalactosamine (GalNAc) attached to some of the 9 Ser / Thr residues. The pathogenic potential of IgG autoantibodies specific for Gd-IgA1 was confirmed in an experimental murine model of IgAN. It is thought that the same or similar mechanisms may be involved in other immune-complex glomerular diseases, such as IgA vasculitis with nephritis (IgAVN).

[0006] As disclosed herein, removal of circulatory IgG autoantibodies specific for Gd- IgA1 would represent a disease-specific treatment of IgAN and related diseases, such as IgAVN. Removal of such autoantibodies from the circulation may be accomplished by affinity methods using a Gd-IgA1 (autoantigen)-mimicking molecules. In addition, these Gd-IgA1- mimicking molecules can be used for targeting cells producing such autoantibodies.

[0007] Therefore, disclosed herein are glycoforms and mutants of IgA1 and their uses for detection, epitope determination, and removal of pathogenic autoantibodies.

[0008] For example, disclosed herein is a glycoform of IgA1 antibodies or fragments thereof having 1, 2, 3, 4, 5, or 6 terminal N-acetylgalactosamine (GalNAc) O-glycans at amino acids T225, T228, S230, S232, T233, and T236. In some embodiments none of these O- glycans are sialated, i.e., modified with Į2,6-linked sialic acid. In some embodiments 1, 2, 3, 4, 5, or 6 of these O-glycans are sialated.

[0009] In some embodiments the IgA1 glycoform has 1, 2, 3, 4, or 5 Gal-ȕ1,3-GalNAc (core 1) O-glycans at amino acids T225, T228, S230, S232, T233, and T236. In some embodiments none of these O-glycans are sialylated, i.e., modified with Į2,6-linked sialic acid and / or Į2,3-linked sialic acid. In some embodiments 1, 2, 3, 4, or 5 of these O-glycans are sialylated.

[0010] Also disclosed are mutant IgA1 antibodies or fragments thereof having one or more mutations at amino acids T225, T228, S230, S232, T233, and T236. For example, the mutation can be a Ser to Ala (S to A) or Thr to Val (T to V) mutation. Therefore, in some embodiments, the mutant IgA1 has one or more T225V, T228V, S230A, S232A, T233V, and / or T236V mutations. In some embodiments, the mutant IgA1 includes a hinge region containing the amino acid sequence PVPSX1PPX2PX3PX4X5PPX6PSPS (SEQ ID NO:5), where at least one of the following is true: X1is not T, X2is not T, X3is not S, X4is not S, X5is not T, and X6is not T.

[0011] In some embodiments, the glycoform and / or mutant antibodies are polymeric. Therefore, in some embodiments, the glycoform and / or mutant antibodies have a J chain.

[0012] In some embodiments the IgA1 glycoform and / or mutant antibody has one or more additional mutations, such as altering the cis or trans conformation of a proline (Pro, P) amino acid. In some embodiments, the J chain is modified to include a tag (e.g., avitag, C-tag,TH Docket No.222120-2040 myc tag) to provide additional functionality for binding to active surfaces (ELISA, plates, beads for bead arrays etc).

[0013] Also disclosed are immune complexes comprising a glycoform and / or mutant antibody disclosed herein bound by an IgG autoantibody specific for galactose-deficient hinge- region O-glycans (Gd-IgA1). The Gd-IgA1 autoantibody can be natural or recombinant.

[0014] Also disclosed are glycoengineered cell lines that can be used for expression of the disclosed glycoforms as well as other glycoproteins with defined O-glycans (e.g., MUC1 glycoforms for vaccination trials in breast cancer and other adenocarcinomas wherein MUC1 is aberrantly glycosylated).

[0015] In some embodiments, the cell line has been engineered with knocked out or silenced expression of C1GalT1. In some embodiments, the cell line has been engineered with knocked out or silenced expression of ST3Gal1. Also disclosed are glycoforms of IgA1 antibodies produced by an engineered cell line disclosed herein.

[0016] Also disclosed herein are immunohistochemical methods for determining amounts and specificities of Gd-IgA1 autoantibodies in a sample from a subject using the disclosed glycoform and / or mutant IgA antibodies. In some embodiments a panel of glycoforms is used to determine the specificity of the autoantibody. For example, in some embodiments, each glycoform has an exposed GalNAc at only 1, 2, 3, 4, or 5 of T225, T228, S230, S232, T233, and T236. In other embodiments, a glycoform having no galactose-containing O-glycans, i.e., having an exposed GalNAc at each of T225, T228, S230, S232, T233, and T236, is used as a pan antigen.

[0017] The disclosed glycoform and / or mutant antibodies (e.g. membrane-bound antibodies) can be used to screen for blocking reagents that prevent the formation of immune complexes. In some embodiments, the antibodies can be used to screen serum, plasma, or urine for autoantibodies or recombinant autoantibodies for specificity, affinity, etc. Readout is based on flow (FACS) analysis and binding can be expressed as MFI. In some embodiments, the antibodies can be used to screen serum, plasma, or urine for Gd-IgA1 variants recognized by BCR or recombinant Gd-IgA1 variants for specificity, affinity, etc.

[0018] In some embodiments, the disclosed glycoform and / or mutant antibodies can be used to target Gd-IgA1-specific autoantibodies and / or B cells that produce Gd-IgA1-specific autoantibodies. This can be used, for example, to selectively deplete Gd-IgA1-specific autoantibodies from a subject’s serum, e.g., by immunoadsorption apheresis. In other embodiments, the disclosed glycoform and / or mutant antibodies are conjugated to a therapeutic agent that is cytotoxic to B cells that produce Gd-IgA1-specific autoantibodies or cause theseTH Docket No.222120-2040 cells to be targeted by the immune system. Various approaches, including bispecific Abs, abs with toxins, or CAR-T cells can be used.

[0019] The disclosed glycoform and / or mutant antibodies can also be used as bait to isolate Gd-IgA1-specific autoantibodies from a sample. These autoantibodies can then be cloned and characterized. In addition, these autoantibodies can be used to isolate and clone B cells producing Gd-IgA1-specific autoantibodies in a subject.

[0020] In some embodiments, the disclosed compositions and methods can also be used in the development of reagents to identify components associated with pathogenic IgA1- immune complexes. These compositions and methods can also be used to form model immune complexes for in vitro (cultured cells, such as mesangial cells) or in vivo (animal model) experiments to assess inhibitors of the complexes in pre-clinical testing.

[0021] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0022] FIG.1 shows a pathogenesis model of IgAN. Polymeric IgA1 with some O- glycans deficient in galactose (Gd-IgA1), often elevated in the circulation of patients with IgAN, is recognized by IgG autoantibodies specific for Gd-IgA1. IgG bound to Gd-IgA1 forms an immune complex to which additional serum proteins are added (e.g., complement). Some of these immune complexes deposit in the kidneys and activate mesangial cells. The activated mesangial cells proliferate and overproduce biologically active compounds such as cytokines and extracellular matrix proteins (ECM), leading to glomerular injury.

[0023] FIG.2 shows the structure and O-glycosylation of human IgA1. A model of dimeric IgA1 with the six most-often utilized O-glycosylation sites in each hinge region of IgA1 are marked with red spheres (Ser / Thr residues 225, 228, 230, 232, 233, 236). Amino-acid sequence (SEQ ID NO:1) below the model of IgA1 shows those residues. Each of those six sites (*) can be utilized or some may remain unglycosylated; IgA1 usually has 3-6 O-glycans per hinge region (1). Each glycosylated site of circulatory IgA1 can contain N-acetylgalactosamine (GalNAc; square) without or with Į2,6-linked sialic acid (diamond; bottom figure, upper right). The latter, sialylated GalNAc, cannot be further modified. However, GalNAc can be modified by Į1,3-linked galactose (Gal; circle), forming core 1 O-glycan (bottom figure, lower left). This disaccharide can be sialylated by Į2,6-linked sialic acid at GalNAc and / or Į2,3-linked sialic acid at Gal (bottom figure, lower right). Enzymes involved in the stepwise biosynthesis of IgA1 O-TH Docket No.222120-2040 glycans in IgA1-producing cells are noted in the figure in blue font. Each heavy chain of IgA1 also contains two N-glycans.

[0024] FIGs.3A to 3D show recombinant polymeric IgA1 produced in parental Expi293F cell line had variable number of O-glycans (0-6) per hinge region (HR) with variable galactosylation (0-5) and sialylation (0-4). Polymeric IgA1 was isolated from supernatants of cell cultures and HR (glyco)peptides generated by proteolytic digestion were analyzed by mass spectrometry. FIG.3A shows the distribution of HR glycoforms: terminal GalNAc, Core 1 glycans (Gal-ȕ1,3-GalNAc), or GalNAc-Gal with Į2,6-linked sialic acid at GalNAc and / or Į2,3- linked sialic acid at Gal. More than 70% of IgA1 produced in the parental Expi293F cell line contained GalNAc with galactose moieties, with sialic acid on one or more glycans. FIG.3B shows the distribution of sialylated HR-glycans. Sialic acid is observed at one to four glycan sites in the distribution. FIG.3C shows distribution of HR-glycans without GalNAc (GalNAc0) or with GalNAc on one to five sites (GalNAc1 to GalNAc5). FIG.3D shows the distribution of HR- glycans with Gal-ȕ1,3-GalNAc.

[0025] FIGs.4A to 4C show treatment with neuraminidase, the recombinant polymeric IgA1 produced in parental Expi293F cell line had a predominance of Core 1 glycans (Gal-ȕ1,3- GalNAc)-containing hinge-region (HR) glycopeptides. Polymeric IgA1 was isolated from supernatants of cell cultures, treated with neuraminidase, and HR (glyco)peptides generated by proteolytic digestion were analyzed by mass spectrometry. FIG.4A shows the percentage of HR (glyco)peptides with terminal GalNAc or (Gal-ȕ1,3-GalNAc) as determined by our mass spectrometry workflow. FIG.4B shows the distribution of glycan residues on recombinant IgA1 containing only the monosaccharide GalNAc. FIG.4C shows the distribution of GalNAc-Gal- containing HR (glyco)peptides with the predominant variants having four or five GalNAc residues with three / four or four / five Gal residues, respectively.

[0026] FIG.5 shows recombinant polymeric IgA1 produced in Expi293F cell line without C1GalT1. Recombinant polymeric IgA1 produced in Expi293F cell line without C1GALT1 (C1GALT1 KO) has O-glycans without Gal or sialic acid. IgA1 hinge-region (HR) clustered O- glycans were analyzed with our optimized high-resolution mass spectrometry workflow. The number of GalNAc residues per HR (glyco)peptide is shown below the graph: the most common glycoforms contained 4 or 5 O-glycans (i.e., GalNAc residues).

[0027] FIGs.6A to 6C show recombinant polymeric IgA1 produced in Expi293F cell line without ST3Gal1. Recombinant IgA1 was produced in Expi293F cell line without ST3Gal1 (ST3Gal1 KO), and purified IgA1was analyzed by mass spectrometry. FIG.6A shows IgA1 hinge-region (HR) has O-glycans as glycoforms with GalNAc and Gal-ȕ1,3-GalNAc without anyTH Docket No.222120-2040 sialic acid residues; GalNAc-Gal-containing glycoforms represent approximately 80 percent of IgA1 HR glycoforms. Individual distribution of GalNAc and mixed GalNAc / GalNAcGal isolates are shown in FIG.6B and FIG.6C, respectively.

[0028] FIGs.7A and 7B show assessing galactosylation and sialylation and immune- complex formation of two glycoforms of recombinant polymeric IgA1. FIG.7A shows a Helix pomatia (HPA) lectin reactivity assay was performed to determine the degree of galactose deficiency and sialylation of recombinant IgA1 expressed in wild-type Expi293F (WT) and Expi293F C1GALT1 knock-out (KO) cells. HPA lectin binding is specific for GalNAc. Assays were performed on IgA1 purified from each cell line directly or after treatment with neuraminidase. IgA1 from WT cells contain glycoforms (GalNAc, GalNAc-Gal, and sialylated GalNAc-Gal), while IgA1 from C1GALT1 KO have GalNAc exclusively. WT reactivity has OD readings one-third to that of C1GALT1 KO IgA1 prior to neuraminidase treatment. Following removal of sialic acid on WT IgA1, reactivity is marginally increased, while IgA1 from C1GALT1 KO does not change. This correlates with the presence of the monosaccharide GalNAc on IgA1 from C1GALT1 KO. FIG.7B shows that to determine the potential of recombinant IgA1 to form engineered immune complexes (EIC) with IgG autoantibodies, EIC were formed in solution and IgA-IgG formed EIC were determined by ELISA. Serial dilutions of EIC formed in solution showed that IgA1 from C1GALT1 KO cell line produced more IgA1-IgG EIC, 157% versus that of IgA1 from the WT cell line.

[0029] FIGs.8A to 8D show amino-acid sequence of the hinge region of IgA1 with amino-acid replacements and O-glycan variants. FIG.8A shows up to six sites (*) that may be O-glycosylated (T225, T228, S230, S232, T233, T236) on SEQ ID NO:1. Threonine (T) and Serine (S) residues have been replaced with Valine (V) and Alanine (A), respectively. FIG.8B shows individual amino acid site that have been mutated with the individual site mutation shown below with arrow. HPA lectin ELISA was performed with IgA1 (wild type IgA1 and IgA1 with single point mutants) expressed in C1GALT1 KO cells as noted in the table. Mutation of T236V resulted in loss of lectin binding. FIG.8C shows EIC ELISA performed with rIgA1 and rIgA1 single point mutants and recombinant IgG autoantibody 1123. IgA1 WT and, to a lesser degree, T228V formed EIC with recombinant IgG autoantibody 1123. FIG.8D shows a panel of mutants where the 6 main glycosylation sites were removed (Mu6) and where one site was reverted at a time, e.g., Mu5(T225). These IgA1 variants were expressed in cells with C1GALT1 KO to produce IgA1 with only GalNAc at the respective hinge-region sequence. The assay with HPA lectin showed that a variant Mu5(T228) was substantially glycosylated whereas other variants were not glycosylated (or the sites were accessible for the HPA lectin) or glycosylated onlyTH Docket No.222120-2040 marginally. HPA assays show that a single-site T236V and multi-site IgA1 mutants Mu6, Mu5(T233), and Mu5(T236) have not be recognized by HPA.

[0030] FIG.9 shows dilution curves of Gd-IgA1-specific antibodies. Plots of the mean fluorescence intensity (MFI) of cytometric beads coated with recombinant Gd-IgA1 produced in C1GALT1 KO cells and as a function of the concentration of the indicated recombinant monoclonal IgG autoantibodies.

[0031] FIG.10 shows quantitative morphometric analysis of engineered immune complexes injected into immunodeficient mice. For the mouse experiments, EIC were formed by mixing recombinant IgG autoantibody 1123150 ^g) and recombinant polymeric IgA1-GalNAc (300 ^g) in PBS (volume ~100 ^l). EIC were injected through a tail vein into nude mice using one of two dosing regimens: 1) mice (n=4) were injected every day for 3 days, receiving a total of 3 injections (3X), 2) mice (n=4) were injected every other day, receiving a total of 6 injections (6X). Control mice (n=4) did not receive any EIC. Mice were sacrificed and kidneys were collected 1 day after the last injection of EIC. Number of nuclei per glomerulus is reported based on quantitative morphometric analyses (as described in Moldoveanu et al. J Autoimmun.2021 Mar:118:102593). Results of analysis showed that EIC injections increased glomerular cellularity compared to the control mice (p < 0.0001 for each EIC group vs. control mice). Notably, the two tested EIC-injection regimens induced similar mesangioproliferative pathologic changes.

[0032] FIG.11 shows glycoengineering of recombinant IgA1. Examples of approaches for production of a specific glycoform of recombinant IgA1 using glycoengineered cell lines with gene knock-out (KO) and knock-in (KI; i.e., overexpressing a specific gene). We have generated the first two cell lines and used enzymes for sialylation (example of ST6GalNAc for sialylation of GalNAc are shown). To produce such glycoforms directly, if desired, we will generate additional cell lines with a desired glycosyltransferase gene KI. GalNAc (square); Gal (circle); sialic acid (diamond). These approaches can be combined with changing hinge-region amino-acid sequences, as shown before in Fig.8.

[0033] FIG.12 showsz expression of Gd-IgA1 variants on the surface of Expi293F C1GALT1 KO cells as BCR. Expi293F C1GALT1 KO cells were transiently co-transfected with heavy and light chains corresponding to WT or variants of IgA1. Heavy chain clone was with the splicing variant encoding the transmembrane domain and intracellular tail of IgA BCR. Two days post-transfection, cells were stained for viability with live / dead cell marker Zombie (Biolegend; YL1-A) and for the presence of cell-surface IgA with mouse monoclonal anti-human IgA1-PE from Southern Biotech (cat# 9130-09; IgA-R-PE-A). Cells were sorted and analyzed by flowTH Docket No.222120-2040 cytometer. This figure shows the IgA1 with hinge-region WT sequence and two variants (T225V and T236V); all showed a good cell-surface presentation. DETAILED DESCRIPTION

[0034] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0035] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0037] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0038] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. AnyTH Docket No.222120-2040 recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0039] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0040] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0041] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible. Definitions

[0042] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0043] The term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0044] The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplary embodiments,TH Docket No.222120-2040 antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.

[0045] The term “antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fabƍ, F(abƍ)2, scFv, Fv, dsFv diabody, Fc, and Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0046] The term “antigen binding site” refers to a region of an antibody that specifically binds an epitope on an antigen.

[0047] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0048] The term “chimeric molecule” refers to a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecules is a fusion protein.

[0049] The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, andTH Docket No.222120-2040 histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0050] The term “engineered antibody” refers to a recombinant molecule that comprises at least an antibody fragment comprising an antigen binding site derived from the variable domain of the heavy chain and / or light chain of an antibody and may optionally comprise the entire or part of the variable and / or constant domains of an antibody from any of the Ig classes (for example IgA, IgD, IgE, IgG, IgM and IgY).

[0051] The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by a multi-specific antibody such as a bi-specific antibody that specifically bind CD71 on T cells.

[0052] The term “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0053] The term “Fab fragment” refers to a fragment of an antibody comprising an antigen-binding site generated by cleavage of the antibody with the enzyme papain, which cuts at the hinge region N-terminally to the inter-H-chain disulfide bond and generates two Fab fragments from one antibody molecule.

[0054] The term “F(abƍ)2 fragment” refers to a fragment of an antibody containing two antigen-binding sites, generated by cleavage of the antibody molecule with the enzyme pepsin which cuts at the hinge region C-terminally to the inter-H-chain disulfide bond.

[0055] The term “Fc fragment” refers to the fragment of an antibody comprising the constant domain of its heavy chain.

[0056] The term “Fv fragment” refers to the fragment of an antibody comprising the variable domains of its heavy chain and light chain.TH Docket No.222120-2040

[0057] “Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc), may be transfected into cells, e.g. in certain embodiments mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0058] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0059] The term “linker” is art-recognized and refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.TH Docket No.222120-2040

[0060] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0061] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0062] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0063] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0064] The terms “polypeptide fragment” or “fragment”, when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to that of the reference polypeptide. Such deletions may occur at the amino- terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least about 5, 6, 8 or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40 or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, a fragment may comprise an enzymatic activity and / or an interaction site of the reference polypeptide. In another embodiment, a fragment may have immunogenic properties.

[0065] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.TH Docket No.222120-2040

[0066] The term “single chain variable fragment or scFv” refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.

[0067] A “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally, a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0068] The term “specifically binds”, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under designated conditions (e.g., immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M–1(e.g., 106M–1, 107M–1, 108M–1, 109M–1, 10101011and 1012M–1or more) with that second molecule.

[0069] The term “specifically deliver” as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non- target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically, specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule.

[0070] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0071] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.TH Docket No.222120-2040

[0072] The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0073] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0074] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e., a degenerate variant), substitutions within the wobble position of each codon (i.e., DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0075] The term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid, or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). Glycoengineered IgA1

[0076] Glycoengineered IgA1 that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the IgA1 hinge domain disclosed herein.

[0077] Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-TH Docket No.222120-2040 line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993)). Human antibodies can also be produced in phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). The techniques of Cote et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991)).

[0078] Optionally, the glycoengineered IgA can be generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992))

[0079] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed following the method of Winter and co- workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327TH Docket No.222120-2040 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or fragment (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0080] Also disclosed are fragments of glycoengineered IgA which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0081] In some embodiments, human IgA1 heavy chain has the amino acid sequence EVQLVQSGGDLVQPGGSLRLSCAASGFPFSNKAMSWVRQAPGKGLEWVASISASSSGTYYA DSLRGRFTISRDNSNSTLFLDMNILRAEDTATYYCASRSETIQPVgkyyyymDVWGKGTTVTVSS ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDL YTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR LSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVL PGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLAR GFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMV GHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO:2).

[0082] For defining individual amino acid sequences within the IgA1, we used the standardized numbering defined in Putnam et al. (Putnam FW, Liu YS, Low TL. Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain. J Biol Chem. 1979 Apr 25;254(8):2865-74. PMID: 107164.) CDR3 of the above construct is longer than the originating sequence in Putnam et al. In order to maintain common numbering with that manuscript and the accepted numbering in the field, the additional lowercase sequence residues in CDR3 have a designation of a-g.

[0083] Commonly utilized sites of heavy-chain O-glycosylation are underlined, i.e., amino acids T225, T228, S230, S232, T233, and T236. VHand VLsequences are italicized. In some embodiments, the hinge domain of human IgA1 has the amino acid sequence PVPSTPPTPSPSTPPTPSPS (SEQ ID NO:1), again with the potential sites of O-glycosylation underlined.TH Docket No.222120-2040

[0084] Also disclosed are membrane bound recombinant IgA. For example, in some embodiments, the secretory tail segment is replaced with an anchoring tail segment. For example, in some embodiments, the membrane-bound human IgA1 heavy chain has the amino acid sequence EVQLVQSGGDLVQPGGSLRLSCAASGFPFSNKAMSWVRQAPGKGLEWVASISASSSGTYYA DSLRGRFTISRDNSNSTLFLDMNILRAEDTATYYCASRSETIQPVgkyyyymDVWGKGTTVTVSS ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDL YTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR LSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVL PGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLAR GFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMV GHEALPLAFTQKTIDRLADWQMPPPYVVLDLPQETLEEETPGANLWPTTITFLTLFLLSLFYSTA LTVTSVRGPSGNREGPQY (SEQ ID NO:6). The membrane anchoring tail segment is underlined.

[0085] Disclosed herein are glycoforms of IgA antibodies or fragments thereof having 1, 2, 3, 4, 5, or 6 terminal N-acetylgalactosamine (GalNAc) O-glycans at amino acids T225, T228, S230, S232, T233, and T236. In some embodiments none of these of these O-glycans are sialated, i.e., modified with Į2,6-linked sialic acid. In some embodiments 1, 2, 3, 4, 5, or 6 of these O-glycans are sialylated.

[0086] In some embodiments the disclosed glycoform of IgA antibodies has 1, 2, 3, 4, or 5 Gal-ȕ1,3-GalNAc (core 1) O-glycans at amino acids T225, T228, S230, S232, T233, and T236. In some embodiments none of these O-glycans are sialylated, i.e., modified with Į2,6- linked sialic acid and / or Į2,3-linked sialic acid. In some embodiments 1, 2, 3, 4, or 5 of these O- glycans are sialylated.

[0087] Also disclosed are mutant IgA1 antibodies or fragments thereof having one or more mutations at amino acids T225, T228, S230, S232, T233, and T236. In some embodiments, the mutation is a conservative mutation. For example, the mutation can be a Ser to Ala or Thr to Val mutation. Therefore, in some embodiments, the mutant IgA1 has one or more T225V, T228V, S230A, S232A, T233V, and / or T236V mutations. In some embodiments, the mutant IgA includes a hinge region containing the amino acid sequence PVPSX1PPX2PX3PX4X5PPX6PSPS (SEQ ID NO:5), where at least one of the following is true: X1is not T, X2is not T, X3is not S, X4is not S, X5is not T, and X6is not T.

[0088] In some embodiments, the glycoform and / or mutant antibodies are polymeric. Therefore, in some embodiments, the glycoform and / or mutant antibodies have a J chain.TH Docket No.222120-2040

[0089] In some embodiments the IgA1 glycoform and / or mutant antibody has one or more additional mutations, such as altering the cis or trans conformation of a proline amino acid. In some embodiments, the J chain is modified to include a tag (e.g., avitag, C-tag, myc tag) to provide additional functionality for binding to active surfaces (ELISA, plates, beads for bead arrays, etc.).

[0090] In some embodiments, human IgA1 light chain has the amino acid sequence DIQLTQSPSSLSASVGDRVTISCRASQGIASYLAWYQQKPGRAPKLLIYAASLLQSGVPSRFSG SGSGTDFTLTISSLQPDDFATYYCQQVNNYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:3). VH and VL sequences are italicized. In some embodiments, the IgA1 has a signal sequence, such as MRVPAQLLGLLLLWLSGARC (SEQ ID NO:4). Glycoengineered Cell Lines

[0091] Also disclosed are glycoengineered cell lines that can be used for expression of the disclosed glycoforms as well as other glycoproteins with defined O-glycans (e.g., MUC1 glycoforms for vaccination trials in breast cancer and other adenocarcinomas wherein MUC1 is aberrantly glycosylated).

[0092] US20190330601 describes mammalian cells that are genetically engineered in a combinatorial approach to differentially express the human glycome. A number of the glycosyltransferase genes have high degree of sequence similarity and these have been classified into subfamilies encoding closely related putative isoenzymes, which have been shown to or predicted to serve related or similar functions in biosynthesis of glycans in cells. Examples of such subfamilies include the polypeptide GalNAc-transferases (GalNT1 to 20), Į2,3sialyltransferases (ST3GAL1-6), Į2,6sialyltransferases (ST6GAL1 and 2), Į2,6sialyltransferases (ST6GALNAC1 to 6), Į2,8sialyltransferases (ST8SIA1 to 6), ȕ4galactosyltransferases (B4GALT1 to 7), ȕ3galactosyltransferases (B3GALT1 to 6), ȕ3GlcNAc-transferases (B3GNT2 to 9), ȕ6GlcNAc-transferases (GCNT1-7 including GCNT2A, 2B and 2C (also known as C2GnT1-7 and IGnT2A to C), ȕ4GalNAc-transferases (B4GALNT1 to 4), 33glucuronyltransferase (B3GAT1 to 3), Į3 / 4-fucosyltransferases (FUT1 to 11), O- fucosyltransferases (POFUT1 and 2), O-glucosyltransferases (POGLUT 1 and 2), ȕ4GlcNAc- transferases (MGAT4A to C), 36GlcNAc-transferases (MGAT5 and 5B), and hyaluronan synthases (HAS1 to 3) (Hansen 2014) (see Table 1). For example, genes that can be inactivated in the disclosed cell lines include C1GALT1, ALG3 / 9, A4GALT, B3GNT2 / 4 / 5 / 8, B4GALNT3 / 4, B4GALT1 / 2 / 3 / 4 / 5 / 6 / 7, DPY19L2 / 3 / 4, FUT4 / 8,TH Docket No.222120-2040 GALNT1 / 2 / 3 / 6 / 7 / 10 / 11 / 12 / 13 / 14 / 16 / 18, GCNT1, GLT1D1, GLT8D1 / 2, GTDC1, MGAT1 / 2 / 3 / 4A / 4B, POMGNT1, POMT1 / 2, ST3GAL1 / 2 / 3 / 4 / 5 / 6, ST6GAL1 / 2, ST6GALNAC1 / 2 / 3 / 4, ST8SIA2 / 4 / 5, STT3A / 3B, TMTC1 / 2 / 3 / 4, UGT8, FUT8, B3GNT2, GNPTAB, GNPTG, ST6GALNACT1, COSMC, MGAT5 and genes that can be introduced include ST6GALNACT1, B4GALT1 / T2 / T3 / T4, B3GNT6, GCNT1, ST3GAL4, ST6GAL1, ST6GALNAC1, TMTC3, FUT8, ST3GAL4 / 6, ST6GAL1 / 2, MGAT3 / 4A / 5, B3GNT2, GNPTAB, GNPTG, GALNT1 / 3, CHST1 / 3 / 15 / , GAL3ST2 / 4.TH Docket No.222120-2040TH Docket No.222120-2040TH Docket No.222120-2040TH Docket No.222120-2040TH Docket No.222120-2040

[0093] In some embodiments, the cell line has been engineered with knocked out or silenced expression of C1GalT1. In some embodiments, the cell line has been engineered with knocked out or silenced expression of ST3Gal1. Also disclosed are glycoform IgA antibodies produced by an engineered cell line disclosed herein.

[0094] Thus, in another aspect, provided herein are methods for inactivating one or more cellular glycosyltransferase and related genes (e.g., MGAT1, MGAT2, MGAT3, MGAT4A, MGAT4B, MGAT4C, MGAT5, MGAT5B, B4GALT1, B4GALT2, B4GALT3, B4GALT4, B3GNT2, B3GNT8, ST3GAL3, ST3GAL4, ST3GAL6, FUT8, GNPTAB, ALG9, ALG12, ALG8, NAGPA, ALG3, MOGS, ST6GALNAC1, ST6GALNAC2, and ST6GAL1 genes) in a cell, by use of methods comprising genome perturbation, gene-editing and / or gene disruption capability such as nucleic acid vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof, nucleic acid vector systems encoding fusion proteins comprising zinc finger DNA-binding domains (ZF) and at least one cleavage domain or at least one cleavage half-domain (ZFN) and / or nucleic acid vector systems encoding a first transcription activator-like (TAL) effector endonuclease monomer and a nucleic acid encoding a second cleavage domain or at least one cleavage half-domain (TALEN). Introduction into a cell of either of the above mentioned nucleic acid cleaving agents (CRISPR, TALEN, ZFN) are capable of specifically cleaving a glycosyltransferase gene target site as a result of cellular introduction of: (Rillahan 2011) a nucleic acid encoding pair of either ZF or TALTH Docket No.222120-2040 glycosyltransferase gene target binding proteins each fused to Fok1 endonuclease, wherein at least one of said ZF or TAL polypeptides is capable of specifically binding to a nucleotide sequence located upstream from said target cleavage site, and the other ZF or TAL protein is capable of specifically binding to a nucleotide sequence located downstream from the target cleavage site, whereby each of the zinc finger proteins are independently bound to and surround the nucleic acid target followed by target nucleic acid disruption by double stranded breakage mediated by the fused endonuclease cleaving moieties, (Hansen 2015) a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes glycosyltransferase gene target sequence, and b) a second nucleotide sequence encoding a Type-II Cas9 protein, wherein components (a) and (b) are located on same or different vectors of the system, wherein the guide RNA is comprised of a chimeric RNA and includes a guide sequence and a trans- activating cr (tracr) sequence, whereby the guide RNA targets the glycosyltransferase gene target sequence and the Cas9 protein cleaves the glycosyltransferase gene target site.

[0095] In yet other embodiments, this invention provides mammalian cells with inactivation of one or more glycosyltransferase genes and with stable introduction of one or more glycosyltransferases to enhance fidelity of desirable glycosylation features and / or introduce improved glycosylation features and / or novel glycosylation features.

[0096] In certain embodiments mammalian cells with inactivation of one or more sialyltransferases, and / or galactosyltransferases, and / or glucosyltransferases, and / or GlcNAc- transferases, and / or GalNAc-transferases, and / or xylosyl-transferase, and / or glucuronosyltransferases, mannosyltransferases, and / or fucosyltransferases, and in which one or more glycosyltransferases have been stably introduced are provided.

[0097] There are a number of methods available for introduction of exogenous genes such as glycosyltransferase genes in mammalian cells and selecting stable clonal mammalian cells that harbor and express the gene of interest. Typically, the gene of interest is co- transfected with a selection marker gene that favors mammalian cells expressing the selection marker under certain defined media culture conditions. The media could contain an inhibitor of the selection marker protein or the media composition could stress cell metabolism and thus require increased expression of the selection marker. The selection marker gene may be present on same plasmid as gene of interest or on another plasmid.

[0098] In some embodiments, introduction of one or more exogenous glycosyltransferase(s) is performed by plasmid transfection with a plasmid encoding constitutive promotor driven expression of both the glycosyltransferase gene and a selectable antibiotic marker, where the selectable marker could also represent an essential gene not present in theTH Docket No.222120-2040 host cell such as GS system (Sigma / Lonza), and / or separate plasmids encoding the constitutive promotor driven glycosyltransferase gene or the selectable marker. For example, plasmids encoding ST6GalNAc-I and Zeocin have been transfected into cells and stable ST6Gal-I expressing lines have been selected based on zeocin resistance.

[0099] In some other embodiments, introduction of one or more exogenous glycosyltransferase(s) is performed by site-directed nuclease-mediated insertion.

[0100] Also disclosed is a method of producing a recombinant glycoengineered IgA that involves the steps of: (a) providing a host cell with knocked out or silenced C1GalT1; (b) introducing an expression vector comprising a transgene, the transgene comprising a sequence encoding IgA, into the host cell, thereby producing the glycoengineered IgA.

[0101] In any of the cells and methods described herein, the cell or cell line can be a HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T, HEK293-6E, Expi293F™), COS, VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2 / 0-Ag14, HeLa, or PERC6 cell.

[0102]

[0103]

[0104] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES Example 1: Background:

[0105] IgA nephropathy (IgAN) is a chronic kidney disease with an autoimmune mechanism wherein the kidneys are injured as innocent bystanders. Patients with IgAN develop characteristic glomerular IgA1-containing immunodeposits in the kidneys. These glomerular immunodeposits are enriched for IgA1 glycoforms with galactose-deficient hinge-region O- glycans (Gd-IgA1) and IgG autoantibodies specific for Gd-IgA1. Complement C3 is also present. It is thought that these immunodeposits originate from the circulating immune complexes (CICs). CICs of patients with IgAN contain Gd-IgA1 (autoantigen) bound by the corresponding IgG autoantibodies; other serum proteins may be also associated with these CICs (e.g., complement C3). Some of these CICs may deposit in the glomeruli to induce kidney injury (Fig.1).

[0106] Gd-IgA1 in CICs is predominantly polymeric, with a high degree of galactose deficiency and minimal sialylation; these molecular glycoforms represent a very small proportionTH Docket No.222120-2040 of total IgA1 in the circulation. In contrast, the predominant molecular form of circulatory IgA1 is monomeric and mostly fully glycosylated. Multiple lines of evidence suggest that elevated levels of circulatory Gd-IgA1 are not sufficient for development of IgAN and that IgG autoantibodies specific for Gd-IgA1 are necessary to form the pathogenic immune complexes. These IgG autoantibodies are thought to target glycoforms of Gd-IgA1 molecules that have some of the 3 to 6 O-glycans in the hinge region without galactose, i.e., having exposed residue(s) consisting of N-acetylgalactosamine (GalNAc) attached to some of the 9 Ser / Thr residues (Fig.2). We have confirmed the pathogenic potential of IgG autoantibodies specific for Gd-IgA1 in an experimental murine model of IgAN. It is thought that the same or similar mechanisms may be involved in other immune-complex glomerular diseases, such as IgA vasculitis with nephritis (IgAVN).

[0107] We postulate that removal of circulatory IgG autoantibodies specific for Gd-IgA1 would represent a disease-specific treatment of IgAN and related diseases, such as IgAVN. Removal of such autoantibodies from the circulation may be accomplished by affinity methods using a Gd-IgA1 (autoantigen)-mimicking molecules. In addition, these Gd-IgA1-mimicking molecules can be used for targeting cells producing such autoantibodies. Results

[0108] Herein, we describe novel methods for detection, epitope determination, and removal of pathogenic autoantibodies or cells producing those autoantibodies in patients with IgA nephropathy and related diseases.

[0109] VH and VL of IgA1 were cloned from single-cell clones that secreted Gd-IgA1 recognized by IgG autoantibodies. VH and VL amino-acid sequences are provided in Appendix 1. IgA1 was then expressed as a recombinant polymeric IgA1 protein in cell lines that were engineered to produce specific glycoforms (e.g., not to galactosylate and / or sialylate O- glycans). For comparison, we provide the information on polymeric IgA1 glycoforms produced in parental cell lines without (Fig.3) and with neuraminidase treatment (Fig.4).

[0110] In other experiments, we have shown that immune complexes formed from recombinant polymeric IgA1-GalNAc and a recombinant IgG autoantibody induce pathogenic glomerular changes in our mouse model of IgAN. Thus, this glycoform (polymeric IgA1-GalNAc) has pathogenic potential.

[0111] Figs.5 and 6 shows molecular-level analyses of different glycoforms of hinge region of polymeric IgA1 – whereas WT cells produce IgA1 with high degree of glycosylation heterogeneity, and with high content galactosylated and sialylated O-glycans, cells with C1GALT1 KO produce hinge region with only GalNAc (Fig.5) and cells with ST3GAL1 KOTH Docket No.222120-2040 produce hinge region with GalNAc and variable degree of galactosylation but no sialic acid (Fig. 6).

[0112] Fig.7 shows Helix pomatia (HPA) lectin reactivity of IgA1 from WT and C1GALT1 KO cell lines. HPA lectin binding is specific for GalNAc. The table in Fig.7A shows reactivity without and with neuraminidase treatment, while the table in Fig.7B shows the ability of each IgA1 to form immune complexes with recombinant IgG autoantibodies.

[0113] Fig.8 shows examples of amino-acid replacement in the hinge region (one Thr or Ser residue removed at a time and replaced with Val or Ala; Fig.8A). Fig.8B shows a panel of mutants with single-site replacements that were expressed in cells with C1GALT1 KO to produce hinge region with only GalNAc. The assay with HPA lectin (table in Fig.8B) showed that a mutation T236V resulted in substantially reduced glycosylation compared to other single- site mutants and WT IgA1 hinge region. IgA1 WT and, to a lesser degree, T228V formed immune complexes with recombinant IgG autoantibody 1123 (table in Fig.8C).

[0114] Fig.8D shows a panel of mutants where the 6 main glycosylation sites were removed (Mu6) and where one site was added at a time, e.g., Mu5(T225). These IgA1 variants were expressed in cells with C1GALT1 KO to produce hinge region with only GalNAc. The assay with HPA lectin (table in Fig.8D) showed that a variant Mu5(T228) was substantially glycosylated whereas other variants were not glycosylated or glycosylated only marginally.

[0115] Fig.9 shows data from cytometric bead arrays where IgA1 expressed in cells with C1GALT1 KO bind to a panel of recombinant IgG autoantibodies derived from IgAN patients. Testing the pathogenic potential of recombinant dimeric IgA1-GalNAc in an animal model

[0116] Pathogenic potential of recombinant dimeric IgA1-GalNAc (rdIgA1-GalNAc) described here was tested using our published animal model of IgA nephropathy (IgAN) (Moldoveanu Z, et al. J Autoimmun.2021118:102593). In this model, immunodeficient mice are injected with preformed engineered immune complexes (EIC). EIC are formed using recombinant human IgG autoantibody (rIgG) specific for galactose-deficient IgA1 (Gd-IgA1) and human Gd-IgA1 protein. Previously, we used myeloma protein IgA1 (Ale) that has a high content of Gd-IgA1 and showed that EIC consisting of rIgG and IgA1 (Ale) induced mesangioproliferative glomerular changes (Moldoveanu Z, et al. J Autoimmun.2021 118:102593). These pathologic changes were induced by EIC but not by the individual components alone, i.e., IgA1 or rIgG.TH Docket No.222120-2040

[0117] rdIgA1-GalNAc was expressed in a cell line that was glycoengineered as not to attach galactose to O-linked GalNAc residues of IgA1; thus, the produced IgA1 is galactose- deficient (rdIgA1-GalNAc). rdIgA1-GalNAc was purified by affinity chromatography. rIgG was produced in Expi293F cell line (Moldoveanu Z, et al. J Autoimmun.2021118:102593). ELISA confirmed that rdIgA1-GalNAc was recognized by rIgG and formed EIC.

[0118] For the mouse experiments, EIC were formed by mixing rIgG (150 ug) and rdIgA1-GalNAc (300 ug) in PBS (volume ~100 ^l). Then, these EIC were injected through a tail vein into nude mice. Two different dosing regimens were used. In the first group, mice (n=4) were injected every day for 3 days, thus receiving total of 3 injections. In the second group, mice (n=4) were injected every other day, thus receiving total of 6 injections. In each group, mice were sacrificed and kidneys were collected 1 day after the last injection of EIC. Control mice (n=4) did not receive any EIC.

[0119] One half of a kidney from each mouse was fixed in phosphate-buffered formalin, processed, and embedded in paraffin. Tissue sections (4-μm thick) were stained with hematoxylin-eosin (H&E). Images of 25 glomeruli per each mouse kidney stained with H&E were acquired (magnification 200x). Quantitative morphometric analysis was used to evaluate the pathological changes induced by the administration of EIC and compared to control mice. The number of nuclei per glomerulus was calculated using Image J-Fiji software.

[0120] Results of quantitative morphometric analysis showed that EIC injections increased glomerular cellularity compared to the control mice (Fig.10) (P < 0.0001 for each EIC group vs. control mice). Notably, the two tested EIC-injection regimens induced similar mesangioproliferative pathologic changes.

[0121] These results together indicate that rdIgA1-GalNAc is recognized by rIgG autoantibodies to form pathogenic immune complexes. Thus, this recombinant glycoengineered IgA1 represents a renewable source of pathogenic IgA1 glycoforms and can be used to determine which features are critical for the recognition by autoantibodies. Procedures that include site-specific mutagenesis to alter hinge-region glycosylation sites, glycosyltransferases to modify glycans by, or tools to modify proline residues (e.g., alter their location and / or cis / trans isomerization) may be used. Furthermore, similar approaches can be used to modify rIgG to define the key epitope-binding regions of the autoantibodies. Ultimately, the definition of antigenic features of Gd-IgA1 and the epitope-binding regions of rIgG will inform development of disease-specific treatments of IgAN. Additional description of glycoengineering of current and future recombinant IgA1TH Docket No.222120-2040

[0122] Fig.11 shows examples of approaches for production of a specific glycoform of recombinant IgA1 using glycoengineered cell lines with gene knock-out (KO) and knock-in (KI; i.e., overexpressing a specific gene). As noted above, we have generated the first two KO cell lines (C1GALT1 and ST3GAL1) and can use enzymes for sialylation (example of ST6GalNAc for sialylation of GalNAc are shown) for these IgA1 glycovariants. We have the potential to make these glycovariants directly by generating additional cell lines with a desired glycosyltransferase gene KI. Examples of such KI cell lines would be the addition of the ST6GALNAC1 or 2 gene (i.e., KI) to cell lines with KO of C1GALT1 and ST3GAL1 genes to yield IgA1 with hinge region glycoforms GalNAc sialylated by D2,6-linked sialic acid and GalNAcGal sialylated D2,6-linked sialic acid at GalNAc, respectively. Producing recombinant polymeric IgA1 with distinct hinge-region O-glycans

[0123] Heavy- and light-chain-encoding plasmids and a plasmid encoding J chain (native or modified with a tag) are used to transfect glycoengineered cells to produce polymeric IgA1 of a desired glycoform. Polymeric IgA1 with J chain expressed in glycoengineered cells modified by CRISPR-Cas9 to produce specific O-glycoforms (not galactosylating GalNAc or not sialylating Gal residues: e.g., Expi cells with C1GALT KO or ST3GAL1 KO) Production and testing

[0124] Expression protocol: Expi293F cells were cultured at 37°C on orbital shaker platform (125 rpm) in a humidified 8% CO2incubator to reach a density of 3-5x106viable cells / mL to be used for transfection. One day before transfection, the Expi293 (F / WT or KO) cell culture was split to final density of 2.0x106viable cells / mL. After 24 h, cells were again split to a final density of 2.9x106viable cells / mL with fresh, pre-warmed medium. Opti-MEM I medium was mixed with DNA (plasmids encoding heavy, light, and J-chain genes) and ExpiFectamine [ExpiFectamine™ 293 Transfection Kit (Gibco)] separately, then combined. After a 30 min incubation at RT, DNA+Fectin complexes were added by drop transfer to the Expi293F cell culture while gently swirling the flask. Cultures were incubated in the humidified 8% CO2incubator on an orbital shaker. Transfection Enhancer 1 and 2 were added after 20 h. Four days post transfection, cells and debris were removed by centrifugation. Supernatants were harvested.

[0125] Purification protocol: rIgA1 was purified by affinity chromatography with Kappa XL matrix (Thermo Scientific™ CaptureSelect™ KappaXL Affinity Matrix, Catalog number: 194321005). Supernatants were mixed with the slurry in dPBS, then placed on a bottle roller at slow speed overnight at 4°C. Beads were collected, then washed with dPBS. rIgA1 was eluted with IgG Elution buffer (Thermo Scientific™ Pierce™ IgG Elution Buffer, Fisher cat # PI21004).TH Docket No.222120-2040 Elution fractions were immediately neutralized with a 1:10 volume of 1 M Tris. Elution was repeated for a total of three times. rIgA was pooled and concentrated. Aliquots were frozen at - 80°C in sterile, 1.5 mL screw-cap O-ring tubes. Testing – HPA + / - neu and EIC assays.

[0126] HPA assay: 96-well plates were coated with F(ab’)2fragment of goat IgG anti- human IgA at 3 ^g / ml. Plates were blocked with 1% BSA in PBS containing 0.05% Tween 20, and serial two-fold dilutions of samples and standards in blocking solution were incubated overnight at room temperature. To remove terminal sialic acid, the samples were treated with 100 ^L (1 mU) per well of neuraminidase (Arthrobacter) for 3 h at 37°C. Next, the samples were incubated with GalNAc-specific biotinylated HPA lectin for 3 h at 37°C. The bound lectin was detected with avidin-horseradish peroxidase conjugate, followed by the peroxidase substrate, o- phenylenediamine-H2O2(Sigma); the reaction was stopped with 1 M sulfuric acid. The concentration of Gd-IgA1 was calculated by interpolating the optical densities at 490 nm on calibration curves constructed using a Gd-IgA1 standard.

[0127] EIC Assay: IgA1 and IgG proteins were incubated in a solution for 1 h at 37oC or o / n at 4oC to form EIC. The samples, serially diluted, we analyzed by ELISA.96-well ELISA plates were coated overnight at 4°C with 1 Pg / mL AffiniPure F(ab’)2fragment of goat anti-human IgG, Fc fragment specific in PBS+0.02% NaN3. The next day, plates were blocked for 3 hours at 25°C with blocking buffer: PBS with 1% BSA and 0.05% Tween20. Serial dilutions of EIC samples (in blocking buffer) were loaded. Bound IgG-IgA EIC were detected by using a biotin- conjugated anti-human IgA goat F(ab’)2(alpha chain specific). The bound biotin-labeled antibody was detected using ExtraAvidin-HRP and reaction developed with the peroxidase chromogenic substrate o-phenylenediamine (OPD)-H2O2(Sigma). After 30 minutes at room temperature, the reaction was stopped with 1 M sulfuric acid and optical density was measured at 490 nm using a Synergy2 plate reader with Gen5 software. IgA1 O-glycan profiling by mass spectrometry

[0128] Preparation of HR glycopeptides: Purified recombinant polymeric IgA1 samples (5 ug each, based on A280 via nanodrop) were treated with neuraminidase (N) or not, and then digested with IgA1 protease AK183 (AK) followed by reduction (DTT) and trypsin (TRP). Samples were purified over C18 column and dried in a Speed Vac O / N, no heat, then stored at - 80ºC.

[0129] MS Analysis: All samples were diluted to 40 ng / uL (200 ng total loaded onto the MS per run; two biological replicates, each analyzed in two technical replicates). IgA1 with modified hinge region to alter O-glycosylation sites.TH Docket No.222120-2040

[0130] Mutagenesis of hinge-region glycosylation site residues was carried out using a Q5 site directed mutagenesis kit (New England BioLabs, Ipswich, MA) and the parental heavy chain. The Mu6 plasmid which encode the mutation of all six hinge-region glycosylation sites, was cloned with the aid of a gene block (Purchased from Integrated DNA Technology) encoding the modified hinge region gene sequence. Individual reversion mutations, e.g., Mu5(T225), were generated with the Q5 site directed mutagenesis kit. All sequences were verified in the UAB sequencing core. IgA1 with modified J chain (adding a tag, e.g., avitag, C-tag, myc tag, etc).

[0131] J chain has been modified by addition of C-terminal AviTag for in vitro biotinylation, and C-tag or Strep tag II for immobilization and detection. Example 2: Expi293F C1GALT1 KO cells expressing membrane-bound IgA1- GalNAc, i.e., B-cell receptor (BCR)

[0132] To present membrane-bound galactose-deficient IgA1 on Expi293F C1GALT1 KO cells, a plasmid was generated in which the IgA1 heavy chain tail segment of the secreted IgA1 splicing variant was replaced with the membrane anchor segment. Sequences are listed below. In addition to the WT hinge-region (HR), the IgA1 membrane anchoring variants were generated with mutated HR sequences (T225V and T236V). Variant T236V serves as a negative control as it is not recognized by HPA lectin or by IgG autoantibody 1123 in ELISA tests.

[0133] IgA1 heavy chain splicing variant with tail segment for secreted IgA1 (secretory tail segment underlined): MELGLSWIFLLAILKGVQCEVQLVQSGGDLVQPGGSLRLSCAASGFPFSNKAMSWVRQAPGK GLEWVASISASSSGTYYADSLRGRFTISRDNSNSTLFLDMNILRAEDTATYYCASRSETIQPVGK YYYYMDVWGKGTTVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSES GQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPST PPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSA VQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLL PPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSI LRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO:7). Note that this version includes the signal sequence which is removed.

[0134] IgA1 heavy chain splicing variant with tail segment for anchoring IgA1 to membrane of the cell (membrane anchoring tail segment underlined; GenBank: ACA51870.1): MELGLSWIFLLAILKGVQCEVQLVQSGGDLVQPGGSLRLSCAASGFPFSNKAMSWVRQAPGK GLEWVASISASSSGTYYADSLRGRFTISRDNSNSTLFLDMNILRAEDTATYYCASRSETIQPVGKTH Docket No.222120-2040 YYYYMDVWGKGTTVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSES GQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPST PPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSA VQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLL PPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSI LRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLADWQMPPPYVVLDLPQETLEEETPGANL WPTTITFLTLFLLSLFYSTALTVTSVRGPSGNREGPQY (SEQ ID NO:8).

[0135] Light chain sequence: MDMRVPAQLLGLLLLWLSGARCDIQLTQSPSSLSASVGDRVTISCRASQGIASYLAWYQQKPG RAPKLLIYAASLLQSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQVNNYPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:9).

[0136] To produce the cell lines, plasmids encoding heavy and light chain sequences were transiently transfected to Expi293F C1GALT1 KO cells, according to protocols described herein. Following IgA1 expression, cells were stained with mouse monoclonal anti-human IgA1- PE from Southern Biotech (cat# 9130-09), at a concentration of 5 ^g / mL in 100 uL of FACS buffer for 15 min on ice. This was followed by 2x wash with 1xPBS / 1%BSA FACS buffer, fixed using BD fixation buffer for 30 min, washed 2x with 1xPBS / 1%BSA FACS buffer. Cells were then stained with live / dead cell marker Zombie (BioLegend) and analyzed on by flow cytometer. Figure 12 shows analysis of cells expressing of WT and two variants (T225V and T236V) of IgA1. Each shows presence surface available IgA1.

[0137] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0138] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

TH Docket No.222120-2040 CLAIMS 1. A recombinant IgA1 glycoform antibody comprising 1, 2, 3, 4, 5, or 6 terminal N- acetylgalactosamine (GalNAc) O-glycans at amino acids T225, T228, S230, S232, T233, and T236.

2. The recombinant IgA1 glycoform antibody of claim 1, wherein none of the terminal GalNAc O-glycans are sialylated.

3. The recombinant IgA1 glycoform antibody of claim 1, wherein 1, 2, 3, 4, 5, or 6 of the terminal GalNAc O-glycans are sialylated.

4. The recombinant IgA1 glycoform antibody of any one of claims 1 to 3, comprising 1, 2, 3, 4, or 5 Gal-ȕ1,3-GalNAc (core 1) O-glycans at amino acids T225, T228, S230, S232, T233, and T236.

5. The recombinant IgA1 glycoform antibody of claim 1 or 2, wherein none of the Gal-ȕ1,3- GalNAc (core 1) O-glycans are sialylated.

6. The recombinant IgA1 glycoform antibody of claim 1 or 2, wherein 1, 2, 3, 4, or 5 of the Gal-ȕ1,3-GalNAc (core 1) O-glycans are sialylated.

7. A recombinant IgA1 antibody comprising one or more mutations at amino acids T225, T228, S230, S232, T233, and T236.

8. The mutant IgA1 antibody of claim 7, wherein the mutant IgA1 has one or more T225V, T228V, S230A, S232A, T233V, and / or T236V mutations.

9. The mutant IgA1 antibody of claim 7 comprising the amino acid sequence PVPSX1PPX2PX3PX4X5PPX6PSPS (SEQ ID NO:5), where X1is not T, X2is not T, X3is not S, X4is not S, X5is not T, X6is not T, or any combination thereof.

10. The recombinant IgA1 antibody of any one of claims 1 to 9, wherein the antibody is a polymer of two or more antibodies joined by a J chain.

11. The recombinant IgA1 antibody of claim 10, wherein the J chain is modified to comprise a tag.

12. The recombinant IgA1 antibody of any one of claims 1 to 9, wherein the antibody is a monomeric antibody.

13. The recombinant IgA1 antibody of claim 11, wherein the antibody is an antibody fragment comprising at least the hinge region.

14. A method for producing IgA1 glycoform antibodies, comprising (a) providing a glycoengineered cell line engineered with knocked out or silenced expression of C1GalT1;TH Docket No.222120-2040 (b) introducing an expression vector comprising a transgene encoding an IgA1 into the glycoengineered cell line; and (c) culturing the cell line under conditions suitable for protein expression, thereby producing the glycoengineered IgA1.

15. The method of claim 14, wherein the cell line is further engineered with knocked out or silenced expression of ST3Gal1.

16. An isolated IgA1 glycoform antibodies produced by the method of claim 14 or 15.

17. A method for detecting a galactose-deficient IgA1 (Gd-IgA1) autoantibodies in a sample, comprising contacting the sample with the recombinant IgA1 antibody of any one of claims 1 to 13, detecting binding of an IgG antibody in the sample to the recombinant IgA1 antibody, thereby detecting a Gd-IgA1 autoantibody.

18. The method of claim 17, wherein the recombinant IgA1 antibody comprises an exposed N-acetylgalactosamine (GalNAc) at only one of T225, T228, S230, S232, T233, and T236, wherein detection of binding of an IgG antibody determines the specificity of the Gd-IgA1 autoantibody.

19. A method for screening candidate agents for an agent that prevents the formation of Gd- IgA1 autoantibody immune complexes, comprising (a) providing in a sample container the recombinant IgA1 antibody of any one of claims 1 to 13, a Gd-IgA1 autoantibody, and a candidate agent under conditions suitable for immune complex formation; and (c) assaying the sample for immune complex formation, wherein lack of immune complex formation is an indication that the candidate agent prevents the formation of Gd-IgA1 autoantibody immune complexes 20. A method for treating IgA nephropathy in a subject, comprising (a) selectively deplete Gd-IgA1 autoantibodies from a subject’s serum by immunoadsorption apheresis using the recombinant IgA1 antibody of any one of claims 1 to 13; or (b) administering to the subject the recombinant IgA1 antibody of any one of claims 1 to 13 conjugated to cytotoxic agent.

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