A bifunctional degrading agent for galactose-deficient immunoglobulins.

Abstract

A composition of a substance comprising a deglycosylated IgA binding moiety, a cell receptor binding moiety that binds to hepatocytes or other degrading cells via asialoglycoprotein receptors (ASGPRs) on the surface of hepatocytes or other degrading cells in a patient or subject, and optionally a linker moiety connecting the deglycosylated IgA binding moiety and the cell receptor binding moiety, wherein the composition of the substance is useful for removing galactose-deficient IgA1 in a patient or subject.

Description

[Technical Field]

[0001] The present invention relates to a bifunctional molecule containing a circulating protein binding moiety linked to a cell receptor binding moiety via a linker group. Specifically, the present invention relates to a bifunctional molecule containing a circulating protein binding moiety that binds to galactose-deficient immunoglobulin. [Background technology]

[0002] IgA nephropathy (IgAN) is a heterogeneous autoimmune disease characterized by (1) increased levels of circulating galactose-deficient IgA1 (Gd-IgA1) antibodies, (2) production of galactose-deficient IgA1-specific IgG antibodies, and (3) the formation of circulating nephritis-inducing immune complexes composed of galactose-deficient IgA1 antibodies and galactose-deficient IgA1-specific IgG antibodies. These immune complexes accumulate and deposit in the glomerular mesangium, inducing mesangial proliferative glomerulonephritis characteristic of IgAN, which leads to kidney damage. Overproduction of galactose-deficient IgA1 and the formation of Gd-IgA1-IgG immune complexes are major drivers of this four-hit pathogenic cascade. See Lai et al., Nature Rev. Dis. Primers, 2, 16001 (2016). In renal immunodeposition of patients with IgA neuropathy, IgG is enriched with galactose-deficient IgA1-specific antibodies.

[0003] Serum levels of galactose-deficient IgA1 autoantigen and corresponding autoantibodies were found to correlate with the severity and progression of IgAN, respectively. Suzuki et al., J. Clin. Invest., 119(6), 1668-1677 (2009) (the entire paper is incorporated herein by reference).

[0004] Moldoveanu et al. provided in vivo evidence for the role of IgG autoantibodies in inducing glomerulonephritis in IgAN. Moldoveanu et al., J. Autoimmun., 118, 102593 (2021). Immune complexes formed from galactose-deficient IgA and human IgG autoantibodies were intravenously injected into mice to induce glomerular injury. Histopathological changes in the injured tissue were characteristic of IgAN. Exploratory renal transcriptome profiling showed that these immune complexes altered gene expression in multiple pathways consistent with changes observed in kidney biopsies from patients with IgAN.

[0005] IgAN is the most common form of primary glomerulonephritis worldwide, and currently there is no cure. There remains a need for new medicines that can treat this disease or slow its progression. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This invention relates to a bifunctional molecule (drug) that can bind to and degrade galactose-deficient IgA1 (Gd-IgA1) immunoglobulin. [Means for solving the problem]

[0007] In one embodiment, the present invention is a composition of a substance (pharmaceutical), Galactose-deficient IgA1 binding site, A cell receptor binding portion that can bind to hepatocytes or other degrading cells via hepatocyte asialoglycoprotein receptors (ASGPR) or other cell receptors on surface degrading cells (for example, in a patient or subject), The present invention provides a composition of a substance (pharmaceutical) comprising a linker portion connecting a galactose-deficient IgA1 binding portion and a cell receptor binding portion, wherein the linker portion may be a single peptide bond or a larger linker portion.

[0008] In another embodiment, the binding moiety capable of binding to galactose-deficient IgA1 is an anti-human rat Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof.

[0009] In another embodiment, the binding moiety capable of binding to galactose-deficient IgA1 is a polypeptide having a complementarity-determining region (CDR) of the Km55 antibody. In a particular embodiment, the complementarity-determining region comprises six CDRs following a Kabat numbering scheme having the structures of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

[0010] In some embodiments, the present invention has the following structure: R CN -(Xaa)yR CC , [ka] [AGN101] [ka] [AGN102] [ka] [AGN103], or [ka] [AGN104] The present invention provides a composition (pharmaceutical) of a substance having [a certain characteristic], or a salt thereof.

[0011] In some embodiments, the present invention relates to the following formula AGN105: [ka] [AGN105] The present invention provides a composition (pharmaceutical) of a substance or a salt thereof, wherein the composition of the substance has additional elements as described herein.

[0012] In some embodiments, the present invention relates to the following formula AGN106: [ka] [AGN106] The present invention provides a composition of a substance comprising a salt thereof, wherein the composition of the substance has additional elements as described herein.

[0013] In another embodiment, the cell receptor binding moiety has the following chemical structure: [ka] [TBT101], or [ka] [TBT102] The cell receptor binding group comprises an ASGPR binding group, and the cell receptor binding portion has additional elements as described herein.

[0014] In another embodiment, the present invention provides a method for producing a composition (pharmaceutical) of a bifunctional substance that can bind to and degrade galactose-deficient IgA1 (Gd-IgA1) immunoglobulin.

[0015] In another embodiment, the present invention provides a method for removing galactose-deficient IgA1 in a patient or subject by administering a drug to the patient or subject who requires the removal of galactose-deficient IgA1.

[0016] In another embodiment, the present invention provides a method for preparing a composition of a substance (a pharmaceutical).

[0017] In another embodiment, the present invention provides a method for treating a disease state or condition associated with the upregulation of galactose-deficient IgA1 in a patient or subject by administering an effective amount of a drug to the patient or subject in need.

[0018] In another embodiment, the present invention provides a composition comprising a drug and at least one additional drug having a portion that can bind to an antibody forming an antibody portion of a first compound.

[0019] In another embodiment, the present invention provides a composition comprising a drug and at least one pharmaceutically acceptable excipient.

[0020] In one embodiment, the composition of the substance (pharmaceutical) binds to galactose-deficient IgA1, but most non-defective IgA in the patient or subject is spared.

[0039] By reducing the levels of galactose-deficient IgA1 and IgG:galactose-deficient IgA1 immune complexes in the circulating system, mesangial deposition can be reduced and renal function can be improved.

[0021] In one embodiment, the composition of the substance (pharmaceutical) has a balanced binding strength (balanced binding) to liver receptors by the TBT moiety compared to the binding of the ABT moiety to degalactosylated IgA antibody.

[0022] Various purposes, features, embodiments, and advantages of the present invention will become more apparent from the following detailed description of embodiments of the invention, in conjunction with the drawings.

[0023] For illustrative purposes, several embodiments of the present invention are shown in the drawings described below. Similar figures in the drawings indicate similar elements throughout. The present invention is not limited to the exact arrangements, dimensions, and equipment shown. [Brief explanation of the drawing]

[0024] [Figure 1] This is a list showing how IgA nephropathy (IgAN) is treated using Gd-IgA1 degradation. [Figure 2]A pair of figures showing the results of preclinical trials, illustrating that a low-concentration embodiment of the present invention (Gd-IgA-specific MoDE®) selectively degrades Gd-IgA1 present in IgA nephropathy (IgAN), while ignoring total IgA, thus limiting its impact on the immune system. [Figure 3] This document describes a process for preparing one embodiment of a bifunctional molecule (drug) by conjugating a chimeric Km55 antibody to a reagent that has affinity for the chimeric Km55 antibody. [Figure 4] This is a sequence comparison of the constant heavy chains of human IgG1 (SEQ ID NO: 16), IgG2 (SEQ ID NO: 17), and IgG4 (SEQ ID NO: 18), which form the basis of human and humanized therapeutic antibodies. Sequence differences between IgG2 and IgG4 from IgG1 are shown. Hinge and lower hinge regions with the N-glycosylation site N297 are shown. Numbering follows the EU numbering scheme. Sequences important for FcgR binding, C1q, and FcRn are indicated by shading. Several enhanced ADCC variants are shown above or below the sequences, as shown below: S239D, A330L, I332E of Xencor [red, sequence above], S298A, E333A, K334A of Genentech [blue, sequence below], and P247I, A339D / Q of Eli Lilly / AME [green, sequence above]. Examples of FcRn binding variants for half-life extension are also shown, as shown below. The YTE variants of MedImmune (M252Y, S254T, T256E), the T250Q and M428L variants of PDL, the H433K and N434Y variants of Sally Ward, and the N434A variant of Genentech are shown as red, orange, green, and blue squares, respectively. [Figure 5] Table 1 shows the R group of the MATE reagent and the bifunctional MoDE final compound. [Figure 6] This chart, from IBL-America, #27600, Pamphlet 2, shows galactose-deficient IgA1 in serum. An ELISA assay using the Km55 antibody specifically detects galactose-deficient IgA1. [Figure 7] This graph shows the ASPGR1-dg-IgA TCF, expressed as the FRET R ratio relative to log[AGN03A] and nM. Circles indicate the formation of a viable ternary complex containing the drug AGN03A. Squares indicate results controlled by phosphate-buffered saline. [Figure 8] This graph shows the detection level of galactose-deficient IgA1 in human plasma samples. Compare with Figure 7. [Figure 9] This graph shows the results of a dg-IgA intracellular reintegration assay. HEK293 cells transfected with ASGPR1 were used to measure endocytosis of 1 μg / mL dg-IgA and total IgA conjugated with Alexa Fluor 594. MFI = mean fluorescence intensity, S / N = signal-to-noise ratio. [Figure 10] This graph shows the isolation of dg-IgA aggregates by size exclusion chromatography. See Table 10. [Figure 11] This graph shows the results of intracellular relocation of the dg-IgA complex. Endocytosis of 1 μg / mL monomeric, dimeric, and tetrameric dg-IgA conjugated with Alexa Fluor 594 was measured using HEK293 cells transfected with ASGPR1. See Table 11. [Figure 12]This is a pair of line graphs showing the results of drug administration to mice. The line graph on the left shows the pharmacokinetics resulting from administration to wild-type mice and ASGPR knockout (KO) mice. Lines with filled triangles show the results of AGN03A administration to wild-type mice. Lines with open triangles show the results of AGN03A administration to ASGPR knockout (KO) mice. Lines with filled circles show the results of antibody ABT030 administration to wild-type mice. The line graph on the right shows the pharmacodynamics of dg-IgA clearance in wild-type mice. Lines with triangles show the clearance of AGN03A. Lines with squares show the clearance of ABT030. Lines with circles show the clearance of dg-IgA. The dosages were 200 μg / mouse = 8 mpk intravenously for AGN03A and 100 μg = 4 mpk intravenously for dg-IgA (5 times greater than the median galactose-deficient IgA1 level in patients). [Modes for carrying out the invention]

[0025] To assist those skilled in the biomedical field, the following detailed description is provided. Exemplary embodiments are described. These embodiments are illustrative only. This disclosure is defined by the scope of the appended claims, but is not limited to them. Those skilled in the biomedical field may modify and alter the embodiments described without departing from the spirit or scope of this disclosure.

[0026] Industrial applicability The present invention provides a composition (pharmaceutical) of a medically useful substance for treating or slowing the progression of IgA nephropathy (IgAN).

[0027] Galactose-deficient IgA1 is attracting attention as a key effector molecule in the pathogenesis and progression of IgA nephropathy (IgAN). Several O-linked glycan modification regions are present in the heavy chain hinge region of the human IgA1 molecule. See Sequence ID No. 83. Galactose-deficient IgA1 circulates in the bloodstream of patients with the pathological condition of IgAN. Deposition of galactose-deficient IgA1 in the glomeruli is involved in IgAN (the Gd-IgA1 multi-hit hypothesis).

[0028] Levels of galactose-deficient IgA in IgAN are significantly elevated compared to levels in healthy subjects or patients with other renal diseases. Measurement systems using Helix aspersa snail lectin (HAA) have been used in research and diagnostic procedures. Commercial ELISA assay kits using Km55 antibody have also been used in research and diagnostic procedures. See IBL-America, Minneapolis, MN, USA, 55432, Catalog Number: 10777. See also IBL-America, Code 27600 galactose-deficient IgA1 assay. IHC protocols are provided with the assay kit. Km55 antibody can detect galactose-deficient IgA1 in tissues by immunohistochemistry (IHC) techniques distinct from those of HAA lectin. Galactose-deficient IgA1 is specifically present in the glomeruli of patients with IgAN, as determined in research studies using this antibody. Figure 6 shows a diagram of one result from an assay using the assay kit.

[0029] Reducing levels of circulating galactose-deficient IgA1 and IgG:galactose-deficient IgA1 immune complexes can reduce mesangial deposition and improve renal function. Reduction of circulating galactose-deficient IgA1 has been achieved using clinical-stage immunomodulatory BlyS / APRIL inhibitors, with potential improvements in proteinuria and renal function. Barratt et al., Kidney Int. Rep., 7(8), 1831-1841 (2022). Selective proteolysis of circulating galactose-deficient IgA1 and its complexes can stabilize or reverse the progression of IgAN without causing widespread immunosuppression.

[0030] In contrast, a bifunctional substance composition (drug) selectively binds to circulating galactose-deficient IgA1, forming a protein complex that binds to a cell receptor. The protein complex is endocytotic and degraded. Galactose-deficient IgA1 is removed from circulation by hepatocytes, macrophages, or other cell types, reducing the level of galactose-deficient IgA1 and potentially weakening IgAN symptoms. The removal of galactose-deficient IgA1 may result in a substantial reduction, or even cure or elimination, of IgAN symptoms.

[0031] Non-glycosylated proteins (e.g., immunoglobulins) are not known to be natural targets of ASGPR on hepatocytes and other degrading cells. The present invention provides a bifunctional agent for degrading circulating galactose-deficient IgA1 that utilizes ASGPR as a protein entry point into the endosomal-lysosomal degradation pathway.

[0032] Proper protein synthesis, secretion, and turnover are necessary to maintain homeostasis. Newly synthesized proteins targeting secretion are first trafficked into the endoplasmic reticulum, where they are post-translationally modified with N-linked glycan chains terminated with sialic acid. N-acetylgalactosamine is the endogenous ligand. As proteins age, terminal sialic acid residues are removed by circulating endogenous glycosidases. This innate protein aging process removes the masking of galactose and N-acetylgalactose (GalNAc) residues that bind to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes. ASGPR is a type C lectin that removes aged circulating proteins with exposed GalNAc residues from circulation by trafficking them into lysosomes. Multiple galactose or GalNAc residues presented on the protein surface are necessary for high-affinity binding to ASGPR and subsequent endocytosis by ASGPR. Avidity is obtained by incorporating a bidental or tridental ligand.

[0033] After these proteins are endocytized, they are released from ASGPR due to calcium depletion from endosomes and changes in binding site amino acid protonation resulting from a decrease in pH. ASGPR is then recycled back to the hepatocyte surface. The endocytized proteins are trafficked into late endosomes, which fuse with lysosomes. Lysosomal proteases then degrade the endocytized proteins, permanently removing them from circulation. See Caianiello et al., Nature Chemical Biology, 17(9), 947-953 (2021).

[0034] There is a need in the biomedical technology field for better treatments of IgAN nephropathy to replace or complement the use of immunosuppressants to treat the symptoms of IgAN. See Agenix, Cochrane Database Syst Rev.2020,2020(3)CD003965(March 12,2020).

[0035] The present invention advantageously provides a therapeutic agent having some or all of the following features. ● Highly water-soluble (>100 mg / mL) to aid in intravenous or subcutaneous delivery. ●Good chemical stability in aqueous solution at room temperature (for more than t days or t weeks). ● High stability in human plasma (t 1 / 2 >120 minutes). ● Rapid in vivo clearance driven by hepatic uptake and the ASGPR pathway (t 1 / 2 (Approximately 30 minutes). ●Breakdown into harmless metabolites in the liver.

[0036] definition For convenience, the meanings of some terms and phrases used in this specification, the examples, and the appended claims are provided below. Unless otherwise specified or implied in the context, these terms and phrases shall have the following meanings. These definitions are useful for describing specific embodiments but are not intended to limit the claimed invention.

[0037] When used in this application, unless otherwise expressly provided herein, each of these terms shall have the meanings set forth below. Additional definitions are provided throughout this application. If a term is not specifically defined, it shall be given the meaning recognized in the biomedical art to which it is applied in connection with its use in describing the present invention.

[0038] The articles "a" and "an" have the plain meaning of one or more (i.e., at least one) of the grammatical objects of the article, unless otherwise indicated by context. For example, "an element" means one element or more than one element.

[0039] The term "ABT" has the meaning recognized in the biomedical art of antigen-binding portions. In some embodiments herein, ABT binds to galactose-deficient IgA1. In one embodiment, ABT has a linker attachment point that does not reduce binding affinity.

[0040] The term "active ingredient" has the meaning provided by the United States Food and Drug Administration of any ingredient that produces a pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or that affects the structure or any function of the body of a human or animal.

[0041] The term "ADCC" has the meaning recognized in the biomedical art of antibody-dependent cell-mediated cytotoxicity, which is a mechanism of cell-mediated immune defense in which effector cells of the immune system kill target cells whose membrane surface antigens are bound by specific antibodies.

[0042] The term "ADCP" has the meaning of antibody-dependent cell-mediated phagocytosis recognized in the biomedical art, which is an immunological mechanism of removal in which tumor cells are targeted with antibodies to facilitate their clearance from the body by phagocytic immune cells.

[0043] The term "agent" has the meaning recognized in the biomedical art of compositions of substances useful for performing functions. This specification describes several useful biomedical functions.

[0044] The term "alleviate" has the meaning recognized in the biomedical art of the process by which the severity of a sign or symptom of a disorder is reduced. A sign or symptom can be alleviated without being eliminated. Administration of a composition or pharmaceutical composition of the invention can lead to or can lead to the elimination of a sign or symptom, although elimination is not required. An effective dosage amount should be expected to reduce the severity of the sign or symptom.

[0045] The terms "effective amount" and "therapeutically effective amount" have the meaning recognized in the biomedical art of an amount effective to achieve its intended purpose. The effect can be detected by any assay method known in the art. The exact effective amount for a subject will depend upon the subject's weight, size, and health, the nature and extent of the medical condition, and the therapeutic agent or combination of therapeutic agents selected for administration. A therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician. In some embodiments, the disease or medical condition being treated is a tendon disorder.

[0046] The term "anti-galactose-deficient IgA1 (Gd-IgA1) antibody" has the meaning recognized in the biomedical art of an antibody that selectively binds to galactose-deficient IgA1. In some embodiments of the invention, the anti-galactose-deficient IgA1 antibody is the published Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof.

[0047] The term "anti-galactose-deficient IgA1 (Gd-IgA1) IgG antibody" has the meaning recognized in the biomedical art of an IgG antibody or fragment thereof that binds to galactose-deficient IgA1. In some embodiments of the invention, the anti-galactose-deficient IgA1 antibody is the published Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof. In some embodiments, the present specification describes "glycan-specific IgG antibody binding" as anti-galactose-deficient IgA1:IgG antibody.

[0048] The term "antigen-binding fragment" has the biomedical field meaning of (1) an intact antibody fragment that binds to the same antigen recognized by a full-length antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, sFv, or other fragments consisting of a variable region, or (2) any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex. The term antigen-binding portion of an antibody includes single-chain antibodies.

[0049] The term “asiaroglycoprotein receptor (ASGPR) binding group” has a recognized meaning in the biomedical technology of a binding group that binds to hepatocyte asiaroglycoprotein receptors. The ASGPR binding group selectively binds to hepatocyte asiaroglycoprotein receptors on the surface of hepatocytes. In some embodiments herein, the ASGPR binding group is a component of a bifunctional drug as a cell receptor binding moiety that is covalently bound to an antibody binding moiety via a linker group. This ASGPR moiety is a bifunctional drug complexed with a circulating protein (e.g., galactose-deficient IgA1) that binds to hepatocytes. After the bifunctional drug complexed with the circulating protein binds to hepatocytes or other cells, the circulating protein is taken up by the hepatocytes or other cells via phagocytic mechanisms and is degraded by lysosomal degradation.

[0050] The term "asialoclycoprotein receptor (ASGPR)" has a recognized meaning in the biomedical technology field of lectins that bind to asialoclycoproteins and glycoproteins from which sialic acid has been removed to expose a galactose residue. These cellular receptors are located on mammalian hepatocytes and other cells such as gallbladder and gastric glandular cells. ASGPRs remove target glycoproteins from circulation.

[0051] When preceding a list of elements, terms such as "at least one of" modify the entire list of elements, not the individual elements of the list.

[0052] The term "AT" has the meaning recognized in the biomedical technology field for the antibody portion. In some embodiments herein, AT binds to galactose-deficient IgA1.

[0053] The term “cell receptor binding site” has the meaning recognized in the biomedical technology field. In some embodiments herein, the cell receptor binding site is an asialoglycoprotein receptor (ASGPR) binding site.

[0054] The term "cell receptor" has a recognized meaning in the biomedical art of binding compounds, e.g., ligands, e.g., proteins, in solution, or onto other cells, to proteins on the surface of cells. Generally, ligand-receptor binding induces one or more biological responses. In this specification, asialoglycoprotein receptors (ASGPRs) are cell receptors on the surface of hepatocytes or other cells that bind to asialoglycoproteins or derivatives thereof.

[0055] The term "chimerization" has a recognized meaning in the biomedical technology field. Chimeric antibodies are created by fusing a variable domain from one species, such as a mouse, with a constant domain from another species, such as a human. In such a biotechnical operation, the chimeric antibody possesses the antigen specificity and affinity of the exogenous antibody.

[0056] The terms “combination therapy” and “co-treatment” have the meanings recognized in the biomedical art of administering the compositions described herein with at least a second agent as part of a particular treatment regimen intended to provide beneficial effects from the co-actions of these therapeutic agents. Beneficial effects of combination may include, but are not limited to, pharmacokinetic or pharmacodynamic synergies resulting from the combination of therapeutic agents. These therapeutic agents are typically administered in combination over a defined period of time (usually minutes, hours, days, or weeks, depending on the chosen combination). The term combination therapy also includes administering therapeutic agents in further combination with other biologically active ingredients and non-pharmacological therapies (e.g., surgery or radiation therapy). Where combination therapy further includes non-pharmacological therapy, the non-pharmacological therapy may be administered at any appropriate time, provided that beneficial effects are achieved from the co-actions of the combination of therapeutic agents. Where appropriate, beneficial effects can still be achieved even if the non-pharmacological therapy is temporarily, perhaps several days or weeks, away from the administration of the therapeutic agent.

[0057] The term "complementarity-determining regions (CDRs)" has a recognized meaning in the biomedical field of the polypeptide region of an antibody heavy chain, or of the antibody light chain, which is the determinant of an antibody's antigen binding. Each antibody heavy chain contains three complementarity-determining regions. Each antibody light chain typically has three complementarity-determining regions distinct from the three CDRs on the antibody heavy chain. Those skilled in the biomedical field calculate these using a standardized numbering method known as the Kabat numbering scheme. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition (Public Health Service, National Institutes of Health, Bethesda, MD., USA, 1991)). However, other numbering schemes such as Chothia and IMGT are also used by those skilled in the biomedical field.

[0058] The terms “comprises,” “comprising,” “includes,” and “including” specify the described features, areas, items, steps, actions, elements, or components, but do not exclude the presence or addition of one or more other features, areas, items, steps, actions, elements, components, or groups thereof.

[0059] The term "dd-IgA1" has the recognized meaning in the biomedical technology field of desialylated IgA1 and degalactosylated IgA1.

[0060] The term "Fc-III-4c" has a recognized meaning in the biomedical field of the polypeptide region (tail region) within the crystallizable region (Fc region) of an antibody fragment.

[0061] The term "Fc-M" has a recognized meaning in the biomedical field of the polypeptide region (tail region) within the crystallizable region (Fc region) of an antibody fragment.

[0062] The term "FcB-1" has a recognized meaning in the biomedical field of the polypeptide region (tail region) within the crystallizable region (Fc region) of an antibody fragment.

[0063] The term "FcB-2" has a recognized meaning in the biomedical field of the polypeptide region (tail region) within the crystallizable region (Fc region) of an antibody fragment.

[0064] Terms such as "first", "second", "third", etc. have a clear meaning for describing several elements, components, regions, layers, or sections. These terms do not limit these elements, components, regions, layers, or sections. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. The first element, component, region, layer, or section may be referred to as the second element, component, region, layer, or section without departing from the teachings of the embodiments of the present invention.

[0065] The term "Gal" has the meaning of galactose recognized in the field of biomedical technology.

[0066] The term "galactose-deficient IgA1 (Gd-IgA1) binding portion" has the meaning recognized in the field of biomedical technology of a binding protein that binds to galactose-deficient IgA1, for example, a portion on an IgG antibody or its fragment. In some embodiments described herein, the "glycan-specific IgG antibody binding portion" is a galactose-deficient IgA1 binding portion on an IgG antibody such as the publicly available Km55 antibody, Km55 variant, or its antigen-binding fragment.

[0067] The term "GalNAc" has the meaning recognized in the field of biomedical technology of N-acetylgalactosamine.

[0068] The term "glomerular mesangium" has the meaning recognized in the field of biomedical technology of a component of the renal glomerulus and forms a supporting framework in which glomerular mesangial capillaries are formed in a dendritic manner. The mesangium contains an extracellular matrix including type IV collagen, proteoglycan, other proteins, and two cell types.

[0069] The term "HAA" has the meaning recognized in the field of biomedical technology of Helix aspersa lectin.

[0070] The term "hepatocyte" has a recognized meaning in the biomedical field as referring to the cells of the main parenchymal tissue of the liver. Hepatocytes make up 55–65% of the liver's mass.

[0071] The term "hepatocyte" has a recognized meaning in the biomedical field as referring to the cells of the main parenchymal tissue of the liver. Hepatocytes make up 55–65% of the liver's mass.

[0072] The term "humanization" has a recognized meaning in the biomedical technology field when a protein, such as an antibody, is genetically engineered to closely resemble the polypeptide structure of its human homolog. The variable domain of a rodent-derived antibody can be fused to a constant domain of human origin while maintaining the specificity of the rodent antibody. The human-derived domain does not need to be directly derived from humans in the sense that it is first synthesized in humans. Instead, the human domain can be generated in a rodent having a genome incorporating human immunoglobulin genes. Antibodies can be partially or completely humanized. One approach uses four common steps to humanize a monoclonal antibody. These steps are: (1) determining the nucleotide and predicted amino acid sequences of the lightly and heavily variable domains of the initiating antibody; (2) designing the humanized antibody, i.e., determining which antibody framework regions to use during the humanization process; (3) the actual humanization method / technology; and (4) transfection and expression of the humanized antibody.

[0073] The term "IgA antibody" has a recognized meaning in the biomedical technology field. Two IgA molecules are linked together, and the newly formed IgA molecule associates with a protein, allowing it to be secreted across epithelial cells lining several tubules and organs.

[0074] The term "IgA nephropathy (IgAN)," also known as Berger's disease, is recognized in the biomedical field as the most common form of primary glomerulonephritis worldwide. This condition is named based on the pathological features of IgA deposition in the glomeruli. An estimated 15-20% of patients with IgAN progress to end-stage renal disease within 20 years of the onset of the disease. (Kuroyanagi et al., Galactose-deficient IgA1 is involved in IgA deposition in renal grafts biopsied one hour after kidney transplantation. Intern Med. (October 26, 2022)). IgA in mesangial deposits originates solely from the IgA1 subclass and is an abnormal glycosyl form in which the hinge region O-linked glycan lacks galactose (Gal). Circulating IgA1 in patients with IgAN also harbors Gal-deficient O-glycans, although Gal-deficient variants are rarely found in IgA1 from the serum of normal individuals. The production of these variants is attributed to altered expression of specific glycosyltransferases in IgA1-producing cells. Binding of IgA1-containing immune complexes with abnormally glycosylated IgA1 to mesangial cells induces renal expression characteristic of IgAN. See Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009).

[0075] The term "IgG" antibody has a recognized meaning in the biomedical technology field. Each IgG molecule consists of a basic four-chain immunoglobulin structure (two gamma heavy chains and two identical light chains, either kappa or lambda) and possesses two identical antigen-binding sites. There are four subclasses of IgG, each subclass having slightly different biological properties, although they differ in their H chains.

[0076] The term "IgG1" antibody has a recognized meaning in the biomedical technology field of IgG antibodies, and the Ig gamma-1 chain C region is the protein encoded by the IGGH1 gene in humans.

[0077] The term "IgG2" antibody has a recognized meaning in the biomedical technology field of IgG antibodies, and the Ig gamma-2 chain C region is a human protein encoded by the IGGH2 gene.

[0078] The term "IgG4" antibody has a recognized meaning in the biomedical technology field of IgG antibodies, and the Ig gamma-4 chain C region is the protein encoded by the IGGH4 gene in humans.

[0079] The term "IVIG" has a recognized meaning in the biomedical field of administration of intravenous immunoglobulin (IVIG).

[0080] In this specification, the term "Km55" refers to a group of anti-galactose-deficient IgA1 antibodies. In some embodiments, Km55 may be a publicly available Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof. In some embodiments, Km55 may be a chimeric, partially humanized, or humanized Km55 variant thereof, or an antigen-binding fragment thereof.

[0081] The term “linker portion” has the meaning recognized in the biomedical art of a portion of a chemical compound that links one portion of a chemical compound to another portion of the same compound. In some embodiments of the present invention, the linker portion connects an anti-galactose-deficient IgA1 IgG antibody to a cell receptor binding portion. In one embodiment, the linker is composed mainly of PEG units. In another embodiment, the linker is suitable for presenting multiple ASGPR binders. In yet another embodiment, the linker is long enough to accommodate the formation of a ternary complex. See also U.S. Patent Publication 2020 / 0190165 (each of its cleavable linkers and cleavable portions is incorporated herein by reference).

[0082] The term "Markush group" has the meaning recognized in patent law.

[0083] The term "MoDE" has a unique meaning as a molecular decomposition agent. See International Patent Publication No. 2019 / 199634 (Yale University) and No. 2019 / 199621 (Yale University).

[0084] The term "part" has a biomedical meaning as a defined chemical group or entity having a specific structure or activity.

[0085] The term "monotherapy" has a recognized meaning in the biomedical field of administering a single active or therapeutic compound to a target that requires it. Monotherapy typically involves administering a therapeutically effective dose of the active composition.

[0086] The term "multimodal antibody therapy enhancer (MATE or MATES)" has its own meaning. See International Patent Publication No. 2021 / 102052 (Kleo Pharmaceuticals).

[0087] The term "N-acetyl-D-galactosamine (GalNAc) moiety" has a recognized meaning in the biomedical technology field.

[0088] The term "on" has a clear meaning. When an element is described as existing "on" another element, it can either be in direct contact with the other element or there may be an intervening element between them. When an element is described as existing "directly on" another element, there is no intervening element.

[0089] The term “one or more” has a clear meaning. As used in this disclosure, in some embodiments, “at least one” or “one or more” means 1 to 1000, 1 to 500, 1 to 200, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, it is 1. In some embodiments, it is 2 or more. In some embodiments, it is about 3. In some embodiments, it is about 4. In some embodiments, it is about 5. In some embodiments, it is about 6. In some embodiments, it is about 7. In some embodiments, it is about 8. In some embodiments, it is about 9. In some embodiments, it is about 10. In some embodiments, it is about 10 or more.

[0090] As used herein, the term "or" means "or." As used herein, the term "or" includes any combination of one or more of the related enumerated items.

[0091] The term "other degrading cells" has a recognized meaning in the biomedical technology field. Asialoglycoprotein receptors (ASGPRs) are located on glandular cells of the gallbladder and stomach.

[0092] The term "partial humanization" has a recognized meaning in the biomedical technology field when a protein, such as an antibody, is genetically engineered to more closely resemble the polypeptide structure of its human homolog. Variable domains of rodent-derived antibodies can be fused to human-derived constant domains while maintaining the specificity of the rodent antibody. The human-derived domain does not need to be directly derived from humans in the sense that it is first synthesized in humans. Instead, the human domain can be produced in rodents with a genome incorporating human immunoglobulin genes. Antibodies can be partially or fully humanized.

[0093] The term “pharmaceutically acceptable excipient” generally has the recognized meaning in the biomedical art of excipients that are safe, non-toxic, and useful in the preparation of biologically and otherwise undesirable pharmaceutical compositions, and includes excipients that are permitted for veterinary and human medicinal use. As used herein and in the claims, “pharmaceutically acceptable carrier” includes both one such carrier and more than one such carrier. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences, 23rd edition (Elsevier, 2020).

[0094] The term "pharmaceutically acceptable" has the meaning recognized in the biomedical art of a compound, anion, cation, substance, composition, carrier, or dosage form that is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, with a reasonable benefit / risk ratio, within the bounds of sound medical judgment.

[0095] The term "production trial" has an obvious business meaning: a set of processes required to produce a predetermined quantity of individual or multiple batches of a product. In business, production trials are conducted according to a manufacturing plan for producing a product for research and development or clinical use. A production trial is a standard manufacturing process for mass-producing a product. The objective of a production trial is to efficiently produce a consistent, high-quality output within a specific timeframe.

[0096] The term "protein-binding portion" has a recognized meaning in the biomedical technology field, specifically in the area of ​​chemical compositions, such as polypeptides of chemical compositions, where the portion specifically binds to a protein, for example, a particular protein.

[0097] The term "rIgG" has a recognized meaning in the biomedical technology field of recombinant human IgG.

[0098] The term "ROC" has a recognized meaning in the biomedical field as a receiver that operates on a typical curve.

[0099] The terms “subject” and “patient” have their recognized meanings in the biomedical technology field. The term patient includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0100] The term "TBT" has the meanings recognized in the biomedical art of target binding and cell receptor binding. In some embodiments herein, TBT binds to ASGPR.

[0101] The terms “to treat” and “to heal” have the recognized meaning in the biomedical technology field of managing and caring for a patient in order to combat a disease, condition, or disorder. This treatment includes the administration of compositions described to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder.

[0102] The term "universal antibody-binding region" has a recognized meaning in the biomedical technology field for the polypeptide region of an antibody-binding protein that binds to a class of antibodies, rather than a specific set of antibodies.

[0103] The term "UP / Cr" refers to the urinary protein / urinary creatinine (ratio) as recognized in the biomedical technology field.

[0104] Several embodiments are described below by reference to structures and schemes in order to illustrate parts of this specification.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the biomedical field. The technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting. Terms such as those defined in commonly used dictionaries should be construed to be consistent with their meanings in the relevant technical field and in this disclosure. They should not be construed in an idealized or overly formal sense unless expressly defined.

[0106] This specification does not relate to processes for cloning humans, methods for altering the genetic identity of human germline cells, the use of human embryos for industrial or commercial purposes, or procedures for altering the genetic identity of animals that may suffer without providing any substantial medical benefit to humans or animals resulting from such processes.

[0107] How to diagnose IgA nephropathy. Previously, diagnosing IgAN was complicated because it often required a kidney biopsy, which could lead to fatal complications.

[0108] There is growing evidence that galactose-deficient IgA is a triggering factor in the pathogenesis of IgAN. High levels of galactose-deficient IgA1 have been reported to be associated with disease progression. Zhang et al., Kidney Blood Pressure Res., 44, 1196-1206 (2019).

[0109] Many researchers have used snail Helix aspersa agglutinin (HAA) lectin-based assays to measure serum levels of galactose-deficient IgA, but lectin-dependent assays needed to be more robust.

[0110] More recently, a more robust and stable enzyme-linked immunosorbent assay (ELISA) has been developed using the Km55 antibody to recognize the hinge region in human galactose-deficient IgA (Gd-IgA1 ELISA). Km55 has high affinity for galactose-deficient IgA and has become a diagnostic tool for detecting IgAN. Yasutake et al., Nephrol. Dial. Transplant., 30, 1315-1321 (2015). See also Japanese Patent Publication No. 2010-285419, published December 24, 2010, and International Patent Publication No. 2015 / 064348, published May 7, 2015 (each of these publications is incorporated herein by reference in its entirety).

[0111] A method for removing galactose-deficient IgA1 antibodies from a patient or subject. The inventors of this invention hypothesized that reducing galactose-deficient IgA1 levels in IgAN patients could lead to disease treatment. By reducing galactose-deficient IgA1 in subjects predisposed to IgAN, the disease could be prevented. To this end, the inventors designed and developed a multifunctional galactose-deficient IgA1 degrading agent incorporating a galactose-deficient IgA1 binding moiety, such as the Km55 variant, and a cell receptor binding moiety having affinity for hepatocytes and other hepatogenic cells. Galactose-deficient IgA1 bound via the anti-galactose-deficient IgA1 binding moiety is then delivered to hepatogenic cells, where it is internalized and degraded via asialoglycoprotein receptors (ASGPRs) or other cell receptors located on the surface of hepatocytes or other degrading cells in the patient or subject.

[0112] Method of administration. In one embodiment, the administration method involves subcutaneously administering a composition of the substance (pharmaceutical) at a dose of 50 mg / ml to the patient or subject over a period of 5 to 7 days. In a more specific embodiment, the composition of the substance (pharmaceutical) is AGN03A.

[0113] A method for measuring the removal of galactose-deficient IgA1 antibodies from a patient or subject. Guidance on the extent to which administration of the agents of the present invention to patients or subjects reduces IgA levels in those patients or subjects is provided in several publications, e.g., Nihei, Suzuki, & Suzuki, Current understanding of IgA antibodies in the pathogenesis of IgA nephropathy. Front. Immunol., 14, 1165394 (2023). Analysis based on lectin spectroscopy and mass spectrometry showed that IgAN patients showed elevated serum levels of abnormally glycosylated, specifically galactose-deficient IgA1, in O-linked glycans in their hinge regions. More than 70% of patients with IgAN showed elevated serum galactose-deficient IgA1 levels above the 90th percentile in a healthy control group. Moldoveanu et al., Kidney International, 71(11), 1148-54 (2007), demonstrated the development of an enzyme-linked immunosorbent assay for measuring serum galactose-deficient IgA1 using a lectin from Helix aspersa that recognizes N-acetylgalactosamine. Median serum lectin-bound IgA1 levels were significantly higher in patients with IgA nephropathy that had not progressed to end-stage renal disease compared to healthy adult controls. The diagnostic test showed a sensitivity of 76.5%, specificity of 94%, a positive predictive value of 88.6%, and a negative predictive value of 78.9%.

[0114] Statistical analysis. Quantitative variables distributed normally could be expressed as mean and standard deviation and were compared by independent sample t-tests, as performed by Zhang et al. (October 2019). The inventors of this invention used the median and interquartile range of non-normally distributed variables and analyzed them with the Mann-Whitney U test. Categorical data were summarized as percentages. A two-tailed p-value < 0.05 was considered statistically significant. All statistical tests were performed using SPSS version 16.0.

[0115] The chemical structure of a substance's composition (pharmaceutical). In one embodiment, the present invention is The anti-galactose-deficient IgA1 binding site, A cell receptor binding portion that binds to hepatocytes or other degrading cells via hepatocyte asialoglycoprotein receptors (ASGPRs) or other cell receptors on surface degrading cells in a patient or subject, It consists of a substance (drug) that includes an antibody portion and a linker portion that connects the antibody portion and the cell receptor binding portion.

[0116] In some embodiments, the present invention relates to a substance (pharmaceutical), which is as follows: R CN -(Xaa)yR CC , [ka] [AGN101] [ka] [AGN102] [ka] [AGN103], or [ka] [AGN104] The substance (drug) is composed of a structure selected from the Markush group of structures consisting of a salt thereof or a salt thereof. In these structures, a, b, and c may be independently integers of 1 or more. In some embodiments, each cell receptor binding moiety is independently -(R CN -(Xaa)yR CC It has the structure or salt form of ).

[0117] In some embodiments, the present invention relates to the following formula AGN105: [ka] [AGN105] a substance (drug) or a salt thereof, wherein, each Xaa is, independently, a residue of an amino acid or an amino acid analogue, t is from 0 to 50, z is from 1 to 50, L is a linker moiety, TBT is a cell receptor binding moiety, each R c is, independently, -L a -R’, each of a and b is, independently, from 1 to 200, each L a is, independently, a covalent bond, or an optionally substituted divalent group selected from a C1-C 20 aliphatic group or a C1-C with 1 to 5 heteroatoms 20 heteroaliphatic group, wherein one or more methylene units of the group are optionally and independently replaced by -C(R’)2-, -Cy-, -O-, -S-, -S-S-, -N(R’)-, -C(O)-, -C(S)-, -C(NR’)-, -C(O)N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R’)-, -C(O)S-, or -C(O)O-, each -Cy- is, independently, an optionally substituted divalent monocyclic group, bicyclic group, or polycyclic group, and each monocyclic ring is independently selected from a C 3~20 alicyclic ring, a C 6~20 aryl ring, a 5- to 20-membered heteroaryl ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3- to 20-membered heterocyclyl ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, each R’ is, independently, -R, -C(O)R, -CO2R, or -SO2R, each R is, independently, -H or a C 1~30 aliphatic, a C having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon 1~30 heteroaliphatic, C 6~30Ariel, C 6~30 Aryl aliphatic C12C, having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. 6~30 The group is an optionally substituted group selected from aryl heteroaliphatic groups, 5-30 membered heteroaryls having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclils having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or The two R groups either selectively and independently come together to form a covalent bond, or Two or more R groups on the same atom may, optionally and independently, combine with that atom to form an optionally substituted 3-30 member monocyclic, bicyclic, or polycyclic ring having, in addition to that atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or The substance (drug) consists of two or more R groups on two or more atoms, which are optionally and independently combined with their intervening atoms to form an optionally substituted monocyclic, bicyclic, or polycyclic ring of 3 to 30 members, which has 0 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon in addition to the intervening atoms.

[0118] In some embodiments, a is 1. In some embodiments, b is 3. In some embodiments, a is 1, b is 3, and the composition of the substance of formula AGN107 is [ka] It has the structure of [AGN107].

[0119] Anti-galactose-deficient IgA1 binding site The anti-galactose-deficient IgA1 binding moiety can be a publicly available Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof. The moiety may also be a partially humanized galactose-deficient IgA1 antibody Km55 or its antigen-binding fragment. Alternatively, the moiety may be a chimeric galactose-deficient IgA1 antibody Km55 or its antigen-binding fragment.

[0120] In some embodiments, the galactose-deficient IgA1 binding moiety includes a moiety selected from the Markush group, which consists of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide containing one or more native amino acid residues, and a peptide containing one or more non-native native amino acid residues.

[0121] The galactose-deficient IgA1 binding site is [ka] [ABT101] Alternatively, it may be the salt, or it may contain them.

[0122] In some embodiments, ABT101 is a galactose-deficient IgA1 binding moiety. Each galactose-deficient IgA1 binding moiety in the drug may be the same galactose-deficient IgA1 binding moiety or a salt thereof.

[0123] The anti-galactose-deficient IgA1 binding site serves as a universal antibody binding site. In some embodiments, ABT101 is a universal antibody-binding moiety. In some embodiments, ABT101 is a universal antibody-binding moiety that can bind to glycan-specific IgG antibodies having different Fab regions. In some embodiments, ABT101 is a universal antibody-binding moiety that binds to an Fc region, for example, an Fc region that binds to an Fc receptor.

[0124] Galactose-deficient IgA1 binding site. In some embodiments, a galactose-deficient IgA1 binding moiety, for example, a galactose-deficient IgA1 binding moiety having the structure of ABT101, [ka] [ABT102] or [ka] It has the structure of [ABT103].

[0125] Other embodiments of the drug. In some embodiments, the present invention relates to the following formula AGN106: [ka] [AGN106] or a substance containing a salt thereof, in the formula, R 1 , R 3 , and R 5 Each of them is independently either hydrogen or C 1~6 An optionally substituted group selected from aliphatic, 3-8 member saturated or partially unsaturated monocyclic carbocyclic rings, phenyl, 8-10 member bicyclic aromatic carbocyclic rings, 4-8 member saturated or partially unsaturated monocyclic heterocyclic rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 member monocyclic heteroaromatic rings having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted group selected from an aliphatic, 3-8 member saturated or partially unsaturated monocyclic carbocyclic ring, 8-10 member bicyclic heteroaromatic rings having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 1 and R 1’ However, optionally, together with the intervening carbon atoms, they form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring, or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R 3 and R 3’However, optionally, together with the intervening carbon atoms, they form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring, or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R bonded to the same carbon atom 5 base and R 5’ The groups may optionally combine with the intervening carbon atoms to form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring, or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or Two R's 5 The groups, optionally together with those intervening atoms, C 1~10 The optionally substituted divalent linear or branched saturated or unsaturated hydrocarbon chains are formed, and 1 to 3 methylene units in the chain are independently and optionally -S-, -SS-, -N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -S(O)-, -S(O)2-, or -Cy 1 - is replaced by each -Cy 1 - is a 5-6 member heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R 1’ , R 3’ , and R 5’ Each of them is independently either hydrogen or optionally substituted with C 1~3 It is aliphatic, R 2 , R 4 , and R 6 Each of them is independently either hydrogen or optionally substituted with C 1~4 Are they aliphatic, or R 2 and R 1However, optionally, together with these intervening atoms, they form 4-8 member optionally substituted saturated or partially unsaturated monocyclic heterocycles having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R 4 and R 3 However, they may optionally combine with these intervening atoms to form a 4-8 member optionally substituted saturated or partially unsaturated monocyclic heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 6 Base and adjacent R 5 The groups, optionally together with their intervening atoms, form an optionally substituted saturated or partially unsaturated monocyclic heterocycle of 4 to 8 members, each having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. L 1 but, [ka] This is the trivalent linker portion that connects the components. L 2 However, is it a covalent bond, or C 1~30 A divalent linear or branched saturated or unsaturated hydrocarbon chain in which 1 to 10 methylene units are independently and selectively substituted with -S-, -N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -S(O)-, -S(O)2-, -(CH2OCH2)n-, -(OCH2CH2)n-, or -Cy 1 - is replaced by each -Cy 1 - is a 5-6 member heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. TBT is the cell receptor binding site, It is composed of a substance in which each of m and n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Other embodiments utilize components described in International Patent Application Publication No. 2019 / 023501.

[0126] In some embodiments, the present invention is The antibody portion, The cell receptor binding site, The present invention provides a drug comprising an antibody portion and a linker portion (optionally a single peptide bond) that connects the antibody portion and the cell receptor binding portion.

[0127] In some embodiments, the drug is given by the following formula AGN101: [ka] [AGN101] has the structure or a salt thereof, in which, Each of a, b, and c is independently between 1 and 200. Each AT is independently a galactose-deficient IgA1 binding site. L is the linker part, Each TBT is independently a cell receptor binding site. The galactose-deficient IgA1 binding site is a Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof.

[0128] In some embodiments, the drug is given by the following formula AGN102: [ka] Having the structure of [AGN102] or a salt thereof, in the formula, Each of a and b is independently between 1 and 200. Each AT is independently a galactose-deficient IgA1 binding site. L is the linker part, Each TBT is independently a cell receptor binding site. The galactose-deficient IgA1 binding site is a Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof.

[0129] In some embodiments, the drug comprises one and one or fewer galactose-deficient IgA1 binding moieties. In some embodiments, one or one or fewer galactose-deficient IgA1 binding moieties are bound to a linker moiety. In some embodiments, a is 1. In some embodiments, the drug comprises two or more glycan-specific IgG antibody moieties. In some embodiments, two or more glycan-specific IgG antibody moieties are bound to a single linker moiety. In some embodiments, a is 2 or more. In some embodiments, one and one or fewer cell receptor binding moieties are bound to a linker moiety. In some embodiments, b is 1. In some embodiments, two or more cell receptor binding moieties are bound to a single linker moiety. In some embodiments, b is 2 or more. In some embodiments, the drug comprises one and one or fewer cell receptor binding moieties. In some embodiments, c is 1. In some embodiments, b is 1 and c is 1. In some embodiments, a is 1, b is 1, and c is 1. In some embodiments, the drug comprises two or more target binding moieties. In some embodiments, b is 2 or more and c is 1. In some embodiments, b is 2 or more and c is 2 or more. In some embodiments, b is 1 and c is 2 or more.

[0130] In some embodiments, c is 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, c is selected from a group of markers in a size range, such that c is 1 to 15, c is 1 to 10, c is 1 to 9, c is 1 to 8, c is 1 to 7, c is 1 to 6, c is 1 to 5, c is 1 to 4, c is 1 to 3, c is 1 to 2. In some embodiments, c is a size selected from a group of markers with sizes 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0131] In some embodiments, each cell receptor binding portion in the drug is the same. In some embodiments, each linker portion connecting the cell receptor binding portion to the antibody portion is the same. In some embodiments, the TBT in the drug is the same. In some embodiments, several -L-(TBT) b They are the same.

[0132] In some embodiments, b is 1. In some embodiments, c is 1. In some embodiments, c is 2 or more. In some embodiments, c is 2. Those skilled in the biomedical art know that certain techniques, e.g., specific techniques used to prepare antibody-drug conjugates under this disclosure, can conjugate an antibody moiety with a target-binding moiety. In some embodiments, the target-binding moiety is conjugated to the antibody moiety via a specific type of group or amino acid residue. In some embodiments, the target-binding moiety is optionally conjugated to a lysine residue via a linker moiety. In some embodiments, the target-binding moiety is optionally conjugated to a cysteine ​​residue via a linker moiety. In some embodiments, the target-binding moiety is optionally conjugated to a non-natural amino acid residue via a linker moiety. In some embodiments, the present invention provides techniques for selectively linking a target-binding moiety to a specific amino acid residue via a linker moiety. In some embodiments, the provided techniques selectively link a target-binding moiety to a specific type of amino acid residue (e.g., a lysine residue) via a linker moiety. In some embodiments, the provided techniques selectively link a target-binding moiety to a specific site on the antibody moiety via a linker moiety. In some embodiments, the provided technology selectively connects a target-binding moiety to a specific type of amino acid residue at a particular site via a linker moiety. In some embodiments, the target-binding moiety is selectively connected via a linker moiety to K246 and K248 of the IgG1 heavy chain, and their corresponding amino acid residues. In some embodiments, the target-binding moiety is selectively connected via a linker moiety to K251 and K253 of the IgG2 heavy chain, and their corresponding amino acid residues. In some embodiments, the target-binding moiety is selectively connected via a linker moiety to K239 and K241 of the IgG4 heavy chain, and their corresponding amino acid residues.In some embodiments, the cell receptor binding portion is optionally connected to a specific amino acid residue or site via a linker. In some embodiments, each cell receptor binding portion is independently and optionally connected to a specific amino acid residue or site via a linker.

[0133] As is known to those skilled in the biomedical field, antibody drugs may contain more than one specific site (e.g., more than one chain (e.g., one or one in each of each heavy chain)). In some embodiments, the antibody moiety contains two heavy chains, and one or both of the amino acid residues or their corresponding amino acid residues are independently and optionally linked to the cell receptor binding moiety via linkers. In some embodiments, one and one or fewer are linked. In some embodiments, c is 1. In some embodiments, both are linked. In some embodiments, c is 2. In some embodiments, both target binding moieties or both linker moieties (if present) are the same.

[0134] antibody binding part Several antibody-binding moieties, including universal antibody-binding moieties, can be used under the teachings of this specification. Specific antibody-binding moieties, and techniques for identifying or evaluating them, are described in WO2019 / 023501 and WO2019 / 136442, each of which is incorporated herein by reference in its entirety. Those skilled in the biomedical art will know that additional techniques in the biomedical art may be preferred for identifying or evaluating antibody-binding moieties under this disclosure. In some embodiments, each antibody-binding moiety independently comprises one or more natural or non-natural amino acid residues.

[0135] In some embodiments, the galactose-deficient IgA1 binding moiety, for example, the protein binding moiety, for example, the antibody binding moiety, for example, the universal antibody binding moiety, has the structure or a salt form thereof, in the formula, R 1 , R3 , and R 5 Each of them is independently either hydrogen or C 1~6 An optionally substituted group selected from aliphatic, 3-8 member saturated or partially unsaturated monocyclic carbocyclic rings, phenyl, 8-10 member bicyclic aromatic carbocyclic rings, 4-8 member saturated or partially unsaturated monocyclic heterocyclic rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 member monocyclic heteroaromatic rings having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted group selected from an aliphatic, 3-8 member saturated or partially unsaturated monocyclic carbocyclic ring, 8-10 member bicyclic heteroaromatic rings having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 1 and R 1’ However, optionally, together with the intervening carbon atoms, they form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring, or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R 3 and R 3’ However, optionally, together with the intervening carbon atoms, they form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring, or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R bonded to the same carbon atom 5 base and R 5’ The groups may optionally combine with the intervening carbon atoms to form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring, or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or Two R's 5 The groups, optionally together with those intervening atoms, C 1~10The optionally substituted divalent linear or branched saturated or unsaturated hydrocarbon chains are formed, and 1 to 3 methylene units in the chain are independently and optionally -S-, -SS-, -N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -S(O)-, -S(O)2-, or -Cy 1 - is replaced by each -Cy 1 - is a 5-6 member heteroarylenyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R 1’ , R 3’ , and R 5’ Each of them is independently either hydrogen or optionally substituted with C 1~3 It is aliphatic, R 2 , R 4 , and R 6 Each of them is independently either hydrogen or optionally substituted with C 1~4 Are they aliphatic, or R 2 and R 1 However, optionally, together with these intervening atoms, they form 4-8 member optionally substituted saturated or partially unsaturated monocyclic heterocycles having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R 4 and R 3 However, they may optionally combine with these intervening atoms to form a 4-8 member optionally substituted saturated or partially unsaturated monocyclic heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or R 6 Base and adjacent R 5 The groups, optionally together with their intervening atoms, form an optionally substituted saturated or partially unsaturated monocyclic heterocycle of 4 to 8 members, each having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. L 1 However, this is the trivalent linker portion. Each of m and n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0136] In some embodiments, L 1 is a trivalent group optionally selected from C1-C 20 aliphatic or C1-C having 1 to 5 heteroatoms 20 heteroaliphatic, wherein one or more methylene units of the group are optionally and independently -C(R')2-, -Cy-, -O-, -S-, -S-S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-. In some embodiments, L 1 is -(CH2CH2O) 2~4 - or -(CH2CH2O)2-.

[0137] In some embodiments, the galactose-deficient IgA1 binding moiety, such as a protein binding moiety, such as an antibody binding moiety, such as a universal antibody binding moiety, has its structure or its salt form, wherein each of R 7 is independently hydrogen or is an optionally substituted group selected from C 1~6 aliphatic, a 3- to 8-membered saturated or partially unsaturated monocyclic carbocycle, phenyl, an 8- to 10-membered bicyclic aromatic carbocycle, a 4- to 8-membered saturated or partially unsaturated monocyclic heterocycle having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5- to 6-membered monocyclic heteroaromatic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8- to 10-membered bicyclic heteroaromatic ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or the R 7 groups and R 7’The groups, optionally together with the intervening carbon atoms, form a 3- to 8-membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring, or independently form a 3- to 8-membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1 to 2 heteroatoms selected from nitrogen, oxygen, or sulfur, R 7’ each of which is independently hydrogen or optionally substituted C 1~3 is aliphatic, R 8 each of which is independently hydrogen or optionally substituted C 1~4 is aliphatic or R 8 the group and the adjacent R 7 group, optionally together with their intervening atoms, independently form a 4- to 8-membered optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms selected from nitrogen, oxygen, or sulfur, R 9 is hydrogen, optionally substituted C 1-3 is aliphatic, or -C(O)-.

[0138] In some embodiments, the galactose-deficient IgA1 binding moiety, e.g., the universal antibody binding moiety, is a peptide moiety (e.g., a moiety having the structure of R c -(Xaa)z-), or a salt form thereof, or includes it, wherein each of R c , z, and Xaa is as described herein. One or more Xaa may independently be non-natural amino acid residues. The side chains of two or more amino acid residues may be linked to form a bridge. The side chains of two cysteine residues may form a disulfide bridge including -S-S- (which can be formed by two -SH groups as in many proteins).

[0139] In some embodiments, the galactose-deficient IgA1 binding moiety, for example, the protein binding moiety, for example, the antibody binding moiety, for example, the universal antibody binding moiety, is a cyclic peptide moiety, for example, a moiety having the structure or a salt form thereof, or comprises the same, in the formula, Each Xaa is independently an amino acid or amino acid analog residue. t is between 0 and 50. z is between 1 and 50. Each R c -L is independent of -L a -R', Each L a These can be independent, covalently bonded, or C1-C 20 Aliphatic or C1-C atoms containing 1-5 heteroatoms 20 A divalent group that is optionally substituted from heteroaliphatic groups, in which one or more methylene units of the group are optionally and independently replaced with C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-. Each -Cy- is independently and optionally substituted divalent monocyclic, bicyclic, or polycyclic group, and each monocyclic ring is independently C 3~20 Alicyclic ring, C 6~20 Selected from aryl rings, 5-20 membered heteroaryl rings having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-20 membered heterocyclyl rings having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, Each R' is independently -R, -C(O)R, -CO2R, or -SO2R. Each R is independently either -H or C 1~30 Aliphatic carbon atoms having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. 1~30 Heteroliphatic, C6~30 Ariel, C 6~30 Aryl aliphatic C12C, having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. 6~30 The group is an optionally substituted group selected from aryl heteroaliphatic groups, 5-30 membered heteroaryls having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclils having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or The two R groups either selectively and independently come together to form a covalent bond, or Two or more R groups on the same atom may, optionally and independently, combine with that atom to form an optionally substituted 3-30 member monocyclic, bicyclic, or polycyclic ring having, in addition to that atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or Two or more R groups on two or more atoms, optionally and independently, combine with their intercalating atoms to form an optionally substituted 3-30 member monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the intercalating atoms.

[0140] In some embodiments, the heteroatoms are independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.

[0141] MATE As used herein, the term "MATE" refers to a multimodal antibody therapy enhancer. This next-generation antibody conjugation technology enables site-specific pairing with therapeutic monoclonal antibodies (mAbs) or therapeutic immunoglobulins (IGs) pooled from a donor. Those skilled in the biomedical field can use MATE materials and methods as guidance to predictable results when preparing and using the present invention.

[0142] This drug can be a MATE drug or a MATE. MATE drugs are described, for example, in international application PCT / US2020 / 061127, filed on November 18, 2020, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the drug comprises an antibody portion, a cell receptor binding portion, and a linker portion connecting the antibody portion and the cell receptor binding portion.

[0143] The galactose-deficient IgA1 binding moiety may be configured to bind to galactose-deficient IgA1. In one embodiment, the galactose-deficient IgA1 binding moiety may be a recombinant IgG antibody moiety (rIgG). In another embodiment, the galactose-deficient IgA1 binding moiety may be isolated from the serum of a patient with IgA nephropathy. In another embodiment, the galactose-deficient IgA1 binding moiety may be chimeric Km55 or its antigen-binding fragment. In another embodiment, the galactose-deficient IgA1 binding moiety may be a partially humanized galactose-deficient IgA1 antibody Km55 or its antigen-binding fragment. In another embodiment, the galactose-deficient IgA1 binding moiety may be the Km55 antibody described in Yasutake et al., Nephrol. Dial. Transplant., 30, 1315-1321 (2015).

[0144] Galactose-deficient IgA1 binding site In some embodiments, the galactose-deficient IgA1 binding moiety is R c -(Xaa)z-, or a salt form thereof, or comprising the same, each variable as described herein. In some embodiments, the galactose-deficient IgA1 binding moiety is [ka] [ABT101], or a salt thereof, or comprising the same, each variable as described herein.

[0145] In some embodiments, the protein binding portion is R c The protein-binding moiety comprises -(Xaa)z- or a salt form thereof, each variable as described herein. In some embodiments, the protein-binding moiety is ABT101 or a salt form thereof, or comprises the same, each variable as described herein.

[0146] In some embodiments, the galactose-deficient IgA1 binding moiety, for example, the universal antibody binding moiety, is R c -(Xaa)z-, or a salt thereof, or comprising the same, each variable as described herein. In some embodiments, the galactose-deficient IgA1 binding moiety, for example, the universal antibody binding moiety, is ABT101, or a salt thereof, or comprising the same, each variable as described herein. In some embodiments, the galactose-deficient IgA1 binding moiety, for example, the universal antibody binding moiety, is Rc-(Xaa)z- or ABT101, or a salt thereof, and is a peptide unit, or comprises the same.

[0147] In some embodiments, -(Xaa)z- is a peptide unit or contains one.

[0148] In some embodiments, amino acid residues may form crosslinks, for example, links formed by side chains via an optional linker moiety (e.g., L). Like many polypeptides, cysteine ​​residues may form disulfide crosslinks.

[0149] In some embodiments, the peptide unit is an amino acid residue (for example, a positively charged amino acid residue, Xaa at a physiological pH of approximately 7.4) P ), for example, the following formula NH(R a1 )-L a1 -C(R a2 )(R a3 )-L a2 -COOH [LNK101] It contains amino acid residues and has a positively charged side chain, lysine (Lys, K), arginine (Arg, R), and histidine (His, H) (basic side chain). In some embodiments, the peptide unit contains R. In some embodiments, at least one Xaa is R.

[0150] In some embodiments, the peptide unit includes an amino acid residue with a functional group that can react with a functional group of another amino acid residue. In some embodiments, the peptide unit includes an amino acid residue having a side chain containing a functional group that can react with another functional group of the side chain of another amino acid residue to form a linkage (see, for example, the portions listed in Table 1). In some embodiments, a functional group of one amino acid residue is linked to a functional group of another amino acid residue to form a linkage (or crosslink). The linkage is bonded to a skeletal atom of the peptide unit and does not contain a skeletal atom. In some embodiments, the peptide unit includes a linkage formed by two side chains of non-adjacent amino acid residues. In some embodiments, the linkage is bonded to two skeletal atoms of two non-adjacent amino acid residues. In some embodiments, both skeletal atoms bonded to the linkage are carbon atoms.

[0151] In some embodiments, the galactose-deficient IgA1 binding moiety includes an optionally substituted moiety from Table 2. In some embodiments, the protein binding moiety is or includes an optionally substituted moiety from Table 2. In some embodiments, the galactose-deficient IgA1 binding moiety, e.g., the universal antibody binding moiety, is or includes an optionally substituted moiety from Table 2. In some embodiments, the galactose-deficient IgA1 binding moiety is selected from Table 2. In some embodiments, the protein binding moiety is selected from Table 2. In some embodiments, the galactose-deficient IgA1 binding moiety, e.g., the universal antibody binding moiety, is selected from Table 2. In some embodiments, the C-terminus or N-terminus is optionally capped, for example, by converting -COOH to -C(O)N(R')2 such as -C(O)NH2 for the C-terminus, or by adding R'C(O)- such as CH3C(O)- to the amino group for the N-terminus. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0152] In some embodiments, antibody-binding moieties, e.g., antibody-binding moieties, and techniques useful for developing or evaluating such moieties are, for example, Alves, Langmuir, 28, 9640-9648 (2012), Choe et al., Materials, 9, 994 (2016), Gupta et al., Nature Biomedical Engineering, 3, 917-929 (2019), Muguruma et al., ACS Omega, 4, 14390-14397 (2019), Yamada et al., Angew Chem. Int., Ed Engl.; 58(17), 5592-5597 (April 16, 2019), Kruljec et al., Bioconjug Chem., 28(8):2009-2030 (2017), e.g., Fabsorbent, triazine, etc., Kruljec et al. These are described in *al., Bioconjugate Chem., 29(8), 2763-2775(2018), WO2012017021A2, etc., and each of these binding sites (e.g., antibody binding sites) is incorporated herein by reference in its entirety.

[0153] In some embodiments, the antibody-binding portion, for example, the protein-binding portion (e.g., the antibody-binding portion), is an affinity substance described in AU2018259856 or WO2018199337, each of which is incorporated herein by reference.

[0154] In some embodiments, the antibody-binding moiety, for example, is an adapter protein agent, such as those described in Hui et al., Bioconjugate Chem., 26, 1456-1460 (2015), or includes such an adapter protein agent. In some embodiments, when used under the present disclosure, the adapter protein does not require reactive residues to achieve one or more benefits.

[0155] In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, is or includes a triazine moiety, for example, one described in US2009 / 0286693. In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, is an antibody-binding moiety having a structure such that its corresponding compound is a compound described in U.S. Patent Publication 2009 / 0286693, and these compounds are independently incorporated herein by reference. In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, is ABT. In some embodiments, the ABT is a structure such that H-ABT is a compound described in US2009 / 0286693, and these compounds are independently incorporated by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to the Fc region of an antibody.

[0156] In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, is or includes a triazine moiety, for example, one described in Teng et al., J. Mol. Recognit., 12, 67-75 (1999). In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, is an antibody-binding moiety having a structure such that its corresponding compound is a compound described in Teng et al., J. Mol. Recognit., 12, 67-75 (1999), and these compounds are incorporated independently by reference. In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, has a structure such that H-ABT is a compound described in Teng, and these compounds are incorporated independently by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to the Fc region of an antibody.

[0157] In some embodiments, the antibody-binding portion, for example, is a triazine portion, for example, one described in Uttamchandani et al., J. Comb. Chem., 6(6), 862-8 (November-December 2004). In some embodiments, the antibody-binding portion, for example, has a structure such that its corresponding compound is a compound described by Uttamchandani, and these compounds are incorporated independently by reference. In some embodiments, the antibody-binding portion, for example, has a structure such that H-ABT is a compound described by Uttamchandani, and these compounds are incorporated independently by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to the Fc region of an antibody.

[0158] In some embodiments, the antibody-binding moiety binds to one or more binding sites of proteins selected from the protein marker group consisting of protein A, protein G, protein L, protein Z, protein LG, protein LA, and protein AG. Some antibody-binding moieties are described in Choe, Durgannavar, & Chung, Materials, 9(12)(2016).

[0159] In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, can bind to a nucleotide-binding site. In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, is a small molecule that can bind to a nucleotide-binding site. In some embodiments, the small molecule is tryptamine. In some embodiments, the antibody-binding moiety, for example, the antibody-binding moiety, has a structure such that H-ABT is tryptamine. A specific useful technique was described in Mustafaoglu et al., Analyst, 141(24), 6571-6582 (November 28, 2016).

[0160] The antibody binding properties are determined. Numerous techniques for identifying, evaluating, or characterizing antibody-binding moieties, including protein-binding moieties (e.g., antibody-binding moieties such as universal antibody-binding moieties), or their use in provided techniques, such as those described in WO / 2019 / 023501, are available and incorporated herein by reference. In some embodiments, the antibody-binding moiety is a moiety (e.g., a small molecule moiety, a peptide moiety, a nucleic acid moiety, etc.) that can selectively bind to IgG and, when used in provided techniques, can provide or stimulate ADCC or ADCP. In some embodiments, peptide display techniques (e.g., phase displays, non-cell displays, etc.) can identify the antibody-binding moiety. In some embodiments, the antibody-binding moiety is a moiety (e.g., a small molecule moiety, a peptide moiety, a nucleic acid moiety, etc.) that can bind to IgG and, optionally, compete with known antibody-binding agents (e.g., protein A, protein G, protein L, etc.).

[0161] Characterization of Gal-deficient IgA1-specific antibodies secreted by cloned cell lines can be performed using the method described by Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009). Capture ELISA measures the level of antigen-specific IgG produced by IgG-secreting cell lines. The results are expressed as optical density (OD) measured at 490 nm. Levels of IgG directed towards dd-IgA1 and Fab-IgA1 are higher in IgAN patients than in the control group. IgG secreted by cell lines from IgAN patients and healthy control groups is tested for binding to hinge region glycopeptide (HR-GalNAc-BSA) or HR-BSA, with or without HAA blockade. IgG produced by IgAN patient-derived cell lines bound to HR-GalNAc in a HAA-inhibitory manner.

[0162] The ability of glycan-specific antibodies to form immune complexes with Gal-deficient IgA1 can be determined in vitro by incubation of purified IgG protein with Gal-deficient IgA1 myeloma protein (Ale mono) in a 1:1 molar ratio, according to the method of Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009). The reaction mixture was then fractionated by HPLC with IgA1-IgG immune complexes identified by cross-capture ELISA. Incubation of Gal-deficient IgA1 with IgG produced by cells derived from IgAN patients produced more immune complexes than incubation with IgG produced by cells derived from healthy controls. Analysis of the size and composition of the immune complexes suggested that they consisted of one molecule of IgG bound to either one or two molecules of IgA1.

[0163] The characterization of immune complex formation can be determined in vitro using the method of Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009). Size exclusion chromatography and ELISA analysis measure immune complexes formed in vitro with monomeric Gal-deficient IgA1 (50 μg) and glycan-specific IgG (50 μg) from patients with IgAN or healthy control groups. Columns were calibrated using IgG and monomeric (m) and dimeric (d) IgA1 standards. Glycan-specific IgG from IgAN patients shows greater binding to Gal-deficient IgA1 than bound IgG from healthy control groups. Immune complexes are likely to contain one or two molecules of IgA1 bound to two IgG molecules. Dot blot analysis shows that IgG secreted by cell lines derived from IgAN patients shows high binding to Gal-deficient IgA1.

[0164] Ig and immune complex levels can be determined in vitro using the method described by Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009). The isotype of immunoglobulins secreted by immortalized cells is determined by capture ELISA. ELISA plates were coated with 1 μg / ml of F(ab')2 fragments of goat IgG specific to human IgA, IgG, or IgM (Jackson ImmunoResearch Laboratories Inc.). Captured immunoglobulins were detected with biotin-labeled F(ab')2 fragments of goat IgG anti-human IgA, IgG, or IgM antibody (BioSource). Avidin-horseradish peroxidase conjugate (ExtrAvidin, Sigma-Aldrich) and the peroxidase chromogenic substrate o-phenylenediamine-H2O2 (Sigma-Aldrich) were added. The color reaction was stopped with 1M sulfuric acid, and the absorbance at 490 nm was measured using an EL312 BioKinetics Microplate Reader (BioTek). A standard curve for immunoglobulins was generated from a pool of normal human serum calibrated for all Ig isotypes (binding sites). The results were calculated using the DeltaSoft III computer program (BioMetallics). Urinary IgA-IgG immune complexes were measured using cross-capture ELISA.

[0165] ELISA characterization of antigen-specific IgG antibodies. Following the method of Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009), a panel of antigens (dd-IgA1, Fab-IgA1) generated using IgA-specific proteases derived from Haemophilus influenzae HK50, HR-BSA, and HR-GalNAc-BSA was used to analyze the binding of serum IgG from IgAN patients and healthy controls, as well as IgG secreted by EBV-immobilized cells from the same subjects, by ELISA. HR-GalNAc was synthesized by Bachem (asterisks indicate sites containing GalNAc). VPSTPP-*TP-*SP-*STPPTPSPSC-NH2 [SEQ ID NO: 15]. The hinge region peptide was the same peptide, but GalNAc was absent. Both preparations were crosslinked to bovine serum albumin. For the ELISA, a flat-bottomed 96-well plate (MaxiSorp, Nunc) was coated with a 1 μg / ml solution of the antigen described above. Serum or culture supernatant samples diluted in phosphate-buffered saline were added to each well. The total IgG used for analysis was normalized for all samples. Captured IgG was detected with the biotin-labeled F(ab')2 fragment of goat IgG anti-human IgG antibody (BioSource, Invitrogen). Subsequently, avidin-horseradish peroxidase conjugate (ExtrAvidin, Sigma-Aldrich) was added, and the reaction was carried out as described above.

[0166] SDS-PAGE and Western blotting were performed according to the method of Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009). SDS-PAGE allows for the separation of serum and culture supernatant under reducing conditions using a 4%–20% gradient slab gel (Bio-Rad). The amount of loaded protein was adjusted to achieve equal amounts of IgA protein in each lane. The gel was blotted on a PVDF membrane and incubated with antibodies specific to IgA heavy chains (Vector Laboratories) or biotin-labeled HAA lectin. HAA reacts with terminal GalNAc but not with sialylated GalNAc or GalNAc-Gal disaccharide. Gal-deficient IgA1 myeloma protein (Mce or Ale poly) was separated by SDS-PAGE under reducing conditions and, after electroblotting on a PVDF membrane, served as an antigen for glycan-specific IgG analysis. The bound IgG is detected with an IgG-specific antibody, and visualization of the positive band is performed by subsequent membrane incubation with an avidin-peroxidase conjugate, followed by enhanced chemiluminescence detection (Pierce, Thermo Scientific).

[0167] Dot blot analysis. Gal-deficient IgA1 cells were placed in the wells of a 96-well plate (MultiScreenHTS IP Filer Plate, Millipore) equipped with a PVDF membrane and blocked with a SuperBlock (Pierce, Thermo Scientific). Serum or cell culture supernatant (normalized to 0.5 μg of IgG in each sample) was added and incubated overnight at 4°C. 0.5 μg of rIgG from IgAN patients was used as a positive control. Binding was detected with an IgG-specific antibody, and subsequently, the membrane was incubated with an avidin-peroxidase conjugate. The reaction was visualized using enhanced chemiluminescence (Pierce, Thermo Scientific) as described in the method for Western blotting by Suzuki et al., J. Clin. Invest., 119, 1668-1677 (2009). Results were evaluated by density measurement. A value of 100% was assigned to the strength of rIgG binding to Gal-deficient IgA.

[0168] IgA antibody binding portion. Those skilled in the biomedical field know that antibodies of certain properties and activities, such as antibodies that recognize different antigens and have selective mutations, can be targeted by the antibody-binding moieties described herein. In some embodiments, such antibodies include, for example, antibodies administered to a subject for therapeutic purposes. In some embodiments, the antibody-binding moieties described may bind to antibodies against different antigens and are useful for conjugating the moiety of interest with various antibodies.

[0169] In some embodiments, the antibody-binding moiety is, for example, a meditope drug moiety, or includes one. Meditop drugs are described in US2019 / 0111149.

[0170] In some embodiments, the antibody-binding portion, for example, can bind to human IgG. In some embodiments, the antibody-binding portion, for example, can bind to an antibody selected from the Markush group of antibodies consisting of rabbit IgG, IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody-binding portion, for example, can bind to IgG1, IgG2, and IgG4.

[0171] Methods for manufacturing pharmaceuticals. The agents of this disclosure may be prepared or isolated by synthetic, semi-synthetic, or recombinant methods under this disclosure. Specific techniques are described in the examples. In some embodiments, polypeptide agents, such as cell receptor-binding partial peptide agents, may be prepared using a biological expression system. In some embodiments, the agents provided are prepared synthetically. In some embodiments, the agents provided are prepared using specific techniques described in WO2019 / 023501, which is incorporated herein in whole by reference.

[0172] Several techniques (e.g., techniques for preparing antibody-drug conjugates) can be used to prepare MATE drugs. In many such techniques, conjugation is not selective with respect to amino acid residue sites, and the product composition typically has several types of drugs, which may differ from one another with respect to several conjugated target binding sites or conjugation sites. In some embodiments, the present invention provides techniques that can be used for selective conjugation of target binding sites at specific amino acid residue sites.

[0173] In some embodiments, the present invention is a synthesis method, A step of contacting a first drug, which includes a cell receptor binding moiety optionally linked to a first reactive group via a first linker, with a second drug, which includes an antibody moiety optionally linked to a second reactive group via a second linker, wherein the first reactive group reacts with the second reactive group; The present invention provides a synthesis method comprising the step of optionally forming a drug product containing a cell receptor binding portion and an antibody binding portion via a linker.

[0174] In some embodiments, the present invention is a synthesis method, The process includes the step of contacting a first composition comprising a plurality of first drugs, each comprising a cell receptor binding moiety independently and optionally linked to a first reactive group via a first linker moiety, and a second composition comprising a plurality of second drugs, each comprising an antibody moiety independently and optionally linked to a second reactive group via a second linker moiety. The present invention provides a synthesis method in which a product composition is formed, comprising multiple product agents, each independently and selectively via a linker, each containing a cell receptor binding portion and an antibody binding portion.

[0175] The second drug. In another embodiment, the present invention provides a composition comprising a drug and at least one additional drug having a portion that can bind to an antibody forming an antibody portion of a first compound.

[0176] In some embodiments, the first composition comprises a first agent as described herein. In some embodiments, the second agent independently comprises a second reactive group. In some embodiments, the second composition comprises a plurality of agents, each cell receptor binding moiety independently comprising a reactive group as described herein. In some embodiments, the second composition is an antibody composition, and the antibody in the composition is chemically unaltered. In some embodiments, the second composition is an IVIG preparation. In some embodiments, the product composition comprises a plurality of agents, each cell receptor binding moiety independently comprising a cell receptor binding moiety as described herein.

[0177] In some embodiments, the cell receptor binding moiety in the product drug is the cell receptor binding moiety in the first drug. In some embodiments, the antibody moiety in the product drug is the antibody moiety in the second drug. In some embodiments, the second drug is an antibody drug, such as a monoclonal antibody, an antibody in a polyclonal antibody, or an antibody in an IVIG preparation. In some embodiments, the second reactive group is a functional group of an amino acid residue, such as -NH2 of Lys or -SH of Cys. In some embodiments, the second reactive group is -NH2 of a Lys residue, for example, residues selected from K246 and K248 of the IgG1 heavy chain, K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues, and K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues. In some embodiments, the present invention provides a selective reaction at specific amino acid residues of the antibody moiety.

[0178] In some embodiments, the second reactive group is optionally attached to the antibody moiety via a linker. In some embodiments, the second reactive group is optionally attached to the antibody moiety via a linker. In some embodiments, the second reactive group is selectively linked to specific positions(s) of the antibody moiety, for example, K246 and K248 of the IgG1 heavy chain, K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues, and K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues. In some embodiments, the present invention provides selective reaction at specific amino acid residues of the antibody moiety.

[0179] In some embodiments, the present invention provides agents comprising, independently, an antibody-binding moiety that binds to an antibody drug, a reactive group, a cell receptor-binding moiety, and one or more linker moieties that optionally link such group / moiety. In some embodiments, such agents are useful as reaction partners (e.g., a first agent) for conjugating an agent (e.g., a second agent) comprising a target-binding moiety, an antibody moiety, to a reactive group (e.g., a second reactive group). In some embodiments, the present invention provides agents for conjugating a target moiety to an antibody moiety in several agents or antibody drugs, such as monoclonal antibody drugs, polyclonal antibody drugs, antibody drugs of IVIG preparations, etc. In embodiments, each of the provided agents comprises a cell receptor-binding moiety, a reactive group, an antibody-binding moiety, and one or more linker moieties (linkers) that optionally link such moiety. In some embodiments, the antibody-binding moiety is a portion of the leaving groups released after contacting the drug (e.g., a first drug) with the antibody moiety (e.g., a second drug) and reacting the reactive group of the drug (e.g., a first reactive group of the first drug) with the reactive group of the antibody moiety (e.g., a second reactive group of the second drug, such as -NH2 on a Lys residue of the antibody protein). In some embodiments, the provided technique can provide improved conjugation efficiency, high selectivity, or fewer steps (in some cases, a single step) for the conjugated drug product. In some embodiments, the provided drug, e.g., a first drug, is a composition of a substance of formula AGN301. LG-RG-LRM-TBT (AGN301) or a salt thereof, in the formula, LG is a group that contains an antibody-binding portion. RG is a reactive group, LRM is a linker, TBT is the cell receptor binding site.

[0180] Those skilled in the biomedical art know that the provided agents may contain one or more stereocenters and may exist as racemic or diastereomer mixtures. Those skilled in the biomedical art know that many methods exist for isomer separation to obtain stereochemically enriched or stereochemically pure isomers of those compounds, including, but not limited to, HPLC, chiral HPLC, fractional crystallization of diastereomer salts, kinetic enzymatic resolution (e.g., lipases or esterases of fungal, bacterial, or animal origin), and formation of covalently bonded diastereomer derivatives using enantioenriched reagents.

[0181] Those skilled in the biomedical field know that some functional groups present in the compounds of this disclosure, such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles, can be interconverted by techniques well known in the biomedical field, including, but not limited to, reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. (Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5) th edition (John Wiley & Sons, 2001) (the entire edition is incorporated herein by reference). Such interconversions may require one or more techniques. Several methods for synthesizing the compounds of this disclosure are described below.

[0182] As is known to those skilled in the biomedical field, reaction partners generally come into contact with each other under conditions and for a time sufficient to produce the desired result, for example, to form a product drug and its composition to a desired degree. Many reaction conditions and reaction times may be evaluated and used to determine whether they are suitable for the purposes described herein. Several conditions, reaction times, evaluations, etc., are described in the examples.

[0183] In some embodiments, the present invention provides a product of a provided process having a lower level of damage to the antibody moiety compared to a process that includes steps taken to remove the antibody-binding moiety but not to substantially conjugate the moiety of interest (e.g., the target-binding moiety). In some embodiments, the provided product drug composition has higher uniformity (e.g., with respect to the number of cell-receptor-binding moieties per antibody moiety, or the position of amino acid residues in the antibody moiety conjugated to the moiety of interest) compared to a reference product composition (e.g., from a technique that does not use an antibody-binding moiety or does not utilize an additional step for removal of the antibody-binding moiety (e.g., does not utilize the described reaction partner which includes a reactive group between the antibody-binding moiety and the cell-receptor-binding moiety)).

[0184] In some embodiments, the present invention provides a drug product comprising an antibody moiety, a cell receptor binding moiety, and a linker moiety that optionally connects the antibody binding moiety and the cell receptor binding moiety.

[0185] In some embodiments, the present invention relates to a composition comprising a plurality of agents, wherein each agent is independent of the other. The antibody portion, The cell receptor binding site, The present invention provides a composition comprising, optionally, a linker portion that connects an antibody-binding portion and a cell-receptor-binding portion.

[0186] In some embodiments, the product drug is a MATE drug. In some embodiments, the antibody drug moiety includes an IgG Fc region. In some embodiments, the antibody moiety is optionally linked to a cell receptor binding moiety via an amino group through a linker. In some embodiments, the amino group of the side chain is optionally linked to a cell receptor binding moiety via a lysine residue (forming -NH-C(O) as part of an amide group, carbamate group, etc., via a linker).

[0187] In some embodiments, a selected site of the antibody moiety is used for conjugation. In some embodiments, K246 or K248 (EU numbered, or the corresponding residue) of the antibody drug is the conjugation site. In some embodiments, the conjugation site is K246 of the heavy chain (unless otherwise specified, the sites herein include, for example, the corresponding residue in a modified sequence (e.g., a longer, shorter, rearranged sequence). In some embodiments, the site is K248 of the heavy chain. In some embodiments, the site is K288 or K290 of the heavy chain. In some embodiments, the site is K288 of the heavy chain. In some embodiments, the site is K290 of the heavy chain. In some embodiments, the site is K317. In some embodiments, the antibody moiety is a portion of an IgG1 antibody or its antigen-binding fragment. In some embodiments, the antibody moiety is a portion of an IgG2 antibody or its antigen-binding fragment. In some embodiments, the antibody moiety is a portion of an IgG4 antibody or its antigen-binding fragment. In some embodiments, the composition comprises a plurality of MATE agents, where the antibody moiety of the MATE agent is independently an antibody moiety of an IgG1, IgG2, or IgG4 antibody or its antigen-binding fragment.

[0188] In some embodiments, the antibody heavy chain is selectively conjugated / labeled across the light chain.

[0189] In some embodiments, the present invention relates to a composition comprising a plurality of agents, each of which independently The antibody portion, The cell receptor binding site, It comprises, optionally, a linker portion that connects the antibody portion and the cell receptor binding portion, The antibody portions of multiple drugs contain a common amino acid sequence, and the multiple drugs share a common cell receptor binding region independently of at least one common amino acid residue in the common amino acid sequence. The present invention provides a composition in which approximately 1% to 100% of all drugs, including an antibody portion containing a common amino acid sequence and a cell receptor binding portion, are multiple drugs.

[0190] In some embodiments, the present invention relates to a composition comprising a plurality of agents, each of which independently The antibody portion, The cell receptor binding site, It comprises, optionally, a linker portion that connects the antibody portion and the cell receptor binding portion, Multiple drugs share the same or substantially the same antibody moiety and cell receptor binding moiety at at least one common site. The present invention provides a composition in which approximately 1% to 100% of the total drug, including the antibody portion and the cell receptor binding portion, consists of multiple drugs.

[0191] Reactive group In some embodiments, the provided drug or compound (e.g., one useful as a reaction partner, such as a first drug) comprises a reactive group (e.g., RG). In some embodiments, the reactive group (e.g., RG) is located between an antibody-binding moiety (e.g., ABT) and a moiety of interest (e.g., MOI) and is optionally and independently linked to the antibody-binding moiety and the moiety of interest via a linker. In some embodiments, the RG is the reactive group described herein.

[0192] In some embodiments, when used with drugs that do not contain an antibody-binding moiety, the reactive group reacts slowly, resulting in a low level of conjugation of the moiety of interest and the target drug, and in some embodiments, substantially no conjugation of the moiety of interest and the target drug. For example, a combination of a reactive group and an antibody-binding moiety in the same drug, such as a compound of formula AGN301 or its salts, can accelerate the reaction between the reactive group and the target drug, enhance reaction efficiency, reduce side reactions, or improve reaction selectivity (e.g., with respect to the target site where conjugation of the moiety of interest and the target drug occurs).

[0193] Reactive groups in a drug can react with several types of groups in a target drug. In some embodiments, reactive groups in a drug selectively react with amino groups of the target drug, for example, the -NH2 group on the side chain of a lysine residue of a protein. In some embodiments, when used in a drug (e.g., those of formula AGN301 or a salt thereof), the reactive group selectively reacts with one or more of specific sites of the target drug, for example, K246, K248, K288, K290, K317 of IgG1, K251, K253 of IgG2, K239, K241 of IgG4, as shown in the examples herein. In some embodiments, the site is K246 or K248 of the antibody heavy chain. In some embodiments, the site is K246 and / or K248 of the antibody heavy chain. In some embodiments, the site is K246 of the antibody heavy chain. In some embodiments, the site is K248 of the antibody heavy chain. In some embodiments, the site is K288 or K290 of the antibody heavy chain. In some embodiments, the site is K288 of the antibody heavy chain. In some embodiments, the site is K290 of the antibody heavy chain. In some embodiments, the site is K317. In some embodiments, the site is K414 of the antibody heavy chain. In some embodiments, the site is K185 of the antibody light chain. In some embodiments, the site is K187 of the antibody light chain. In some embodiments, the site is K251 or K253 of the IgG2 heavy chain. In some embodiments, the site is K251 of the IgG2 heavy chain. In some embodiments, the site is K253 of the IgG2 heavy chain. In some embodiments, the site is K239 or K241 of the IgG4 heavy chain. In some embodiments, the site is K239 of the IgG4 heavy chain. In some embodiments, the site is K241 of the IgG4 heavy chain. In some embodiments, conjugation occurs selectively at one or more heavy chain sites across light chain sites. In some embodiments, for techniques without antibody-binding sites, conjugation occurs more at light chain sites than at heavy chain sites.

[0194] In some embodiments, the reactive group (e.g., RG) is or contains an ester group. In some embodiments, the reactive group (e.g., RG) is or contains an electrophile (e.g., a Michael acceptor).

[0195] In some embodiments, the reactive group (e.g., RG) is -L RG1 -L RG2 - is or includes it, L RG1 and L RG2 Each of these is independently L as described herein. In some embodiments, the reactive group (e.g., RG) is -L LG4 -L RG1 -L RG2 - or including, each variable as described herein. In some embodiments, the reactive group (e.g., RG) is -L LG3 -L LG4 -L RG1 -L RG2 - or including, each variable as described herein. In some embodiments, the reactive group (e.g., RG) is -L LG2 -L LG3 -L LG4 -L RG1 -L RG2 - or including, each variable as described herein. In some embodiments, the reactive group (e.g., RG) is -L LG4 -L RG2 - or including, each variable as described herein. In some embodiments, the reactive group (e.g., RG) is -L LG3 -L LG4 -L RG2 - or including, each variable as described herein. In some embodiments, the reactive group (e.g., RG) is -L LG2 -L LG3 -L LG4 -L RG2 - or including such variables, each variable as described herein.

[0196] In some embodiments, L LG4 is -O-. In some embodiments, L LG4 is -N(R)-. In some embodiments, L LG4 It is -NH-.

[0197] In some embodiments, L LG3 is an optionally substituted aryl ring, or contains one. In some embodiments, L LG3 The ring is a phenyl ring or contains one. In some embodiments, the aryl or phenyl ring is substituted. In some embodiments, the substituent is an electron-absorbing group, such as -NO2, -F, etc.

[0198] In some embodiments, L RG1 It is a covalent bond. In some embodiments, L RG1 It is not a covalent bond. In some embodiments, L RG1 It is -S(O)2-.

[0199] In some embodiments, L RG2 is -C(O)-. In some embodiments, the reactive group is -L LG4 It is -C(O)- or contains it, and each variable is as described herein. In some embodiments, the reactive group is -L LG3 -L LG4 It is -C(O)- or contains it, and each variable is as described herein. In some embodiments, the reactive group is -L LG2 -L LG3 -L LG4 It includes -C(O)-, and each variable is as described herein.

[0200] In some embodiments, L RG2 is, -L RG3 -C(=CR RG1 R RG2 )-CR RG3 R RG4- and R RG1 , R RG2 , R RG3 and R RG4 Each of them is independently -L-R', and L RG3 R is -C(O)-, -C(O)O-, -C(O)N(R')-, -S(O)-, -S(O)2-, -P(O)(OR')-, -P(O)(SR')-, or -P(O)(N(R')2)-. In some embodiments, R RG1 , R RG2 , R RG3 and R RG4 Each of these is independently R'. In some embodiments, R RG1 , R RG2 , R RG3 and R RG4 One or more of these are independently -H. In some embodiments, L RG3 is -C(O)-. In some embodiments, L RG3 L is -C(O)O-. In some embodiments, L RG3 -O-, -N(R')- etc. are L PM It is connected to

[0201] In some embodiments, R RG1 is -H. In some embodiments, R RG3 It is -H.

[0202] In some embodiments, L RG2 This is an optionally substituted -L RG3 -C(=CHR RG2 )-CHR RG4 - and each variable is as described herein.

[0203] In some embodiments, R RG2 and R RG4These intercalating atoms, together with the intercalating atoms, form the optionally substituted rings described herein. In some embodiments, the formed ring is an optionally substituted 3-10 member monocyclic or bicyclic ring having 0-5 heteroatoms. In some embodiments, the formed ring is an optionally substituted 3-10 member alicyclic ring. In some embodiments, the formed ring is selected from the Marcush group, which consists of optionally substituted alicyclic rings comprising 3-8 member alicyclic rings, 5-8 member alicyclic rings, 5 member alicyclic rings, 6 member alicyclic rings, and 7 member alicyclic rings. In some embodiments, the formed ring is substituted. In some embodiments, the formed ring is not substituted. In some embodiments, the formed ring is C(=CHR RG2 ) or C (=CR RG1 R RG2 In addition to the double bond in the compound, it does not contain any further unsaturated regions.

[0204] In some embodiments, -C(=CHR RG2 )-CHR RG4 Or -C (=CR RG1 R RG2 )-CR RG3 R RG4 is optionally replaced [ka] is, or [ka] In some embodiments, -[C(=CHR RG2 )-CHR RG4 ]-L RG3 -or-[C(=CR RG1 R RG2 )-CR RG3 R RG4 ]-L RG3 - is an optional replacement. [ka] is, or [ka] In some embodiments, -L RG1 -[C(=CHR RG2 )-CHR RG4 ]-L RG3 - or -L RG1 -[C(=CR RG1 R RG2 )-CR RG3 R RG4 ]-L RG3 - is an optional replacement. [ka] In some embodiments, -L RG1 -[C(=CHR RG2 )-CHR RG4 ]-L RG3 - or -L RG1 -[C(=CR RG1 R RG2 )-CR RG3 R RG4 ]-L RG3 - is being substituted at will.

[0205] In some embodiments, the reactive group is a structure selected from Table 3 below. In some embodiments, -L LG2 -L LG3 -L LG4 -L RG1 -L RG2 - is a structure selected from Table 3 below. In some embodiments, -L LG2 -L LG3 -L LG4 -L RG1 - is a structure selected from Table 3 below. [Table 2]

[0206] In some embodiments, -L LG4 -L RG2- is -OC(O)- or -SC(O)-. In some embodiments, -L LG4 -L RG1 -L RG2 - is -SC(O)-.

[0207] In some embodiments, -L LG4 -L RG2 - is -N(-)-C(O)-, where N is a ring atom of an optionally substituted heteroaryl ring. In some embodiments, -L LG4 -L RG2 - is -N(-)-C(O)-, where N is an optionally substituted heteroaryl ring or L containing it. LG4 It is a ring atom. In some embodiments, -L LG4 -L RG2 - is -N(-)-C(O)-O-, where N is an optionally substituted heteroaryl ring or L containing it. LG4 It is a ring atom.

[0208] In some embodiments, L RG2 is an optionally substituted -CH2-C(O)-, where -CH2- contains an antibody-binding moiety or is bound to an electron-withdrawing group connected to it. In some embodiments, L RG2 is an optionally substituted -CH2- which contains an antibody-binding moiety or is bonded to an electron-withdrawing group connected thereto. In some embodiments, L RG1 L is an electron-withdrawing group. In some embodiments, L RG1 The selected element is from the Markush group consisting of -C(O)-, -S(O)-, -S(O)2-, -P(O(OR)-, -P(O(SR)-, -P(O(N(R)2)-, -OP(O(OR)-, -OP(O(SR)-, and -OP(O(N(R)2)-).

[0209] In some embodiments, L RG2is an optionally substituted -CH2-C(O)-, where -CH2- contains an antibody-binding moiety or is bound to a leaving group connected to it. In some embodiments, L RG2 is an optionally substituted -CH2- which contains an antibody-binding moiety or is attached to a leaving group connected to it. In some embodiments, L RG1 The selected element is from the Markush group consisting of -OC(O)-, -OS(O)2-, -OP(O(OR)-, -OP(O(SR)-, and -OP(O(N(R)2)-.

[0210] In some embodiments, the reactive group reacts with the amino group of the target drug. In some embodiments, the amino group is the -NH2 side chain of a lysine residue.

[0211] In some embodiments, the target drug is a protein drug. In some embodiments, the target drug is an antibody drug. In some embodiments, the reactive group reacts with an amino acid residue of such protein or antibody drug. In some embodiments, the amino acid residue is a lysine residue. In some embodiments, the reactive group reacts with the -NH2 of the side chain of the lysine residue. In some embodiments, the reactive group is -C(O)-O- or contains it, which reacts with -NH2 (e.g., of the side chain of the lysine residue) to form an amide group -C(O)-O- having -NH2.

[0212] In some embodiments, the reactive group is located at a terminal position, for example, a first reactive group, a second reactive group, etc. In some embodiments, the drug comprises a first reactive group optionally linked to a target-binding moiety via a linker moiety, and does not have an antibody-binding moiety.

[0213] In some embodiments, the present invention relates to a method for preparing a composition comprising a plurality of agents, wherein each agent is independently, The antibody portion, The cell receptor binding site, It comprises, optionally, a linker portion that connects the antibody portion and the cell receptor binding portion, Book 2, The present invention provides a method comprising contacting multiple drugs, each independently containing a reactive group, with multiple antibody drugs.

[0214] In some embodiments, the drug containing the reactive group comprises an antibody-binding moiety, a cell receptor-binding moiety, and optionally a linker. In some embodiments, the drug containing the reactive group shares the same cell receptor-binding moiety. In some embodiments, the drug containing the reactive group shares the same structure. In some embodiments, the antibody molecule has a structure, properties, or activity that provides the antibody moiety in the described drug. In some embodiments, the plurality of antibody molecules comprise two or more IgG subclasses. In some embodiments, the plurality of antibody molecules comprise IgG1. In some embodiments, the plurality of antibody molecules comprise IgG2. In some embodiments, the plurality of antibody molecules comprise IgG4. In some embodiments, the plurality of antibody molecules comprise IgG1 and IgG2. In some embodiments, the plurality of antibody molecules comprise IgG1, IgG2, and IgG4. In some embodiments, the plurality of antibody molecules comprise IgG1, IgG2, IgG3, and IgG4. In some embodiments, the plurality of antibody molecules are IVIG antibody molecules.

[0215] In some embodiments, the provided agent has a reactive group, for example, [ka] This includes. In some embodiments, -C(O)- is optionally connected via a linker to a cell receptor binding moiety or a moiety containing -(Xaa)y-, and the other end is connected to an antibody binding moiety. In some embodiments, [ka] It reacts with an amino group of another part, for example, an antibody part, to form an amide group with that part and release the part containing the antibody binding part. In some embodiments, the amino group is -NH2 of the lysine side chain. In some embodiments, -C(O)- is optionally connected via a linker to a cell receptor binding part or a part containing -(Xaa)y-, and the other end is connected to R' or an optionally substituted substituent. In some embodiments, the provided drug is optionally substituted [ka] This includes such reactive groups. Such reactive groups may be useful for conjugation with detection, diagnostic, or therapeutic agents. Those skilled in the field of biomedical technology know that several agents and technologies, such as click chemical reactions based on functional groups like amino groups, can be used for conjugation.

[0216] In some embodiments, the antibody-binding moiety binds to the Fc region of the antibody. In some embodiments, the reaction occurs at a residue in the Fc region. In some embodiments, the target-binding moiety is optionally conjugated to a residue in the Fc region via a linker moiety. In some embodiments, the residue is a Lys residue. In some embodiments, the antibody is IgG1 or contains it. In some embodiments, the antibody is IgG2 or contains it. In some embodiments, the antibody is IgG4 or contains it. In some embodiments, the antibody composition used in the method contains IgG1 and IgG2. In some embodiments, the antibody composition used in the method contains IgG1, IgG2, and IgG4. In some embodiments, the antibody composition used in the method contains IgG1, IgG2, IgG3, and IgG4.

[0217] In some embodiments, the product comprises IgG1, IgG2, IgG3, and IgG4. In some embodiments, the product composition comprises IgG1, IgG2, IgG2, and IgG4.

[0218] In some embodiments, the drug containing the antibody moiety provides one or more antibody immune activities, for example, to mobilize one or more types of immune cells, or to provide short-term and long-term immune activities. In some embodiments, the provided drug containing the antibody moiety does not significantly reduce one or more, or substantially all, of the relevant antibody immune activities. In some embodiments, the provided drug containing the antibody moiety improves one or more, or substantially all, of the relevant antibody immune activities (for example, compared to the antibody moiety itself). In some embodiments, the drug provides equivalent or better stability (e.g., residence time in the blood) compared to the antibody moiety itself. In some embodiments, the antibody moiety in the provided drug can bind to FcRy cells (e.g., several FcRy cells of immune effector cells for the desired immune activity, typically at equivalent or better levels). In some embodiments, the antibody moiety in the provided drug has equivalent Fab / antigen binding ability. In some embodiments, the antibody moiety in the provided drug has equivalent Fab / antigen binding ability. In some embodiments, the antibody moiety in the provided drug provides FcRn binding. In some embodiments, the antibody moiety in the provided drug provides FcRn binding, for example, antibody recycling or extension of half-life. In some embodiments, the provided technology is suitable for all IgG subclasses and can be used for them, making it useful for modifying blood-derived IgG products.

[0219] In some embodiments, the method provided includes one step described below. [ka] It reacts with the amino group of the lysine side chain to form an amide bond with the antibody molecule and release it, or releases a salt form thereof.

[0220] Linker section In some embodiments, the parts are optionally connected to one another via linker parts. In some embodiments, a reactive group (e.g., RG) is connected to a linker (e.g., L RM It is connected to a cell receptor binding portion (e.g., TBT) via a linker. In some embodiments, a portion (e.g., LG) is also connected to several portions by one or more linkers, e.g., L LG1 , L LG2 , L LG3 , L LG4 These may include, for example, L LG L is the linker portion described. In some embodiments, L LG1 L is the linker portion described. In some embodiments, L LG2 L is the linker portion described. In some embodiments, L LG3 L is the linker portion described. In some embodiments, L LG4 L is the linker portion described. In some embodiments, L RM L is the linker portion described. In some embodiments, L PM L is as described herein. In some embodiments, L PM L is the linker portion described. In some embodiments, L PM L is as defined in this specification.

[0221] Under this disclosure, several types of linker portions, or linker portions for several purposes (e.g., linker portions used in antibody-drug conjugates), may be used.

[0222] Linker portions can be divalent or polyvalent, depending on how they are used. In some embodiments, the linker portions are divalent. In some embodiments, the linker is polyvalent and connects two or more portions.

[0223] In some embodiments, the linker portion (for example, Lz (In the formula, z represents a superscript, for example, L PM , L RM , L LG , L LG1 (etc.) are either L or include L.

[0224] In some embodiments, L is a divalent or trivalent C that is optionally substituted with a covalent bond, or one or more aliphatic, aryl, heteroaliphatic having 1 to 20 heteroatoms, heteroaromatic having 1 to 20 heteroatoms, or any combination thereof. 1~100 The group is a methylene group, and one or more methylene groups of the group are optionally and independently C 1~6 Alkylene, C 1~6 Alkenylenes, divalent carbon atoms containing 1 to 5 heteroatoms 1~6 Heteroaliphatic group, -C≡C-, -Cy-, -C(R')2-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')- , -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S- , -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P(S)(SR')-, -P(S )(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, amino acid residue, or -[(-OC(R')2-C(R')2-) n ] is replaced by -N(R a1 )-L a1 -C(R a2 )(R a3 )-L a2 It has a structure that is -CO- or a salt form of it.

[0225] In some embodiments, L is divalent. In some embodiments, L is covalent.

[0226] In some embodiments, L is C 1~00 Aliphatic and carbon atoms containing 1 to 50 heteroatoms 1~100 A divalent or optionally substituted group selected from heteroaliphatic groups, wherein one or more methylene units of the group are optionally and independently C 1~6 Alkylene, C 1~6 Alkenylenes are divalent carbon atoms having 1 to 5 heteroatoms. 1~6 Heteroaliphatic group, -C≡C-, -Cy-, -C(R')2-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')- , -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S- , -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P(S)(SR')-, -P(S )(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, amino acid residue, or -[(-OC(R')2-C(R')2-) n ] is replaced by C 1~20 Aliphatic and carbon atoms containing 1 to 10 heteroatoms 1~20 A divalent or optionally substituted group selected from heteroaliphatic groups, wherein one or more methylene units of the group are optionally and independently C 1~6 Alkylene, C 1~6 Alkenylenes are divalent carbon atoms having 1 to 5 heteroatoms. 1~6Heteroaliphatic group, -C≡C-, -Cy-, -C(R')2-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')- , -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S- , -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P(S)(SR')-, -P(S )(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, amino acid residue, or -[(-OC(R')2-C(R')2-) n ] is replaced by C. In some embodiments, L is C 1~20 A divalent or optionally substituted group selected from aliphatic groups, wherein one or more methylene units of the group are optionally and independently -C≡C-, -Cy-, -C(R')2-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S (O)2-, -S(O)2N(R')-, -C(O)S-, -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P( S)(SR')-, -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, amino acid residue, or -[(-OC(R')2-C(R')2-) n ] is replaced by C. In some embodiments, L is replaced by a divalent or optionally substituted C 1~20It is aliphatic, and one or more methylene units of the group are optionally and independently -C≡C-, -Cy-, -C(R')2-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, -C(O)O-, amino acid residues, or -[(-OC(R')2-C(R')2-) n ] is replaced by C. In some embodiments, L is replaced by a divalent or optionally substituted C 1~100 It is an aliphatic group, and one or more methylene units of the group can be optionally and independently -C≡C-, -Cy-, -C(R')2-, -O-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, amino acid residue, or -[(-OC(R')2-C(R')2-) n ] is replaced by C. In some embodiments, L is replaced by a divalent or optionally substituted C 1~50 It is an aliphatic group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted with C 1~40 It is an aliphatic group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted with C 1~20 It is an aliphatic group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted with C 1~10It is an aliphatic group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted with C 1~100 The group is an alkylene group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted C 1~50 The group is an alkylene group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted C 1~40 The group is an alkylene group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted C 1~20 The group is an alkylene group, and one or more methylene units of the group are optionally and independently replaced as described herein. In some embodiments, L is divalent or optionally substituted C 1~10 The alkylene group is such that one or more methylene units of the group are optionally and independently replaced as described herein.

[0227] In some embodiments, the linker portion (for example, L, L) PM , L RM These include acidic groups, such as -S(O)2OH.

[0228] In some embodiments, L is -[(-OC(R')2-C(R')2-) n ]- or including it. In some embodiments, L is -[(-O-CH2-CH2-) n]- or containing it. In some embodiments, L is -[(-CH2-CH2-O)6]-CH2-CH2-. In some embodiments, L is -[(-CH2-CH2-O)8]-CH2-CH2-. In some embodiments, -CH2-CH2-O- is bound to the antibody binding site with -CH2-. In some embodiments, -CH2-CH2-O- is bound to the cell receptor binding site with -CH2-. In some embodiments, L PM L is L as described herein. In some embodiments, L RM L is as described herein.

[0229] In some embodiments, the linker portion (e.g., L) is one or more -(CH2)nO-, where each n is independently 1 to 20. In some embodiments, it is one or more -[(CH2)nO]m-, where each n is independently 1 to 20 and m is 1 to 100. In some embodiments, it is two or more -[(CH2)nO]m-, where each n is independently 1 to 20 and each m is 1 to 100. In some embodiments, it is one or more -(O)C-[(CH2)nO]m(CH2)nNH-, -[(CH2)nO]mNHC(O)[(CH2)nO]mNH-, -[(CH2)nO]m{NHC(O)[(CH2)nO]m}pNH--, where each n is independently 1 to 20, each m is independently 1 to 100, and each p is independently 1 to 10. In some embodiments, n is 1 to 10. In some embodiments, n is 1 to 5. In some embodiments, each n is 2. In some embodiments, m is 1 to 50. In some embodiments, m is 1 to 40. In some embodiments, m is 1 to 30. In some embodiments, m is 1 to 20. In some embodiments, m is 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18.In some embodiments, m is 19. In some embodiments, m is 20.

[0230] In some embodiments, the linker portion, or L, is -(CH2CH2O)n- or contains thereof, where each -CH2- is independently and optionally substituted, and n is between 1 and 20. In some embodiments, the linker portion, or L, is -(CH2)nO-(CH2CH2O)n-(CH2)n- or contains thereof, where each n is independently between 1 and 10, and each -CH2- is independently and optionally substituted.

[0231] In some embodiments, the linker portion is trivalent or polyvalent. In some embodiments, the linker portion is L as described herein, where L is trivalent or polyvalent. In some embodiments, L is trivalent. For example, in some embodiments, L is -CH2-N(-CH2-)-C(O)-.

[0232] In some embodiments, the linker portion (e.g., L) comprises one or more amino acid residues or analogues thereof.

[0233] In some embodiments, the linker portion (for example, L, L) RM(etc.) are or contain the reactive groups described herein. In some embodiments, the drug comprises an antibody-binding moiety and a cell-receptor-binding moiety linked via a linker containing the reactive group. In some embodiments, the reactive group can be reacted with a lysine residue of the antibody in an aqueous buffer described herein. In some embodiments, the reactive group comprises -C(O)-O-. In some embodiments, the reactive group comprises -C(O)-O-, where -O- is bonded to an optionally substituted aryl group. In some embodiments, the reactive group comprises -C(O)-O-, where -O- is bonded to an aryl group substituted with one or more electron-absorbing groups. In some embodiments, one or more of each electron-withdrawing group are independently selected from -NO2 and -F. In some embodiments, the aryl group is [ka] It has the structure, in the formula R s is a halogen, -NO2, -F, -L-R', -C(O)-L-R', -S(O)-L-R', -S(O)2-L-R', or -P(O)(-L-R')2. In some embodiments, the aryl group is [ka] It has the structure, in the formula, each R s These are independently halogen, -NO2, -F, -L-R', -C(O)-L-R', -S(O)-L-R', -S(O)2-L-R', or -P(O)(-L-R')2. In some embodiments, the aryl group is [ka] In some embodiments, the aryl group is [ka] In some embodiments, C1 is bound to the -O- of -C(O)-O-. In some embodiments, the cell receptor binding site is on the -C(O)- side and the antibody binding site is on the -O- side.

[0234] In some embodiments, the linker portion (for example, L, L) RM (etc.) contains a reactive group, and upon contact with an antibody, the reactive group reacts with the antibody's group, selectively conjugating the antibody via a linker to a cell receptor binding moiety or a moiety containing -(Xaa)y-. In some embodiments, the reactive group is [ka] The -C(O)- is optionally linked via a linker to a cell receptor binding moiety or a moiety containing -(Xaa)y-. In some embodiments, the reactive group is [ka] The -C(O)- group is optionally linked via a linker to either a cell receptor binding moiety or a moiety containing -(Xaa)y-, and the other end of the reactive group is linked to an antibody binding moiety.

[0235] In some embodiments, the linker portion (e.g., L) is one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) polyethylene glycol units, or comprises them. In some embodiments, the linker portion is -(CH2CH2O) n -Includes the formula, where n is as described herein. In some embodiments, one or more methylene units of L are independently -(CH2CH2O) n It is replaced with -.

[0236] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is 11. In some embodiments, n is 12. In some embodiments, n is 13. In some embodiments, n is 14. In some embodiments, n is 15. In some embodiments, n is 16. In some embodiments, n is 17. In some embodiments, n is 18. In some embodiments, n is 19. In some embodiments, n is 20.

[0237] In some embodiments, the linker portion (e.g., L) is or contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acid residues. In some embodiments, one or more methylene units of L are independently replaced by amino acid residues. In some embodiments, one or more methylene units of L are independently replaced by amino acid residues, and each amino acid residue is independently -N(R a1 )-L a1 -C(R a2 )(R a3 )-L a2 It has a structure that is -CO- or a salt form of it.

[0238] In some embodiments, the linker portion comprises one or more parts (e.g., amino, carbonyl, etc.) that can be used for connection with other parts. In some embodiments, the linker portion comprises one or more -NR'-, where R' is as described herein. In some embodiments, -NR'- improves solubility. In some embodiments, -NR'- functions as a connection point to another part. In some embodiments, R' is -H. In embodiments, one or more methylene units of L are independently replaced by -NR'-, where R' is as described herein.

[0239] In some embodiments, the linker portion (e.g., L) includes a -C(O)- group that can be used for connection with the portion. In some embodiments, one or more methylene units of L are independently replaced with -C(O)-.

[0240] In some embodiments, the linker portion (e.g., L) includes an -NR'- group that can be used for connection with the portion. In some embodiments, one or more methylene units of L are independently replaced with -N(R')-.

[0241] In some embodiments, the linker portion (e.g., L) includes a -C(O)NR'- group that can be used for connection with the portion. In some embodiments, one or more methylene units of L are independently replaced with -C(O)N(R')-.

[0242] In some embodiments, the linker portion (e.g., L) contains a -C(R')2- group. In some embodiments, one or more methylene units of L are independently replaced by -C(R')2-. In some embodiments, -C(R')2- is -CHR'-. In some embodiments, R' is -(CH2)2C(O)NH(CH2) 11It is COOH. In some embodiments, R' is -(CH2)2COOH. In some embodiments, R' is -COOH.

[0243] In some embodiments, the linker portion is or comprises one or more ring portions, for example, one or more methylene units of L being replaced by -Cy-. In some embodiments, the linker portion (e.g., L) comprises an aryl ring. In some embodiments, the linker portion (e.g., L) comprises a heteroaryl ring. In some embodiments, the linker portion (e.g., L) comprises an aliphatic ring. In some embodiments, the linker portion (e.g., L) comprises a heterocyclyl ring. In some embodiments, the linker portion (e.g., L) comprises a polycyclic ring. In some embodiments, the ring of the linker portion (e.g., L) has 3 to 20 members. In some embodiments, the ring has 5 members. In some embodiments, the ring has 6 members. In some embodiments, the ring of the linker is the product of a cycloaddition reaction (e.g., click chemistry, and its variations) used to link different portions.

[0244] In some embodiments, the linker portion (e.g., L) is [ka] It is or includes it. In some embodiments, the methylene unit of L is [ka] It is replaced by. In some embodiments, the methylene unit of L is replaced by -Cy-. In some embodiments, -Cy- is [ka] That is the case.

[0245] In some embodiments, the linker portion (e.g., L) is -CO)y- or contains it. In some embodiments, L is -[(CH2)nO]mCy[(CH2)nO]mNH- or contains it, or L is -[(CH2)nO]mCy[(CH2)nO]mNHC(O)[(CH2)nO]mNH- or L is -[(CH2)nO]mCy[(CH2)nO]m{NHC(O)[(CH2)nO]m}pNH-, where n, m, and p are independently 1 to 20, 1 to 12, or 2 to 10 at each occurrence. In some embodiments, each n is 2 and m is independently selected from an integer 2 to 10 at each occurrence, or in some embodiments, m is independently selected from an integer 2 to 6 and Cy is [ka] In some embodiments, the methylene unit of L is replaced with -Cy-. In some embodiments, -Cy- is [ka] In some embodiments, -Cy- is [ka] In some embodiments, -Cy- is [ka] That is the case.

[0246] In some embodiments, the linker portion is as shown in Table 2. In some embodiments, L is as described in this disclosure. 1 In some embodiments, L is as described in this disclosure. b That is the case.

[0247] In some embodiments, L RMIt is a covalent bond. In some embodiments, L RM It is not a covalent bond. In some embodiments, L RM is -(CH2CH2O)n- or contains it. In some embodiments, L RM is either -(CH2)nO-(CH2CH2O)n-(CH2)n- or contains thereof, where each n is independent and each -CH2- is independently and optionally substituted. In some embodiments, L RM The formula is -(CH2)nO-(CH2CH2O)n-(CH2)n-, where each n is independent and each -CH2- is independently and optionally substituted. In some embodiments, L RM The formula is -(CH2)2-O-(CH2CH2O)n-(CH2)2-, where n is and each -CH2- is independently and optionally substituted. In some embodiments, L RM The formula is -(CH2)2-O-(CH2CH2O)n-(CH2)2-, where n is as described herein.

[0248] In some embodiments, L PM It is a covalent bond. In some embodiments, L PM It is not a covalent bond. In some embodiments, L PM is -(CH2CH2O)n- or contains it. In some embodiments, L PM is either -(CH2)nO-(CH2CH2O)n-(CH2)n- or contains thereof, where each n is independent and each -CH2- is independently and optionally substituted. In some embodiments, L PM The formula is -(CH2)nO-(CH2CH2O)n-(CH2)n-, where each n is independent and each -CH2- is independently and optionally substituted. In some embodiments, L PMThe formula is -(CH2)2-O-(CH2CH2O)n-(CH2)2-, where n is and each -CH2- is independently and optionally substituted. In some embodiments, L PM The formula is -(CH2)2-O-(CH2CH2O)n-(CH2)2-, where n is as described herein.

[0249] In some embodiments, L PM (For example, in the products of the first drug and the second drug) is a reaction product portion formed by the first reactive portion and the second reactive portion, or includes such portion.

[0250] In some embodiments, the linker portion (for example, L in the product of the first agent and the second agent) PM )teeth, [ka] or including the linker portion (e.g., L) is a methylene unit or L (e.g., L RM , L PM Linker portions that can be such as are replaced with -Cy-. In some embodiments, -Cy- is optionally replaced. [ka] In some embodiments, -Cy- is [ka] In some embodiments, L is -[(CH2)nO]mCH2Cy[(CH2)nO]m- or ~~. In some embodiments, -Cy- is [ka] In some embodiments, -Cy- is [ka] In some embodiments, -Cy- is [ka] That is the case.

[0251] Cell receptor binding site According to embodiments of the present invention, several receptor-binding moieties are described in WO2019 / 199621A1, published on 17 October 2019; WO2019 / 199634, published on 17 October 2019; International Application PCT / US2020 / 055053, filed on 9 October 2020; and International Application PCT / US2020 / 055053, filed on 9 October 2020, each of which is incorporated herein by reference in its entirety.

[0252] In one embodiment, the cell receptor binding moiety may include an asialoglycoprotein receptor (ASGPR) binding group connected to the linker moiety via an amine group.

[0253] The amine group may be a primary alkylamine group or a secondary alkylamine group, in which case the amine group is optionally substituted with a C1-C3 alkyl group.

[0254] The cell receptor binding site has the following chemical structure: [ka] [TBT101] or [ka] It may also contain an ASGPR binding group that follows [TBT102]. In the formula, X is 1 to 4 atomic lengths, and O, S, N(R) N1 ) or C(R N1 )(R N1 ) contains a group, and as a result, If X has a single atomic length, then X is O, S, N(R) N1 ) or C(RN1 )(R N1 ) and If X has a length of 2 atoms, then one or fewer atoms of X are O, S, or N(R) N1 ) and If X has 3 or 4 atomic lengths, then two or fewer atoms of X are O, S, or N(R) N1 ) and R N1 is either H or a C1-C3 alkyl group optionally substituted with 1-3 halo groups. R1 and R3 operate independently of each other. H, -(CH2) K OH, optionally substituted with 1 to 3 halo groups, -(CH2) K OC1-C4 alkyl, optionally substituted with 1-3 halo groups, C1-C4 alkyl, -(CH2) K -Vinyl, O-(CH2) K -Vinyl, -(CH2) K -Alkinyl, -(CH2) K -COOH, -(CH2) is optionally substituted with 1 to 3 halo groups. K C(O)O-C1~C4 alkyl, optionally substituted with 1~3 halo groups, OC(O)-C1~C4 alkyl, optionally substituted with 1~3 halo groups, -C(O)-C1~C4 alkyl, or R1 and R3 are each independently and selectively substituted with up to three halo groups. [ka] A C1-C4 alkyl group in which each of the alkyl groups is optionally substituted with 1-3 halo groups or 1 or 2 hydroxyl groups, or an O-C1-C4 alkyl group in which each of the alkyl groups is optionally substituted with 1-3 halo groups or 1 or 2 hydroxyl groups. K is independently an integer between 0 and 4, or R1 and R3 each have the following chemical structures independently: [ka] It is a base that follows the formula, where R 7 It is an O-C1-C4 alkyl group, or R, which is optionally substituted with 1-3 halo groups and 1 or 2 hydroxyl groups. 7 -NR N3 R N4 base or [ka] is it, or R1 and R3 each have the following independent structure: [ka] A group that follows the following chemical structure, or a group that follows the following chemical structure. [ka] Base: [ka] [ka] is it, or R1 and R3 operate independently of each other. [ka] It is the basis, [ka] It is a C3-C8 saturated carbocyclic group, R C It is either absent, or a C1-C4 alkyl group optionally substituted with H, 1-3 halo groups, or 1-2 hydroxyl groups, or the following structure: [ka] It is a group that follows the formula, where R4, R5, and R6 are each independently H, halo(F, Cl, Br, I), CN, and NR. N1 R N2,-(CH2) K OH, -(CH2) is optionally substituted with 1 to 3 halo groups. K OC1-C4 alkyl, optionally substituted with 1-3 halo groups, C1-C3 alkyl, optionally substituted with 1-3 halo groups, -O-C1-C3-alkyl, -(CH2) K COOH, -(CH2) optionally substituted with 1 to 3 halo groups K C(O)O-C1~C4 alkyl, optionally substituted with 1~3 halo groups, OC(O)-C1~C4 alkyl, optionally substituted with 1~3 halo groups, -C(O)-C1~C4 alkyl, or R C teeth, [ka] basis, [ka] base [ka] base or [ka] It is the basis, R N , R N1 and R N2 Each of these is independently either H or a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxyl groups. K is an independent integer between 0 and 4. K' is an integer between 1 and 4. R N3 is a C1-C3 alkyl group optionally substituted with H, or 1-3 halo groups or 1 or 2 hydroxyl groups. R N4 is a C1-C3 alkyl group optionally substituted with H, 1-3 halo groups or 1 or 2 hydroxyl groups, or RN4 is the base, and K is preferably 1. [ka] This is a linker group comprising at least one galactose-deficient IgA1 binding moiety, which links at least one galactose-deficient IgA1 binding moiety to a cell receptor binding moiety via an optional linker moiety, or [ka] This is a linker group containing at least one functional group that can be used to covalently bond the linker group to at least one galactose-deficient IgA1 binding moiety or an optional linker moiety. R 2 teeth, [ka] It is a base, R N1 And K is the same as above, R AM This is a C1-C4 alkyl group optionally substituted with H, up to 3 halo groups and 1 or 2 hydroxyl groups, -(CH2) K -(CH2) is optionally substituted with a COOH group and 1 to 3 halo groups. K C(O)O-C1~C4 alkyl group, optionally substituted with 1~3 halo-F groups, OC(O)-C1~C4 alkyl group, optionally substituted with 1~3 halo groups, -C(O)-C1~C4 alkyl group, -(CH2) K -NR N3 R N4 group (in the formula, R N3 (is either H or a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxyl groups), R N4 is a C1-C3 alkyl group or group optionally substituted with H, 1-3 halo groups or 1 or 2 hydroxyl groups, or R2 is [ka] It is the basis, R TA H, CN, NR N1 R N2 ,-(CH2) K OH, optionally substituted with 1 to 3 halo groups, -(CH2) K C1-C4 alkyl, optionally substituted with 1-3 halo groups, C1-C4 alkyl, -(CH2) K COOH, -(CH2) optionally substituted with 1 to 3 halo groups K C(O)O-C1~C4 alkyl, optionally substituted with 1~3 halo groups, OC(O)-C1~C4 alkyl, optionally substituted with 1~3 halo groups, -C(O)-C1~C4 alkyl, or R TA This is a heteroaryl group containing 3 to 10 membered aryl or up to 5 heteroaryl atoms, and each of the above aryl or heteroaryl groups optionally contains up to 3 (preferably 1) CN, NR N1 R N2 ,-(CH2) K OH, optionally substituted with 1 to 3 halo groups, -(CH2) K OC1-C4 alkyl, optionally substituted with 1-3 halo groups or 1 or 2 hydroxyl groups, C1-C3 alkyl, optionally substituted with 1-3 halo groups, -O-C1-C3-alkyl, -(CH2) K COOH, -(CH2) optionally substituted with 1 to 3 halo groups K C(O)O-C1~C4 alkyl, optionally substituted with 1~3 halo groups, OC(O)-C1~C4 alkyl, or -(CH2) optionally substituted with 1~3 halo groups K It is substituted with C(O)-C1~C4 alkyl, or R TA teeth, [ka] basis, [ka] basis, [ka] base [ka] The group is optionally substituted with up to three halo groups, and optionally substituted with up to three C1-C3 alkyl groups. [ka] It is the basis, or R TA is the base, R N , R N1 and R N2 Each is independently either H or a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxyl groups, and each is -(CH2) K The group is optionally substituted with C1-C3 alkyl groups which are optionally substituted with 1-4, preferably 1 or 2, 1-3 fluoro groups or 1-2 hydroxyl groups. K is independently between 0 and 4.

[0255] The cell receptor binding site has the following chemical structure: [ka] [TBT101], or [ka] It may also contain an ASGPR binding group that follows [TBT102]. In the formula, X is 1 to 4 atomic lengths, and O, S, N(R) N1 ) or C(R N1 )(R N1 ) contains a group, and as a result, If X has a single atomic length, then X is O, S, N(R) N1 ) or C(R N1 )(R N1 ) and If X has a length of 2 atoms, then one or fewer atoms of X are O, S, or N(R) N1 ) and If X has 3 or 4 atomic lengths, then two or fewer atoms of X are O, S, or N(R) N1 ) and Each R N1 (R) is independently H, or a C1-C3 alkyl group optionally substituted with 1-3 halo groups, preferably F. N1 (Is preferably H or methyl, and more frequently H), R1 and R3 are independently H, -(CH2) K -(CH2) K OC1-C4 alkyl, optionally substituted with 1-3 halo (F, Cl, Br, I, preferably F) groups, C1-C4 alkyl, -(CH2) K Vinyl, O-(CH2) K vinyl, -(CH2) K Alkinyl, -(CH2) K COOH, 1 to 3 halos, preferably optionally substituted with an F group -(CH2) K C(O)O-C1~C4 alkyl, optionally substituted with 1 to 3 halos, preferably with F groups, OC(O)-C1~C4 alkyl, optionally substituted with 1 to 3 halos, preferably with F groups, -C(O)-C1~C4 alkyl, or R1 and R3 are each independently and optionally substituted with up to three (preferably one) halo groups (preferably F). [ka] A C1-C4 alkyl group, each of which is optionally substituted with 1-3 halo groups, preferably F, or 1 or 2 hydroxyl groups, or Each of the alkyl groups is an O-C1-C4 alkyl group, optionally substituted with 1-3 halo groups, preferably F, or 1 or 2 hydroxyl groups, and K is independently 0-4 (0, 1, 2, 3 or 4), or R1 and R3 each independently have the following chemical structures: [ka] It is a base that follows the formula, where R 7 is an O-C1-C4 alkyl group or R, which is optionally substituted with 1-3 halo groups, preferably F, and 1 or 2 hydroxyl groups. 7 teeth, -NR N3 R N4 base, or [ka] is it, or R1 and R3 each have the following structure independently: [ka] A group that follows the following chemical structure, or a group that follows the following chemical structure. [ka] The following basis: [ka] [ka] is it, or R1 and R3 operate independently of each other. [ka] It is the basis, [ka] It is a C3-C8 saturated carbocyclic group, R Cis either absent, or a C1-C4 alkyl group optionally substituted with H, 1-3 halo (preferably fluoro) groups, or 1-2 hydroxyl groups, or the following structure: [ka] It is a group that follows the formula, where R4, R5, and R6 are each independently H, halo(F, Cl, Br, I), CN, and NR. N1 R N2 , -(CH2) K -(CH2) K OC1-C4 alkyl, optionally substituted with 1-3 halo (F, Cl, Br, I, preferably F) groups, C1-C3 alkyl, optionally substituted with 1-3 halo, preferably F groups, -O-C1-C3-alkyl, -(CH2) K COOH, 1 to 3 halos, preferably optionally substituted with an F group -(CH2) K C(O)O-C1~C4 alkyl, optionally substituted with 1 to 3 halos, preferably with F groups, OC(O)-C1~C4 alkyl, optionally substituted with 1 to 3 halos, preferably with F groups, -C(O)-C1~C4 alkyl, or R C teeth, [ka] basis, [ka] basis, [ka] base, or [ka] It is a base, R N , R N1 and R N2Each of these is independently a C1-C3 alkyl group that is optionally substituted with H, or 1-3 halo groups, preferably F, or 1 or 2 hydroxyl groups. K is independently 0 to 4 (0, 1, 2, 3 or 4), preferably 0 or 1. K' is 1 to 4, preferably 1. R N3 is either H, or a C1-C3 alkyl group optionally substituted with 1-3 halo groups, preferably F, or 1 or 2 hydroxyl groups. R N4 is a C1-C3 alkyl group optionally substituted with H, 1-3 halo groups, preferably F, or 1 or 2 hydroxyl groups, or R N4 is the base, and K is preferably 1. [ka] This is a linker group comprising at least one galactose-deficient IgA1 binding moiety, which links at least one galactose-deficient IgA1 binding moiety to a cell receptor binding moiety via an optional linker moiety, or [ka] This is a linker group containing at least one functional group that can be used to covalently bond the linker group to at least one galactose-deficient IgA1 binding moiety or an optional linker moiety. R 2 teeth, [ka] It is a base, R N1 And K is the same as above, R AM C1-C4 alkyl, -(CH2) optionally substituted with H, up to 3 halo groups (preferably F), and 1 or 2 hydroxyl groups. K -(CH2) is optionally substituted with a COOH group, 1 to 3 halos, preferably fluorine groups.K C(O)O-C1~C4 alkyl groups, OC(O)-C1~C4 alkyl group, which is optionally substituted with 1 to 3 halos, preferably with an F group; -C(O)-C1~C4 alkyl group, which is optionally substituted with 1 to 3 halos, preferably with an F group; R N3 H is -(CH2) K -NR N3 R N4 A C1-C3 alkyl group optionally substituted with a group, or 1-3 halo groups, preferably F, or 1 or 2 hydroxyl groups, or R2 is [ka] It is the basis, R TA H, CN, NR N1 R N2 ,-(CH2) K -(CH2) K OC1-C4 alkyl, optionally substituted with 1-3 halo (F, Cl, Br, I, preferably F) groups, C1-C4 alkyl, -(CH2) K COOH, 1 to 3 halos, preferably optionally substituted with an F group -(CH2) K C(O)O-C1~C4 alkyl, optionally substituted with 1 to 3 halos, preferably with F groups, OC(O)-C1~C4 alkyl, optionally substituted with 1 to 3 halos, preferably with F groups, -C(O)-C1~C4 alkyl, or R TA C3~C 10 An aryl or 3- to 10-membered heteroaryl group containing up to 5 heteroaryl atoms, wherein each of the above-mentioned aryl or heteroaryl groups contains up to 3 (preferably 1) CN, NR atoms. N1 R N2 ,-(CH2) K -(CH2) KOC1-C4 alkyl, optionally substituted with 1-3 halo (F, Cl, Br, I, preferably F) groups or 1 or 2 hydroxyl groups, C1-C3 alkyl, optionally substituted with 1-3 halo, preferably F groups, -O-C1-C3 alkyl, -(CH2) K COOH, 1 to 3 halos, preferably optionally substituted with an F group -(CH2) K C(O)O-C1~C4 alkyl, optionally substituted with 1~3 halos, preferably with F groups, OC(O)-C1~C4 alkyl, or -(CH2) optionally substituted with 1~3 halos, preferably with F groups K It is optionally substituted with C(O)-C1~C4 alkyl groups, or R TA The following is based [ka] , [ka] basis, [ka] base, or [ka] is, or R TA It is optionally substituted with up to three C1-C3 alkyl groups, preferably one C1-C3 alkyl group, which are optionally substituted with up to three halo (preferably F) groups. [ka] It is the basis, or R TA teeth, [ka] It is the basis, R N , R N1 and R N2Each of these is independently a C1-C3 alkyl group that is optionally substituted with H, or 1-3 halo groups, preferably F, or 1 or 2 hydroxyl groups. Each-(CH2) K The group is optionally substituted with C1-C3 alkyl groups that are optionally substituted with 1-4, preferably 1 or 2, 1-3 fluoro groups or 1-2 hydroxyl groups. K is independently 0 to 4 (0, 1, 2, 3 or 4), preferably 0 or 1, or It may contain pharmaceutically acceptable salts, stereoisomers, solvates, or polymorphs thereof.

[0256] In one embodiment, If X has a length of 2 atoms, then X is -OC(R N1 )(R N1 ), C(R N1 )(R N1 )-O-, -SC(R N1 )(R N1 ), C(R N1 )(R N1 )-S-, N(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-N(R N1 ) or C(R N1 )(R N1 )-C(R N1 )(R N1 ) and If X has a length of 3 atoms, then X is -OC(R N1 )(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-OC(R N1 )(R N1 )-, -OC(R N1 )(R N1 )-O-, -OC(R N1 )(R N1)-S-, -OC(R N1 )(R N1 )-N(R N1 )-, -SC(R N1 )(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-SC(R N1 )(R N1 )-, C(R N1 )(R N1 )-C(R N1 )(R N1 )-S, -SC(R N1 )(R N1 )-S-, -SC(R N1 )(R N1 )-O-, -SC(R N1 )(R N1 )-N(R N1 )-, N(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-N(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-C(R N1 )(R N1 )-N(R N1 ), N(R N1 )-C(R N1 )(R N1 )-N(R N1 ) or C(R N1 )(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 ) and If X has a length of 4 atoms, then X is -OC(R N1 )(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 ), C(RN1 )(R N1 )-OC(R N1 )(R N1 )-(R N1 )(R N1 )-、-OC(R N1 )(R N1 )-OC(R N1 )(R N1 )-、-SC(R N1 )(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 )-、C(R N1 )(R N1 )-SC(R N1 )(R N1 )-C(R N1 )(R N1 )-、C(R N1 )(R N1 )-(R N1 )(R N1 )-SC(R N1 )(R N1 )-、-SC(R N1 )(R N1 )-SC(R N1 )(R N1 )-、N(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 )-、C(R N1 )(R N1 )-N(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 )、C(R N1 )(R N1 )-C(R N1 )(R N1 )-N(R N1 )、N(R N1 )-C(R N1 )(R N1 )-N(R N1 )、またはC(R N1 )(R N1 )-C(RN1 )(R N1 )-C(R N1 )(R N1 ) and R N1 The same applies to the above.

[0257] In one embodiment, X is OCH2 or CH2O, and R N1 H is H.

[0258] The cell receptor binding site has the following chemical structure: [ka] [TBT101] or [ka] [TBT102] It may also contain an ASGPR group that conforms to the following rules. In the formula, R1, R2, and R3 may be the substance itself, or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof.

[0259] The cell receptor binding site has the following structure: [ka] It may have, In the formula, R A is a C1-C3 alkyl group optionally substituted with 1-5 halo (preferably fluoro) groups (preferably R A (These are methyl or ethyl groups optionally substituted with 1 to 3 fluoro groups.) Z A is, -(CH2) IM -O-(CH2) IM , S-(CH2) IM , NR M -(CH2) IM , C(O)-(CH2) IM -, a PEG group containing 1 to 8, preferably 1 to 4, ethylene glycol residues, or -C(O)(CH2) IM NRM The group (preferably a PEG-containing group containing 1 to 8 ethylene glycol residues, preferably 2 to 4 ethylene glycol residues), and IM and R M However, the same applies, Z B (CH2) IM , C(O)-(CH2) IM -, or C(O)-(CH2) IM -NR M IM and R M However, the same applies.

[0260] In one embodiment, R A This may be a methyl or ethyl group optionally substituted with 1 to 3 fluoro groups.

[0261] In one embodiment, Z A This may be a PEG group containing 1 to 4 ethylene glycol residues.

[0262] In one embodiment, the methyl or ethyl group may be substituted with 1 to 3 fluoro groups.

[0263] In one embodiment, the ASGPR binding group may be N-acetyl-D-galactosamine.

[0264] In one embodiment, the cell receptor binding moiety may be low-density lipoprotein receptor-related protein 1 (LRP1), low-density lipoprotein receptor (LDLR), FcγRI binding group, FcRN binding group, transferrin receptor binding group, or macrophage scavenger receptor binding group.

[0265] A pharmaceutically acceptable excipient. For example, suitable formulations for parenteral administration via intra-articular, intravenous, intramuscular, intratumor, intradermal, intraperitoneal, and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the intended recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the embodiment of the present invention, the composition may be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or subarachnoidally. Parenteral, oral, and intra-articular administration are preferred methods of administration. Formulations of the compound may be presented in unit-dose or multi-dose sealed containers such as ampoules and vials.

[0266] Solutions or suspensions used for parenteral, intradermal, or subcutaneous applications may contain the following components: sterile diluents such as water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonicity adjusters such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. [Examples]

[0267] The present invention is further illustrated by non-limiting embodiments.

[0268] Example 1 Antibody-drug conjugates In one embodiment, the antibody-drug conjugate may have the structure shown in Figure 3, which illustrates the process of preparing an antibody-drug conjugate from a chimeric Km55 antibody and a reagent having affinity for the chimeric Km55 antibody by conjugating the chimeric Km55 antibody to the reagent.

[0269] In one embodiment, ABT010 is an antibody in which the galactose-deficient IgA1 binding moiety has the heavy chain polypeptide sequence of SEQ ID NO: 1 (HC010) and the light chain polypeptide sequence of SEQ ID NO: 2 (LC010).

[0270] In another embodiment, the chimeric Km55 antibody may include amino acid mutations (chimeric mutant Km55 antibody). In one example, the chimeric mutant Km55 antibody (ABT020) may include a sequence in which the galactose-deficient IgA1 binding moiety has the heavy chain polypeptide sequence of SEQ ID NO: 3 (HC020) and the light chain polypeptide sequence of SEQ ID NO: 2 (LC010).

[0271] In another example, the chimeric mutant Km55 antibody (ABT030) may contain a sequence in which the galactose-deficient IgA1 binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 4 (HC030) and the light chain polypeptide sequence of SEQ ID NO: 2 (LC010).

[0272] In another example, the chimeric mutant Km55 antibody (ABT040) may contain a galactose-deficient IgA1 binding moiety that has the heavy chain polypeptide sequence of SEQ ID NO: 5 (HC040) and the light chain polypeptide sequence of SEQ ID NO: 2 (LC010).

[0273] In another example, the chimeric mutant Km55 antibody (ABT050) may contain a sequence in which the galactose-deficient IgA1 binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 6 (HC050) and the light chain polypeptide sequence of SEQ ID NO: 2 (LC010).

[0274] In another example, the chimeric mutant Km55 antibody (ABT060) may contain a sequence in which the galactose-deficient IgA1 binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 7 (HC060) and the light chain polypeptide sequence of SEQ ID NO: 2 (LC010).

[0275] In another example, the chimeric mutant Km55 antibody (ABT070) may contain a galactose-deficient IgA1 binding moiety that has the heavy chain polypeptide sequence of SEQ ID NO: 8 and the light chain polypeptide sequence of SEQ ID NO: 2 (LC010).

[0276] Specific mutational strategies to reduce the binding and effector function of FcγR and c1q, and enhance antibody FcRn binding / exposure. Fc mutation strategies to reduce FcγR and c1q binding. The inventors of this application mutated Km55 and inserted an LALA mutation using a method recognized in the biomedical technology field, first described by the Winter group in the 1990s. In this example, LALA = L234A / L235A.

[0277] The inventors of this invention mutated Km55 and inserted LALA mutations using a biomedical technology-recognized method first described by the Winter group in the 1990s, and then inserted P mutations using a biomedical technology-recognized method introduced by the Roche team in 2016, including techniques for adding P329G and combinations of P329G with LALA. In this example, LALA / PA = L234A / L235A / P329A. LALA / PG = L234A / L235A / P329G. See Tilman, Schlothauer and others, Protein Engineering, Design and Selection, Volume 29, Issue 10, October 2016, Pages 457-466. This paper shows that even LALA itself lacks c1q binding. Only P329A has been tested. P329A lacks c1q binding and reduces FcgR binding. Although P329A / LALA has not been tested, it has been shown that P329G / LALA further reduces FcgR binding compared to LALA alone. [Table 3]

[0278] The inventors of this application also mutated Km55 in other regions. N297A / Q removes the native N-bond glycosylation site within the hinge region.

[0279] Fc mutation strategies to enhance FcRn binding for prolonged exposure. Many publicly available approaches and mutation sets exist to increase Fc binding to FcRn. See Figure 4 and Table 5. An older overview is provided by Strohl, Current Opinion in Biotechnology, 20(6), 685-691 (2009). [Table 4]

[0280] For examples of introducing YTE regions into proteins (M252Y / S254T / T256E), see Acqua et al., The Journal of Immunology 169(9), 5171-5180 (2002). For recent examples of combining LALA and YTE, see Cobb et al., bioRxiv, 2021-09 (2021). Further references regarding the details of the procedure are also available.

[0281] For an example of introducing an LS domain into a protein (M428L / N434S), please refer to Zalevsky et al., Nature Biotechnology, 28(2), 157-159 (2010).

[0282] Example 2 Antibody-drug conjugate production Antibody expression. Approximately 6×10 6 ExpiCHO-S cells at a density of cells / mL were cultured in ExpiCHO Expression Medium and transiently co-transfected with equal heavy and light chain vectors using an ExpiCHO transfection kit with a final concentration of vector DNA of 1.0 μg / mL. Transfected cells were cultured at 37°C at 125 rpm in a shaking flask in an incubator containing 8% carbon dioxide. ExpiCHO medium was added 18–22 hours after transfection.

[0283] Antibody purification A mixed cell culture was collected on day 6. The collected cell culture medium was replaced by tangential flow filtration before dialyzing against 50 mM MES, pH 6.5, and 5 mM sodium chloride. The sample was then applied to a HiTrap SP HP column (Cytiva Life Sciences), and the column was washed with 50 mM MES, pH 6.5, and 5 mM sodium chloride. The antibody was eluted with a 15 C.V. gradient against 50 mM MES, pH 6.5, and 1 M sodium chloride. The eluted protein was dialyzed against 50 mM sodium phosphate, pH 7.5, and 150 mM sodium chloride. The purity of the sample was analyzed by SDS-PAGE and SEC-HPLC.

[0284] Binding affinity of purified antibody to dg-IgA In one method, 10 μg / mL of deglycosylated IgA (dg-IgA) was loaded onto a Protein L chip (Sartorius) in 50 mM sodium phosphate, pH 7.5, 150 mM sodium chloride, and 0.05% bovine serum albumin. After equilibration, purified antibodies at various concentrations from 200 to 3.125 nM were analyzed for binding by association (300 seconds) and dissociation (600 seconds). The data were globally fitted and automatically generated using Octet User Software (ForteBio, Inc.). Binding kinetics were analyzed using Prism (GraphPad Software, Inc.). obs The values ​​were extracted using a synchronicity formula. off The values ​​were extracted using the phase decay formula. on to, k obs =k on ×[ligand]+k off Derive using, where [ligand] is the concentration of the free ligand in solution. D to, k off / k on The calculation was performed as follows: To correct for drift during the meeting, the slope during the drift period was fitted using linear regression, and the product of the slope and time (in seconds) was subtracted from the signal.

[0285] In an alternative method, 5 μg / mL of deglycosylated IgA (dg-IgA, in-house prepared) was loaded onto a Protein L tip (Sartorius) in phosphate-buffered saline for 600 seconds in the presence of 0.05% Tween-20 and 0.1% bovine serum albumin. After 60 seconds of equilibration, purified antibody / ADCs at various concentrations from 50.0 to 0.8 nM were analyzed for binding by association (300 seconds) and dissociation (600 seconds) using serial 2-fold dilutions. The data were globally fitted and analyzed using Octet User Software (ForteBio, Inc.). D The value was generated automatically.

[0286] Antibody-drug conjugate production A purified antibody at a concentration of 5 mg / mL in 50 mM sodium phosphate, pH 7.5, and 150 mM sodium chloride was incubated with a 3-fold molar excess of Tris-GalNAc conjugate in 5% DMSO at 25°C for 72 hours. After incubation, the mixture was diluted 25-fold in 50 mM MES, pH 7.5, and 150 mM sodium chloride, and then applied to a HiTrap SP HP column (Cytiva Life Sciences). The column was washed with 50 mM MES, pH 6.5, and 5 mM sodium chloride. The antibody was eluted with a 15 C.V. gradient to 50 mM MES, pH 6.5, and 1 M sodium chloride. The eluted protein was dialyzed against 50 mM sodium phosphate, pH 7.5, and 150 mM sodium chloride. The purity of the samples was analyzed by SDS-PAGE and SEC-HPLC.

[0287] Example 3: Increased production of antibody-drug conjugates Development of antibody-based degrading agents for galactose-deficient IgA. A specific embodiment of the composition of the substance (pharmaceutical) (Gd-IgA-specific MoDE AGN01A) removes Gd-IgA for ASGPR-dependent degradation. To produce this composition of substance, the Gd-IgA antibody ABT010 is conjugated to the ASGPR conjugate MAT001 to obtain MoDE AGN01A.

[0288] The objective of this embodiment is to optimize the production of the antibody ABT010 for in vivo testing and its subsequent conjugation.

[0289] Overview of antibody production - initial scale. A specific embodiment (ABT010) of the composition (pharmaceutical) of the substance removes Gd-IgA for ASGPR-dependent degradation.

[0290] The production strategies were as follows: (1) Expression by transient transfection in the ExpiCHO system. (2) Separation and purification of biomolecules by tangential flow (cross-flow) filtration. (3) Ion exchange chromatography using a sulfopropyl (SP HiTrap) column. (4) Dialysis against conjugated (phosphate-buffered saline) buffer. Fraction 23 was dialyzed against phosphate-buffered saline (PBS) for conjugation.

[0291] Antibody expression. Approximately 6 x 10 6 ExpiCHO-S cells at a density of cells / mL were cultured in ExpiCHO Expression Medium and transiently co-transfected with equal heavy and light chain vectors using an ExpiCHO transfection kit with a final concentration of vector DNA of 1.0 μg / mL. Transfected cells were cultured at 37°C at 125 rpm in a shaking flask in an incubator containing 8% carbon dioxide, and ExpiCHO medium was added 18–22 hours after transfection.

[0292] Antibody purification. A mixed cell culture was collected on day 6. The collected cell culture medium was replaced by tangential flow filtration before dialyzing against 50 mM MES, pH 6.5, and 5 mM sodium chloride. The sample was then applied to a HiTrap SP HP column (Cytiva Life Sciences), and the column was washed with 50 mM MES, pH 6.5, and 5 mM sodium chloride. The antibody was eluted with a 15 C.V. gradient against 50 mM MES, pH 6.5, and 1 M sodium chloride. The eluted protein was dialyzed against 50 mM sodium phosphate, pH 7.5, and 150 mM sodium chloride. The purity of the sample was analyzed by SDS-PAGE and SEC-HPLC.

[0293] The cation exchange chromatography (SP HiTrap) step yielded approximately 90% pure protein, as estimated by SDS-PAGE analysis. The estimated yield was approximately 50 mg / L for ExpiCHO expression. Binding affinity was measured to dg-IgA KD(BLI) = 23 ± 3 nM. No aggregation was observed by SEC-HPLC.

[0294] Overview of antibody production - intermediate scale. To produce AGN01A, approximately 100 μg of reaction (5 mg / mL antibody) was generated. This reaction was carried out at 25°C for 72 hours in a 3-fold molar excess of triGalNAc conjugate MAT001.

[0295] The production of conjugate products was observed by electrophoresis, specifically by an upward shift in species.

[0296] Biophysical characterization using biolayer interferometry (BLI), an activity assay, showed a binding affinity of conjugated AGN01A to dg-IgA consistent with that of the unconjugated product ABT010 (Kd approximately 18 nM). Biolayer interferometry is an optical technique for measuring macromolecule interactions by analyzing the white light interference pattern reflected from the surface of a biosensor tip.

[0297] The batch was ready to be tested for endotoxin removal and mouse studies.

[0298] Optimization of conjugation. This example demonstrates that conjugating an ASGPR conjugate to an antibody results in an improved "hetero-bifunctional degradation agent." Optimization was determined to ensure sufficient conjugation.

[0299] The binder-to-antibody ratio (BAR) measures the number of "binders," i.e., ASGPR-binders, per antibody. A BAR value of 1.8 indicates 0.9 ASGPR-binders per heavy chain.

[0300] A BAR value of approximately 1.5, determined by CE-SDS, was achieved for the initial scale of AGN01A production.

[0301] Plasmid preparation: The target DNA sequence was designed, optimized, and synthesized. The complete sequence was subcloned into an optimized vector. Transfection-grade plasmids were maximally prepared for TubroCHO cell expression.

[0302] Cell culture and transient transfection: TubroCHO cells were grown in serum-free expression medium. The cells were maintained in an Ellenmeyer flask at 37°C using 8% CO2 on an orbital shaker. One day before transfection, the cells were seeded at an appropriate density in the Ellenmeyer flask. On the day of transfection, DNA was added to the flask using cells ready for transfection. Recombinant plasmids encoding the target protein were transiently transfected into the suspension TubroCHO cell culture.

[0303] Purification and Analysis: The cell culture broth was centrifuged and then filtered. The filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and elution with buffer, the eluted fraction was pooled. The buffer was replaced with the final formulation buffer. The purified protein was analyzed by SDS-PAGE to determine its molecular weight and purity. The concentration was determined by the A280 method.

[0304] One liter is produced using the CHO-HP stable pool process. The expected yield is approximately 1.5 grams / L. onset = 53℃, T m1 = 63.25℃.

[0305] In one production batch of ABT010, this process produced 2.77 grams. The results were divided equally into 10 × 10 mL tubes, each containing 276.8 mg. [Table 5]

[0306] A binder-antibody ratio of approximately 1.8, determined by CE-SDS, was achieved for large-scale AGN01A production.

[0307] Antibody-drug conjugate production. The purified antibody ABT030 was exchanged in 20 mM phosphate buffer (pH 6.5) and concentrated to over 12 mg / mL. The antibody was then incubated at 25°C for 72 hours with a 4-fold molar excess of MATE reagent in 2.7% DMSO for a final antibody concentration of 10 mg / mL. After incubation, the conjugated product was purified by TFF ultrafiltration into phosphate-buffered saline (pH 7.4) in the presence of 50 DV 10% DMSO. Finally, the conjugated product was buffer-exchanged with phosphate-buffered saline (pH 7.4) for sample formulation.

[0308] Antibody expression and purification. The DNA sequences of the HC010 heavy chain (HC) and light chain (LC) were optimized and subcloned into a vector. TurboCHO cells were cultured in serum-free expression medium and maintained at 37°C with 8% CO2. Cells were seeded at density one day before transfection. On the day of transfection, the heavy chain and light chain vectors were transiently co-transfected into TurboCHO cells. After expression, the cell culture broth was centrifuged and then filtered. The filtered cell culture supernatant was loaded onto a MabSelect PrismA Protein A column (Cytiva Life Sciences), followed by standard washing and elution steps. The eluted proteins were replaced with phosphate-buffered saline (pH 7.2) and analyzed by SDS-PAGE, SEC-HPLC, and reduced mass spectrometry. [Table 6]

[0309] Biophysical / biochemical efficacy assays for measuring target engagement. Biomolecules are bound to the surface of the sensor chip as ligands. When the analyte flows in solution over the immobilized ligand, binding to the sensor chip surface induces a change in refractive index (RU) proportional to the bound mass. This was performed using a Biacore S200 instrument with high sensitivity and low to moderate throughput. Immediately before assay, deglycosylated IgA was immobilized on the CM5 chip via amine coupling. The deglycosylated IgA-bound analyte AGN03A was administered from 1 nM to 1 μM via six 4-fold serial dilutions in a single-cycle kinetics experimental setting. Binding affinity (Kd) was derived by a steady-state approach, and RU was plotted against analyte concentration. AGN03 Kd values ​​of 19 nM ± 1 nM were extracted from two independent biological replicas.

[0310] ASGPR-dependent uptake assay for measuring endocytosis. We evaluated ASGPR-dependent uptake by cells in vitro using a HEK293 bioluminescent cell-based assay.

[0311] A common assay format. Harvest HEK293 (+ASGPR) cells - rinse twice with phosphate-buffered saline.

[0312] 3 ml / T182 flask, 37°C, 5-10 minutes.

[0313] Cells: 1.85×10 6 cells / mL, 96% viable cells, 15.23 μm.

[0314] Add 1% P / S to DMEM + 200 μg / mL G418 + 10% fetal bovine serum, and set the cells in 40k / 100 μl / well overnight to grow.

[0315] dg-IgA endocytosis assay.

[0316] Dilute BH 3845, BH 5820, and BH 5305 to prepare a 10-fold stock solution of 150 μL (0.0938 mg / mL = 0.625 μM).

[0317] 0.625 μM BH 3845 (7.14 mg / mL) = 2 μL + 148 μL optimization.

[0318] We will optimize the mixture of 0.625 μM BH 5820 (9 mg / mL) = 1.6 μL + 148.4 μL.

[0319] 0.625 μM BH 5305 (5.26 mg / mL) = 2.7 μL + 147.3 μL optimized.

[0320] Dilute 1 / 3 of the 100% optimized + 50 μl sample for 6 different concentrations.

[0321] #7: 1 / 10 15μL + 135μL optimization.

[0322] Finally, there is the blank antibody.

[0323] Prepare a detection antibody to add to the well stock.

[0324] Columns 1-6: 1.1 ug / mL dg-IgA-594-5 mL (5.5 μL of dg-IgA).

[0325] Columns 7-12: 1.1 ug / mL IgA-594-5 mL (2.75 μL of IgA).

[0326] After overnight growth, manually remove the culture medium.

[0327] For the control group, 90 μl of dg-IgA / IgA mAb or 0Ab will be added.

[0328] Add 10 μl of serially diluted compound to the first 9 columns, and then add the optimal 10 μl to the remaining columns.

[0329] Incubate overnight - read the Incucyte in the red channel at 4 hours, 12 hours, and 20 hours. [Table 7] [Table 8]

[0330] Example 4 Targeted degradation of circulating galactose-deficient IgA1 using a lysosome-targeted bifunctional conjugate AGN03A is an antibody-based bifunctional conjugate designed to bind to and degrade pathogenic circulating galactose-deficient IgA1 and IgG:Gd-IgA1 immune complexes via ASGPR-mediated hepatocyte integration. Compelling preclinical evidence has been obtained in cellular and rodent experiments for the use of AGN03A for rapid and robust degradation of deglycosylated IgA and associated protein aggregates. AGN03A recognizes pathogenic galactose-deficient IgA1 in human IgAN and renal disease patient plasma samples. AGN03A has therapeutic potential as a transformative non-immunosuppressive therapy for patients with IgAN.

[0331] Drug synthesis. The MoDE platform develops bifunctional molecules that degrade extracellular protein targets via the asialoglycoprotein receptor (ASGPR)-mediated endosomal / lysosomal pathway. The inventors of this invention manipulated an anti-human galactose-deficient IgA1 chimeric antibody containing the human IgG1 Fc region. An ASGPR-conjugated bifunctional conjugate (AGN03A) was assembled in one step from antibody ABT030 with four equivalent amounts of MATE reagent in intravenous buffer containing 10% DMSO, using proprietary FcIII-targeted MATE® technology (disclosed in Table 2). The linker and ASGPR conjugate were conjugated via a stable amide linkage. AGN03A was isolated in good yield and homogeneity (conjugate-to-antibody ratio (BAR) = 2, approximately 87%). Site-specific conjugation of the bi-Lys 248 residue was confirmed by peptide mapping.

[0332] Synthesis of deglycosylated IgA. Deglycosylated IgA (dg-IgA) is a semi-synthetic galactose-deficient IgA1 surrogate prepared from pooled human serum IgA in three enzymatic steps. Human serum IgA was treated with PNGase, neuraminidase, and galactosidase to produce deglycosylated IgA (dg-IgA).

[0333] AGN03A demonstrated the ability to simultaneously engage ASGPR with the target protein (POI). AGN03A showed strong affinity for dg-IgA (KD=4nM) and the carbohydrate recognition domain (148-291) (KD=9nM) of ASGPR1. Proximity-based time-resolved fluorescence resonance energy transfer assays provided evidence for the formation of a viable ternary complex with a hook effect. See Figure 7.

[0334] Detection of galactose-deficient IgA1 levels in human plasma samples. Galactose-deficient IgA1 antibody concentrations in patient plasma samples were measured using Meso Scale Discovery with immobilized AGN03A and SULFO-TAG anti-IgA antibodies. Galactose-deficient IgA1 levels in samples from patients with renal disease and IgAN were approximately four times higher than in healthy volunteers. The results were consistent with literature reports using the KM55 diagnostic. See Figure 6. The results confirm that AGN03A recognizes significant levels of circulating galactose-deficient IgA1 and corresponding immune complexes. See Figure 8. [Table 9]

[0335] In vitro intracellular internalization of dg-IgA by AGN03A. In human embryonic kidney (HEK) cells transfected with human ASGPR1 (hASGPR1), dose-dependent, selective endocytosis of dg-IgA versus IgA was observed with AGN03A. Low nanomolar half-effect concentrations and robust mean fluorescence were observed at 12 hours for the internalization of dg-IgA conjugated with Alexa Fluor 594. AGN03A internalizes the target protein for lysosomal degradation, while normal IgA is spared. See Figure 9.

[0336] Intracellular intercellularization of the dg-IgA1 antibody complex. In human embryonic kidney (HEK) cells transfected with human ASGPR1 (hASGPR1), dose-dependent, selective dg-IgA vs. IgA endocytosis was observed with AGN03A. Low nanomolar half-effect concentrations and robust mean fluorescence were observed at 12 hours for the internalization of dg-IgA conjugated with Alexa Fluor 594. AGN03A internalizes the target protein for lysosomal degradation, while normal IgA is spared. [Table 10]

[0337] Endocytosis of 1 μg / mL dg-IgA and total IgA conjugated with Alexa Fluor 594 was measured using HEK293 cells transfected with ASGPR1. MFI = mean fluorescence intensity, S / N = signal-to-noise ratio.

[0338] Isolation of dg-IgA aggregates by size exclusion chromatography. dg-IgA conjugated to Alexa Fluor A594 was separated into three fractions corresponding to tetramer, dimer, and monomeric forms. All three forms of dg-IgA were subjected to intracellular reintegration assays. See Figure 10. [Table 11]

[0339] Intracellular internalization of dg-IgA1 antibody complexes. AGN03A demonstrated efficient internalization of dimeric and tetrameric DG-IgA complexes in HEK(hASGPR1) cells within 12 hours, similar to the internalization of DG-IgA monomers. AGN03A can internalize and degrade large IgA antibody complexes. See Figure 11. [Table 12]

[0340] Endocytosis of 1 μg / mL monomeric, dimeric, and tetrameric dg-IgA conjugated with Alexa Fluor 594 was measured using HEK293 cells transfected with ASGPR1.

[0341] AGN03A achieves robust degradation of exogenous dg-IgA in mice. Following intravenous administration, AGN03A exhibited ASGPR-dependent clearance in wild-type mice compared to ASGPR1 knockout (KO) mice. The results were consistent with the mechanism of action of AGN03A. AGN03A depleted exogenous dg-IgA in wild-type mice to 58% of the area under the curve of the control at a 2:1 (drug:target) ratio after sequential intravenous (IV) administration. The parental control antibody ABT030 did not affect the degradation of exogenous dg-IgA in wild-type mice, as expected. AGN03A rapidly and robustly degrades the enzymatically deglycosylated form of human IgA administered to mice. See Figure 12.

[0342] Example 5 Improved conjugation method Cysteine-based conjugation of the Km55 variant does not present an opportunity for selective conjugation.

[0343] Site-specific conjugation of Lys248 on the HC of Fc via a proprietary FcIII-targeted MATE (disclosed in Table 2) yields an overall DAR value (DAR2) of approximately 2.0 in over 80% of the target DAR (DAR2). Four equivalents of the MATE reagent (FcIII, a 13-membered cyclic peptide in some examples) were incubated with Km55 variant antibody in phosphate-buffered saline containing 10% DMSO at pH 7.4 for 48 hours. The product was then incubated in TFF buffer exchange to remove the FcIII moiety and obtain the composition of the substance (pharmaceutical).

[0344] In some cases, mAb conjugation with the MATE reagent exhibits site specificity at Lys248. Approximately 91–92% of conjugation occurred at the expected site, heavy chain K248 (or heavy chain K247 in linear numbering). Approximately 0.3% occurred at heavy chain K246 (or heavy chain in linear numbering), which is a lysine proximal to the MATE reagent binding site. The second most abundant site was the heavy chain, with an occupancy of approximately 7–9%, located at the C-terminus of the CH1 domain and several residues prior to the start of the hinge region. The third most abundant site was the N-terminal amine of LC, with an occupancy of 0.7%. The remaining 15 sites had occupancies of less than 0.5%.

[0345] The inventors of this application performed the following conjugation steps using the Km55 variant. ● Clarification. ● Direct Product Capture (DPC). ● Virus inactivation. ● Filtration to an uncertain depth. ● Uncertain ultrafiltration / diafiltration (UF / DF). ● Conjugation. Development of conjugation in downstream processes. Conjugation occurs as part of the mAb manufacturing process. ● Cation exchange chromatography (CEX). MATE reagent and hydrolyzed GalNac can be removed by CEX chromatography, but FcIII peptide cannot. ● Affinity chromatography (AC). The conjugation method incorporates subsequent protein A-based affinity chromatography before the ion exchange step. The protein A column removes excess MATE reagent. Capture chromatography is effective in completely removing FcIII peptides. The capture chromatography (protein A) column is reused after conjugation. ● Anion exchange chromatography (AEX). ● Ultrafiltration / diafiltration (UF / DF). FcIII peptides can be removed on a small scale by UF / DF, but large-scale purification is difficult. Aggregation with DMSO cosolvent results in a large diavolute volume. ●Formulation.

[0346] The inventors of this application developed highly sensitive analytical methods (HPLC) for each of the following. The expected LOQ is approximately 400 ppm.

[0347] Typical bioconjugation for antibody-drug conjugates is usually performed with fully purified and release-tested mAbs at a separate facility. The use of non-cytotoxic payloads increases the opportunities for a “integrated” approach.

[0348] The inventors of this application streamlined the purification and testing processes. An improvement of more than 2m in overall cycle time was anticipated.

[0349] Conjugation conditions. A range of suitable assays evaluating pH and temperature for conjugation. The target is >80% DAR2. Higher temperatures increase DAR2 over 24 hours. Lower pH (approximately pH 6.0–6.2) favors DAR1, while higher pH (approximately pH 6.2–6.4) favors DAR3. [Table 13]

[0350] Next, the inventors of the present invention pursued an improved method for selectivity in bioconjugate formation using MATE technology. The inventors evaluated three types of buffer solutions. All three yielded comparable results. A 20 nM phosphate buffer solution was selected.

[0351] The inventors tested several conditions using an experimental design approach. They collected size exclusion chromatography (SEC) data and mass spectrometry DAR data. After the results of the first round of one-of-a-kind (OFAT) analysis showed that a DAR2 of over 80% was achievable by the analysis, they further optimized the conditions. After conducting a suitable range analysis, the inventors selected the final conditions and achieved a target DAR2 of 88%. [Table 14]

[0352] The inventors performed peptide mapping to confirm the selectivity of the conjugate site. They also confirmed that controlling the pH is important to prevent "off-target" conjugates. pH=6.0 yields preferred conjugates only with the desired lysine. The amount of LP can be reduced to 2.5 equivalents, but a preferred reaction time requires a high temperature of 35°C. [Table 15]

[0353] Medium scale purification. The results were comparable to those obtained from smaller-scale experiments using the same conjugation conditions.

[0354] A 3L production trial was conducted to generate a Km55 variant for conjugation, and the substance composition (pharmaceutical) was prepared. The 3L MCB trial showed comparable cell growth and metabolic profiles to the 15L trial.

[0355] A 15L production trial was conducted to generate a Km55 variant for conjugation, and the substance composition (pharmaceutical) was prepared. A 3L confirmation trial of MCB achieved cell growth and productivity equivalent to that of the 15L trial. ●mAb: Buffer solution: 20mM PB, pH 6.5 ●Concentration: 16.40mg / mL ●UF / DF: DF buffer: 20mM PB, pH 6.2 ●Concentration: 23.81mg / mL ●Conjugated concentration: 20 mg / mL, ●LP / mAb molar ratio: 2.5, LP batch number: MC01432-4-P-L1 ●Temperature: 33℃ ● pH: 6.2 ●Time: 48 hours ● Quench: Adjust the pH to 5.1 with 1M acetic acid.

[0356] After quenching and sampling, the product was stored at -70°C.

[0357] DAR1 decreased after CEX under low loading density (loading density: 32.3 g / L, lower than PD 45-50 g / L), while DAR3 decreased after AEX in 15 L of material production.

[0358] Larger-scale purification (robust process). A 500L production trial was conducted to generate the Km55 variant for conjugation, and the substance composition (pharmaceutical) was prepared. The titers of the 500L and 3L satellite trials were equivalent to those of the 15L trial. After 14 days of incubation, upstream production was successfully completed with a titer of 6.15 g / L and 511.90L of culture medium. The process parameters were as follows: ●Filter: 90ZB: Loading density: 93L / m³ 2 (≦100L / m 2 ). ●Resin: MabSelect PrismA: Column inner diameter: 30.0cm, BH: 21.5cm (15~25cm), CV: 15.197L Loading density: 46.4 (trial 1), 43.6 (trial 2), 46.9 (trial 3), 45.7 (trial 4) g / L resin (20~50 g / L resin). ●Low pH: pH3.59 (3.50~3.70). ●Neutralization pH: pH5.45 (5.3~5.7). ●Filter: 90ZB: Loading density: 1667g / m2 (≤2000g / m2) 2 ). ●Filter: Pellicon 3, PES, MWCO 30kDa, A screen: Loading density: 550g / m2 (≤1000g / m2) 2 The DF / OC concentrations are 25.2 g / L and 35.2 g / L, respectively. ●DF buffer: 20mM PB, pH 6.1; UF / DF pool concentration: 25.7 (22.5~27.5) g / L. ●mAb: 20 mg / mL; MATE reagent / mAb molar ratio: 2.5; Conjugated buffer: 20 mM PB, pH 6.1; 33°C, 2 days. ●Purification after conjugation. Resin: POROS XS; Column inner diameter: 30.0 cm, BH: 22.9 cm (15-25 cm), CV: 16.187 L. ●Loading density: 43.0 (trial 1), 41.1 (trial 2) g / L resin (28~45 g / L resin). ●Resin: MabSelect PrismA: Column inner diameter: 30.0cm, BH: 25.1cm (15~25cm), CV: 17.742L. ● Loading density: 37.3 (trial 1), 37.4 (trial 2) g / L resin (20~40 g / L resin). ●Resin: POROS 50HQ: Column inner diameter: 20.0cm, BH: 21.6cm (15~25cm), CV: 6.785L. ● Loading density: 169.8g / L resin (50~300g / L resin). ●Virus filter: Planova BioEX: 1 m2. ●Loading density: 54L / m2 (≦288L / m2). ●Filter: Pellicon 3, CRC, MWCO 30kDa, C screen: Loading density: 501g / m2 (≤800g / m2). ●DF buffer: 20mM His, pH 5.7; UF / DF pool concentration: 77.5g / L. ●Formulation buffer: 20 mM His, 8% (w / v) sucrose, 0.02% (w / v) PS80, 5 mM methionine, pH 5.7. ●Target AGC concentration: 50.0mg / mL. ●Total 101.063 L of mAb intermediate in 20 mM PB, pH 6.1. ●Formulation buffer: 20 mM His, 8% (w / v) sucrose, 0.02% (w / v) PS80, 5 mM methionine, pH 5.7.

[0359] The 500L non-GMP production was completed successfully without any unexpected events. The overall yield was 81.4%, and the step yields for individual operating units were comparable to the PD data (see above). The affinity chromatography chromatogram was also comparable to the PD data.

[0360] Cumulative yield of purification after conjugation: 87% (from CEX to UF / DF). Protein concentrations of Protein A, measured by HPLC, were 4.59 g / L at 312.48 L and 4.57 g / L at 304.36 L after clarification, with a yield of 89.7% for the clarification step. 14.63 L of UF / DF pool (77.5 mg / mL, 1134.1 g of compound 1) was used for formulation, and 22.55 L of DS (1127.5 g of compound 1) was obtained for fractionation.

[0361] AGC DS formulation: 50.0 mg / mL (target) AGC, 20 mM His, 8% (w / v) sucrose, 0.02% (w / v) PS80, 5 mM methionine, pH 5.7.

[0362] The production downstream process for 500L of the non-GMP mAb intermediate was successfully completed with an overall yield of 81.4% (from clarification to intermediate UF / DF).

[0363] The step yields for individual operating units and chromatograms are comparable to those of the PD data.

[0364] The final composition (pharmaceutical) produced by this larger-scale purification process weighed approximately 1 kg. Therefore, the amount of composition (pharmaceutical) that could be produced was 1 kg or more.

[0365] Example 6 Surface plasmon resonance (SPR) activity assay. The inventors of this application performed surface plasmon resonance to confirm the binding of AGN03A to dg-IgA. CM5 amine coupling showed a binding response but was not viable for batch releases (KD > 100 nM, low-activity surface). The assay provides robust and repeatable results due to orientation-specific protein-L capture.

[0366] TR-FRET assay. Time-resolved fluorescence resonance energy transfer (TR-FRET) assays were used to evaluate the formation of a ternary complex between ASGPR-1, AGN03A, and dg-IgA. AGN03A was diluted 2-fold over 12 points in phosphate-buffered saline pH 7.4 and transferred to 384-well ProxiPlate (Revvity) using an Echo 650 liquid handler (Beckman Coulter) to achieve final test concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.1 nM, 1.6 nM, 0.78 nM, 0.39 nM, and 0 nM. Only phosphate-buffered saline solvent was added to the plates as a negative control because there was no expected induction of protein-protein interactions.

[0367] A solution containing 25 nM biotin-ASGPR-1 (Viva Biotech) and 5.5 nM dg-IgA was prepared in 1× assay buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1 mM DTT, 0.01% Tween-20) and added to an assay plate containing AGN03A diluent and phosphate-buffered saline. After incubation at room temperature for 60 minutes, a solution containing 3 nM streptavidin-europium cryptate (Revvity) and 25 nM goat anti-human IgA-AlexaFluor 647 conjugate (Jackson ImmunoResearch) was prepared from 1x assay buffer and added to the plate. The reaction mixture was incubated for a further 60 minutes. TR-FRET was measured using an Envision multi-label reader (Revvity) equipped with filters to excite at 320 nm and emit at 665 nm and 615 nm. The TR-FRET value was calculated as the ratio of emission at 665 nm and 615 nm, normalized to [(665 / 615)*1000], and plotted as the response at each test concentration of AGN03A. The increase in the TR-FRET ratio as a function of test concentration indicates induced proximity of labeled ASGPR and dg-IgA, thereby indicating the formation of an AGN03A-mediated ternary complex.

[0368] The TR-FRET signal depends on the AGN03A-induced proximity of ASGPR and dg-IgA. For assay information, see Pettersson & Crews, Drug Discovery Today: Technologies, Vol. 31, pp. 15-27 (2019). The induced proximity between ASGPR and dg-IgA generates a specific energy transfer signal.

[0369] The inventors titrated ASGPR and dg-IgA on immobilized [Compound 1] and immobilized donor / acceptor fluorophores. The signal "hot spot" on the matrix confirmed that the PPI was detectable and identified the ideal concentrations of dg-IgA and ASGPR1. [Table 16]

[0370] After optimizing protein and fluorophore concentrations, AGN03A was administered in the range of 0.4–400 nM. A ternary complex and hook effect specific to the presence of AGN03A were observed. The response is specific to the induced proximity between ASGPR and dg-IgA. AGN03A at 6–12 nM produces approximately 3.5 times the signal / background peak response in this system. The response is specific to galactose-deficient IgA compared to native IgA.

[0371] The resulting signal emission from the acceptor is proportional to the level of interaction.

[0372] In summary, a ternary complex was observed between ASGPR1, compound 1, and dg-IgA.

[0373] Equal portions Those skilled in the field of biomedical technology will be able to recognize or confirm numerous equivalents to the specific procedures described using only routine experiments. Such equivalents are considered to be within the scope of the invention and are encompassed by the claims. Other pharmaceutically acceptable salts not specifically revealed herein may be used. Furthermore, specific items within the item list, or subsets of items within a larger group of items, are intended to be combined with other specific items, subsets of items, or larger groups of items, with or without specific disclosure herein specifying such combinations.

[0374] References Those skilled in the field of biomedical technology may use these patents, patent applications, and scientific literature as guidance to predictable results when creating and using the present invention.

[0375] Patent Documents U.S. Patent No. 7,083,784 (Dall'Acqua et al.), Molecules with extended half-lives, compositions and uses thereof. U.S. Patent No. 7,658,921 (Dall'Acqua et al.), Molecules with extended half-lives, compositions and uses thereof. U.S. Patent No. 8,088,376 (Chamberlain et al.), Fc variants with changed binding to FcRn. U.S. Patent No. 8,969,526 (Baehner), Antibody Fc variants. U.S. Patent No. 9,562,100 (Dall'Acqua et al.), Molecules with extended half-lives, compositions and uses thereof. U.S. Patent Publication No. 2020 / 0190165 (Yamada et al.), Compound having affinity substance to soluble protein, cleavable portion and reactive group, or salt thereof. European Patent No. EP1355919 (MedImmune LLC et al.), Molecules with extended half-lives, compositions and uses thereof. International Patent Publication No. WO2002 / 060919 (MedImmune LLC et al.), Molecules with extended half-lives, compositions and uses thereof. International Patent Publication No. WO2012 / 017021 (Graffinity Pharmaceuticals GmbH), Ligands for antibody and Fc-fusion protein purification by affinity chromatography. International Patent Publication No. WO2015 / 064348, Monoclonal antibody that recognizes sugar-chain-deficient human IgA1 hinge region, and use therefor, published May 7, 2015. International Patent Publication No. WO2019 / 023501 (Kleo Pharmaceuticals, Inc.), Universal ABT compounds and uses thereof, published January 31, 2019. Japanese Patent Publication No. JP2010-285419, "Antibody recognizing sugar chain deficient human IgA1 hinge region and use thereof," published December 24, 2010.

[0376] Non-patent literature Acqua et al., Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences. The Journal of Immunology, 169(9), 5171-5180(2002). Alegre et al., A non-activating humanized anti-CD3 monoclonal antibody keeps immunosuppressive properties in vivo. Transplantation 57.11(1994):1537-1543. Alves et al.,Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection,Langmuir,28,9640-9648(2012). Barratt et al.,Randomized phase II JANUS study of atacicept in patients with IgA nephropathy and persistent proteinuria.Kidney Int.Rep.,7(8),1831-1841(2022). Bolt et al.,The generation of a humanized,non-mitogenic CD3 monoclonal antibody which keeps in vitro immunosuppressive properties.European Journal of Immunology 23(2),403-411(1993). Caianiello et al.,Bifunctional small molecules that mediate the degradation of extracellular proteins.Nature Chemical Biology,17(9),947-953(2021)describes ASGPR-dependent mechanism of Molecular Degraders of Extracellular targets(MoDE)-induced degradation. Choe,Durgannavar,& Chung,Fc-binding ligands of immunoglobulin G:An overview of high affinity proteins and peptides.Materials,9(12)(2016). Cobb et al., A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in rhesus macaques. bioRxiv, 2021-09 (2021). Further references regarding the procedure are also available. Gupta et al., Computationally designed antibody-drug conjugates self-assembled via affinity ligands.Nature Biomedical Engineering, vol.3, 917-929(2019). Hui et al., LASIC:Light activated site-specific conjugation of native IgGs.Bioconjugate Chem.,26,1456-1460(2015). IBL-America,Novel ELISA(using KM55)specifically detects Gd-IgA1 #27600 Gd-IgA1(Galactose-deficient IgA1),Pamphlet 2. 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Lund et al.,Multiple binding sites on the CH2 domain of IgG for mouse FcγR11.Molecular immunology 29.1(1992):53-59. Martin-Penagos et al.,Measurement of galactosyl-deficient IgA1 by the monoclonal antibody Km55 contributes to predicting patients with IgA nephropathy with high risk of long-term progression.Nefrologia,41(3),311-320(May-June 2021). Moldoveanu et al.Experimental evidence of pathogenic role of IgG autoantibodies in IgG nephropathy.J.Autoimmun.,118,102593(2021). Moldoveanu et al.,Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney International,71(11),1148-54(2007). Muguruma et al.,Kinetics-based structural requirements of human immunoglobulin G binding peptides.ACS Omega,4,14390-14397(2019). Mustafaoglu et al.,Antibody purification via affinity membrane chromatography method using nucleotide binding site targeting with a small molecule,Analyst,141(24),6571-6582(November 28,2016). Neufeld et al.,Galactose-deficient IgA1(GD-IgA1)in skin and serum from patients with skin-limited and systemic IgA Vasculitis.J.Am.Acad.Dermatol.(March 19,2019). Nihei,Suzuki,and Suzuki,Current understanding of IgA antibodies in the pathogenesis of IgA nephropathy.Front.Immunol.,14,1165394(2023). Nishioka et al.Glomerulonephritis with severe nephrotic syndrome induced by immune complexes composed of galactose-deficient IgA1 in primary Sjogren’s syndrome:A case report.BMC Nephrol.,22(1),108(March 25,2021). Pettersson & Crews,PROteolysis TArgeting Chimeras(PROTACs)-Past,present,and future.Drug Discovery Today:Technologies,Vol.31,pp.15-27(Elsevier BV,2019). Rajasekaran,Julian,& Rizk,IgA nephropathy:An interesting autoimmune kidney disease.Am.J.Med.Sci.,361(2),176-194(2021). Saxena & Wu,Advances in therapeutic Fc engineering-modulation of IgG-associated effector functions and serum half-life.Frontiers in immunology,7,580(2016)(review). 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Tilman et al., Novel human IgG1 and IgG4 Fc-engineered antibodies with abolished immune effector functions. Protein Engineering, Design and Selection, Volume 29, Issue 10, pages 457-466 (October 2016). This paper shows that even LALA itself lacks c1q binding. Only P329A has been tested. P329A lacks c1q binding and reduces FcgR binding. P329A / LALA has not been tested, but it has been shown that P329G / LALA further reduces FcgR binding than LALA alone. Yamada et al.,AJICAP:Affinity peptide mediated regiodivergent functionalization of native antibodies.Angew.Chem.Int.Ed.Engl.,58(17),5592-5597(April 16,2019). Yamasaki et al., Galactose-deficient IgA1-specific antibody recognizes GalNAc-changed unique epitope on hinge region of IgA1. Monoclonal Antibody Immunodiagn. Immunother., 37(6), 252-256 (December 2018). Yasutake et al.,Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy Nephrol Dial Transplant,30,1315-1321(2015). 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[0377] Thanks for watching this Current Protocols in Immunology (CPI), Colligan, Kruisbeek, Margulies, Shevach, and Strobe, (eds.) (John Wiley and Sons, Inc., 2003).(ISBN 0471142735,9780471142737). Current Protocols in Molecular Biology (CPMB), (2014). Current Protocols in Protein Science(CPPS),(2005).John E.Coligan(ed.),John Wiley and Sons,Inc. Immunology(2006).Werner Luttmann,published by Elsevier. Janeway’s Immunobiology,(2014).Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),Taylor & Francis Limited,(ISBN 0815345305,9780815345305). Laboratory Methods in Enzymology:DNA,(2013).Jon Lorsch(ed.)Elsevier(ISBN 0124199542). Lewin’s Genes XI,(2014).published by Jones & Bartlett Publishers(ISBN-1449659055). Molecular Biology and Biotechnology:A Comprehensive Desk Reference,(1995).Robert A.Meyers(ed.),published by VCH Publishers,Inc.(ISBN 1-56081-569-8). Molecular Cloning:A Laboratory Manual,4th edition,Green & Sambrook,(2012).Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,USA(ISBN 1936113414). The Encyclopedia of Molecular Cell Biology and Molecular Medicine,Porter et al.(eds.),published by Blackwell Science Ltd.,1999-2012(ISBN 9783527600908). The Merck Manual of Diagnosis and Therapy,19 th edition(Merck Sharp & Dohme Corp.,2018). Remington's,Pharmaceutical Sciences 23 rd edition (Elsevier, 2020). Smith & March,March's Advanced Organic Chemistry:Reactions,Mechanisms,and Structure,5 th edition(John Wiley & Sons,2001). Larock,Comprehensive Organic Transformations,2 nd edition(John Wiley & Sons, 1999). Greene & Wuts,Protecting Groups in Organic Synthesis,3 rd edition(John Wiley & Sons, 1999).

[0378] Throughout this application, various publications are referenced by author name and date, or by patent number or patent publication number. The disclosures of these publications are described herein and incorporated in their entirety by reference to more fully illustrate the most advanced technology known to those skilled in the art, as claimed herein. However, the references of references herein should not be construed as an acknowledgment that these references constitute prior art to the present invention.

[0379] All patents and publications cited throughout this specification are incorporated by reference to clarify and illustrate materials and methods that may be used in conjunction with the technology described herein. The publications discussed are provided for prior disclosure only. They should not be construed as acknowledging that the inventors of this application should not precede this disclosure under prior inventions or for any other reason. In the event of any clear conflict between prior patents or publications and the descriptions contained herein, the specification (including any definitions) and claims shall prevail. All statements regarding the dates or content of these documents are based on information available to the applicant. These statements do not constitute an endorsement of the accuracy of the dates or content of these documents. The publication dates shown herein may differ from the actual publication dates. In the event of any clear conflict between the publication date of this application and the actual publication date provided by the publisher, the actual publication date shall prevail. [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6] [Table 17-7] Table 17-8 Table 17-9 Table 17-10

Claims

1. A composition of a substance, Galactose-deficient IgA1 (Gd-IgA1) binding site, A cell receptor binding portion that can bind to hepatocytes or other degrading cells via hepatocyte asialoglycoprotein receptors (ASGPR) or other cell receptors on surface degrading cells, A composition of a substance comprising a linker portion connecting the galactose-deficient IgA1 binding portion and the cell receptor binding portion.

2. The following structure: 【Chemistry 1】 [AGN101] 【Chemistry 2】 [AGN102] 【Transformation 3】 [AGN103], or 【Chemistry 4】 [AGN104] It has, in the formula, a and b are independently integers greater than or equal to 1. Each AT or ABT is a galactose-deficient IgA1 binding site or a fragment thereof. L is the linker part, A composition of the substance according to claim 1, or a pharmaceutically acceptable salt thereof, wherein each TBT is a cell receptor binding moiety that independently binds to hepatocytes or other degrading cells via hepatocyte asialoglycoprotein receptors (ASGPR) or other cell receptors on surface degrading cells in a patient or subject.

3. A composition of the substance according to claim 2, wherein a is 1, b is 3, and each TBT contains an N-acetyl-D-galactosamine (GalNAc) moiety.

4. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding portion is a Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof.

5. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding portion is a polypeptide having a complementarity-determining region (CDR) of the anti-galactose-deficient IgA1 antibody Km55.

6. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding moiety comprises six regions according to the Kabat numbering scheme having the structures of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO:

14.

7. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding portion is a partially humanized Km55 variant or its antigen-binding fragment.

8. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding portion is a chimeric Km55 variant or its antigen-binding fragment.

9. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding portion is a chimeric Km55 variant or an antigen-binding fragment thereof having a heavy chain sequence containing LALA peptide substitutions at sites L234A and L235A.

10. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding site is a chimeric Km55 variant or an antigen-binding fragment thereof having a heavy chain sequence containing LALA-PA peptide substitutions at sites L234A, L235A, and P329A.

11. The galactose-deficient IgA1 binding moiety includes IgG1 or an antigen-binding fragment attached to the linker L at amino acid residues selected from K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues, or The galactose-deficient IgA1 binding site includes IgG2 or a fragment thereof, and the IgG2 or fragment thereof is linked to the linker at amino acid residues selected from K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues, or The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 binding portion comprises IgG4 or an antigen-binding fragment thereof, which is linked to the linker at amino acid residues selected from K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues.

12. The composition of the substance according to claim 11, wherein the galactose-deficient IgA1 binding site contains IgG1, and more than 90% of it is connected to the linker L at the K248 amino acid residue, compared to any other binding to lysine of the antibody.

13. The composition of the substance according to claim 11, wherein the galactose-deficient IgA1 binding portion contains IgG1 and has two linkers L at amino acids connected to K246 and K248 of the IgG1 heavy chain and residues selected from the corresponding amino acid residues.

14. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the polypeptide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO:

14.

15. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 1 and the light chain polypeptide sequence of SEQ ID NO:

2.

16. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 3 and the light chain polypeptide sequence of SEQ ID NO:

2.

17. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 4 and the light chain polypeptide sequence of SEQ ID NO:

2.

18. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 5 and the light chain polypeptide sequence of SEQ ID NO:

2.

19. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 6 and the light chain polypeptide sequence of SEQ ID NO:

2.

20. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the heavy chain polypeptide sequence of SEQ ID NO: 7 and the light chain polypeptide sequence of SEQ ID NO:

2.

21. The composition of the substance according to claim 1, wherein the galactose-deficient IgA1 (Gd-IgA1) binding portion has the heavy-chain polypeptide sequence of SEQ ID NO: 8 and the light-chain polypeptide sequence of SEQ ID NO:

2.

22. The composition of the substance according to claim 1, wherein the linker comprises a single peptide bond.

23. The linker contains one or more -[(CH ２ ) ｎ -O] ｍ A composition of the substance according to claim 1, comprising -, wherein each n is independently 1 to 20 and m is 1 to 100.

24. The composition of the substance according to claim 1, wherein the cell receptor binding portion includes an ASGPR binding group connected via an amine group.

25. The cell receptor binding portion has the following chemical structure: 【Transformation 5】 ASGPR binding group according to A composition of the substance according to claim 1, comprising a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof.

26. The cell receptor binding portion has the following structure: 【Transformation 6】 It has, In the formula, R Ａ However, C is optionally substituted with 1 to 5 halo (preferably fluoro) groups. １ ~C ３ It is an alkyl group (preferably R Ａ (These are methyl or ethyl groups optionally substituted with 1 to 3 fluoro groups.) Z Ａ is -(CH ２ ), -O-(CH ＩＭ ), S-(CH ２ ), NR ＩＭ -(CH ２ ), C(O)-(CH ＩＭ ), 1 to 8, preferably 1 to 4 ethylene glycol residues-containing PEG group, or -C(O)(CH Ｍ ), NR ２ ), C(O)-(CH ＩＭ ), C(O)-(CH ２ ), 1 to 8, preferably 1 to 4 ethylene glycol residues-containing PEG group, or -C(O)(CH ＩＭ ), NR ２ ), C(O)-(CH ＩＭ ), NR Ｍ group (preferably, 1 to 8 ethylene glycols, preferably 2 to 4 ethylene glycol residues-containing PEG-containing group), IM and R Ｍ are as defined above, Z Ｂ However, it does not exist, (CH ２ ) ＩＭ , C(O)-(CH ２ ) ＩＭ - or C(O)-(CH ２ ) ＩＭ -NR Ｍ IM and R Ｍ However, the composition of the substance according to claim 17 is the same as above.

27. The composition of the substance according to claim 26, wherein the ASGPR binding group is N-acetyl-D-galactosamine.

28. A pharmaceutical composition comprising a composition of a substance according to any one of the preceding claims 1 to 27, and a pharmaceutically acceptable excipient.

29. A composition, A composition of the first substance, Galactose-deficient IgA1 (Gd-IgA1) binding site, A cell receptor binding portion that can bind to hepatocytes or other degrading cells via hepatocyte asialoglycoprotein receptors (ASGPR) or other cell receptors on surface degrading cells, and A composition of a first substance comprising a linker portion connecting the galactose-deficient IgA1 binding portion and the cell receptor binding portion, A composition of at least one additional substance, A cell receptor binding portion that can bind to hepatocytes or other degrading cells via hepatocyte asialoglycoprotein receptors (ASGPR) or other cell receptors on surface degrading cells, and A composition comprising at least one additional substance composition having a linker portion that can connect the cell receptor binding portion and bind to the galactose-deficient IgA1 binding portion.

30. A method for producing a composition of a substance comprising: a galactose-deficient IgA1 (Gd-IgA1) binding portion; a cell receptor binding portion capable of binding to hepatocytes or other degrading cells via the asialoglycoprotein receptor (ASGPR) or other cell receptors on hepatocytes on surface degrading cells; and a linker portion connecting the galactose-deficient IgA1 binding portion and the cell receptor binding portion, wherein the galactose-deficient IgA1 binding portion is a Km55 antibody, a Km55 variant, or an antigen-binding fragment thereof. (1) Steps to obtain the Km55 variant, (2) A method comprising the step of conjugating the Km55 variant with a MATE reagent to produce a composition (pharmaceutical) of a certain amount of substance.

31. The method according to claim 30, wherein the amount of the composition of the substance produced is 1 kg or more per production trial.

32. A method for removing galactose-deficient IgA1 in a patient or subject in need, comprising administering to the patient or subject an agent described in any one of the preceding claims 1 to 27.

33. A method for treating a disease state or condition in a patient or subject related to the upregulation of galactose-deficient IgA1, A method comprising the step of administering an effective amount of the drug described in any one of the preceding claims 1 to 27 to the patient or subject.

34. The method according to claim 33, wherein the disease state or condition associated with the upregulation of galactose-deficient IgA1 is an autoimmune disease.

35. The method according to claim 34, wherein the autoimmune disease is IgA nephropathy.

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