Antigen-specific degraders of immunoglobulin a and their uses

Abstract

Provided is a method of removal a pathogenic species from a system of a patient having Immunoglobulin A ("IgA") nephropathy by administering to the patient a degrader of galactose-deficient Immunoglobulin A ("Gd-IgA"), wherein the pathogenic species is a monomeric Gd-IgA, a dimeric Gd-IgA complex, a trimeric Gd-IgA complex, a tetrameric Gd-IgA complex, or a combination thereof.

Description

[0001] ANTIGEN-SPECIFIC DEGRADERS OF IMMUNOGLOBULIN A AND THEIR USES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to bifunctional molecules that contain a circulating protein- binding moiety linked through a linker group to a cellular receptor-binding moiety. Specifically, the present invention relates to bifunctional molecules containing a circulating protein-binding moiety that binds to galactose-deficient immunoglobulins.

[0004] BACKGROUND OF THE INVENTION

[0005] IgA nephropathy (IgAN) is a heterogeneous autoimmune disease characterized by (1) increased levels of circulating galactose-deficient IgA1 (Gd-lgA1) antibodies, (2) production of galactose-deficient IgA1-specific IgG antibodies, and (3) formation of circulating nephritogenic immune complexes composed of galactose-deficient IgA1 antibodies and a galactose-deficient IgA1-specific IgG antibodies. These immune complexes accumulate and are deposited in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgAN, a cause of kidney injury. Overproduction of galactose-deficient IgA1 and the formation of Gd-lgA1-IgG immune complexes are key drivers of this 4-hit pathogenic cascade. See Lai et al., Nature Rev. Dis. Primers, 2,16001 (2016). IgG is enriched for galactose-deficient IgA1-specific antibodies in renal immunodeposits of IgA neuropathy patients.

[0006] Serum levels of galactose-deficient IgA1 autoantigen and the corresponding autoantibodies were each found to correlate with IgAN severity and progression. Suzuki et al., J. Clin. Invest., 119(6), 1668-1677 (2009), which is incorporated herein in its entirety by reference.

[0007] Moldoveanu et al. provided in vivo evidence for the nephritogenic role of the IgG autoantibodies in IgAN. Moldoveanu et al., J. Autoimmun., 118, 102593 (2021). Immune complexes formed from galactose-deficient IgA and human IgG autoantibody injected intravenously into mice produced glomerular injury. The histopathological changes in the injured tissues were characteristic of IgAN. Exploratory kidney-transcriptome profiling indicated these immune complexes changed the gene expression of multiple pathways, consistent with the changes seen in kidney biopsies of patients with IgAN. IgAN is the most common form of primary glomerulonephritis in the world and currently has no treatment. There remains a need for new medicines capable of treating or slowing down the progression of the disease.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention is directed to multifunctional molecules (agents) capable of binding and degrading galactose-deficient IgA1 (Gd-lgA1) immunoglobulins.

[0010] In an embodiment, provided is provided a method of removal a pathogenic species from a system of a patient having Immunoglobulin A ("IgA") nephropathy. The method includes administering to the patient a degrader of galactose-deficient Immunoglobulin A ("Gd-lgA"), wherein the pathogenic species is a monomeric Gd-lgA, a dimeric Gd-lgA complex, a trimeric Gd-lgA complex, a tetrameric Gd- lgA complex, or a combination thereof.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] These and / or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

[0013] FIG. 1 is a scheme illustrating pathogenesis of IgA nephropathy ("IgAN");

[0014] FIG. 2 illustrates mean Gd-lgA levels in plasma samples;

[0015] FIG. 3A is a diagram illustrating production of deglycosylated IgA (dg-lgA, surrogate Gd-lgA1);

[0016] FIG. 3B is a graph of mean fluorescence intensity (arbitrary units) versus concentration (nanomolar) showing results of the cellular dg-lgA and total IgA internalization mediated by BHV-1400 (nanomolar);

[0017] FIG. 4A is a graph of relative signal (arbitrary units) versus time after wash (hours) illustrating intracellular degradation of endocytosed dg-lgA as quantified by western blot;

[0018] FIG. 4B is a graph of relative signal (arbitrary units) versus time after wash (hours) illustrating intracellular degradation of BHV-1400 as quantified by western blot; FIG. 5 is a graph of mean fluorescence intensity (arbitrary units) versus concentration (nanomolar) showing results of cellular internalization of dg-lgA monomers, dimers, and tetramers mediated by BHV-1400 (nanomolar);

[0019] FIG. 6A is a graph of concentration (micrograms per milliliter) versus time (hours post dose) showing the change in dg-lgA levels in plasma across different groups;

[0020] FIG. 6B is a reproduction showing dg-lgA clearance in the blood circulation and kidney;

[0021] FIG. 7A is a reproduction showing cellular internalization of tetrameric dg-lgA complexes;

[0022] FIG. 7B is a diagram showing the measured mean fluorescence intensity of the dg-lgA signal in lysosomes at the 1-hour time point for BH-5305 (control) and BHV-1400; and

[0023] FIG. 8 shows the chemical structure of BHV-1400.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] The following detailed description is provided to aid those skilled in the art in practicing the present invention. Exemplary embodiments will hereinafter be described in detail. However, these embodiments are only exemplary, and the present disclosure is not limited thereto but rather is defined by the scope of the appended claims. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure.

[0026] Accordingly, the embodiments are merely described below, by referring to structures and schemes, to explain aspects of the present description. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. The term "or" means "and / or." Expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0027] It will be understood that when an element is referred to as being "on" another element, it can be directly in contact with the other element or intervening elements may be present therebetween. In contrast, when an element is referred to as being "directly on" another element, there are no intervening elements present.

[0028] It will be understood that, although the terms first, second, third etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a first element, component, region, layer, or section discussed below could be termed a second element, component, region, layer, or section without departing from the teachings of the present embodiments.

[0029] It is understood that the terms "comprises" and / or "comprising," or "includes" and / or "including" when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting. It will be further understood that the terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0031] As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. In instances where a term is not specifically defined herein, that term is given an art- recognized meaning by those of ordinary skill applying that term in context to its use in describing the present invention.

[0032] The articles "a" and "an" refer to one or to more than one ( / .e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0033] Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description.

[0034] The starting materials useful for making the pharmaceutical compositions of the present invention are readily commercially available or can be prepared by those skilled in the art. Solvates are compounds formed by solvation, which is the combination of solvent molecules with molecules or ions of the solute. Solvation is an interaction of a solute with the solvent, which leads to stabilization of the solute species in the solution. One may also refer to the solvated state, whereby an ion in a solution is complexed by solvent molecules. The difference in the physical properties of different solvates and polymorph ic forms thereof results from different orientation and intermolecular interactions of adjacent molecules in the solid. Polymorphic forms of compounds or solvates can be distinguished by X-ray diffraction and by other methods such as, infrared spectroscopy or Raman spectroscopy.

[0035] The term "ABT" has the biomedical art-recognized meaning of an antigen-binding moiety. In some embodiments of this specification, the ABT binds to galactose-deficient IgA1. In an embodiment, the ABT has a linker attachment point that does not diminish binding affinity.

[0036] The term "agent" has the biomedical art-recognized meaning of a composition of matter useful for performing a function. The specification describes several useful biomedical functions.

[0037] The terms "an effective amount" and "a therapeutically effective amount" have the biomedical art-recognized meaning of an amount effective to achieve its intended purpose. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

[0038] The term "anti-galactose-deficient IgA1 (Gd-lgA1) antibody" has the biomedical art-recognized meaning of an antibody that selectively binds galactose-deficient IgA1. In several embodiments of the invention, the anti-galactose-deficient IgA1 antibody is the published Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof.

[0039] The term "anti-galactose-deficient IgA1 (Gd-lgA1) IgG antibody" has the biomedical art- recognized meaning of an IgG antibody or a fragment thereof that binds to a galactose-deficient IgA1. In several embodiments of the invention, the anti-galactose-deficient IgA1 antibody is a published Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof. In some embodiments, this specification describes a "glycan-specific IgG antibody-binding" as an anti-galactose-deficient IgA1dgG antibody. The term "antigen-binding fragment thereof" has the biomedical art-recognized meaning of (1) a fragment of an intact antibody that binds to the same antigen recognized by the full-length antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, sFv, or other fragments consisting of the variable regions, or (2) any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The term antigen-binding part of an antibody encompasses single-chain antibodies.

[0040] The term "asialoglycoprotein receptor (ASGPR) binding group" has the biomedical art- recognized meaning of a binding group that binds to a hepatocyte asialoglycoprotein receptor. The ASGPR binding group selectively binds to hepatocyte asialoglycoprotein receptors on the surface of hepatocytes. In several embodiments of this specification, an ASGPR binding group is a component of a bifunctional agent as a cellular receptor-binding moiety covalently bound to the antibody-binding moiety through a linker group. Through this ASGPR moiety, bifunctional agents complexed with a circulating protein, e.g., galactose-deficient IgA1, bind to hepatocytes. After the bifunctional agent complexed with a circulating protein is bound to a hepatocyte or other cell, the circulating protein is taken into the hepatocyte or other cell via a phagocytosis mechanism, wherein the circulating protein is degraded through lysosomal degradation.

[0041] The term "asialoglycoprotein receptor (ASGPR) has the biomedical art-recognized meaning of lectins, which bind asialoglycoprotein and glycoproteins from which a sialic acid has been removed to expose galactose residues. These cellular receptors are located on mammalian hepatocytes and other cells, such as glandular cells of the gallbladder and the stomach. ASGPR removes target glycoproteins from circulation.

[0042] The term "AT" has the biomedical art-recognized meaning of an antibody moiety. In some embodiments of this specification, the AT binds to galactose-deficient IgA1.

[0043] The term "cellular receptor-binding moiety" has the biomedical art-recognized meaning. In several embodiments of this specification, the cellular receptor-binding moiety is an asialoglycoprotein receptor (ASGPR) binding group.

[0044] The term "cellular receptor" has the biomedical art-recognized meaning of a protein on the surface of a cell that binds to a compound, e.g., a ligand, e.g., a protein, in solution, or on another cell. Generally, ligand-receptor binding induces one or more biological responses. In this specification, an asialoglycoprotein receptor (ASGPR) is a cellular receptor on the surface of hepatocytes or other cells that binds to an asialoglycoprotein or a derivative thereof. The term "chimerized" has the biomedical art-recognized meaning. Chimeric antibodies are made by fusing variable domains from one species, such as a mouse, with constant domains from another species, such as a human being. With such biotechnical manipulation, chimeric antibodies have the foreign antibody's antigen specificity and affinity.

[0045] The term "complementarity determining region (CDR)" has the biomedical art-recognized meaning of a polypeptide region of an antibody heavy chin or an antibody light chain that is a determinant of the antibody to antigen binding. Each antibody heavy chain contains three complementarity determining regions. Each antibody light chain has three complementarity determining regions, usually different from the three CDRs on an antibody heavy chain. Persons having ordinary skill in the biomedical art calculate the using a standardized numbering method known as the Kabat numbering scheme. Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition (Public Health Service, National Institutes of Health, Bethesda, MD., USA, 1991), although other numbering schemes such as Chothia and IMGT are also used by persons having ordinary skill in the biomedical art.

[0046] The term "galactose-deficient IgA1 ("Gd-lgA1") binding moiety" has the biomedical art- recognized meaning of a moiety on a binding protein, e.g., an IgG antibody or a fragment thereof, that binds to a galactose-deficient IgA1. In some embodiments described in this specification, a "glycan- specific IgG antibody-binding moiety" is a galactose-deficient IgA1 binding moiety on an IgG antibody, such as the published Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof.

[0047] The term "GalNAc" has the biomedical art-recognized meaning of N-acetylgalactosamine.

[0048] The term "glomerular mesangium" has the biomedical art-recognized meaning of a component of the kidney glomerulus, forming the supporting framework in which the glomerular tuft capillaries ramify. The mesangium includes an extracellular matrix comprising Type IV collagen, proteoglycans, other proteins, and two cell types.

[0049] The term "IgA antibody" has the biomedical art-recognized meaning. Two molecules of IgA are joined and associated with a protein that enables the newly formed IgA molecule to be secreted across epithelial cells that line several ducts and organs.

[0050] The term "IgA nephropathy (IgAN)," also known as Berger disease, has the biomedical art- recognized meaning of the most common form of primary glomerulonephritis worldwide. The condition was named based on the pathologic characteristics of IgA deposition in the glomeruli. An estimated 15- 20% of the patients with IgAN progress to end-stage renal disease within 20 years of the disease onset. Kuroyanagi et al., Galactose-deficient IgA1 is involved in IgA deposition in renal grafts biopsied one hour after kidney transplantation. Intern Med. (October 26, 2022). The IgA in the mesangial deposits is exclusively from the IgA1 subclass and is aberrantly glycosyl with the hinge-region O-linked glycans being deficient in galactose (Gal). The IgA1 in the circulation of patients with IgAN also carries Gal-deficient O- glycans, although Gal-deficient variants are rarely found in the IgA1 in sera from normal individuals. The production of these variants is due to altered expression of specific glycosyltransferases in the IgA1- producing cells. The binding of IgA1-containing immune complexes with aberrantly glycosylated IgA1 to mesangial cells induces the renal manifestations characteristic of IgAN. See Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009).

[0051] The term "IgG" antibody has the biomedical art-recognized meaning. Each IgG molecule consists of the basic four-chain immunoglobulin structure— two y (gamma) heavy chains and two identical light chains (either kappa or lambda)— and carries two identical antigen-binding sites. There are four subclasses of IgG, each with minor differences in its H chains but with distinct biological properties.

[0052] The term "IgGl" antibody has the biomedical art-recognized meaning of an IgG antibody, where the Ig gamma-1 chain C region is a protein encoded by the IGHG1 gene in humans.

[0053] The term "lgG2" antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-2 chain C region is a protein that in humans is encoded by the IGHG2 gene.

[0054] The term "lgG4" antibody has the biomedical art-recognized meaning of an IgG antibody, where the Ig gamma-4 chain C region is a protein encoded by the IGHG4 gene in humans.

[0055] The term "Km55" in this specification refers to a group of anti-galactose-deficient IgA1 antibodies. In some embodiments, Km55 may be the published Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof. In some embodiments, Km55 may be chimerized, partially humanized, or humanized Km55 variants thereof, or an antigen-binding fragment thereof.

[0056] The term "linker" or "linker moiety" has the biomedical art-recognized meaning of a moiety of a chemical compound that links one moiety of the chemical compound to another moiety of the same compound. In several embodiments of this invention, the linker moiety connects an anti-galactose- deficient IgA1 IgG antibody to a cellular receptor-binding moiety. In an embodiment, a linker is largely comprised of PEG units. In another embodiment, a linker is amenable to presenting multiple ASGPR binders. In another embodiment, a linker is long enough to accommodate ternary complex formation. See also U.S. Pat. Publ. 2020 / 0190165, describing cleavable linkers and cleavable parts of each of which is incorporated herein by reference.

[0057] The term "moiety" has the biomedical meaning of a defined chemical group or entity with a particular structure or activity.

[0058] The term "MoDE" has the proprietary meaning of molecular degraders. See International Pat. Publ. WO 2019 / 199634 (Yale University) and WO 2019 / 199621 (Yale University).

[0059] The term "Multimodal Antibody Therapy Enhancers (MATE or MATES)" has the proprietary meaning. See International Pat. Publ. WO 2021 / 102052 (Kleo Pharmaceuticals).

[0060] The term "pharmaceutically acceptable excipient" has the biomedical art-recognized meaning of an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use and human pharmaceutical use. A "pharmaceutically acceptable excipient," as used in the specification and claims, includes both one and more than one such excipient. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences, 23rd edition (Elsevier, 2020).

[0061] The term "pharmaceutically acceptable" has the biomedical art-recognized meaning of those compounds, anions, cations, materials, compositions, carriers, or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0062] The terms "subject" and "patient" have the biomedical art-recognized meanings. The term patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0063] The term "TBT" has the biomedical art-recognized meaning of a target-binding moiety, a cellular receptor-binding moiety. In some embodiments of this specification, the TBT binds to ASGPR.

[0064] Methods of diagnosing IgA nephropathy

[0065] Previously, the diagnosis of IgAN was complicated because it often required renal biopsy, which may cause deadly complications Increasing evidence has indicated that galactose-deficient IgA is a trigger factor for the pathogenesis of IgAN. High levels of galactose-deficient IgA1 were reported to be associated with the disease progression. Zhang et al., Kidney Blood Pressure Res., 44, 1196-1206 (2019).

[0066] Although many researchers measured serum levels of galactose-deficient IgA using snail Helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay needed to be more robust.

[0067] More recently, a more robust and stable enzyme-linked immunosorbent assay (ELISA) method was developed that uses the Km55 antibody to recognize a hinge region in human galactose-deficient IgA (Gd-lgA1 ELISA). Km55 has a high affinity towards galactose-deficient IgA and has been a diagnostic tool for detecting IgAN. Yasutake et al., Nephrol. Dial. Transplant., 30, 1315-1321 (2015). See also Japanese Pat. Publ. JP 2010-285419 published December 24, 2010, and International Pat. Publ. WO 2015 / 064348 published May 7, 2015, each of which publication is incorporated herein in its entirety by reference.

[0068] Methods of removing galactose-deficient IgAl antibody from a patient or subject

[0069] The inventors conceived that reducing galactose-deficient IgA levels in IgAN patients results in disease treatment. The reduction of galactose-deficient IgA1 in subjects predisposed to IgAN may prevent disease. Towards this goal, the inventors designed and developed multifunctional degraders of galactose-deficient IgA1 that incorporate a galactose-deficient IgA1 binding moiety, such as a Km55 variant, and a cellular receptor-binding moiety with affinity to hepatocytes and other liver degrading cells. Galactose-deficient IgA1 bound through the anti-galactose-deficient IgA1 binding moiety is then delivered to the liver-degrading cells, where it is internalized and degraded through asialoglycoprotein receptors (ASGPR), or other cell receptors located on the surface of hepatocytes or other degrading cells in a patient or subject.

[0070] In an embodiment, provided is a method of removal a pathogenic species from a system of a patient having Immunoglobulin A ("IgA") nephropathy. The method includes administering to the patient a degrader of galactose-deficient Immunoglobulin A ("Gd-lgA"), wherein the pathogenic species is a monomeric Gd-lgA, a dimeric Gd-lgA complex, a trimeric Gd-lgA complex, a tetrameric Gd-lgA complex, or a combination thereof.

[0071] Administration of the degrader may result in the reduction of the area under the curve ("AUC") of control of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8% at least 9% at least 10% at least 11% at least 12% at least 13% at least 14% at least 15% at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0072] Method of administration

[0073] In an embodiment, the method of administration is by subcutaneous administration to a patient or subject of 50 mg / ml of the composition of matter (agent) for five to seven days. In a more specific embodiment, the composition of matter (agent) is BHV-1400.

[0074] Methods of measuring the removal of galactose-deficient IgAl antibody from a patient or subject

[0075] Guidance as to how much the administration of the agents of the invention to a patient or subject decreases IgA levels in the patient or subject is provided by several publications, e.g., Nihei, Suzuki, & Suzuki, Current understanding of IgA antibodies in the pathogenesis of IgA nephropathy. Front. Immunol., 14, 1165394 (2023). Lectin-spectrometry and mass-spectrometry-based analyses showed that IgAN patients showed elevated serum levels of aberrantly glycosylated, specifically galactose-deficient, IgA1 in O-linked glycans of its hinge region. Over 70% of patients with IgAN showed increased serum galactose-deficient IgA1 levels above the 90th percentile in healthy controls. Moldoveanu et al., Kidney International, 71(11), 1148-54 (2007) showed that lectin from Helix aspersa, recognizing N-acetylgalactosamine, was used to develop an enzyme-linked immunosorbent assay that measures galactose-deficient IgA1 in serum. The median serum lectin-binding IgA1 level was significantly higher for patients with IgA nephropathy without progression to end-stage renal disease as compared with that for healthy adult controls. The sensitivity as a diagnostic test was 76.5%, with a specificity of 94%; the positive predictive value was 88.6%, and the negative predictive value was 78.9%. Statistical analyses

[0076] Normal distribution quantitative variables can be expressed as means and SDs and compared by an independent-sample t-test, as was done by Zhang et al. (October 2019). The inventors used median and interquartile ranges for non-normally distributed variables and analyzed them with the Mann- Whitney U test. Categorical data was summarized by percentages. A two-sided p-value <0.05 was considered statistically significant. All statistical tests were performed using SPSS version 16.0.

[0077] The chemical structure

[0078] In an embodiment, the composition of matter (agent) may have the chemical formula [AGN101]: wherein, s a means for binding galactose-deficient Immunoglobulin A1 (Gd-lgA1); s a cellular receptor-binding moiety comprising an N-acetyl-D-galactosamine (GalNAc) group; and

[0079] L is a linking group, a is 1, b is 1, 2, or 3, and c is 1 or 2.

[0080] In an aspect may be an antibody or an antigen-binding fragment thereof capable of binding to Gd-lgA1.

[0081] In an aspect may include the polypeptide sequences of SEQ ID NO: 9, SEQ ID NO: 10,

[0082] SEQ ID NO: 11, SEQ 2, SEQ ID NO: 13, and SEQ ID NO: 14.

[0083] In an aspect may include a heavy chain polypeptide sequence of SEQ ID NO: 1 and light chain polypeptide sequence SEQ ID NO: 2. may include a heavy chain polypeptide sequence of SEQ ID NO: 3 and light chain polypeptide sequence SEQ ID NO: 2.

[0084] In an aspect, may include a heavy chain polypeptide sequence of SEQ ID NO: 4 and light chain polypeptide sequence SEQ ID NO: 2.

[0085] In an aspect, L may include one or more -(O)C-[(CH2)nO]m(CH2)nNH-, -

[0086] [(CH2)nO]mN HC(O) [(CH2)nO]mNH-, and -[(CH2)nO]m{NHC(O)[(CH2)nO]m}pNH-, wherein each m and n are independently 1 to 10.

[0087] In an aspect, the linker may include one or more -[(CH2)n-0]m-, wherein each m and n are independently 1 to 10.

[0088] In an aspect, L may include -(CH2)n-O-(CH2CH2O)n-(CH2)n-, wherein each n is independently 1-

[0089] 10.

[0090] In an aspect, AT may include an IgGl heavy chain or an antigen-binding fragment thereof that is connected to L at an amino acid residue selected from K246 and K248 of the IgGl heavy chain.

[0091] In an aspect, AT may be connected to L at the amino acid residue K248 of the IgGl heavy chain.

[0092] In an aspect, b may be 3, and each TBT may include an N-acetyl-D-galactosamine (GalNAc) group.

[0093] In an aspect, b may be 3, and may include the chemical formula: wherein,

[0094] ZBis absent, -(CH2)IM-, -C(=O)-(CH2)IM-, or -C(=O)-(CH2)IM-NRM-; RMis H or C1-C3alkyl; and each occurrence of IM is independently 1, 2, or 3.

[0095]

[0096] In an aspect, c may be 2.

[0097] In an aspect, the composition of matter (agent) may have the chemical formula shown in FIG. 8.

[0098] Exemplary Gd-lgA degraders are disclosed in International Patent Application No. PCT / US2022 / 019658 filed March 10, 2022 (published as WO 2022 / 192478 A1 on September 15, 2022), and International Patent Application No. PCT / US2024 / 026733 filed April 29, 2024 (published as WO 2024 / 228935 A1 on November 7, 2024), the contents of which applications are incorporated herein in their entireties by reference.

[0099] Formulations suitable for parenteral administration, such as by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can have antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral, oral, and administration are preferred administration methods. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0100] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include these components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the change in tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. SEQUENCE LISTING

[0101] Residues

[0102] ELQLQESGPGLVKPSQSLSLTCSVTGYSIRSNYWGWIRKFPGNKMEWMGYITYSGGTYYNPSLKSRISIT

[0103] RDTSKNQFFLQLTSVTTEDTATYYCTRWGDWYFDFWGPGTKVTVSPASTKGPSVFPLAPSSKSTSGGTAA

[0104] LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV

[0105] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDG

[0106] VEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

[0107] Residues

[0108] DWLTQTPPPLSVAIGQSVSISCKSSQSLVHTDGKTYLHWLLQSPGQSPKLLIYQVSNLGSGVPDRFSGT

[0109] GSQKDFTLKISRVEAEDLGVYYCVQATHFPLTFGIGTKLELQRTVAAPSVFI FPPSDEQLKSGTASWCL

[0110] LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

[0111] Residues

[0112] ELQLQESGPGLVKPSQSLSLTCSVTGYSIRSNYWGWIRKFPGNKMEWMGYITYSGGTYYNPSLKSRISIT

[0113] RDTSKNQFFLQLTSVTTEDTATYYCTRWGDWYFDFWGPGTKVTVSPASTKGPSVFPLAPSSKSTSGGTAA

[0114] LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV

[0115] DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDG

[0116] VEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

[0117] R id > >

[0118] The invention is further illustrated by the following non-limiting examples.

[0119] EXAMPLE 1. Detection of Gd-lgA1 Levels in Human Plasma Samples. Plasma concentrations of Gd-lgA1 antibody in healthy controls, disease controls, and patients with IgAN were measured using Meso Scale Discovery by capture with immobilized BHV-1400 lacking ASGPR-binding moiety (BH-5305) and detection with SULFO-TAG anti-lgA antibody.

[0120] Gd-lgA1 mean levels in IgAN patient samples were ~7-fold higher than mean levels in healthy volunteers and ~3-fold higher than mean levels in disease controls. See Table 1 and FIG. 2. Table 1. Mean Gd-lgA levels in plasma samples.

[0121] Consistent with literature reports using diagnostic KM55 (a rat antibody raised against Gd-lgA hinge peptide), these findings confirm that the anti-Gd-lgA1 antibody recognizes significant levels of circulating human Gd-lgA1.

[0122] EXAMPLE 2 In Vitro Cellular Internalization of dg-lgA with BHV-1400.

[0123] Deglycosylated IgA (dg-lgA) is a semi-synthetic Gd-lgA1 surrogate prepared from pooled human serum IgA in 3 enzymatic steps. See FIG. 3A.

[0124] Human embryonic kidney (HEK) cells transfected with human ASGPR1 (hASGPRl) were incubated with 1 μg / mL dg-lgA or total IgA conjugated to a fluorescent tag (Alexa Fluor 594 - A594), along with a dose curve of BV-1400. Cellular internalization was measured with live cell imaging, measuring the accumulation of intracellular A594 fluorescence signal. BHV-1400 was shown to cause dose-dependent, selective endocytosis of dg-lgA as opposed to total IgA. See FIG. 3B.

[0125] Low-nanomolar half-maximal effective concentration and robust mean fluorescence were observed at 12 hours for internalization of dg-lgA, illustrating that BHV-1400 internalized dg-lgA for lysosomal degradation but spared normal IgA.

[0126] EXAMPLE 3 Cells Efficiently Degrade dg-lgA and BHV-1400.

[0127] ASGPRl-expressing cells were incubated with 2 μg / mL dg-lgA and 200 nM BHV-1400 for 3 hours, washed to remove exogenous ligand and compound, added to fresh media, and harvested for intracellular protein analysis at multiple time points.

[0128] The presence of dg-lgA (see FIG. 4A) and BHV-1400 (see FIG. 4B) were assessed via western blot analysis of IgA and IgG heavy chain, which showed a decrease in signal intensity over time, suggesting a gradual degradation of both antibody and compound every hour and almost complete degradation after 20 hours.

[0129] These data confirm that dg-lgA and BHV-1400 do not aggregate within cells but are degraded to almost completion within 20 hours.

[0130] EXAMPLE 4 Cellular Internalization of Surrogate Gd-lgA1 Antibody Complexes.

[0131] Size exclusion chromatography was used to isolate tetramers, dimers, and monomers of dg-lgA- A594. See Table 2 and FIG. 5. HEK293 cells transfected with ASGPR1 were used to measure endocytosis of 1 μg / mL monomeric, dimeric, and tetrameric dg-lgA-A594 along with a dose curve of BHV-1400. Cellular internalization was quantified after 12 hours of incubation with live cell imaging measuring accumulation of intracellular A594.

[0132] Table 2. Cell internalization of dg-lgA complexes.

[0133] EC5o, half-maximal effective concentration; Max MFI, maximum mean fluorescence intensity; S / N, signal-to-noise ratio.

[0134] At 12 hours, BHV-1400 demonstrated efficient internalization of both dimeric and tetrameric dg- lgA complexes in HEK293 (hASGPRl) cells.

[0135] These data confirm that BHV-1400 can target and mediate internalization of large antibody complexes.

[0136] EXAMPLE 5. BHV-1400 Achieves Robust Degradation of Exogenous dg-lgA in Mice.

[0137] To evaluate the in vivo clearance and tissue distribution of deglycosylated IgA (dg-lgA), mice were intravenously administered 100 μg of dg-lgA to model circulating dg-lgA and its renal deposition. Five minutes post dose animals received an intravenous injection of either vehicle 200 μg of BH 5305 or 200 μg of BHV-1400. Plasma, kidney, and liver tissues were collected at the indicated time points for pharmacodynamic and histological analyses.

[0138] Plasma concentrations of dg-lgA were quantified using a mouse IgA MSD assay. The presence and localization of dg-lgA in tissues were assessed by immunofluorescence staining using an anti-lgA antibody.

[0139] FIG. 6A: Sequential intravenous dosing of dg-lgA followed by BHV-1400 (2:1 ratio) resulted in a substantial reduction of circulating dg-lgA, with plasma exposure (AUC) decreased to 58% of that observed in the vehicle-treated group. In contrast, BH-5305 - the parent control antibody lacking the ASGPR-binding moiety - did not facilitate dg-lgA clearance and showed plasma levels comparable to those in the vehicle group.

[0140] FIG. 6B: Immunofluorescence analysis of kidney sections revealed that dg-lgA localized prominently within both tubular and glomerular compartments in BH-5305-treated animals. Treatment with BHV-1400 markedly reduced dg-lgA signal intensity in both regions, demonstrating rapid and robust removal of dg-lgA from the kidneys. By the 6-hour time point, dg-lgA signals were largely depleted in both tubules and glomeruli, indicating efficient clearance.

[0141] The graph in FIG. 6A shows plasma dg-lgA levels over time for each group. Images in FIG. 6B display dg-lgA (red) localization within the kidney, with tubules and glomeruli shown in green. Insets highlight representative glomeruli. Numbers below the insets represent mean ± SD fluorescence intensity values of dg-lgA in glomeruli (n = 22-25). Abbreviations: T, tubule; G, glomerulus.

[0142] EXAMPLE 6. BHV-1400 Depletes Exogenous Tetrameric dg-lgA in Mouse Hepatocytes.

[0143] To assess whether BHV-1400 can promote the degradation of large immune complexes formed by deglycosylated IgA (dg-lgA), tetrameric dg-lgA was isolated and intravenously administered to mice. Five minutes after dosing, animals received either BH-5305 or BHV-1400.

[0144] As shown in FIG. 7A, strong dg-lgA signals persisted within hepatic sinusoids in the BH5305- treated control group, indicating minimal endocytic uptake or degradation of exogenous tetrameric dg- lgA. In contrast, the BHV-1400-treated group exhibited prominent colocalization of dg-lgA signals with the lysosomal marker LAMP-1 at the 1-hour time point, followed by a substantial decrease in signal intensity by 17 hours post-dose. These findings suggest that BHV-1400 actively promotes endocytosis and lysosomal degradation of multimeric dg-lgA complexes in vivo. Fluorescent images in FIG. 7A show dg-lgA signals in red, lysosomes labeled with LAMP-1 in green, and nuclei counterstained with DAPI in blue. The corresponding graph in FIG. 7B quantifies dg- lgA fluorescence intensity colocalized with lysosomes at the 1-hour time point, further supporting BHV- 1400-mediated intracellular processing of dg-lgA immune complexes.

[0145] CONCLUSIONS

[0146] BHV-1400 is a novel, antibody-based, bifunctional conjugate designed to selectively bind and degrade pathogenic circulating Gd-lgA1 and Gd-lgA1-IgG immune complexes via ASGPR-mediated hepatocyte internalization.

[0147] BHV-1400 binds to pathogenic Gd-lgA1 in human IgAN and in plasma samples from patients with kidney disease.

[0148] BHV-1400 demonstrates compelling preclinical evidence for rapid and robust degradation of deglycosylated IgA and deglycosylated IgA - containing immune complexes in cellular and rodent experiments.

[0149] BHV-1400 holds therapeutic potential as a transformative, non-immunosuppressive, disease- modifying treatment for patients with IgAN.

[0150] REFERENCES

[0151] 1. Sukcharoen K, Sharp SA, Thomas NJ, et al. "IgA Nephropathy Genetic Risk Score to Estimate the Prevalence of IgA Nephropathy in UK Biobank." Kidney Int Rep. 2020;5(10):1643-1650.

[0152] 2. Lai KN, Tang SC, Schena FP, et al. "IgA nephropathy." Nat Rev Dis Primers. 2016;2:16001.

[0153] 3. Knoppova B, Reily C, King RG, Julian BA, Novak J, Green TJ. "Pathogenesis of IgA nephropathy: current understanding and implications for development of disease-specific treatment." J Clin Med. 2021;10(19):4501.

[0154] 4. Rajasekaran A, Julian BA, Rizk DV. "IgA nephropathy: an interesting autoimmune kidney disease." Am J MedSci. 2021;361(2):176-194.

[0155] 5. Barratt J, Tumlin J, Suzuki Y, et al. " Randomized phase II JANUS study of atacicept in patients with IgA nephropathy and persistent proteinuria." Kidney Int Rep. 2022;7(8):1831-1841. 6. Zhang M, Zhou W, Ni Z, Liu S. "KM55 Monoclonal Antibody and IgA Variant of Proliferative Glomerulonephritis With Monoclonal Ig Deposits." Kidney Int Rep. 2020;5(6):946-950.

[0156] Throughout this application, various publications are referenced by author name and date, or by patent number or patent publication number. The disclosures of these publications are hereby incorporated in their entireties by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. For example, pharmaceutically acceptable salts other than those specifically disclosed in the description and Examples herein can be employed. Furthermore, it is intended that specific items within lists of items, or subset groups of items within larger groups of items, can be combined with other specific items, subset groups of items or larger groups of items whether or not there is a specific disclosure herein identifying such a combination.

Claims

CLAIMS1. A method of removal a pathogenic species from a system of a patient having Immunoglobulin A ("IgA") nephropathy, comprising administering to the patient a degrader of galactose-deficient Immunoglobulin A ("Gd-lgA"), wherein the pathogenic species is a monomeric Gd- IgA, a dimeric Gd-lgA complex, a trimeric Gd-lgA complex, a tetrameric Gd-lgA complex, or a combination thereof.

2. The method according to Claim 1, wherein the pathogenic species is a dimeric Gd-lgA complex.

3. The method according to Claim 1, wherein the pathogenic species is a trimeric Gd-lgA complex.

4. The method according to Claim 1, wherein the pathogenic species is a tetrameric Gd-lgA complex.

5. The method according to Claim 1, wherein the degrader of the Gd-lgA has the chemical formula [AGN101]:wherein, is a means for binding galactose-deficient Immunoglobulin A1 (Gd-lgA1);s a cellular receptor-binding moiety comprising an N-acetyl-D-galactosamine (GalNAc) group; andL is a linking group, a is 1, b is 1, 2, or 3, and c is 1 or 2.

6. The method according to Claim 5, wherein s an antibody or an antigen-binding fragment thereof capable of binding to Gd-lgA1.

7. The method according to Claim 5, whereinomprises the polypeptide sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

8. The method according to Claim 5, wherein the comprises a heavy chainpolypeptide sequence of SEQ ID NO: 1 and light chain polypeptide sequence SEQ ID NO: 2.

9. The method according to Claim 5, wherein thecomprises a heavy chain polypeptide sequence of SEQ ID NO: 3 and light chain polypeptide sequence SEQ ID NO: 2.

10. The method according to Claim 5, whereincomprises a heavy chain polypeptide sequence of SEQ ID NO: 4 and light chain polypeptide sequence SEQ ID NO: 2.

11. The method according to Claim 5, wherein L comprises one or more -(O)C-[(CH2)nO]m(CH2)nNH-, - [(CH2)nO]mNHC(O)[(CH2)nO]mNH-, and -[(CH2)nO]m{NHC(O)[(CH2)nO]m}pNH-, wherein each m and n are independently 1 to 10.

12. The method according to Claim 1, wherein the linker comprises one or more - [(CH2)n- O]m- , wherein each m and n are independently 1 to 10.

13. The method according to Claim 1, wherein L comprises -(CH2)n-O-(CH2CH2O)n-(CH2)n-, wherein each n is independently 1-10.

14. The method according to Claim 5, whereincomprises an IgGl heavy chain or an antigen-binding fragment thereof that is connected to L at an amino acid residue selected from K246 andK248 of the IgGl heavy chain.

15. The method according to Claim 14, whereinis connected to L at the amino acid residue K248 of the IgGl heavy chain.

16. The method according to Claim 5, wherein b is 3, and each TBT comprises an N-acetyl-D- galactosamine (GalNAc) group.

17. The method according to Claim 5, wherein b is 3, andcomprises the chemical formula:wherein,ZBis absent, -(CH2)IM-, -C(=O)-(CH2)IM-, or -C(=O)-(CH2)IM-NRM-;RMis H or C1-C3alkyl; and each occurrence of IM is independently 1, 2, or 3.

18. The method according to Claim 5, wherein b is 2 or 3, andb has the chemical formula [MAT01A], [MAT01B], [MAT01C], or [MAT01D]:

19. The method according to Claim 5, wherein c is 2.

20. The method according to Claim 1 having the chemical formula shown in FIG. 8.

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