Anti-uremic toxin antibodies and methods of use thereof

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof, specific for a uremic toxin(s), compositions comprising these antibodies or antigen-binding fragments, and methods of use thereof.

Description

ANTI-UREMIC TOXIN ANTIBODIES AND METHODS OF USE THEREOF

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 667,979, filed July 5, 2024, which is hereby incorporated by reference in its entirety.

[0002] The Sequence Listing is being submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 3, 2025, is named 147462.002561. xml and is 26,921 bytes in Size. No new matter is being introduced.FIELD

[0003] The present disclosure relates to antibodies or antigen-binding fragments that are specific for uremic toxins, compositions comprising these antibodies or antigen-binding fragments, and methods of use thereof.BACKGROUND

[0004] Chronic kidney disease (CKD) is a major global health concern, affecting roughly 10% of the world’s population (Webster et al., “Chronic Kidney Disease,” Lancet 389(10075): 1238-1252 (2017) and Jadoul et al., “The Major Global Burden of Chronic Kidney Disease,” Lancet Glob Health 12(3):e342-e343 (2024)). The clinical burden of CKD is severe - patients face heightened risks of cardiovascular disease, immune dysfunction, and other complications, contributing to high morbidity and mortality rates. A key driver of these poor outcomes is the accumulation of uremic toxins (UTs) that are normally excreted by healthy kidneys. In CKD, impaired renal function leads to a buildup of these toxins in blood and tissues, which in turn exacerbates organ damage and systemic illness (Barreto et al., “Serum Indoxyl Sulfate is Associated with Vascular Disease and Mortality in Chronic Kidney Disease Patients,” Clin. J. Am. Soc. Nephrol. 4(10): 1551 - 1558 (2009)). Among the dozens of UTs identified, indoxyl sulfate (IS) and p-cresyl sulfate (pCS) have emerged as particularly harmful protein-bound toxins. These small-molecule metabolites originate from intestinal microbial fermentation of dietary amino acids and circulate tightly bound to serum albumin. High levels of IS and pCS are implicated in vascular disease progression and immune dysregulation in CKD, making them prime targets for therapeutic removal (Barreto et al., “Serum Indoxyl Sulfate is Associated with Vascular Disease and Mortality in Chronic Kidney Disease Patients,” Clin. J. Am. Soc. Nephrol. 4(10): 1551-1558 (2009)). Patients with end-state renal disease (ESRD) must rely on regular dialysis or kidney transplantation for survival.

[0005] Existing clinical strategies for removing uremic toxins have significant limitations. Conventional hemodialysis, the cornerstone of CKD management, effectively clears water-soluble UTs in their free (unbound) form and remains the predominant method of toxin removal. However, albumin-bound toxins like IS and pCS are not filtered by standard dialysis membranes. Indeed, an estimated 90-95% of IS (molecular weight 213.2 Da) and pCS (188.2 Da) in circulation is albumin-bound, leaving only a small free fraction accessible for dialysis (Viaene et ak,m “Albumin is the Main Plasma Binding Protein for Indoxyl Sulfate and P-Cresyl Sulfate,” Biopharm. Drug Dispos. 34(3): 165-175 (2013); Shi et al., “Improved Dialytic Removal of Protein-Bound Uremic Toxins by Intravenous Lipid Emulsion in Chronic Kidney Disease Rats,” Nephrol. Dial. Transplant 34(11): 1842-1852 (2019); and Sanchez-Ospina et al., “Displacing the Burden: A Review of Protein-Bound Uremic Toxin Clearance Strategies in Chronic Kidney Disease,” J. Clin. Med. 13(5): 1428 (2024)). This protein binding raises the effective molecular size of the toxin complexes and sequesters them from dialytic clearance. As a result, conventional dialysis fails to meaningfully reduce total IS and pCS levels in ESRD patients, and these toxins accumulate despite treatment. Alternative approaches have been explored to target the protein-bound fraction of UTs (Saar-Kovrov et al., “Reduction of Protein- Bound Uraemic Toxins in Plasma of Chronic Renal Failure Patients: A Systematic Review.” J. Intern. Med. 290(3):499-526 (2021); Shi et al., “Improved Dialysis Removal of Protein-Bound Uraemic Toxins with a Combined Displacement and Adsorption Technique,” Blood Purif 51(6):548-558 (2022); and Rodrigues and Faria, “Adsorption- and Displacement-Based Approaches for the Removal of Protein-Bound Uremic Toxins,” Toxins (Basel) 15(2): 110 (2023)). Adsorptive therapies, for example, use sorbents (such as activated charcoal columns) during dialysis to bind and remove toxins from blood. While conceptually attractive, adsorptionbased methods have proven challenging due to practical issues like occlusive thrombosis in the columns, procedural complexity, and high costs (Shi et al., “Improved Dialysis Removal of Protein-Bound Uraemic Toxins with a Combined Displacement and Adsorption Technique,” Blood Purif 51(6):548-558 (2022); Kato et al., “Zirconium-Based Metal-Organic Frameworks for the Removal of Protein-Bound Uremic Toxin from Human Serum Albumin,” J. Am. Chem. Soc. 141(6):2568-2576 (2019); Yamamoto et al., “Adsorption of Protein-Bound Uremic Toxins Using Activated Carbon through Direct Hemoperfusion in vitro,” Blood Purif. 48(3):215-222 (2019); and Nagata and Yoshizawa, “Pharmacological Actions of Indoxyl Sulfate and AST-120 That Should Be Recognized for the Strategic Treatment of Patients with Chronic Kidney Disease,” Int. J. Nephrol. Renovasc. Dis. 4: 13:359-365 (2020)). Molecular displacement is another strategy, in which small molecules (e.g., ibuprofen or other binding competitors) areadministered to dislodge UTs from albumin, converting them into a free form that dialysis can remove. Unfortunately, high doses of displacers are often required, leading to significant side effects and toxicity risks (Li et al., “Improved Dialysis Removal of Protein-Bound Uremic Toxins by Salvianolic Acids,” Phytomedicine 57: 166-173 (2019); Maheshwari et al., “Removal of Protein-Bound Uremic Toxins Using Binding Competitors in Hemodialysis: A Narrative Review,” Toxins (Basel) 13(9):622 (2021). Moreover, by releasing large amounts of toxin into the circulation at once, displacers can transiently raise free toxin concentrations and potentially worsen the very toxic effects one aims to mitigate. These drawbacks underscore the complexity of eliminating albumin-bound toxins and highlight an urgent need for novel, safe, and more efficacious approaches.

[0006] One promising avenue is the use of monoclonal antibodies (mAbs) to selectively capture and remove uremic toxins. Antibody-based targeting offers several potential advantages over existing methods. First, mAbs are large (-150 kDa) proteins that can be deployed in extracorporeal circulation (e.g., added to dialysis fluid or coated on a hemoperfusion column) and will not cross dialysis membranes. This containment means the antibodies can bind toxins in the dialysate compartment without entering the patient’s circulation, adding a layer of safety. Second, antibodies exhibit highly specific binding affinity for their target molecules. A toxinspecific mAb can precisely recognize IS or pCS with minimal off-target binding, enabling the tailored removal of individual toxins or combinations of toxins by using a cocktail of mAbs. This specificity contrasts with small-molecule displacers, which interact with albumin or multiple proteins and can have broad pharmacological effects. Third, mAbs are biologically engineerable. Using protein engineering and mutagenesis, their antigen-binding sites can be modified to improve binding potency or alter specificity. It is feasible, for instance, to affinity- mature an antibody for tighter binding to IS, or to re-engineer binding pocket residues to preferentially recognize other albumin-bound toxins such as pCS. Beyond single specificities, antibodies can also be made bi- or tri-specific, allowing one engineered mAb to bind multiple different toxin molecules. A multi-specific antibody or antibody mixture could thus target several uremic toxins simultaneously, potentially reducing treatment complexity and cost by consolidating efficacy into a single therapeutic agent. Together, these features make mAbs an attractive platform for developing a targeted UT removal therapy that directly addresses the shortcomings of conventional dialysis, adsorption, and displacement.

[0007] The present disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY

[0008] One aspect of the present disclosure relates to an antibody, or an antigen-binding fragment thereof, specific for a uremic toxin, where the antibody or antigen-binding fragment thereof comprises:(a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3), where the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, or variants of any of the foregoing; and a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 5, or variants of any of the foregoing;(b) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, where the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 10, or variants of any of the foregoing; and a light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13, or variants of any of the foregoing; or(c) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, where the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, or variants of any of the foregoing; and a light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21, or variants of any of the foregoing.

[0009] Another aspect of the present disclosure relates to indoxyl sulfate x p- cresylsulfate bispecific antibody, or bispecific antigen-binding fragment thereof, where the bispecific antibody or antigen-binding fragment thereof comprises:(i) a first antigen-binding arm comprising a first heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3); and a first light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3);(ii) a second antigen-binding arm comprising a second heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3); and a second light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3); where the first heavy chain variable region and the first light chain variable region pair to form a first antigen-binding site that specifically binds to indoxyl sulfate and the second heavy chain variable region and the second light chain variable region pair to form a second antigen-binding site that specifically binds to / ?-cresylsulfate.

[0010] Also disclosed are nucleic acid molecules and vectors encoding the antibodies or antigen binding fragments thereof according to the present disclosure.

[0011] A further aspect of the disclosure is directed to a cell expressing an anti -uremic toxin antibody or an antigen-binding fragment thereof according to the present disclosure.

[0012] Also disclosed are methods of producing an anti -uremic toxin antibody or antigen-binding fragment thereof according to the present disclosure.

[0013] Another aspect of the presently claimed invention is directed to a dialysis fluid composition comprising an antibody or antigen-binding fragment thereof according to the present disclosure and a pharmaceutically acceptable carrier and / or excipient.

[0014] Another aspect of the present disclosure is directed to a method of removing or reducing one or more uremic toxins from blood. This method involves contacting blood with a dialysis fluid composition comprising an antibody, or antigen-binding fragment thereof, according to the present disclosure; and removing uremic toxins from the blood by dialyzing the blood through a membrane, where the membrane has a molecular weight cutoff smaller than the molecular weight of the uremic toxin antibody or antigen-binding fragment thereof.

[0015] Another aspect of the present disclosure is directed to a method of treating a subject having a kidney disease or condition. This method involves selecting a subject having akidney disease or condition; and dialyzing a portion of the subject’s blood against a composition comprising an antibody, or an antigen-binding fragment thereof, according to the present disclosure, where said dialyzing is sufficient to remove one or more uremic toxins from the subject’s blood, thereby treating the subject having the kidney disease or condition.

[0016] Another aspect of the present disclosure is directed to a method of detecting a uremic toxin. This method involves analyzing a biological sample in an immunoassay comprising an antibody, or an antigen-binding fragment thereof, according to the present disclosure. This method further involves determining, based on said analyzing, whether the biological sample contains one or more uremic toxins.

[0017] Described herein are monoclonal antibodies specific for anti -uremic toxins. These antibodies are specific for indoxyl sulfate (IS) and / or p-cresyl sulfate (pCS), key uremic toxins in Chronic Kidney Disease (CKD). Also described are crystal structures of the disclosed mAbs in complex with IS and pCS, which reveal the specific binding pocket and residues. The Examples infra demonstrate the generation and characterization of a panel of mAbs against indoxyl sulfate (IS), demonstrating their high affinity and specificity for the toxin. The X-ray crystal structure of one lead antibody (aIS-02) bound to IS was determined; the atomic- resolution structure revealed the toxin nestled deep within the antibody’s antigen-binding site, confirming a tight and specific interaction. Because IS and p-cresyl sulfate (pCS) are structurally similar aromatic sulfate molecules, whether the same mAb could also bind pCS was also examined. Remarkably, a co-crystal structure of aIS-02 with pCS showed that the antibody can accommodate the toxin in two distinct conformations within its binding pocket. aIS-02 was evaluated in dialysis-mimicking assays and it was found to competitively reduce IS concentrations in vitro. Together, these results provide a basis for protein-engineering efforts to develope a targeted, safe, and effective regimen to clear albumin-bound uremic toxins in CKD patients.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1 A-1B illustrate the characterization of alS monoclonal antibodies. FIG. 1 A is a graph showing the ELISA binding curves for antibodies aIS-01, aIS-02, and aIS-03 against IS-B SA. Wells coated with BSA alone served as the negative control. FIG. IB provides the amino acid sequences of the heavy and light-chain variable regions of the three antibodies with Kabat-defined complementarity-determining regions (CDRs) shown in bold text.

[0019] FIGS. 2A-2D provide the crystal structure of Fab aIS-02 bound to indoxyl sulfate (IS). FIG. 2A is a side view of the variable domains, with the light chain shown on the right, theheavy chain shown on the left, and IS shown as sticks. FIG. 2B shows the top view of the binding pocket. FIG. 2C is an electrostatic surface representation of the antibody, illustrating the deep, positively charged pocket that accommodates IS. FIG. 2D is a close-up view of antigenantibody contacts; interacting side chains are shown as sticks, and hydrogen bonds or salt bridges are indicated by dashed lines with inter-atomic distances (A).

[0020] FIGS. 3A-3B show the Fab aIS-02 in complex with p-cresyl sulfate (pCS). FIG. 3 A and FIG. 3B provide orthogonal views of the antigen-binding site and reveal pCS in two alternative conformations within the same pocket.

[0021] FIGS. 4A-4B are graphs showing the reduction ratio of antibody-facilitated dialysis in a static mini-dialysis model. FIG. 1 A is a bar graph showing the results of an experiment in which indoxyl-sulfate potassium (ISK, 100 pg mL ') in PBS was dialyzed for 4 hours at room temperature against solutions containing BSA, a mock IgG, or aIS-02 (0.5 mg mL1)- IS remaining inside the dialysis cup was quantified by HPLC; results are expressed as percentage relative to the PBS control (mean ± SD, n = 3). FIG. 4B is a bar graph showing the results of the experiment in FIG. 1 A repeated with ISK dissolved in 100 % fetal bovine serum (FBS) to mimic whole blood; data are presented as in panel A.

[0022] FIGS. 5A-5B are graphs showing the reduction ratio of antibody-facilitated dialysis in a tangential-flow-filtration (TFF) model. FIG. 1 A is a bar graph showing the results of an experiment in which ISK (0.1 mg mL1in 10 % FBS) was dialyzed for 1 hour through a 10 kDa TFF cartridge against BSA, mock IgG, or aIS-02 (34.5 pg mL1)- ISK levels in the feed reservoir were measured by HPLC and expressed as percentage relative to a PBS control (mean ± SD, n = 3). FIG. IB is a bar graph showing the results of a time-course study of ISK clearance by aIS-02 under reduced feed flow (7 mL min1) to simulate clinical hemodialysis; samples were collected at 1, 2, and 3 hours. Data show progressive IS reduction with longer dialysis duration (mean ± SD, n = 3).DETAILED DESCRIPTION

[0023] Described herein are antibodies and antigen-binding fragments thereof, specific for uremic toxin(s), as well as composition comprising uremic toxin-specific antibodies. Also disclosed are methods of removing or reducing one or more uremic toxins from blood, methods of treating a subject having a kidney disease or a condition and methods of detecting a uremic toxin comprising said uremic-toxin specific antibodies.Definitions

[0024] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0025] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods and / or steps of the type described herein and / or which will become apparent to a person of ordinary skill in the art upon reading this disclosure. In another example, reference to “a compound” includes both a single compound and a plurality of different compounds.

[0026] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 50%, or within 20%, or within 10%, or within 5% (or any amount or range within 5-50%) of a given value or range. The allowable variation encompassed by the term “about” or “approximately” may depend on the context.

[0027] The term “and / or” as used herein means that the listed features are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed features is used or present.

[0028] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0029] As will be understood by a person of ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, as well as any value within a range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so on. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so on. As will also be understood by a person of ordinary skill in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges or specific values therein as discussed above. Finally, as will be understood by a person of ordinary skill in the art, and as discussed above, a range includes each individual value.

[0030] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of’ or “consisting essentially of.” The methods, kits, systems, and / or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0031] The term “antibody” refers to a complete immunoglobulin molecule or a functional fragment thereof. Naturally occurring antibodies generally include tetramers, usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy (H) chain includes a heavy chain variable (hereinafter referred to as VH) domain and a heavy chain constant (CH) domain. The heavy chain constant domain includes three CHI, CH2, and CH3 constant domains. The heavy chain can be of any isotype, including IgG (IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM, and IgE. Each light chain includes a light chain variable (hereinafter referred to as VL) domain and a light chain constant (light chain constant, CL) domain. Light chains include kappa (K) chains and lambda (X) chains. The combination of VH domain and VL domain is generally responsible for recognizing antigens, while the CH domain can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first step of the complement system of the classical pathway. VH and VL domains can be subdivided into highly variable (hypervariability) regions, also known as complementarity determining regions (CDRs), and the CDRs are interspersed with more conserved antibody framework regions (FRs). Each VH and VL domain is composed of three CDRs and four FRs respectively. The order fromN-terminus to C-terminus is as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy and light chain variable regions contain binding domains to interact with antigens.

[0032] The term “antigen-binding fragment” refers to the complete structure or part of an antibody, which may include Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, and disulfide-linked fragments. Fv fragments include single chain variable fragments (scFv), single chain variable fragment dimers ((scFv)2, also known as diabodies), single chain variable fragment Trimers ((scFv)s, also known as triabodies), single chain variable region fragment tetramers ((scFv)4, also known as tetrabodies), single domain antibodies (single domain antibodies, dAb), minibodies, nanobodies, and multispecific antibodies formed from antibody fragments.

[0033] The terms “antibody” and “antigen-binding fragment thereof’ encompass any modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0034] The term “complementarity determining region” or “CDR” is defined as the portion(s) of an antibody variable chain that bind to its specific antigen. In this disclosure, the CDRs of the heavy chain generally refer to CDR-H1, CDR-H2, and CDR-H3, and the CDRs of the light chain generally refer to CDR-L1, CDR-L2, and CDR-L3.

[0035] The term “uremic toxin” refers to any organic or inorganic compound that accumulates in the body due to kidney dysfunction and that when present above a threshold causes an adverse effect on the patient. Uremic toxins accumulate in chronic kidney disease (CKD) patients, causing significant health complications like uremic syndrome, cardiovascular disease, and immune dysfunction see, e.g., Sanchez-Ospina et al., “Displacing the Burden: A Review of Protein-Bound Uremic Toxin Clearance Strategies in Chronic Kidney Disease,” J. Clin. Med. 13(5): 1428 (2024), which is hereby incorporated by reference in its entirety). Even in patients receiving dialysis therapy, not all UTs that accumulate in the body can be effectively removed, which contributes to CKD progression (Frak et al., “Role of Uremic Toxins, Oxidative Stress, and Renal Fibrosis in Chronic Kidney Disease,” Antioxidants 13(6):687 (2024), which is hereby incorporated by reference in its entirety).

[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 invention 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, some embodiments of the methods and materials are now described.

[0037] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, 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.Uremic Toxin-Specific Antibodies

[0038] One aspect of the present disclosure relates to an antibody, or an antigen-binding fragment thereof, specific for a uremic toxin, where the antibody or antigen-binding fragment thereof comprises:(a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3), where the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, or variants of any of the foregoing; and a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 5, or variants of any of the foregoing;(b) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, where the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 10, or variants of any of the foregoing; and a light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13, or variants of any of the foregoing; or(c) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, where the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, or variants of any of the foregoing; and a light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, wherein the LCDR1 comprises the aminoacid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21, or variants of any of the foregoing.

[0039] Uremic toxins can be characterized based on their size and physiochemical properties into three groups: small water-soluble uremic toxins (less than 500 Da), middle molecule uremic toxins (500 Da or larger), and protein-bound uremic toxins (mostly less than 500 Da) see, e.g., Fujii et al., “Role of Uremic Toxins for Kidney, Cardiovascular, and Bone Dysfunction,” Toxins 10(5):202 (2018), which is hereby incorporated by reference in its entirety).

[0040] Non-limiting examples of small water-soluble uremic toxins include, without limitation, asymmetric dimethylarginine (ADMA), carbamylated compounds, creatinine, symmetric dimethylarginine (SMDA), trimethylamine-A-oxide (TMAO), urea, and uric acid.

[0041] Non-limiting examples of middle molecule uremic toxins include, without limitation, atrial natriuretic peptide (ANP), P2-microglobulin, endothelin, ghrelin, immunoglobulin light chains, interleukin-6, interleukin-8, interleuckin-18, lipids and lipoproteins, neuropeptide Y, parathyroid hormone (PTH), retinol binding protein, and tumor necrosis factor alpha (TNF-a).

[0042] Non-limiting examples of protein bound uremic toxins (PBUTs) include, without limitation, advanced glycation end products (AGEs), homocysteine, indoxyl sulfate (IS), indole acetic acid, kynurenines, p-cresylsulfate (pCS), and phenyl acetic acid.

[0043] Indoxyl sulfate is a naturally occurring tryptophan metabolite produced in the liver from indole, a tryptophan derivative that is generated by bacteria in the large intestine and binds efficiently to albumin in the blood (Fujii et al., “Role of Uremic Toxins for Kidney, Cardiovascular, and Bone Dysfunction,” Toxins 10(5):202 (2018), which is hereby incorporated by reference in its entirety). Indoxyl sulfate has the following chemical structure

[0044] p-cresylsulfate is generated from -cresol by intestinal bacteria as a result of the metabolism of tyrosine and phenylalanine (Fujii et al., “Role of Uremic Toxins for Kidney,Cardiovascular, and Bone Dysfunction,” Toxins 10(5):202 (2018), which is hereby incorporated by reference in its entirety), p-cresyl sulfate has the following chemical structure

[0045] The binding of protein bound uremic toxins (PBUTs), such as indoxyl sulfate and p-cresyl sulfate to plasma proteins such as albumin presents a challenge for clearance in CKD patients with conventional dialysis methods (Sanchez-Ospina et al., “Displacing the Burden: A Review of Protein-Bound Uremic Toxin Clearance Strategies in Chronic Kidney Disease,” J. Clin. Med. 13(5): 1428 (2024), which is hereby incorporated by reference in its entirety).

[0046] An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration, and / or with greater affinity with a particular target antigen than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., a uremic toxin) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, e.g., an antibody that specifically binds to a first antigen may or may not specifically bind to a second antigen. As such, “specific binding” does not necessarily require (although it can include) exclusive binding.

[0047] As used herein, “antibody specific for a uremic toxin” or “anti-uremic toxin antibody” or “uremic toxin-specific antibody” are used interchangeably and refer to any antibody capable of binding to a uremic toxin or uremic toxins.

[0048] The antibodies, or antigen-binding fragments thereof, specific for a uremic toxin according to the present disclosure may bind to one or more uremic toxins selected from the group consisting of indoxyl sulfate, -cresyl sulfate, and combinations thereof. In some embodiments, the antibody, or antigen-binding fragment thereof, specific for a uremic toxin binds to indoxyl sulfate. In some embodiments, the antibody, or antigen-binding fragment thereof, specific for a uremic toxin binds to p-cresylsulfate. In some embodiments, the antibody, or antigen-binding fragment thereof, specific for a uremic toxin binds to both indoxyl sulfate and p-cresyl sulfate, though it may bind them with different affinities.

[0049] In some embodiments, the antibodies or antigen-binding fragments thereof described herein specifically bind to indoxyl sulfate. In some embodiments, the antibodies or antigen-binding fragments thereof described herein specifically bind to -cresyl sulfate sulfate. In some embodiments, the antibodies or antigen-binding fragments thereof described herein specifically bind to indoxyl sulfate and -cresyl sulfate sulfate.

[0050] In some embodiments, the uremic toxin-specific antibody, or an antigen-binding fragment thereof, can be used therapeutically to remove uremic toxins from a patient (e.g., a patient with CKD or ESRD). In some embodiments, the uremic toxin-specific antibody, or an antigen-binding fragment thereof, may be used in research or may be used in diagnostic / prognostic methods, e.g., for the detection of uremic toxins.

[0051] Uremic toxin-specific antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof). Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes.

[0052] In some embodiments, the antibody, or antigen-binding fragment thereof, according to the present disclosure is a mouse antibody or antigen-binding fragment thereof. In accordance with such embodiments, the antibody or antigen-binding fragment thereof is a mouse IgG antibody, IgM antibody, IgA antibody, IgE antibody, or IgD antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a mouse IgG antibody or antigen -binding fragment thereof, e.g., an IgGl antibody, an IgG2a antibody, an IgG2b antibody, an IgG2c antibody, or an IgG3 antibody.

[0053] In some embodiments, the uremic toxin-specific antibody or antigen-binding fragment thereof is a chimeric antibody or chimeric antigen-binding fragment. In some embodiments, the uremic toxin-specific antibody or antigen-binding fragment thereof is a chimeric antibody or chimeric antigen-binding fragment thereof which may include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, the chimeric antibody or antigen-binding fragment thereof comprises a mouse variable region or part of a mouse variable region and a human constant region or portion of a human constant region. In some embodiments, amino acid modifications can be made in the variable region and / or the constant region.

[0054] In some embodiments, the antibody, or antigen-binding fragment thereof, according to the present disclosure is a human antibody, a humanized antibody, or an antigenbinding fragment of a human antibody or humanized antibody. In accordance with such embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody, an IgM antibody, an IgA antibody, an IgE antibody, or an IgD antibody. In some embodiments, the uremic toxin specific antibody or antigen-binding fragment thereof is an IgG antibody or antigen -binding fragment thereof, e.g., an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0055] Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient 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 region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In some instances, the humanized antibody may 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 or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in PCT Publication No. WO 99 / 58572, which is hereby incorporated by reference in its entirety. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

[0056] Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Set. USA, 86: 10029-10033 (1989), which is hereby incorporated by reference in its entirety. In one example, variable regions of VH and VL of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to beimportant for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.

[0057] The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions can be used to substitute for the corresponding residues in the human acceptor genes.

[0058] As noted above, a typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which can be further subdivided into complementarity determining regions (CDRs), interspersed with framework regions (FRs).

[0059] Antibodies according to the present disclosure may be a full-length antibody. In accordance with such embodiments, the full-length antibody comprises two heavy chains and two light chains, each including a variable domain and a constant domain. Accordingly, the antibody, or antigen-binding fragment thereof, according to the present disclosure may comprise (i) a first heavy chain antibody sequence and a first light chain antibody sequence, which pair to form a first antigen-binding site and (ii) a second heavy chain antibody sequence and a second light chain antibody sequence which pair to form a second antigen-binding site. In some embodiments, the first and second heavy chain amino acid sequences and the first and second light chain amino acid sequences are the same. In other embodiments, the first and second heavy chain amino acid sequences and the first and second light chain amino acid sequences are the different.

[0060] The antigen-binding fragment according to the present disclosure can comprise or be an antigen-binding fragment of a full-length antibody. Examples of antigen-binding fragments encompassed within the term “antigen-binding fragment” include (i) a Fab fragment comprising a variable light chain (VL) domain, a variable heavy chain (VH) domain, constant light chain (CL) domain, and a fist constant heavy chain (CHI) domain; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment comprising the VH and CHI domains of a heavy chain; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli,” Nature 341(6242):544-546 (1989), which is hereby incorporated byreference in its entirety), comprising a VH domain; (vi) an isolated CDR that retains functionality; and (vii) single-chain variable fragment (scFv) comprising the VH and VL domains of an antibody joined together by a flexible polypeptide linker (see, e.g., Bird et al., “Single-Chain Antigen-Binding Proteins,” Science 242(4877):423-426 (1988); and Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an AntiDigoxin Single-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85(16) : 5879-5883 (1988), which are hereby incorporated by reference in their entirety). In some embodiments, the antibody, or antigen-binding fragment thereof, is a fragment antigen binding (Fab) fragment, a Fd fragment, a F(ab’)2 fragment, a variable fragment (Fv), a single chain variable fragment (scFv), or similar constructs utilizing CDRs, VH, and / or VL sequences according to Table 1.

[0061] Table 1 provides sequences of disclosed uremic toxin-specific antibodies and antigen-binding fragments thereof.Table 1. Sequences of Uremic Toxin-Specific Antibodies and Antigen-Binding Fragments Thereof

[0062] The extent of the CDRs and FRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art (see, e.g., Kabat, E.A., et al., “Sequences of Proteins of Immunological Interest,” Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242 (1991); Chothia et al., “Conformations of Immunoglobulin Hypervariable Regions,” Nature 342(6252):877-883 (1989); Chothia et al., “Canonical Structures for the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196(4):901-917 (1987); Al-lazikani et al., “Standard Conformations for the Canonical Structures of Immunoglobulins,” J. Mol. Biol. 273:927-948(1997); and Almagro, J., “Identification of Differences in the Specificity-determining Residues of Antibodies that Recognize Antigens of Different Size: Implications for the Rational Design of Antibody Repertoires,” J. Mol. Recognit. 17(2): 132-143 (2004), which are hereby incorporated by reference in their entirety). Thus, also contemplated are uremic-toxin specific antibodies, or antigen-binding fragments thereof, comprising: (i) the heavy chain CDR sequences of SEQ ID NO: 6 and the light chain CDR sequences of SEQ ID NO: 7; (ii) the heavy chain CDR sequences of SEQ ID NO: 14 and the light chain CDR sequences of SEQ ID NO: 15; or (iii) the heavy chain CDR sequences of SEQ ID NO: 22 and the light chain CDR sequences of SEQ ID NO: 23, as defined by the Kabat, Clothia, or AbM schemes (see, e.g., Zhu et al, “50 years of AntibodyNumbering Schemes: A Statistical and Structural Evaluation Reveals Key Differences and Limitations,” Antibodies (Basel) 13(4):99 (2024), which is hereby incorporated by reference in its entirety).

[0063] In some embodiments, the antibody, or an antigen-binding fragment thereof, according to the present disclosure comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where:(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 5;(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 10, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13; or(c) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21.

[0064] The antibody, or an antigen-binding fragment thereof, according to the present disclosure may comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 4, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 5. In accordance with such embodiments, the anti-uremic toxin antibody, or antigen-binding fragment thereof, specific for a uremic toxin may be aIS-01 or an antigen-binding fragment of aIS-01.

[0065] The antibody, or an antigen-binding fragment thereof, according to the present disclosure may comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LCDR3comprising the amino acid sequence of SEQ ID NO: 13. In accordance with such embodiments, the anti-uremic toxin antibody, or antigen-binding fragment thereof, specific for a uremic toxin may be aIS-02 or an antigen-binding fragment of aIS-02.

[0066] The antibody, or an antigen-binding fragment thereof, according to the present disclosure may comprise an HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 18, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 21. In accordance with such embodiments, the antibody, or antigen-binding fragment thereof, specific for a uremic toxin may be aIS-03 or an antigen-binding fragment of aIS-03.

[0067] The antibody, or antigen-binding fragment, specific for a uremic toxin according to the present disclosure may comprise a heavy chain variable region comprising:(a) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 6, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, and 24, respectively;(b) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 14, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 8, 9, and 10, respectively; or(c) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 22, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 17, and 18, respectively.

[0068] In some embodiments, the antibody, or antigen-binding fragment thereof, specific for a uremic toxin according to the present disclosure comprises a heavy chain variable region comprising:(a) the amino acid sequence of SEQ ID NO: 6, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, and 24, respectively;(b) the amino acid sequence of SEQ ID NO: 14, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 8, 9, and 10, respectively; or(c) the amino acid sequence of SEQ ID NO: 22, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 17, and 18, respectively.

[0069] The antibody, or antigen-binding fragment thereof, specific for a uremic toxin according to the present disclosure may comprise a light chain variable region comprising:(a) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 7, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 3, 4, and 5, respectively;(b) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 15, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 11, 12, and 13, respectively; or(c) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 23, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, and 21, respectively.

[0070] In some embodiments, the antibody, or antigen-binding fragment thereof, specific for a uremic toxin according to the present disclosure comprises:(a) the amino acid sequence of SEQ ID NO: 7, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 3, 5, and 5, respectively;(b) the amino acid sequence of SEQ ID NO: 15, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 11, 12, and 13, respectively; or(c) the amino acid sequence of SEQ ID NO: 23, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, and 21, respectively.

[0071] The antibody, or antigen-binding fragment, specific for a uremic toxin according to the present disclosure may comprise:(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;(b) a heavy chain variable region comprising an amino acid sequence of SEQ IDNO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; or(c) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.Bispecific Antibodies

[0072] Another aspect of the present disclosure relates to indoxyl sulfate x p- cresylsulfate bispecific antibody, or bispecific antigen-binding fragment thereof, where the bispecific antibody or antigen-binding fragment thereof comprises:(i) a first antigen-binding arm comprising a first heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3); and a first light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3);(ii) a second antigen-binding arm comprising a second heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3); and a second light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3); where the first heavy chain variable region and the first light chain variable region pair to form a first antigen-binding site that specifically binds to indoxyl sulfate and the second heavy chain variable region and the second light chain variable region pair to form a second antigen-binding site that specifically binds to / ?-cresylsulfate.

[0073] The first antigen-biding arm and / or the second antigen-binding arm may comprise:(a) an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 24, or variants of any of the foregoing; and the LCDR1 having the amino acid sequence of SEQ ID NO: 3, the LCDR2 having the amino acid sequence of SEQ ID NO: 4, and the LCDR3 having the amino acid sequence of SEQ ID NO: 5, or variants of any of the foregoing;(b) an HCDR1 having the amino acid sequence of SEQ ID NO: 8, an HCDR2 having the amino acid sequence of SEQ ID NO: 9, and an HCDR3 having the amino acid sequence of SEQ ID NO: 10, or variants of any of the foregoing; and an LCDR1 having the amino acid sequence of SEQ ID NO: 11, an LCDR2 having the amino acid sequence of SEQ ID NO: 12, and an LCDR3 having the amino acid sequence of SEQ ID NO: 13, or variants of any of the foregoing; or(c) an HCDR1 having the amino acid sequence of SEQ ID NO: 16, an HCDR2 having the amino acid sequence of SEQ ID NO: 17, and an HCDR3 having the amino acid sequence of SEQ ID NO: 18, or variants of any of the foregoing; and an LCDR1 having the amino acid sequence of SEQ ID NO: 19, an LCDR2 having the amino acid sequence of SEQ ID NO: 20, and an LCDR3 having the amino acid sequence of SEQ ID NO: 21, or variants of any of the foregoing.

[0074] In some embodiments of the indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigen-binding fragment thereof, the first antigen-biding arm and / or the second antigen -binding arm comprise:(a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 3, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 4, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 5;(b) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 10, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 13; or(c) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 18, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 21.

[0075] In some embodiments of the indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigen-binding fragment thereof, the first antigen-biding arm and / or the second antigen -binding arm comprise:(a) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 6, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, and 24, respectively; and an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 7, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ IDS NOs: 3, 4, and 5, respectively;(b) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 14, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 8, 9, and 10, respectively; and an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 15, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 11, 12, and 13, respectively; or(c) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 22, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 17, and 18, respectively; and an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 23, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, and 21, respectively.

[0076] In some embodiments of the indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigen-binding fragment thereof, the first antigen-biding arm and / or the second antigen -binding arm comprise:(a) the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7, provided that the heavy and light chain variable regions comprise the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1, 2, 24, 3, 4, and 5, respectively;(b) the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15, provided that the heavy and light chain variable regions comprise the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively; or(c) the amino acid sequence of SEQ ID NO: 22 and the amino acid sequence of SEQ ID NO: 23, provided that the heavy and light chain variable regions comprise the sequences ofHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively.Antibody aIS-02

[0077] As noted above, “specific binding” does not necessarily require (although it can include) exclusive binding.

[0078] The Examples of the present disclosure demonstrate the unexpected finding that the anti-uremic toxin specific antibody aIS-02 binds both indoxyl sulfate and -cresyl sulfate.

[0079] In some embodiments of the indoxyl sulfate x / 2-cresyl sulfate bispecific antibody, or bispecific antigen-binding fragment thereof, the first antigen-biding arm and / or the second antigen -binding arm are the same and each comprise the heavy chain CDR sequences of SEQ ID NO: 14 and the light chain CDR sequences of SEQ ID NO: 15, as defined by the Kabat, Clothia, or AbM schemes.

[0080] In some embodiments of the indoxyl sulfate x / 2-cresyl sulfate bispecific antibody, or bispecific antigen-binding fragment thereof, the first antigen-biding arm and / or the second antigen -binding arm are the same and comprise: (1) a heavy chain comprising a HCDR1 amino acid sequence of SEQ ID NO: 8, a HCDR2 amino acid sequence of SEQ ID NO: 9, a HCDR3 amino acid sequence of SEQ ID NO: 10; and (2) a light chain comprising a LCDR1 amino acid sequence of SEQ ID NO: 11, an LCDR2 amino acid sequence of SEQ ID NO: 12, and an LCDR3 amino acid sequence of SEQ ID NO: 13.

[0081] In some embodiments of the indoxyl sulfate x / 2-cresyl sulfate bispecific antibody, or bispecific antigen-binding fragment thereof, the first antigen-biding arm and / or the second antigen-binding arm are the same and comprise a heavy chain variable region amino acid sequence of SEQ ID NO: 14 and a light chain variable region of SEQ ID NO: 15.

[0082] Suitable antibody antigen-binding fragments are described in detail infra. In some embodiments, the antibody, or antigen-binding fragment thereof, is a fragment antigen binding (Fab) fragment, a Fd fragment, a F(ab’)2 fragment, a variable fragment (Fv), a single chain variable fragment (scFv), or similar constructs utilizing CDRs, VH, and / or VL sequences of antibody aIS-02 according to Table 1.Nucleic Acid Molecules and Vectors

[0083] Also disclosed are nucleic acid molecules and vectors encoding the antibodies or antigen binding fragments thereof according to the present disclosure.

[0084] A “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The nucleic acidmolecules according to the present disclosure encode an anti -uremic toxin antibody. Suitable exemplary anti -uremic toxin antibodies are disclosed in detail supra.

[0085] The nucleic acid molecules of the present disclosure may be inserted into “vectors.” The term “vector” is widely used and understood by those of skill in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule. Vectors can be linear or circular. Vectors can integrate into a target genome of a host cell or replicate independently in a host cell. Vectors can comprise, e.g., an origin of replication, a multicloning site, and / or a selectable marker.

[0086] The term “vector” also includes both viral and nonviral means for introducing a nucleic acid molecule into a cell in vitro, in vivo, or ex vivo. Vectors may be introduced into the desired host cells by well-known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection.

[0087] Any vector that allows expression of the antibodies of the present disclosure may be used. In certain embodiments, the antibodies or antigen-binding fragments of the present invention may be expressed in vitro (such as using cell-free expression systems) and / or in cultured cells grown in vitro in order to produce the encoded antibodies or antigen binding fragments thereof, which may then be used for various applications such as in the removal of anti -uremic toxins from a biological fluid. For such applications, any vector that allows expression of the antibodies or antigen-binding fragments in vitro and / or in cultured cells may be used.

[0088] The vector may be a recombinant expression vector. The term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.

[0089] Non-limiting examples of expression vectors include, but are not limited to, plasmids and viral vectors, such as replication defective retroviruses, adenoviruses, vesicular stomatitis virus (VSV), herpes viruses, Newcastle disease virus (NDV), vaccinia virus (e.g., Modified Vaccinia Ankara virus), adeno-associated viruses (AAV), plant viruses, and baculoviruses.

[0090] In some embodiments, the vector is an expression vector capable of directing the expression of a nucleic acid sequence encoding an anti-uremic toxin antibody or antigen-binding fragment thereof described herein.

[0091] In some embodiments, the vector is a mammalian vector comprising a nucleotide sequence encoding an anti-uremic toxin antibody or antigen-binding fragment thereof described herein.

[0092] In some embodiments, an expression vector comprises a nucleic acid sequence comprising a nucleotide sequence encoding an anti-uremic toxin antibody or antigen-binding fragment thereof and in a form suitable for expression of the nucleic acid sequence in a cell.

[0093] The expression vector may include one or more regulatory sequences, selected on the basis of the cells to be used for expression, which is operably linked to the nucleic acid to be expressed. Within an expression vector, “operably linked” is intended to mean that a nucleic acid sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid sequence (e.g, in an in vitro transcript! on / translati on system or in a cell when the vector is introduced into the cell). Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleic acid in many types of cells, those which direct expression of the nucleic acid sequence only in certain cells (e.g., tissue specific regulatory sequences), and those which direct the expression of the nucleic acid sequence upon stimulation with a particular agent (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the cell to be transformed, the level of expression of protein desired, etc.

[0094] Expression vectors can be designed for expression of a recombinant anti-uremic toxin antibody or antigen-binding fragment thereof using eukaryotic cells (e.g., mammalian cells, yeast cells, plant cells, or insect cells (using baculovirus expression vectors, see, e.g., Treanor et al., “Safety and Immunogenicity of a Baculovirus-Expressed Hemagglutinin Influenza Vaccine: A Randomized Controlled Trial,” JAMA, 297(14): 1577-1582 (2007) which is hereby incorporated by reference herein in its entirety)) or prokaryotic cells (e.g, E. coli).

[0095] Examples of mammalian cells include, but are not limited to, A549 cells, Crucell Per.C6 cells, Vero cells, CHO cells, VERO cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293 cells, 3T3 cells or WI38 cells. In certain embodiments, the cells are myeloma cells, e.g., NS0 cells, 45.6 TGI.7 cells, AF-2 clone 9B5 cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells, NCI-H929 cells, NP cells, NS0 / 1 cells, P3 NS1 Ag4 cells,P3 / NSl / l-Ag4-l cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U. l cells, RPMI 8226 cells, Sp20-Agl4 cells, U266B1 cells, X63AG8.653 cells, Y3.Ag.1.2.3 cells, and YO cells. In some embodiments, a mammalian cell culture system (e.g. Chinese hamster ovary or baby hamster kidney cells) is used for expression of a recombinant anti-uremic toxin antibody or antigen-binding fragment thereof.

[0096] Examples of yeast cells include, but are not limited to Saccharomyces cerevisiae and Pichia pastor is.

[0097] Non-limiting examples of insect cells include 5 / 9, Sf2A , Trichoplusia ni, Spodoptera frugiperda, and Bombyx mori.

[0098] In some embodiments, a plant cell culture system is used for expression of a recombinant anti -uremic toxin antibody or antigen-binding fragment thereof as described herein. See, e.g, U.S. Patent Nos. 7,504,560; 6,770,799; 6,551,820; 6,136,320; 6,034,298; 5,914,935; 5,612,487; and 5,484,719; which are hereby incorporated by reference in their entirety, and U.S. Patent Application Publication Nos. 2009 / 0208477, 2009 / 0082548, 2009 / 0053762, 2008 / 0038232, 2007 / 0275014, and 2006 / 0204487, which are hereby incorporated by reference in their entirety, for plant cells and methods for the production of proteins utilizing plant cell culture systems.

[0099] In some embodiments, cells are engineered to express a recombinant anti -uremic toxin antibody or antigen-binding fragment thereof.

[0100] As an alternative to recombinant expression of an anti -uremic toxin antibody or antigen-binding fragment thereof using a cell, an expression vector containing a nucleic acid sequence encoding an anti-uremic toxin antibody or antigen-binding fragment thereof can be transcribed and translated in vitro using, e.g., T7 promoter regulatory sequences and T7 polymerase. In a specific embodiment, a coupled transcription / translation system, such as Promega TNT®, or a cell lysate or cell extract comprising the components necessary for transcription and translation may be used to produce an anti-uremic toxin antibody or antigenbinding fragment thereof.

[0101] Once an anti -uremic toxin antibody or antigen-binding fragment thereof has been produced, it may be isolated or purified by any method known in the art for isolation or purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen, by Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the isolation or purification of proteins.Cells Expressing Anti-Uremic Toxin Antibodies

[0102] A further aspect of the disclosure is directed to a cell expressing an anti -uremic toxin antibody or an antigen-binding fragment thereof according to the present disclosure.

[0103] Cells that express an anti -uremic toxin antibody or antigen binding fragment thereof may comprise a nucleic acid molecule or vector encoding the anti -uremic toxin antibody or antigen-binding fragment thereof, as described herein.

[0104] The cells according to the present disclosure may be eukaryotic or prokaryotic cells. Exemplary eukaryotic cells include mammalian cells, yeast cells, insect cells, and plant cells.

[0105] Suitable exemplary mammalian cells include, without limitation, Chinese Hamster Ovary (CHO) cells (e.g, CHO, CHO-S, CHO-K1, and CHO-DG44 cell lines), Human Embryonic Kidney (HEK) 293 cells (e.g, HEK293 cells, HEK293T cells, HEK293F cells, HEK293-6E cells, and Expi293F™ cells), PER.C6® cells, Baby Hamster Kidney (BHK) cells (e.g., BHK21 cells), HeLa cells, Vero cells, and mouse myeloma cells such as NS0 cells and SP2 / 0 cells (see, e.g., Li et al, “Cell Culture Processes for Monoclonal Antibody Production,” Mabs 2(5):466-477 (2010), which is hereby incorporated by reference in its entirety).

[0106] Suitable exemplary yeast cells include, without limitation, Saccharomyces cerevisiae mA Pichia pastoris (see, e.g., Wang et al., “Expression of Antibody Fragments in Saccharomyces cerevisiae Strains Evolved for Enhanced Protein Secretion,” Microbial Cell Factories 134 (2021) and Nylen and Chen, “Production of Full-Length Antibody by Pichia pastoris,” Methods Mol. Biol. 1674:37-48 (2018), which are hereby incorporated by reference in their entirety).

[0107] Suitable exemplary insect cells include, without limitation, Spodoptera frugiperda and cell lines derived from Spodoptera frugiperda (e.g., Sf9 cells, Sf21 cells, and SfSWT-1 (Mimic™) cells insect cell lines derived from Trichoplusia ni cells and cell lines derived from Trichoplusia ni (e.g., BTI-Tn-5Bl-4 (High Five) cells), and Bombyx mori cells (see, e.g., Palmberger et al., “Insect Cells for Antibody Production: Evaluation of An Efficient Alternative,” J. BiotechnoL 153(3-4): 160-166 (2011), which is hereby incorporated by reference in its entirety).

[0108] Suitable exemplary plant cells include, without limitation, Nicotiana benthamiana, Nicotiana tabacum, Lycopersicon esculentum, Arabidopsis thaliana, Oryza sativa, and Zea mays (see, e.g., Shen et al., “Plant-Derived Single Domain COVID-19 Antibodies,” J. Controlled Release 359: 1-11 (2023); Pogrebnyak et al., “Severe acute respiratory syndrome (SARS) S protein production in plants: Development of recombinant vaccine,” Plant Biology102(25):9062-907 (2005); and Yusibov et al., “Antibody Production in Plants and Green Algae,” Annu. Rev. Plant Biol. 29:67:699-701 (2016), which are hereby incorporated by reference in their entirety).

[0109] The prokaryotic cells may be gram-positive cells or gram-negative cells. Suitably exemplary prokaryotic cells include, without limitation, Escherichia coli cells, Aeromonas hydr ophila cells, A. salmonicida cells, Bordetella pertussis cells, Borrelia burgdorferi cells, Borrelia spp. cells, Brucella abortus cells, B. canis cells, B. melitensis cells, B. susi cells, Burkholderie pseudomallei cells, Campylobacter jejuni cells, C. fetus cells, C. intermedis cells, Chlamydia psittaci cells, C. pneumoniae cells, C. trachomatis cells, Francisella tularenisis cells, Haemophilus influenzae cells, H. pleuropneumonias cells, Helicobacter pylori cells, Klebsiella pneumoniae cells, Legionella pneumophila cells, Leptospira interrogans cells, Neisseria gonorrhoeae cells, N. meningitides cells, Pasteurella haemolytica cells, P. mirabilis cells, P. morganii cells, P. multilocida cells, Porphyromonas (Bacteroides) gingivalis cells, Proteus vulgaris cells, Pseudomonas aeruginosa cells, Pseudomonas mallei cells, Rickettsia prowazeki cells, R. richettsii cells, R. typhi cells, Salmonella gastroenteritis (typhimirium) cells, S. dysenteriae cells, S. enter iditis cells, Shigella flexneri cells, S. sonnie cells, S. typhi cells, Spirillum minus cells, Treponema pallidum cells, T. denticola cells, T. orales cells, Vibrio cholerae cells, Vibrio parahaemolyticus cells, and Yersinia pestis cells (see, e.g., Frenzel et al., “Expression of Recombinant Antibodies,” Front. Immunol. 4:217 (2013), which is hereby incorporated by reference in its entirety).

[0110] Methods for transforming / transfecting host cells with expression vectors are well- known in the art and depend on the host system selected, as described in SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 2001), which is hereby incorporated by reference in its entirety. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or any other viral vector. For insect cells, the transfer vector containing the polynucleotide construct of the present invention is co-transfected with baculovirus DNA, such as AcNPV, to facilitate the production of a recombinant virus resulting from homologous recombination between the scaffold-encoding polynucleotide construct in the transfer vector and baculovirus DNA. Subsequent recombinant viral infection of Sf cells results in a high rate of recombinant protein production.[oni] The cells may be stably or transiently transfected with a nucleic acid sequence comprising a nucleotide sequence encoding an anti-uremic toxin antibody or antigen-binding fragment thereof.

[0112] In some embodiments, the cells are engineered to constitutively express an anti- uremic toxin antibody or antigen-binding fragment thereof.

[0113] In some embodiments, the cells are engineered such that expression of the anti- uremic toxin antibody or antigen-binding fragment thereof may be induced.Methods of Producing Anti-Uremic Toxin Antibodies

[0114] Methods of producing an anti-uremic toxin antibody or antigen-binding fragment thereof according to the present disclosure can be carried out using the present disclosure combined with knowledge known and available to persons of skill in the art.

[0115] In some embodiments, the method comprises culturing a cell(s) containing a nucleic acid sequence comprising a nucleotide sequence encoding an antibody or antigenbinding fragment thereof in a suitable medium such that the protein is produced. In some embodiments, the method further comprises isolating the protein from the medium. In certain embodiments, an anti -uremic toxin antibody or antigen-binding fragment thereof is produced in a manner described herein.

[0116] In at least several of the various uses of the polypeptides of the embodiments described herein, it is often desirable for the polypeptides to be produced in substantially purified form, particularly when their administration to a patient is contemplated. Regardless of the expression system and host cell used to facilitate protein production, the expressed polypeptides and fusion proteins of the present disclosure can be readily purified using standard purification methods known in the art and described in PHILIP L. R. BONNER, PROTEIN PURIFICATION (Routledge 2007), which is hereby incorporated by reference in its entirety. Thus, the anti- uremic toxin antibody or antigen-binding fragment thereof may be purified by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen, by Protein A, and sizing column chromatography), centrifugation, differential solubility, and / or by any other standard technique for the isolation or purification of proteins. Accordingly, in some embodiments of the present disclosure, the anti-uremic toxin antibody or antigen-binding fragment thereof described herein is purified.

[0117] In some embodiments, the terms “purified” and “isolated” when used in the context of a protein that is obtained from cells refers to a protein which is substantially free of contaminating materials, e.g. cell debris, cell wall materials, membranes, organelles, the bulk ofthe nucleic acids, carbohydrates, proteins, and / or lipids present in cells. Thus, in some embodiments, an antibody that is isolated includes preparations of an antibody having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellular materials and / or contaminating materials. In some embodiments, an anti -uremic toxin antibody or antigenbinding fragment thereof as described herein is purified or isolated.

[0118] As an alternative to recombinant expression of an anti -uremic toxin antibody or antigen-binding fragment thereof described herein using a cell, an expression vector containing a polynucleotide encoding an anti -uremic toxin antibody or antigen-binding fragment thereof can be transcribed and translated in vitro using, e.g., T7 promoter regulatory sequences and T7 polymerase. In a specific embodiment, a coupled transcription / translation system, such as Promega TNT®, or a cell lysate or cell extract comprising the components necessary for transcription and translation may be used to produce an anti-uremic toxin antibody or antigenbinding fragment thereof.Dialysis Fluid Compositions

[0119] Another aspect of the present disclosure is directed to a dialysis fluid composition comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier and / or excipient.

[0120] The terms “dialysis composition” or “dialysis fluid composition” are used interchangeably and refer to a specialized formulation used in the process of dialysis. A dialysis composition typically includes a solution or medium that facilitates the exchange of solutes and water between the blood and the dialysis fluid across a semipermeable membrane.

[0121] Suitable anti-uremic toxin antibodies and antigen-binding fragments thereof are described in detail infra. In some embodiments, the anti -uremic toxin antibody binds indoxyl sulfate (IS), p-cresyl sulfate (pCS), or both indoxyl sulfate (IS) and p-cresyl sulfate (pCS).

[0122] The dialysis fluid compositions according to the present disclosure may comprise one or more of a variety of pharmaceutically acceptable carriers and / or excipients (see, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (21stEdition) (2005) (Troy, D.B. el al. (Eds.) Lippincott Williams & Wilkins (Pubis.), Baltimore MD), which is hereby incorporated by reference in its entirety). Except insofar as any conventional carrier or excipient is incompatible with the anti-uremic antibody according to the present disclosure, use thereof in the pharmaceutical compositions of the present disclosure is contemplated.

[0123] Suitable pharmaceutically acceptable carriers and excipients include, without limitation, diluents, fillers, salts, electrolytes, buffers, detergents (e.g., a nonionic detergent, suchas Tween-20 or Tween-80), stabilizers e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition. Exemplary electrolytes include sodium, potassium, calcium, magnesium, and chloride. Exemplary diluents are distilled water, physiological phosphate-buffered saline, Ringer’s solutions, dextrose solution, and Hank’s solution. In addition, the dialysis fluid composition may also include other carriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers and the like. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the present disclosure include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, com oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and / or various buffers. Other carriers are well-known in the pharmaceutical arts.

[0124] In some embodiments, the pharmaceutically acceptable carrier or excipient comprises a buffer. Suitable buffers include bicarbonate buffers, lactate buffers, citrate buffers, and acetate buffers.

[0125] In some embodiments, the pharmaceutically acceptable carrier or excipient comprises an electrolyte. Suitable electrolytes may be selected from the group consisting of sodium, potassium, calcium, magnesium, and chloride, in concentrations similar to those found in healthy human blood.

[0126] In some embodiments, the pharmaceutically acceptable carrier or excipient comprises an isotonicity agent. Suitable isotonicity agents include, without limitation, glucose (dextrose), icodextrin, glycerin, mannitol, sorbitol, glycerol, potassium chloride, and sodium chloride.

[0127] The dialysis fluid compositions according to the present disclosure may also contain one or more preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the dialysis fluid composition.

[0128] In some embodiments of the dialysis fluid compositions according to the present disclosure, the pharmaceutically acceptable carriers and / or excipients comprise purified water, electrolytes, buffers, and / or additional molecules such as glucose (see, e.g., McGill and Weiner, “Dialysate Composition for Hemodialysis: Changes and Changing Risk,” Semin. Dial.3Q(iy.112-120 (2017) and Locatelli, “Optimal Composition of the Dialysate, with Emphasis onits Influence on Blood Pressure,” Nephrology Dialysis Transplantation 19(4): 785-796 (2004), which are hereby incorporated by reference in their entirety).Methods of Removing or Reducing Uremic Toxins from Blood

[0129] Another aspect of the present disclosure is directed to a method of removing or reducing one or more uremic toxins from blood. This method involves contacting blood with a dialysis fluid composition comprising an antibody, or antigen-binding fragment thereof, according to the present disclosure; and removing uremic toxins from the blood by dialyzing the blood through a membrane, where the membrane has a molecular weight cutoff smaller than the molecular weight of the uremic toxin antibody or antigen-binding fragment thereof.

[0130] In some embodiments of the methods of removing or reducing one or more uremic toxins from the blood according to the present disclosure, contacting is carried out by dialysis. The term “dialysis” refers to the process in which soluble waste products are separated from the blood using a semipermeable membrane.

[0131] Suitable dialysis fluid compositions and anti -uremic toxin antibodies for use in the methods of removing or reducing one or more uremic toxins from the blood according to the present disclosure are described infra.

[0132] In some embodiments, the anti -uremic toxin antibody binds indoxyl sulfate (IS), p-cresyl sulfate (pCS), or both indoxyl sulfate (IS) and p-cresyl sulfate (pCS).

[0133] In some embodiments, the anti -uremic toxin antibody has a molecular weight of 140 kDa to 160 kDa. In some embodiments, the anti -uremic toxin antibody has a molecular weight of about 150 kDa. Thus, in some embodiments, when the method involves contacting blood with a dialysis composition comprising an anti-uremic toxin antibody according to the present disclosure, the membrane has a molecular weight cutoff (MWCO) smaller than 140 kDa. Accordingly, the membrane may have a molecular weight cutoff of 130 kDa, 120 kDa, 110 kDa, 100 kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, or any amount therebetween.

[0134] In some embodiments, the antigen-binding fragment of the anti -uremic toxin antibody is an (Fab)2 fragment having a molecular weight of about 110 kDa, a minibody having a molecular weight of about 75 kDa, a diabody having a molecular weight of about 50 kDa, a Fab having a molecular weight of about 50 kDa, an scFv having a molecular weight of about 28 kDa, a sdAb having a molecular weight of about 12-15 kDa, or an affibody having a molecular weight of about 6.5 kDa (see, e.g., Herrington- Symes et al., “Antibody Fragments: Prolonging Circulation Half-Life Special Issue-Antibody Research,” Advances in Bioscience andBiotechnology 4(5):689-698 (2013); Tang et al., Application Progress of the Single Domain Antibody in Medicine,” Int. J. Mol. Sci. 24(4):4176 (2023); and Zhang and Zhang, “Recent Advances of Affibody Molecules in Biomedical Applications,” Bioorg. Med. Chem.1 : 113: 117923 (2024), which is hereby incorporated by reference in its entirety). In accordance with such embodiments, the membrane may have a molecular weight cutoff of 100 kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, or any amount therebetween, provided that the MWCO is less than the molecular weight of the antigen-binding fragment of the anti -uremic toxin antibody.

[0135] In some embodiments, the membrane is polymeric membrane. The membrane may comprise bisphenol A (BP A), cellulose triacetate (CTA), ethylene vinyl alcohol copolymer (EVAL), polyamide (PA), polyarylethersulfone (PAES), polyethersulfone (PES), polymethylmethacrylate (PMMA), polyvinylpyrrolidone (PVP), and / or polysulfone (PSf) (see, e.g., Said et al., “A Review of Commercial Developments and Recent Laboratory Research of Dialyzers and Membranes for Hemodialysis Application,” Membranes (Basel) 11(10):767 (2021), which is hereby incorporated by reference in its entirety).

[0136] As described herein, the method is effective to remove one or more uremic toxins from the blood. In some embodiments, the method is effective to remove indoxyl sulfate (IS) and / or p-cresyl sulfate (pCS).Treatment Methods

[0137] Another aspect of the present disclosure is directed to a method of treating a subject having a kidney disease or condition. This method involves selecting a subject having a kidney disease or condition; and dialyzing a portion of the subject’s blood against a composition comprising an antibody, or an antigen-binding fragment thereof, according to the present disclosure, where said dialyzing is sufficient to remove one or more uremic toxins from the subject’s blood, thereby treating the subject having the kidney disease or condition.

[0138] As used herein, the terms “subject” or “patient” are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired. Subjects suitable for treatment in accordance with the methods described herein will vary and may include but are not limited to e.g., subjects suspected of having a disease or condition mediated by uremic toxins such as a kidney disease or condition.

[0139] A “kidney disease or condition” refers to any disorder or dysfunction affecting the kidneys. Kidney diseases can range from acute conditions to chronic diseases such as chronic kidney disease (CKD) and end-stage renal disease (ESRD).

[0140] In some embodiments, the subject has a chronic kidney disease (CKD). CKD is a progressive condition characterized by the gradual loss of kidney function over time. The kidneys, essential for filtering waste and excess fluids from the blood, become less effective, leading to the accumulation of waste products in the body. CKD is classified on the basis of changes in renal architecture and estimated glomerular filtration rate (eGFR) (Foresto-Neto et al., “Immunology of Kidney Disease,” Annu. Rev. Immunol. 42:207-233 (2024), which is hereby incorporated by reference in its entirety). Early stages of CKD are often asymptomatic and later stages present symptoms such as fatigue, swelling, and changes in urination (Chen et al., “Chronic Kidney Disease Diagnosis and Management,” JAMA 322(13): 1294-1304 (2019), which is hereby incorporated by reference in its entirety).

[0141] In some embodiments, the subject has end-stage renal disease (ESRD). ESRD is the final stage of CKD, where the kidneys have lost nearly all their ability to function effectively, resulting in a severely compromised ability to filter waste and excess fluids from the blood. At this stage, renal replacement therapy becomes necessary, either through dialysis or kidney transplantation (Wouk, N., “End-Stage Renal Disease: Medical Management,” Am. Fam. Physician 104(5):493-499 (2021), which is hereby incorporated by reference in its entirety). ESRD presents more pronounced symptoms, including severe fatigue, swelling, shortness of breath, nausea, and confusion.

[0142] In some embodiments, the subject is a mammalian subject. The terms “mammal” or “mammalian subject” for purposes of the methods described herein refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, non-human primates, rodents, etc. In some embodiments, the mammalian subject is a human subject. The human subject may be an infant, a child, an adolescent, an adult, or a geriatric subject.

[0143] As used herein, “treating” a subject having a disease or condition (e.g., a kidney disease or condition) and symptomatic of that disease or condition includes, (i) slowing, stopping or reversing the progression of one or more of the symptoms, (ii) slowing, stopping or reversing the progression of illness underlying such symptoms, (iii) reducing or eliminating the likelihood of the symptom’s recurrence, and / or (iv) slowing the progression of, lowering or eliminating the disease or condition.

[0144] In some embodiments, treating reduces the amount of a uremic toxin in a subject’s blood. For example, treating may reduce the amount of a uremic toxin in a subject’s blood by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any amount therebetween. In some embodiments,treating reduces the amount of a uremic toxin in a subject’s blood by at least about 15%. In some embodiments, treating reduces the amount of a uremic toxin in a subject’s blood by at least about 20%. In some embodiments, treating reduces the amount of a uremic toxin in a subject’s blood by at least about 25%. In some embodiments, treating reduces the amount of a uremic toxin in a subject’s blood by at least about 30%. In some embodiments, treating reduces the amount of a uremic toxin in a subject’s blood by at least about 35%.

[0145] Treating may result in one or more desirable clinical results including reduction of risk of mortality.

[0146] Dialyzing a portion of the subject’s blood against a composition comprising a uremic toxin antibody according to the present disclosure, or antigen-binding fragment thereof, as disclosed herein, can be carried out using peritoneal dialysis or hemodialysis.

[0147] In some embodiments, the dialyzing is carried out by peritoneal dialysis. Peritoneal dialysis operates within the body, utilizing the peritoneal membrane as a semipermeable barrier for blood filtration (Vries et al., “Evidence on Continuous Flow Peritoneal Dialysis: A Review,” Semin. Dial. 35(6):481-497 (2022), which is hereby incorporated by reference in its entirety), which is hereby incorporated by reference in its entirety). In this method, the dialysate is infused directly into the patient's peritoneal cavity through a catheter, with the catheter serving as a reservoir for the dialysate. Toxins in the blood filter through the peritoneal membrane into the cleansing solution, which is subsequently removed from the body through the same catheter and disposed of.

[0148] In some embodiments, said dialyzing is carried out by hemodialysis.Hemodialysis facilitates the extracorporeal removal of water and solutes from the blood through diffusion across a concentration gradient. Blood is pumped along one side of a semi-permeable membrane and a crystalloid solution is pumped in the opposite direction on the other side of the membrane. Solutes with very small molecular weight diffuse across the membrane in an attempt to equilibrate their concentrations. The pore size in the semi-permeable membrane determines its utility in ultrafiltration. Ultrafiltration membranes that are utilized in hemofilters allow the passage of molecules with a molecular weight of less than 20,000 Daltons. Thus, ions and small chemicals present in plasma are filtered freely, including sodium, potassium, phosphate, bicarbonate, glucose and ammonia. So are larger soluble endogenous substances such as myoglobin, insulin, and interleukins, and certain exogenous substances circulating in plasma, including medications (vancomycin, heparin) and toxins (endotoxin, pesticides).

[0149] Suitable anti-uremic toxin antibodies for use in the methods according to the present disclosure are described in detail infra.

[0150] Uremic toxins are described in detail infra. In some embodiments, the methods of treating a subject having a kidney disease or condition is sufficient to remove one or more uremic toxins from the subject’s blood, where the one or more uremic toxins are selected from the group consisting of indoxyl sulfate (IS) and / or p-cresyl sulfate (pCS).Diagnostic Methods

[0151] Another aspect of the present disclosure is directed to a method of detecting a uremic toxin. This method involves analyzing a biological sample in an immunoassay comprising an anti -uremic toxin antibody, or an antigen-binding fragment thereof, according to the present disclosure. This method further involves determining, based on said analyzing, whether the biological sample contains one or more uremic toxins.

[0152] The term “immunoassay” refers to a biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample. It is common to use the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a uremic toxin. Both the presence of antigen and the amount of antigen present can be measured. The presence and amount (z.e., abundance) of an antigen (e.g., a uremic toxin) can determined or measured. Measuring the quantity of an antigen (such as a uremic toxin) can be achieved by a variety of methods. A common method is to label either the antigen or antibody with a detectable label (e.g., a fluorescent tag, enzymatic linkage or radioactive isotope).

[0153] An exemplary diagnostic assay for an anti -uremic toxin comprises, e.g., contacting a sample obtained from a subject (e.g., a subject having CKD or ESRD) with an anti- uremic toxin antibody according to the present disclosure, where the anti-uremic toxin antibody is labeled with a detectable label or reporter molecule. Alternatively, it is used as a capture ligand for the selective isolation of anti-uremic toxins from a patient sample. Alternatively, the unlabeled anti-uremic toxin antibody is used in diagnostic applications in combination with a secondary antibody that is self-detectably labeled. Detectable label or reporter molecules include radioisotopes; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine; enzymes such as alkaline phosphatase, P-galactosidase, horseradish peroxidase, or luciferase. Specific examples of assays that can be used to detect or measure uremic toxins in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-labeled cell fractionation (FACS).

[0154] In some embodiments, the immunoassay is an enzyme-linked immunosorbent assay. The terms “enzyme-linked immunosorbent assay” and “ELISA” are used interchangeably and refer to an assay that uses a solid-phase type of enzyme immunoassay (EIA). In ELISAassays, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates the separation of bound and free-labeled reactants.

[0155] Specific uremic toxins in a sample can be quantified using an ELISA procedure in which a first anti -uremic toxin antibody is bound to a solid phase. A sample may then be contacted with the solid phase to allow binding of any uremic toxins, if present, to bind to the anti -uremic toxin antibodies. Any unbound anti -uremic toxins may be removed by one or more washing steps and bound anti -uremic toxins may be linked to a second anti -uremic toxin antibody comprising an enzyme. In the final step, a substance containing the enzyme’s substrate is added. If there the sample contained an anti -uremic toxin, the subsequent reaction between the enzyme and substrate produces a detectable signal, typically a color change. There are variations of ELISA tests based on how the analytes and antibodies are bonded and used which include Direct ELISA, Sandwich ELISA, Competitive ELISA and Reverse ELISA.

[0156] In some embodiments, the first and / or second anti-uremic toxin antibody is an antibody as disclosed herein.

[0157] Suitable biological samples that can be used in the methods of detecting a uremic toxin according to the present disclosure include any tissue or fluid sample obtainable from a patient. In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, serum, urine, saliva, sputum, bronchial lavage fluid, tears, and stool. In some embodiments, the biological sample is a biofluid sample (e.g., whole blood, plasma, serum, saliva, or urine). In some embodiments, the biological sample is serum.

[0158] In some embodiments, a level of uremic toxin(s) in a particular sample obtained from a healthy subject (e.g., a subject not afflicted with a disease or condition associated with a uremic toxin) is analyzed to determine a baseline, or standard, level of the uremic toxin(s). This baseline level of uremic toxin can then be compared against the levels of uremic toxin measured in samples obtained from individuals suspected of having a uremic toxin-associated condition, or symptoms associated with such condition.

[0159] In some embodiments, a level of uremic toxin(s) in a particular sample obtained from a subject having a disease or condition associated with a uremic toxin at a first time point is analyzed to determine a baseline, or standard, level of the uremic toxin. This baseline level of uremic toxin is compared against a sample obtained from the same subject at a later time point (or samples obtained from the same subject at later time points) to detect the progression of the disease or condition. Thus, in some embodiments, the methods of detecting a uremic toxin conducting an immunoassay described herein (e.g., an ELISA assay) provide methods formonitoring the progression of a disease in a subject having a kidney disease or condition such as CKD or ESRD.

[0160] In some embodiments, a level of uremic toxin(s) in a particular sample obtained from a subject having a disease or condition associated with a uremic toxin at a first time point is analyzed to determine a baseline, or standard, level of the uremic toxin prior to a treatment for the disease or condition. This baseline level of uremic toxin is compared against a sample obtained from the same subject at a later time point (or samples obtained from the same subject at later time points) after the treatment for the disease or condition is administered to the subject, to detect the effectiveness of the treatment for the disease or condition. Thus, in some embodiments, the methods of detecting a uremic toxin conducting an immunoassay described herein (e.g., an ELISA assay) provide methods for monitoring the effectiveness of a treatment for a kidney disease or condition such as CKD or ESRD.

[0161] In some embodiments, conducting an immunoassay described herein provides a method for identifying subject (e.g., human subjects) with specific levels of uremic toxins.

[0162] In some embodiments, a subject identified as having toxins that bind to the anti- uremic toxin antibodies according to the present disclosure may be treated according to the methods described herein, by contacting the patient’s blood with a dialysis composition comprising an anti -uremic toxin antibody according to the present disclosure.

[0163] Conducting an immunoassay described herein provides a method for assessing the efficacy of subjects receiving treatment for CKD or ESRD (e.g., human subjects with CKD or ESRD). For example, conducting an immunoassay described herein provides a method for assessing the efficacy of subjects receiving dialysis treatment with a dialysis composition comprising an anti -uremic toxin antibody according to the present disclosure (e.g., human subjects with CKD or ESRD).

[0164] In some embodiments, conducting an immunoassay described herein provides a method for assessing kidney function in subjects having CKD or ESRD.

[0165] In some embodiments, conducting an immunoassay described herein provides a method for assessing kidney function in a subject being treated for CKD or ESRD.EXAMPLES

[0166] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.Materials and MethodsMaterials

[0167] Slide-A-Lyzer™ MINI dialysis devices (3.5 kDa molecular-weight cut-off, Thermo Scientific) and Minimate™ tangential -flow-filtration (TFF) cartridges (10 kDa cut-off, Pall Corporation) were used for dialysis experiments. Acetonitrile (HPLC grade), indoxylsulfate potassium (ISK), and p-cresol sulfate were purchased from Sigma- Aldrich. A Pharmacia LKB P-1 peristaltic pump (Pharmacia) was employed for continuous dialysis.Generation and Sequencing of Monoclonal Antibodies (mAbs)

[0168] Five hybridoma lines were produced as a fee-for-service by ABclonal (Woburn, MA) using standard hybridoma technology. Total RNA was extracted from each hybridoma, reverse-transcribed to cDNA, and the antibody heavy- and light-chain variable regions were amplified by nested PCR. The heavy-chain variable region was cloned into a human IgGl backbone with Agel and Sall, and the K-chain variable region was cloned into a human K backbone with Agel and BsiWI.Antibody Expression and Purification

[0169] Hybridoma-derived mAbs were harvested from culture supernatants and purified on protein-G agarose (Cytiva, Marlborough, MA) following the manufacturer’s protocol.Chimera (mouse-human) antibodies were expressed in FreeStyle™ 293F cells (Thermo Fisher Scientific, Cat. R790-07). Cells were transiently transfected with equal masses of heavy- and light-chain plasmids using 25-kDa linear polyethyleneimine (PEI; Polysciences, Cat. 23966). Cultures were shaken at 125 rpm in FreeStyle™ 293 expression medium at 37°C / 5 % CO2. Supernatants were harvested five days post-transfection, clarified (0.22 pm), and purified on protein A / G agarose. Binding affinities to IS were assessed by ELISA with IS-conjugated bovine serum albumin (BSA-IS).Enzyme-Linked Immunosorbent Assay (ELISA)

[0170] Ninety-six-well plates were coated overnight at 4°C with BSA-IS (1 pg mL1). After blocking with 5 % w / v non-fat milk (1 h, room temperature), serial three-fold dilutions of antibody (starting at 10 pg mL1) were added and incubated for 1 hour at 37°C. Plates were washed and alkaline-phosphatase-conjugated anti-mouse or anti-human IgG (1 :2000) was applied for 1 hour at 37°C. After washing, p-nitrophenyl phosphate substrate (Thermo Fisher Scientific) was added, and absorbance was read at 405 nm on a BioTek Synergy 2 plate reader.Fab Preparation and Purification

[0171] Fab fragments were generated by papain digestion. IgG and papain (Worthington) were mixed at a 10: 1 molar ratio in 20 mM Tris-HCl, pH 6.8, 100 mM NaCl, supplemented with 20 mM cysteine-HCl and 0.1 M EDTA, pH 8.0. The reaction proceeded for 1 hour at 37°C and was quenched with 10 mM iodoacetamide (Bio-Rad). Fabs were separated from Fc fragments on a HiTrap protein A column, then polished by size-exclusion chromatography and concentrated to > 5 mg mL1for crystallization.Crystallization, Data Collection, and Structure Determination

[0172] Concentrated Fab was mixed with IS at a 1 : 10 molar ratio. Crystals of aIS-02 grew by hanging-drop vapor diffusion in 20 % (w / v) PEG 3350 and 0.2 M potassium sodium tartrate tetrahydrate. Diffraction data were collected at 100 K (X = 0.979 A) on NSLS-II beamline 17-ID-l (AMX). Data were processed with XDS, and structures were solved by molecular replacement (Table 1) and refined iteratively in COOT and PHENIX. Structural analyses used ICM; figures were prepared in Chimera and PyMOL.Static Mini-Dialysis Removal of ISK

[0173] For static dialysis, 100 pL ISK (0.1 mg mL ’) in PBS was loaded into a 3.5 kDa MINI dialysis cup and immersed in 2 mL antibody solution (0.5 mg mL1aIS-02) in a 24-well plate; BSA or isotype-matched IgG served as controls. After 4 hours at room temperature, dialysate samples were analyzed by HPLC. Fractional removal was calculated as:

[0174] Dialysis was also performed with ISK dissolved in fetal bovine serum (FBS) to mimic end-stage renal -disease (ESRD) plasma.TFF Dialysis Removal of ISK

[0175] TFF experiments followed the manufacturer’s protocol. ISK (0.1 mg mL1in 100 mL 10 % FBS; 460 pM) was circulated through the feed line (25 mL min1); aIS-02 (34.5 ug mL1in 100 mL PBS; 0.23 pM) was circulated through the vent line (3 mL min1). Controls used BSA or isotype-matched antibody. Samples were collected for HPLC at the indicated times. A low-flow setting (feed 7 mL min-1, vent 3 mL min1) was also tested to mimic clinical dialysis, with samples taken at 1, 2, and 3 hours.HPLC Sample Preparation and Analysis

[0176] For PBS samples, dialysate was injected directly. For FBS / 10 % FBS samples, proteins were precipitated with three volumes of acetonitrile, centrifuged (10,000xg, 3 min), and the supernatant was extracted with an equal volume of 5 N NaCl; the organic phase was analyzed. Chromatography was employed on a Kinetex 5 pm C18 100 A column (150 x 4.6 mm, Phenomenex). Mobile phase A: 20 mM sodium formate, pH 4; phase B: acetonitrile. Gradient: 15 % B (0-2 min) — 100 % B (2-6 min) — 15 % B (6-8 min) at 1 mL min-1. Injection volume was 30 pL. Each sample was run in triplicate; ISK peak area at 280 nm was quantified against a 1 mg mL1ISK standard.Statistics

[0177] Data were analyzed in GraphPad Prism 10.3.1. ELISA binding curves were fitted with a specific-binding model including Hill slope. ISK-reduction values were expressed as percent change relative to baseline.Example 1 - Development of IS-Specific Monoclonal Antibodies (mAbs)

[0178] Mice were immunized with indoxyl-sulfate-bovine-serum-albumin conjugate (IS- BSA), and hybridoma supernatants were screened by ELISA. The five highest-affinity hybridoma clones (designated aIS-01 to aIS-05) were sequenced, and their variable domains were subcloned into human IgGl expression vectors for recombinant production. After small- scale expression and purification, the three best-performing antibodies (aIS-01, -02, and -03) were selected for detailed study (FIGS. 1 A-1B).Example 2 - Structural Characterization of IS bound to aIS-02

[0179] To elucidate the molecular basis of IS recognition, Fab fragments of aIS-02 were crystallized with IS and the structure solved at 2.26 A resolution (FIGS. 2A-2D). IS is deeply buried in the antigen-binding pocket (FIG. 2C, with ~62 % of its solvent-accessible surface area (168 A2of 269 A2) occluded. The sulfate moiety is anchored by the guanidinium groups of Arg98 (inter-atomic distances 2.85 A and 3.06 A, respectively) and by the amide nitrogen of ArglOO (3.03 A), as well as a conserved water molecule (2.56 A) (FIG. 2D). The indole ring is stabilized by a hydrophobic triad consisting of Leu47 and Tyr50 (from the frame region before CDR L2) and Tyrl02 (CDR H3). These interactions account for the high specificity and affinity of aIS-02 for IS.Example 3 - Complex Structure of aIS-02 with p-cresyl sulfate (pCS)

[0180] Given the chemical similarity between IS and pCS, Fab aIS-02 was next co- crystalized with pCS and obtained a 2.20 A structure (FIGS. 3A-3B). Two pCS conformations occupy the binding site. Conformation 1 overlaps the IS position: the benzene ring replaces the indole ring, and the sulfate group is displaced by ~1.5 A. In conformation 2, the benzene ring rotates -60°, projecting the sulfate moiety toward solvent. ArglOO (CDR H3) re-orients to form a cation-7t interaction that further stabilizes both aromatic rings. The structural data suggest that conformation 1 is energetically favored and provide a blueprint for engineering pCS-selective variants of aIS-02.Example 4 - Removal of ISK in a Static Mini-Dialysis Model

[0181] The ability of aIS-02 to deplete IS was first assessed in a static mini-dialysis assay (FIG. 4A). IS potassium salt (ISK, 100 pg mL1; 460 pM) in PBS was loaded (100 pL) into 3.5 kDa cut-off dialysis cups and dialyzed against 2 mL antibody solution (0.5 mg mL1; 3.3 pM) for 4 hours at room temperature. HPLC quantification showed that aIS-02 reduced ISK to 66 % of its initial level (FIG. 4A), outperforming a mock IgG and BSA controls. Repeating the experiment with 100 % fetal bovine serum (FBS) to mimic plasma yielded a similar 67 % residual ISK (FIG. 4B).Example 5 - Removal of Indoxyl Sulfate Potassium Salt (ISK) in a Tangential-Flow- Filtration (TFF) Model

[0182] To mimic clinical hemodialysis, we employed a 10 kDa Minimate™ TFF cartridge. The feed stream contained 100 mL ISK (0.1 mg mL1; 460 pM) in 10 % FBS; the dialysate stream contained 100 mL aIS-02 (34.5 pg mL '; 0.23 pM) in PBS, giving an ISK:mAb molar ratio of -2000 : 1. At 25 mL min1feed and 3 mL min1dialysate flow (<10 psi), aIS-02 removed 35 % of ISK in 1 hour, significantly better than mock IgG or BSA (FIG. 5A).Reducing the feed flow to 7 mL min1to simulate clinical dwell times showed progressive clearance: -15 %, 44 %, and 60 % removal at 1, 2, and 3 hours, respectively (FIG. 5B). These data indicate that aIS-02 can efficiently capture IS under dialysis-like conditions and highlight its potential for antibody-facilitated toxin removal in CKD therapy.Discussion of Examples 1-5

[0183] Examples 1-5 demonstrate the generation of a novel panel of mAbs that recognize albumin-bound indoxyl sulfate with high affinity and specificity. Three lead candidates (aIS-01, -02, -03) were expressed recombinantly, and detailed structural and functional analyses wereperformed on aIS-02. X-ray crystallography revealed a deep, positively charged pocket that sequesters the sulfate group while hydrophobic residues cradle the indole ring, explaining the nanomolar binding we observed by ELISA. Functionally, aIS-02 removed up to 34 % of IS in a static mini-dialysis assay and ~60 % in a tangential-flow-filtration (TFF) model that mimics clinical hemodialysis, outperforming both BSA and a mock IgG control. Together, these data establish a proof-of-concept for antibody-facilitated clearance of albumin-bound IS under dialysis-like conditions.

[0184] Beyond confirming the molecular basis of IS recognition, the high resolution IS and pCS complex structures furnish atomic-level guidance for antibody redesign. The pCS structure shows two alternative ligand conformations, one nearly isosteric with IS and a second rotated by -60°, stabilized by a cation-7t contact from ArglOO (CDR H3). These observations pinpoint residues whose side-chain repositioning or mutation could either enhance IS affinity (e.g., deepening the hydrophobic triad around Tyrl02) or shift selectivity toward pCS (e.g., enlarging the pocket to favor the benzene ring orientation while realigning basic residues for optimal sulfate coordination). Structure-based saturation mutagenesis, complemented by in- silico affinity predictions, should therefore enable the creation of a pCS-specific mAb as well as bispecific or multi-specific formats targeting several toxins in parallel — an attractive route to broaden therapeutic coverage against the uremic toxin spectrum.

[0185] Antibody-facilitated toxin removal could be integrated into existing dialysis workflows in two principal ways: (i) immobilizing mAbs on cartridges or (ii) adding mAbs to the dialysate bath. In either configuration, the antibody remains extracorporeal, minimizing immunogenicity and simplifying regulatory hurdles compared with systemic biologies. The advantages of using antibodies to improve IS reduction during dialysis include: antibodies are highly specific and can be engineered to improve binding; antibodies should not provoke the side effects seen with small-molecule displacers, nor the clotting complications observed with nonspecific sorbents. Future steps may include affinity maturation to target other albumin-bound toxins, scale-up of GMP-grade antibody production, assessment of column capacity and reuse, and in-vivo efficacy studies in CKD animal models and, ultimately, early-phase clinical trials. If successful, mAb -facilitated sorbent therapy could become a targeted, adjunctive strategy to enhance toxin clearance during dialysis, potentially mitigating cardiovascular and immunological morbidity for ESRD patients.

[0186] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions,substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. An antibody, or antigen-binding fragment thereof, specific for a uremic toxin, wherein the antibody or antigen-binding fragment thereof comprises:(a) a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, or variants of any of the foregoing; and a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 5, or variants of any of the foregoing;(b) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 10, or variants of any of the foregoing; and a light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13, or variants of any of the foregoing; or(c) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, or variants of any of the foregoing; and a light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21, or variants of any of the foregoing.

2. The antibody, or antigen-binding fragment thereof, of claim 1, wherein:(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acidsequence of SEQ ID NO: 24, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 5;(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 10, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13; or(c) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21.

3. The antibody, or antigen-binding fragment thereof, of claim 1 or claim 2, wherein the heavy chain variable region comprises:(a) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 6, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, and 24, respectively;(b) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 14, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 8, 9, and 10, respectively; or(c) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 22, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 17, and 18, respectively.

4. The antibody, or antigen-binding fragment thereof, of claim 3, wherein the heavy chain variable region comprises:(a) the amino acid sequence of SEQ ID NO: 6, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, and 24, respectively;(b) the amino acid sequence of SEQ ID NO: 14, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 8, 9, and 10, respectively; or(c) the amino acid sequence of SEQ ID NO: 22, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 17, and18, respectively.

5. The antibody, or antigen-binding fragment thereof, of any one of claims 1 to 4, wherein the light chain variable region comprises:(a) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 7, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 3, 4, and 5, respectively;(b) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 15, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 11, 12, and 13, respectively; or(c) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 23, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs:19, 20, and 21, respectively.

6. The antibody, or antigen-binding fragment thereof, of claim 5, wherein the light chain variable region comprises:(a) the amino acid sequence of SEQ ID NO: 7, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 3, 5, and 5, respectively;(b) the amino acid sequence of SEQ ID NO: 15, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 11, 12, and 13, respectively; or(c) the amino acid sequence of SEQ ID NO: 23, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, and 21, respectively.

7. The antibody, or antigen-binding fragment thereof, of any one of claims 1-6, comprising:(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;(b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; or(c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.

8. The antibody, or antigen-binding fragment thereof, of any one of claims 1-7, wherein the antibody, or antigen-binding fragment thereof, binds to one or more uremic toxins selected from the group consisting of indoxyl sulfate, -cresyl sulfate, and combinations thereof.

9. The antibody, or antigen-binding fragment thereof, of any one of claims 1-8, wherein the antibody, or antigen-binding fragment thereof is a full-length immunoglobulin molecule.

10. The antibody, or antigen-binding fragment thereof, of any one of claims 1-8, wherein the antibody, or antigen-binding fragment thereof, is a fragment antigen binding (Fab) fragment, a Fd fragment, a F(ab’)2 fragment, a variable fragment (Fv), a single chain variable fragment (scFv), or similar constructs utilizing CDRs, VH, and / or VL sequences according to Table 1.

11. The antibody, or antigen-binding fragment thereof, of any one of claims 1-10, wherein the antibody, or antigen-binding fragment thereof, is a mouse antibody or mouse antigen-binding fragment thereof.

12. The antibody, or antigen-binding fragment thereof, of any one of claims 1-10, wherein the antibody, or antigen-binding fragment thereof, is a humanized antibody or humanized antigen-binding fragment thereof.

13. The antibody, or antigen-binding fragment thereof, of any one of claims 1-12, wherein the antibody, or antigen-binding fragment thereof, is an IgG, IgA, IgM, IgD, or IgE molecule.

14. The antibody, or antigen-binding fragment thereof, of any one of claims 1-12, wherein the antibody, or antigen-binding fragment thereof, is:(a) a mouse IgG molecule selected from the group consisting of IgGl, IgG2a, IgG2b, IgG2c, and IgG3; or(b) a human or humanized IgG molecule selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

15. An indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof, wherein the bispecific antibody or antigen-binding fragment thereof comprises:(i) a first antigen-binding arm comprising a first heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3); and a first light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3);(ii) a second antigen-binding arm comprising a second heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3); and a second light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3); wherein the first heavy chain variable region and the first light chain variable region pair to form a first antigen-binding site that specifically binds to indoxyl sulfate and the second heavy chain variable region and the second light chain variable region pair to form a second antigenbinding site that specifically binds to / ?-cresylsulfate.

16. The indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof of claim 15, wherein the first antigen-biding arm and / or the second antigen -binding arm comprise:(a) an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQID NO: 24, or variants of any of the foregoing; and the LCDR1 having the amino acid sequence of SEQ ID NO: 3, the LCDR2 having the amino acid sequence of SEQ ID NO: 4, and the LCDR3 having the amino acid sequence of SEQ ID NO: 5, or variants of any of the foregoing;(b) an HCDR1 having the amino acid sequence of SEQ ID NO: 8, an HCDR2 having the amino acid sequence of SEQ ID NO: 9, and an HCDR3 having the amino acid sequence of SEQ ID NO: 10, or variants of any of the foregoing; and an LCDR1 having the amino acid sequence of SEQ ID NO: 11, an LCDR2 having the amino acid sequence of SEQ ID NO: 12, and an LCDR3 having the amino acid sequence of SEQ ID NO: 13, or variants of any of the foregoing; or(c) an HCDR1 having the amino acid sequence of SEQ ID NO: 16, an HCDR2 having the amino acid sequence of SEQ ID NO: 17, and an HCDR3 having the amino acid sequence of SEQ ID NO: 18, or variants of any of the foregoing; and an LCDR1 having the amino acid sequence of SEQ ID NO: 19, an LCDR2 having the amino acid sequence of SEQ ID NO: 20, and an LCDR3 having the amino acid sequence of SEQ ID NO: 21, or variants of any of the foregoing.

17. The indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof of claim 16, wherein:(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 5;(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 10, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 13; or(c) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21.

18. The indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof of claim 15, wherein the first antigen-biding arm and / or the second antigen -binding arm comprise:(a) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 6, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 2, and 24, respectively; and an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 7, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ IDS NOs: 3, 4, and 5, respectively;(b) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 14, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 8, 9, and 10, respectively; and an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 15, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 11, 12, and 13, respectively; or(c) an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 22, provided that the heavy chain variable region comprises the sequences of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 17, and 18, respectively; and an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or identical to SEQ ID NO: 23, provided that the light chain variable region comprises the sequences of LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 19, 20, and 21, respectively.

19. The indoxyl sulfate x / ?-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof of claim 18, wherein the first antigen-biding arm and / or the second antigen -binding arm comprise:(a) the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7, provided that the heavy and light chain variable regions comprise the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1, 2, 24, 3, 4, and 5, respectively;(b) the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15, provided that the heavy and light chain variable regions comprise the sequences ofHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 8, 9, 10, 11, 12, and 13, respectively; or(c) the amino acid sequence of SEQ ID NO: 22 and the amino acid sequence of SEQ ID NO: 23, provided that the heavy and light chain variable regions comprise the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively.

20. The indoxyl sulfate x / 2-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof, of claim 17, wherein the first antigen-biding arm and the second antigen -binding arm are the same and each comprise:(1) a heavy chain comprising a HCDR1 amino acid sequence of SEQ ID NO: 8, a HCDR2 amino acid sequence of SEQ ID NO: 9, a HCDR3 amino acid sequence of SEQ ID NO: 10; and(2) a light chain comprising a LCDR1 amino acid sequence of SEQ ID NO: 11, an LCDR2 amino acid sequence of SEQ ID NO: 12, and an LCDR3 amino acid sequence of SEQ ID NO: 13.

21. The indoxyl sulfate x / 2-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof, of claim 20, wherein the heavy chain comprises an heavy chain variable region amino acid sequence of SEQ ID NO: 14 and the light chain comprises a light chain variable region of SEQ ID NO: 15.

22. The indoxyl sulfate x / 2-cresyl sulfate bispecific antibody, or bispecific antigenbinding fragment thereof, of claim 20 or claim 21, wherein the antibody -binding fragment thereof is a fragment antigen binding (Fab) fragment, a Fd fragment, a F(ab’)2 fragment, a variable fragment (Fv), a single chain variable fragment (scFv).

23. The antibody or antigen-binding fragment according to any one of claims 1 to 22, wherein the antibody or antigen-binding fragment is a chimeric antibody or chimeric antigenbinding fragment.

24. A nucleic acid molecule encoding an antibody or antigen-binding fragment according to any one of claims 1 to 23.

25. A vector encoding an antibody according to any one of claims 1 to 23.

26. A cell expressing the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 23.

27. A dialysis fluid composition comprising an antibody according to any one of claims 1 to 23 and a pharmaceutically acceptable carrier and / or excipient.

28. The dialysis fluid composition according to claim 27, wherein the pharmaceutically acceptable carrier and / or excipient includes one or more of a diluent, a filler, a salt, an electrolyte, a buffer, a detergent, a stabilizer, and / or a preservative.

29. A method of producing an antibody, or antigen-binding fragment, specific for a uremic toxin, comprising: culturing a cell(s) according to claim 26 in a suitable medium such that the antibody, or antigen-binding fragment thereof is produced.

30. A method of removing or reducing one or more uremic toxins from blood, said method comprising: contacting blood with a dialysis fluid composition comprising an antibody, or antigenbinding fragment thereof, according to any one of claims 1 to 23; and removing uremic toxins from the blood by dialyzing the blood through a membrane, wherein the membrane has a molecular weight cutoff smaller than the molecular weight of the uremic toxin antibody or antigen-binding fragment thereof.

31. A method of treating a subject having a kidney disease or condition, said method comprising: selecting a subject having a kidney disease or condition; and dialyzing a portion of the subject’s blood against a composition comprising an antibody or antigen-binding fragment thereof, according to any one of claims 1 to 23, wherein said dialyzing is sufficient to remove one or more uremic toxins from the subject’s blood, thereby treating the subject having the kidney disease or condition.

32. A method of detecting a uremic toxin, said method comprising:analyzing a biological sample in an immunoassay comprising an antibody, or antigenbinding fragment thereof, according to any one of claims 1 to 23 and determining, based on said analyzing, whether the biological sample contains one or more uremic toxins.

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