Bifunctional degraders of Anti-PLA2r antibody

Abstract

A composition of matter including an anti-PLA2R antibody-binding moiety, a cellular receptor-binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes or other degrading cells in a patient or subject, and optionally, a linker moiety connecting the anti-PLA2R antibody-binding moiety and the cellular receptor-binding moiety, wherein the composition of matter is useful for removing anti-PLA2R antibody in a patient or subject.

Description

BIFUNCTIONAL DEGRADERS OF ANTI-PLA2R ANTIBODY FIELD OF THE INVENTION

[001] The invention generally relates to medicinal preparations characterized by the non-active ingredients used, e.g., carriers or inert additives, targeting or modifying agents chemically bound to the active ingredient, the non-active ingredient being chemically bound to the active ingredient, e.g., polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g., an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant. Specifically, the invention relates to bifunctional molecules containing a circulating protein-binding moiety that binds to anti-PLA2R antibody for the treatment of membranous nephropathy. BACKGROUND OF THE INVENTION

[002] Membranous nephropathy (primary membranous nephropathy, pMN) is an autoimmune disease characterized by the accumulation of immune complexes in the renal glomeruli. Idiopathic membranous nephropathy (IMN) is a glomerular disease, causing fatigue, swelling, and high cholesterol. The current standard of care is an immunosuppressive therapy. High-risk patients receive immunosuppressive therapy. Cyclophosphamide is used in patients with severe decline in kidney function.

[003] The phospholipase A2 receptor 1 (PLA2R1) is an autoantigen in membranous nephropathy patients. A mouse model of phospholipase A2 receptor 1-associated membranous nephropathy mimics podocyte injury in patients a transgenic mouse line expressing murine full-length PLA2R1 in podocytes. Meyer-Schwesinger et al., Kidney International, 97(5), 913-919 (2020).

[004] No specific therapy exists for Idiopathic membranous nephropathy. About one-third of patients eventually develop kidney failure or end-stage renal disease (ESRD) within five-fifteen years despite standard-of-care immunosuppressive treatment, requiring kidney allograft. Idiopathic membranous nephropathy recurs in 50% of all kidney allograft recipients.

[005] There remains a need in the biomedical art for new medicines capable of treating or slowing down progression of idiopathic membranous nephropathy. SUMMARY OF THE INVENTION

[006] The invention is directed to bifunctional molecules (agents, TRAPs) capable of binding and degrading anti-PLA2R antibody. In one embodiment, the invention provides an anti-PLA2R autoantibody degrader to treat idiopathic membranous nephropathy. An anti-PLA2R degrader could be a safe and effective treatment for idiopathic membranous nephropathy, with a side effect profile better than the current standard of care.

[007] In another embodiment, the invention provides a composition of matter (an agent, a TRAP) comprising:an anti-PLA2R antibody-binding moiety,a cellular receptor-binding moiety that can bind to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, anda linker moiety connecting the anti-PLA2R antibody moiety and the cellular receptor-binding moiety, wherein the linker moiety can be a single peptide bond or a larger linker moiety.

[008] In some embodiments, the invention provides a composition of matter (an agent, a TRAP) having a structure of:RCN−(Xaa)y−RCC,[AGN101],[AGN102],[AGN103], or[AGN104],or a salt thereof.

[009] In some embodiments, the invention provides a composition of matter (an agent, a TRAP) of formula AGN105:[AGN105]or a salt thereof, wherein the composition of matter has additional elements described in this specification.

[010] In more particular embodiments, the invention provides a composition of matter (an agent, a TRAP) selected from the group consisting of the compounds in TABLE 1.TABLE 1 #CharacteristicsAGN30231mer (bicyclic)-TBT103MW5240.9 DaAGN30331mer (bicyclic)-TBT104, MW 5240.9 Da.AGN30431mer (bicyclic)-CF3 pyrazine, MW 4080.61 Da.AGN30631mer-TBT103, MW5258.92 Da.AGN30731mer-TBT104, MW 5258.92 Da.AGN30831mer-CF3 pyrazine, MW 4098.62 DaAGN30931mer- PR1, MW 4036.68 Da.AGN31031mer- GN2 MW 4690.36AGN362ABT816-TBT307AGN363ABT816-TBT443AGN364ABT817 sortase conjugated with TBT544). MW 22436.86 Da.AGN365ABT819 sortase conjugated with TBT544 MW 44914.95 Da.AGN579ABT818 MATE conjugated with TBT307). The product exists as a dimer (Fc). GN3 is conjugated at K248 of each Fc unit. MW 140601 Da.AGN580ABT818-TBT443AGN167ABT740 maleimide conjugated with TBT506. GN3 conjugated to the C-terminal cysteine residue via maleimide conjugation 

[011] In some embodiments, the anti-PLA2R antibody binding moiety is a peptide selected from secretory phospholipase A2 receptor peptides disclosed by Fresquet et al., J. Am. Soc. Nephrol. 26, 302–313 (2015). See SEQ ID NO: 60-145. See corresponding peptides SEQ ID NO: 636-721 from International Patent Application PCT / IB2025 / 050867, filed January 26, 2025.

[012] In more particular embodiments, the anti-PLA2R antibody-binding moiety selected from the group consisting of the moieties in TABLE 2.TABLE 2#ABT301, unconjugated 31mer (bicyclic), 3475.05 Da.ABT305. unconjugated (31-mer) WQDKGIFVIQSESLKK(CIQAGKSVLTLENC)K, 3493.11 Da.ABT309, 24mer VIQSESLKK(CIQAGKSVLTLENC)K, 2618.08 Da.ABT310 (SEQ ID NO: 10) [VIQSES], 661.702 Da.ABT311 (SEQ ID NO: 11) [SVLTLENCK], 1006.175 Da.ABT312,31mer (biotinylated), 4094.87 Da.ABT407 (SEQ ID NO: 146).CysR-CTLD1, GGGGS-His6-GGGGS-Cys.ABT408 (SEQ ID NO: 147).CysR-CTLD1, GGGGS-His6-Cys. Number of amino acids: 359, Molecular weight: 41072.01 Da, Theoretical pI: 5.76.ABT409 (SEQ ID NO: 148).CysR-CTLD1, GGGGS-His6-GGGE-Cys-S, Number of amino acids: 364, Molecular weight: 41459.36 Da, Theoretical pI: 5.70.ABT427 (FnII-CTLD1-his)ABT530 (SEQ ID NO: 14), heavy chain sequence for hIgG1 LALA / PA sequence.ABT603, SEQ ID NO: 19. Light chain, Molecular weight: 22904.35Da, Theoretical pI: 4.72ABT606, Abcam anti-rabbit PLA2R mAb, combined Light chain + Heavy chainABT816 (SEQ ID NO: 15). CysR domain + GGGGS linker, hIgG1 Fc (CA)-LALA / PA. Number of amino acids: 389, Molecular weight: 43224.10 Da, Theoretical pI: 8.91.ABT817 (SEQ ID NO: 16). CysR_Sortase, GGGGS linker, Sortase site, His6 tag. Number of amino acids: 169, Molecular weight: 18669.24 Da, Theoretical pI: 9.46. N-glycoform is present.ABT818 (SEQ ID NO: 17).CysR-CTLD1_Fc-fusion, GGGGS linker, hIgG1 Fc (CA)-LALA / PA. Number of amino acids: 584, Molecular weight: 66078.34 Da, Theoretical pI: 6.23. 2 N-glycoforms are present on each chain.ABT819 (SEQ ID NO: 18).CysR-CTLD1 + Sortase, GGGGS linker, sortase site, His6 tag. Number of amino acids: 364, Molecular weight: 41523.47 Da, Theoretical pI: 6.33. N-glycoform is present.ABT976, CysR-CTLD1 no tag. CysR-CTLD1 (21-367) + Thrombin recognition sequence + His10 tag, Before thrombin cleavage: Number of amino acids: 363, Molecular weight: 41811.88 Da, Theoretical pI: 5.95. After thrombin cleavage: Number of amino acids: 351, Molecular weight: 40296.34 Da, Theoretical pI: 5.55.ABT983 (final mAb product with ABT603, SEQ ID NO: 19 + ABT982, SEQ ID NO: 20)ABT985 (final mAb product with ABT603, SEQ ID NO: 19 + ABT984, SEQ ID NO: 21)

[013] In another embodiment, the cellular receptor-binding moiety comprises an ASGPR-binding group according to the chemical structure:[TBT101], or[TBT102],wherein the cellular receptor-binding moiety has additional elements described in this specification.

[014] In some specific and nonlimiting embodiments, the cellular receptor-binding moiety is selected from the group in TABLE 3.TABLE 3ID NO.Chemical StructureTBT103TBT104TBT105TBT307TBT544TBT506

[015] In another embodiment, the invention provides the composition of matter (agent, TRAP) for use as a medicine.

[016] In another embodiment, the invention provides the composition of matter (agent), TRAP for use in treating or preventing a disease state or condition associated with the presence of anti-PLA2R autoantibody in a subject or patient.

[017] In another embodiment, the invention provides the composition of matter (agent, TRAP) for use in treating a disease membranous nephropathy.

[018] In another embodiment, the invention provides a pharmaceutical composition including the composition of matter and at least one pharmaceutically acceptable excipient.

[019] In another embodiment, the invention provides a method of making a composition of matter wherein the method comprises a conjugation step, wherein the conjugation step results in an anti-PLA2R antibody-binding moiety being linked to a cellular receptor-binding moiety. In another embodiment, the method of making the composition of matter comprises a conjugation step selected from the Markush group comprising a MATE conjugation step, a maleimide conjugation step, and a sortase conjugation step.

[020] In another embodiment, the invention provides a method of making a molecule wherein the anti-PLA2R antibody-binding moiety is linked to another molecule or a device, wherein the method comprises a conjugation step selected from the Markush group comprising a MATE conjugation step, a maleimide conjugation step, a sortase conjugation step, and an AviTag™ conjugation step.

[021] In another embodiment, the invention provides a method of removing anti-PLA2R antibody in a patient or subject in need thereof by administering a composition of matter described herein to the patient or subject.

[022] In another embodiment, the invention provides a method of treating or condition associated with the upregulation of anti-PLA2R autoantibody in a patient or subject in need by administering to the patient or subject an effective amount of a composition of matter described herein.

[023] In another embodiment, the invention provides a composition including a composition of matter described herein and at least one additional molecule comprising a moiety capable of binding to an anti-PLA2R antibody that forms the antibody moiety of the first compound.

[024] Several objects, features, aspects, and advantages of the invention will become more apparent from the following detailed description of embodiments of the invention, along with the drawings. BRIEF description OF THE DRAWINGS

[025] For illustration, some embodiments of the invention are shown in the drawings described below. Like numerals in the drawings indicate like elements throughout. The invention is not limited to the precise arrangements, dimensions, and instruments shown.

[026] FIG. 1 is a chemical drawing showing that azidohomoalanine can be incorporated into a synthetic 31-mer to help with click to ASGPR ligand. CryoEM and epitope mapping of PLA2R have shown that the 31-mer peptide mimics key epitope CysR region. FIG. 1 highlights key amino acids in the binding: V, I, and E. Two key antigenic regions, SVLTLENCK (ABT311, SEQ ID NO: 11) and VIQSES (ABT310; SEQ ID NO: 10) were determined by peptide microarray. See Fresquet, Lockhart-Cairns, Rhoden, & Lennon, Proc. Natl. Acad. Sci, U.S.A., 119 (29), e2202209119) (July 11, 2022). The linker attachment unlikely to interfere with antibody-binding.

[027] FIG. 2 is TABLE 4, showing R-Groups for MATE reagents and bifunctional MoDE final compounds.

[028] FIG. 3 is a flow chart showing the decision-making for the prior art standard-of-care treatment for idiopathic membranous nephropathy. One-third of patients experience spontaneous remission. According to the KDIGO guidelines, “Changes in anti-PLA2R antibodies levels during follow-up likely add to risk estimation. Disappearance of anti-PLA2R antibodies precedes clinical remission.”

[029] FIG. 4 shows the structure of several anti-PLA2R-degrader compounds.

[030] FIG. 5 shows a visual representation of anti-PLA2R degrader conjugation methods.

[031] FIG. 6 shows a visual representation of PLA2R degraders-sortase conjugated.

[032] FIG. 7 shows a visual representation of PLA2R degraders-maleimide conjugated.

[033] FIG. 8 shows a visual representation of PLA2R degraders- MATE conjugated.

[034] FIG. 9 is a chart showing anti-PLA2R binding in ischemic monomelic neuropathy (IMN) samples with AFN364 and AGN365. Anti-PLA2R degraders AGN364 and AGN365 captured the majority of anti-PLA2R antibodies from IMN patient samples via competitive EuroImmun clinical anti-PLA2R ELISA. Most samples brought below diagnostic cut-off for anti-PLA2R positivity (14 RU / ml). detailed description OF THE INVENTION

[035] The following detailed description is provided to aid persons having ordinary skill in the biomedical art. Exemplary embodiments are described. However, these embodiments are only exemplary. This disclosure is not limited thereto but is defined by the scope of the appended claims. Persons having ordinary skill in the biomedical art may make modifications and variations in the embodiments described in this specification without departing from the spirit or scope of this disclosure.Industrial Applicability

[036] The invention provides a medically useful composition of matter (agent, TRAP) for the treating or slowing down progression of diseases that are manifestations of anti-PLA2R antibody disfunction, such as membranous nephropathy.

[037] The Kidney Disease Improving Global Outcome (KDIGO) organization issues guidelines for treatment for physicians. KDIGO recommends using anti-PLA2R titers for treatment guidance. Eighty % of idiopathic membranous nephropathy patients have anti-PLA2R antibodies. High anti-PLA2R titers are associated with poor prognosis, worsened disease, and lower chance of spontaneous remission. Depletion of anti-PLA2R by immunosuppressive therapy is predictive of disease remission. See KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases. Kidney international, Vol. 100, Issue 45, Supplement (October 2021). Changes in anti-PLA2R antibodies levels during follow-up can help risk estimation and patient stratification. Persistent anti-PLA2R titers is predictive of poor outcome.

[038] About 80% of idiopathic membranous nephropathy patients are anti-PLA2R antibody-positive. Despite treatment, titers persist in 30-40% of patients. These patients typically develop end-stage renal disease and require kidney transplant. Idiopathic membranous nephropathy recurs in 50% of allograft recipients.

[039] Chronic membranous nephropathy can cause significant proteinuria and nephrotic syndrome and can progress to kidney failure and nephrotic kidneys in severe cases. Autoantibodies bind to phospholipase A2 receptor (PLA2R) on podocytes in the Bowman’s capsule, injuring glomerular basement membranes. Subepithelial immune complex deposits leads to podocyte injury and thickening of basement membrane, which leads further to proteinuria and hyperlipidemia, resulting in kidney failure. Patients rendered anti-PLA2r negative by immunosuppression have greater disease remission. Lu et al., Medicine (Baltimore), 98(18), e15303 (May 2019).

[040] The standard of care is supportive care includes rituximab, cyclophosphamide, calcineurin INH, glucocorticoids, B-cell therapies, and anticoagulants, as well as therapies that address non-autoantibody-targeted systems including the renin-angiotensin-aldosterone system (RAAS), angiotensin-converting enzyme (ACE), angiotensin receptors, and sodium-glucose cotransporter 2 (SGLT2). Rituximab or cyclophosphamide + glucocorticoids are first-line therapies but have undesirable side effects. Combination of plasmapheresis with standard of care shows more favorable outcomes. See Rovin et al. KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases. Kidney Int (2021) 100(4, Supplement):S1–276, and Bennani et al., J. Pers. Med., 14(3), 249 (2024).

[041] The inventors conceived of an anti-PLA2R degrader that is a safe and effective treatment to address this unmet need. Disease modifying therapies that stop progression to end-stage renal disease. PLA2R antigen-specific MoDEs rapidly remove pathogenic autoantibodies. Deep reductions in anti-PLA2R autoantibodies will prevent further glomerular injury. Definitions

[0035] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are listed below. Unless stated otherwise or implicit from context, these terms and phrases shall have the meanings below. These definitions aid in describing embodiments but are not intended to limit the claimed invention.

[0036] As used in this application, except as otherwise provided in this specification, each term shall have the meaning set forth below. Additional definitions are set forth throughout the application. Where a term is not specifically defined in this specification, that term is given a biomedical art-recognized meaning applying that term in context to its use in describing the invention.

[0037] The articles "a" and "an" have the plain meaning of one or to more than one, i.e., at least one, of the grammatical object of the article unless the context indicates otherwise. For example, "an element" means one element or more than one element.

[0038] The term “ABT” has the biomedical art-recognized meaning of a binding moiety that is itself an antibody, an antibody variant, or an -binding fragment thereof. In some embodiments, the ABT binds to anti-PLA2R antibody.

[0039] The term “active Ingredient” has the United States Food & Drug Administration-provided meaning of any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of a human body or an animal body.

[0042] The term “ADCC” has the biomedical art-recognized meaning of antibody-dependent cell-mediated cytotoxicity, is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system kills a target cell, whose membrane-surface antigens have been bound by specific antibodies.

[0043] The term “ADCP” has the biomedical art-recognized meaning of antibody-dependent cell-mediated phagocytosis, an immunological mechanism of elimination whereby tumor cells are targeted with antibodies to promote their clearance from the body by phagocytic immune cells.

[0040] The term “AF488” has the biomedical art-recognized meaning of Alexa Fluor 488, a bright, green-fluorescent dye with excitation suited for the 488 nm laser line and is used for imaging and flow cytometry.

[0041] The term “AF647” has the biomedical art-recognized meaning of the far-red dye Alexa Fluor 647, which has an excitation peak at 650 nm and an emission peak at 665 nm. Alexa 647 is useful for flow cytometry, microscopy, and super-resolution microscopy.

[0044] The term “agent” has the biomedical art-recognized meaning of a composition of matter useful for performing a function. Several useful biomedically functions are described in this specification.

[0045] The term “alleviate” has the biomedical art-recognized meaning of a 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. The administration of compositions or pharmaceutical compositions of the invention may or can lead to the elimination of a sign or symptom, however, elimination is not required. Effective dosages should be expected to decrease the severity of a sign or symptom.

[0046] The term “an effective amount” and the term “a therapeutically effective amount” has the biomedical art-recognized meaning of an amount effective to achieve its intended purpose. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In embodiments, the disease or condition to be treated is tendinopathy.

[0047] The term “anti-PLA2R antibody-binding moiety” has the biomedical art-recognized meaning a moiety on a binding protein, e.g., an antibody, an antibody variant, or an -binding fragment thereof, that binds to an anti-PLA2R antibody.

[0042] The term “anti-PLA2R antibody” has the biomedical art-recognized meaning. Anti-PLA2R antibodies can be used for the diagnosis idiopathic membranous nephropathy without a kidney biopsy. Anti-PLA2R antibodies are found in ~80% of idiopathic membranous nephropathy patients. Clinically approved anti-PLA2R tests are commercially available. See, e.g., Mayo Clinic Laboratories Test ID: PLA2M ELISA assay, CPT code information: 83520. Anti-PLA2R antibodies are commercially available. A commercial monoclonal antibody from Abcam Limited specific for the PLA2R epitope being planned for the anti-PLA2R degrader program [fragment corresponding to Human PLA2R aa 1-700]: Mouse monoclonal anti-PLA2R antibody [12-6-5] (ab211490).

[0048] The term “antigen-binding fragment thereof” has the biomedical art-recognized meaning of (1) a fragment of an intact antibody that binds to the same antigen recognized by the full-length antibody, such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, sFv, or other fragments including the variable regions, or (2) any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The term antigen-binding portion of an antibody encompasses single chain antibodies.

[0049] The term “asialoglycoprotein receptor (ASGPR) binding group” has the biomedical art-recognized meaning of a binding group which binds to a hepatocyte asialoglycoprotein receptor. The ASGPR-binding group selectively binds to hepatocyte asialoglycoprotein receptor on the surface of hepatocytes. In several embodiments, an ASGPR-binding group is a component of a bifunctional agent as a cellular receptor-binding moiety which is covalently bound to the antibody-binding moiety through a linker group or directly. It is through this ASGPR moiety that bifunctional agents complexed with a circulating protein, e.g., anti-PLA2R antibody, bind to hepatocytes. After the bifunctional agent complexed with a circulating protein is bound to a hepatocyte or other cell, the circulating protein is taken into the hepatocyte or other cell via a phagocytosis mechanism, wherein the circulating protein is degraded through lysosomal degradation.

[0050] The term “asialoglycoprotein receptor (ASGPR) has the biomedical art-recognized meaning of lectins which bind asialoglycoprotein and glycoproteins from which a sialic acid has been removed to expose galactose residues. These cellular receptors are located on mammalian hepatocytes and other cells, such as glandular cells of the gallbladder and the stomach. ASGPR remove target glycoproteins from circulation.

[0051] The term "at least one of," when preceding a list of elements, modifies the entire list of elements and does not modify the individual elements of the list.

[0052] The term “AT” has the biomedical art-recognized meaning of an antibody moiety. In some embodiments, the AT binds to anti-PLA2R antibody.

[0053] The term “cellular receptor-binding moiety” has the biomedical art-recognized meaning. In several embodiments, the cellular receptor-binding moiety is an asialoglycoprotein receptor (ASGPR) binding group.

[0054] The term “cellular receptor” has the biomedical art-recognized meaning of a protein on the surface of a cell that binds to a compound, e.g., a ligand, e.g., a protein, in solution or on another cell. Generally, ligand-receptor binding induces one or more biological responses. In this specification, an asialoglycoprotein receptor (ASGPR) is a cellular receptor on the surface of hepatocytes or other cells that binds to an asialoglycoprotein or a derivative thereof.

[0055] The term “chimerized” has the biomedical art-recognized meaning. Chimeric antibodies are made by fusing variable domains from one species, such as a mouse, with constant domains from another species, such as a human being. With such biotechnical manipulation, chimeric antibodies keep the foreign antibody’s antigen specificity and affinity.

[0056] The term “combination therapy” and the “co-therapy” has the biomedical art-recognized meaning of the administration of a composition described in this specification and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination may include, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time, usually minutes, hours, days, or weeks depending on the combination selected. The term combination therapy” includes the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies, e.g., surgery or radiation treatment. Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time if a beneficial effect from the co-action of the therapeutic agents a is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

[0057] The term “complementarity determining region (CDR)” has the biomedical art-recognized meaning of a polypeptide region of an antibody heavy chin or an antibody light chain that is a determinant of the antibody to antigen-binding. Each antibody heavy chain has three CDRs. Each antibody light chain has three CDRs, usually different from the three CDRs on an antibody heavy chain. persons having ordinary skill in the biomedical art calculate the using a standardized numbering method known as the Kabat numbering scheme. Kabat et al, (1991) Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD., USA), although other numbering schemes such as Chothia and IMGT are also used by persons having ordinary skill in the biomedical art.

[0058] The term "comprises," the term "comprising," the term "includes," and the term "including" specify stated features, regions, integers, steps, operations, elements, or components, but do not prevent the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, or groups thereof.

[0059] The term “Fc-III-4c” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody.

[0060] The term “Fc-M” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody.

[0061] The term “FcB-1” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody.

[0062] The term “FcB-2” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody.

[0063] The term “first,” “second,” “third,” etc. have the plain meaning of describing several elements, components, regions, layers, or sections, these elements, components, regions, layers, or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. A first element, component, region, layer, or section could be called a second element, component, region, layer, or section without departing from the teachings of the present embodiments.

[0064] The term “hepatocyte” has the biomedical art-recognized meaning of a cell of the main parenchymal tissue of the liver. Hepatocytes make up 55-65% of the liver's mass.

[0065] The term “humanized” has the biomedical art-recognized meaning a protein, e.g., an antibody, is genetically engineered so it closely resembles the polypeptide structure of the human homologue. A variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, keeping the specificity of the rodent antibody. The human origin domain need not originate directly from a human in that it is first synthesized in a human. Instead, human domains can be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially humanized. In one approach, there are four general steps used to humanize a monoclonal antibody, These are (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process; (3) the actual humanizing methodologies / techniques; and (4) the transfection and expression of the humanized antibody.

[0066] The term “IC50“ has the biomedical art-recognized meaning of an amount, concentration, or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.

[0067] The term “idiopathic membranous nephropathy (IMN)” has the biomedical art-recognized meaning. Idiopathic membranous nephropathy is caused by autoimmunity to podocytes. Disease severity is characterized by proteinuria, decrease in glomerular filtration rate (eGFR), and anti-PLA2R antibody titers. M-type phospholipase A2 receptor (PLA2R) is the most often targeted antigen, with antibodies being present in about 80% of idiopathic membranous nephropathy patients. Idiopathic membranous nephropathy is driven by autoimmunity to podocytes, causing increased thickness of the glomerular membrane and changes in podocyte morphology.

[0068] The term “IgG” antibody has the biomedical art-recognized meaning. Each IgG molecule includes the basic four-chain immunoglobulin structure—two γ (gamma) heavy chains and two identical light chains (either kappa or lambda)—and carries two identical antigen-binding sites. There are four subclasses of IgG, each with minor differences in its H chains but with distinct biological properties.

[0069] The term “IgG1” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-1 chain C region is a protein that in humans is encoded by the IGHG1 gene.

[0070] The term “IgG2” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-2 chain C region is a protein that in humans is encoded by the IGHG2 gene.

[0071] The term “IgG4” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-4 chain C region is a protein that in humans is encoded by the IGHG4 gene. IgG4 has little effector function. IgG4 cannot fix complement.

[0072] The term “IVIG” has the biomedical art-recognized meaning of the administration of intravenous immunoglobulin (IVIG).

[0042] The term “KD” has the biomedical art-recognized meaning of the measured equilibrium dissociation constant between a compound or ligand and a protein or binding domain of a protein.

[0043] The term “lipoprotein-associated phospholipase A2” has the biomedical art-recognized meaning. See Blackie et al., Bioorg. Med. Chem. Lett., 13(6), 1067-70 (March 24, 2003). The phospholipase A2 receptor 1 (PLA2R1) is the major autoantigen in patients suffering from membranous nephropathy. Meyer-Schwesinger et al., Kidney International, 97 (5), 913-919 (2020).

[0044] The term “linker moiety” has the biomedical art-recognized meaning of a moiety of a chemical compound that links one moiety of the chemical compound to another moiety of the same compound. In several embodiments of this invention, the linker moiety connects an anti-anti-PLA2R antibody IgG antibody moiety to a cellular receptor-binding moiety.

[0045] The term “MoDE” has the proprietary meaning of molecular degraders. See International Pat. Publ. WO 2019 / 199634 (Yale University) and WO 2019 / 199621 (Yale University).

[0046] The term “membranous neuropathy” (primary membranous nephropathy, pMN) has the biomedical art-recognized meaning of an autoimmune disease characterized by the accumulation of immune complexes in the renal glomeruli. It is the most common cause of nephrotic syndrome in adults.

[0047] The term “moiety” has the biomedical meaning of a defined chemical group or entity with a particular structure or activity. A moiety generally refers to a part of a molecule, e.g., in an ester RCOOR’, the alcohol moiety is RO−. In some embodiments, a moiety of an agent keeps one or more or all desirable structural features, properties, functions, or activities of a compound. In some embodiments, a cellular receptor-binding moiety can bind to a target, optionally in a comparable fashion, as its corresponding target-binding agent. In some embodiments, a moiety is monovalent. In some embodiments, a moiety is bivalent. In some embodiments, a moiety is polyvalent.

[0048] The term “monotherapy” has the biomedical art-recognized meaning of the administration of a single active or therapeutic compound to a subject in need thereof. Monotherapy usually is the administration of a therapeutically effective amount of an active composition.

[0049] The term “Multimodal Antibody Therapy Enhancers (MATE or MATES)” has the proprietary meaning. See International Pat. Publ. WO 2021 / 102052 (Kleo Pharmaceuticals).

[0043] The term “nanobody” has the biomedical art-recognized meaning. the term “antibody” refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e., molecules that have an antigen-binding site that specifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains and many forms including full length antibodies and antigen-binding parts thereof; including an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a nanobody, a multi-specific (e.g., tri-specific) antibody, a dual specific antibody, a bispecific antibody, an anti-idiotypic antibody, a functionally active epitope-binding part thereof, or bifunctional hybrid antibodies.

[0050] The term "on" has the plain meaning. When an element is called being on another element, it can be directly in contact with the other element or intervening elements may be present therebetween. When an element is called being "directly on" another element, there are no intervening elements present.

[0051] The term "or" as used in this specification means "or." The term "or" as used in this specification includes all combinations of one or more of the associated listed items.

[0052] The term “other degrading cells” has the biomedical art-recognized meaning. Asialoglycoprotein receptors (ASGPRs) are on the glandular cells of the gallbladder and the stomach.

[0053] The term “partially humanized” has the biomedical art-recognized meaning a protein, e.g., an antibody, is genetically engineered so it more closely resembles the polypeptide structure of the human homologue. A variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, keeping the specificity of the rodent antibody. The domain of human origin need not originate directly from a human in that it is first synthesized in a human. Instead, human domains can be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially humanized.

[0054] The term “pharmaceutically acceptable excipient” has the biomedical art-recognized meaning of an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use and human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient. A thorough discussion of pharmaceutically acceptable excipients is available in Remington’s, Pharmaceutical Sciences 23rd edition (Elsevier, 2020).

[0055] The term “pharmaceutically acceptable” has the biomedical art-recognized meaning of those compounds, anions, cations, materials, compositions, carriers, or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0056] The term “protein-binding moiety” has the biomedical art-recognized meaning of a region of a chemical composition, e.g., a polypeptide region of a chemical composition, that specifically binds to a protein, e.g., a specific protein.

[0057] The term “ROC” has the biomedical art-recognized meaning of receiver operating typical curve.

[0058] The term “subject” and the term “patient” have the biomedical art-recognized meanings. The term patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0059] The term “TBT” has the biomedical art-recognized meaning of a target-binding moiety, a cellular receptor-binding moiety. In some embodiments, the TBT binds to ASGPR.

[0060] The term “treating” and the “treat” has the biomedical art-recognized meaning of the management and care of a patient for combating a disease, condition, or disorder and includes the administration of a composition described in this specification to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder.

[0061] The term “universal antibody-binding moiety” has the biomedical art-recognized meaning of a polypeptide region of an antibody-binding protein that binds a class of antibodies, rather than a specific set of antibodies.

[0062] The term “VHH” has the biomedical art-recognized meaning. VHH has 9 beta-sheets forming a cylindric structure. Several regions may bind. The most important regions 4>1, 2 (greatest difference in uptake). The least important: regions 3, 5 (very minor difference in uptake).

[0063] Some embodiments are described below by referring to structures and schemes, to explain parts of the description.

[0064] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by persons having ordinary skill in the biomedical art.

[0065] This specification does not concern a process for cloning humans, methods for changing the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or procedures for changing the genetic identity of animals likely to cause them suffering with no substantial medical benefit to humans or animals resulting from such processes. Methods of selecting subjects for administration of a composition of matter.

[042] The presence of anti-PLA2R antibodies can diagnose idiopathic membranous nephropathy without a kidney biopsy. Anti-PLA2R antibodies are highly specific to idiopathic membranous nephropathy (~100%) and found in ~80% of patients.

[043] The synthetic 31-mer peptide recognizes the majority of anti-PLA2R antibodies in idiopathic membranous nephropathy patients. The 31-mer peptide can be used in assays to detect anti-PLA2R antibodies in idiopathic membranous nephropathy patients.

[044] Laboratory testing for autoimmune disease uses well validated assays to establish the presence of autoantibodies. For membranous nephropathy, autoantibodies to the M-type phospholipase A2 receptor 1 (PLA2R) were first described in 2009. Beck et al., N. Engl. J. Med. 361, 11–21 (2009). The use of serological testing extends beyond diagnosis to encompass prognosis and assessment of disease activity and treatment response. Quantitative assessment of autoantibody production can provide a biomarker for clinical decision-making, as shown by the antibody response to PLA2R in membranous nephropathy. van de Logt et al., Kidney Int. 93, 1016–1017 (2018); and Wu et al., Medicine 97, e11018 (2018). Methods of measuring the removal of anti-PLA2R antibody from a patient or subject.

[0066] Clinically approved anti-PLA2R tests are commercially available. See, e.g., Mayo Clinic Laboratories Test ID: PLA2M ELISA assay, CPT code information: 83520. Methods of treatmentbyadministering composition of matter.

[0067] The ideal mode of administration depends on where treatment is taking place, whether a hospital or outpatient.

[0068] In untreated patients with active disease, the previously recommended first-line agent for induction of remission is glucocorticoids, e.g., prednisolone 30–40 mg per day for 2–4 weeks, then gradually tapered over 3 to 6 months, unless contraindications exist. Glucocorticoids characteristically result in a rapid improvement in clinical features and often a resolution of radiographic features. However, where advanced fibrotic lesions have resulted in irreversible damage, the response to glucocorticoids and other current treatment options may be poor or even absent.TABLE 5Target product profile for a bifunctional degrader of anti-PLA2R in idiopathic membranous nephropathy. Short-term profileLong-term profileObjectivesImprovement in kidney function and improved side effect profileComplete remission of disease, including in non-responsive patients. Improved side effect profileMechanism of actionClearance of anti-PLA2R to reduce immune complex formation in glomerular membrane / podocytesIndication / Patient populationIdiopathic membranous nephropathyIdiopathic membranous nephropathyRoute of administration / Dosing regimenIntravenous or subcutaneous, once-a-weekSubcutaneous, twice-a-weekClinical efficacy profileEquivalent to standard of care in primary outcome measures. Reduction in anti-PLA2R titer. Reduction in proteinuria. Improvement in eGFRSuperior to standard of care in primary outcome measures and effective in non-responsive patients. Anti-PLA2R below LOD. Proteinuria <3.5 g / d. Normal eGFRSafety & tolerability profileImproved side effect profile relative to standard of care. Well tolerated for chronic treatment. 

[045] Idiopathic membranous nephropathy patients are to be stratified after immunosuppressive treatment. 80% of idiopathic membranous nephropathy patients are anti-PLA2R+, and despite treatment, titers persist in 30-40% of patients. These patients typically develop end-stage renal disease (ESRD) and require kidney transplant. Idiopathic membranous nephropathy recurs in 50% of allograft recipients. The efficacy of immunosuppressive therapy can be predicted by its effect on anti-PLA2R titers.

[046] Reduction of anti-PLA2R levels is followed by partial or complete remission in most patients. Conversely, persistent anti-PLA2R levels are associated with no remission and poor outcome. Faster depletion in anti-PLA2R levels in patients is also associated with earlier reduction in proteinuria and improved nephroprotection.

[047] After the bifunctional degrader of the invention and the bound anti-PLA2R antibody are endocytosed, they are released from the ASGPR through depletion of calcium from the endosome and changes in binding site amino acid protonation changes due to a decrease in pH. The ASGPR is recycled back to the hepatocyte surface. Endocytosed proteins are trafficked to late endosomes, which are fused with lysosomes. Lysosomal proteases then degrade endocytosed proteins, permanently removing them from circulation. The chemical structure of the composition of matter (agent, TRAP).

[0069] In an embodiment, the invention composes matter (agent, TRAP) comprising:an anti-PLA2R antibody-binding moiety,a cellular receptor-binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, anda linker moiety linking the antibody moiety and the cellular receptor-binding moiety.

[0070] In some embodiments, the invention composes matter (an agent, a TRAP) having a structure selected from the Markush group of structures including:RCN−(Xaa)y−RCC,[AGN101],[AGN102],[AGN103], or[AGN104] or a salt thereof. In these structures, a, b, and c may independently be an integer of 1 or greater. In some embodiments, each cellular receptor-binding moiety independently has the structure of −(RCN−(Xaa)y−RCC) or salt form thereof.

[0071] In some embodiments, the invention composes matter (an agent, a TRAP) of formula AGN105:[AGN105] or a salt thereof, wherein:each Xaa is independently a residue of an amino acid or an amino acid analog;t is 0-50;z is 1-50;L is a linker moiety; TBT is a cellular receptor-binding moiety;each Rc is independently −La−R’;each of a and b is independently 1-200;each La is independently a covalent bond, or an optionally substituted bivalent group selected from a C1-C20 aliphatic group or a C1-C20 heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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 bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20-membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20-membered heterocyclyl ring 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 −H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, ortwo R groups are optionally and independently taken together to form a covalent bond, or:two or more R groups on the same atom are optionally and independently taken with the atom to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; ortwo or more R groups on two or more atoms are optionally and independently taken with their intervening atoms to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

[0072] In some embodiments, a is 1. In some embodiments, b is 3. In some embodiments, a is 1 and b is 3.

[0073] In some embodiments, the agent (TRAP) has the structure of formula AGN101:,[AGN101] or a salt thereof, wherein:each of a, b and c is independently 1-200;each AT is independently an anti-PLA2R antibody-binding moiety;L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety,wherein the anti-PLA2R antibody-binding moiety is an anti-PLA2R antibody or an antigen-binding fragment thereof.

[0074] In some embodiments, the agent (TRAP) has the structure of formula AGN102:,[AGN102] or a salt thereof, wherein:each of a and b is independently 1-200;each AT is independently an anti-PLA2R antibody-binding moiety;L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety,wherein the anti-PLA2R antibody-binding moiety is an anti-PLA2R antibody or an antigen-binding fragment thereof.

[0075] In some embodiments, an agent (TRAP) comprises one and no more than one anti-PLA2R antibody-binding moiety. In some embodiments, one or no more than one anti-PLA2R antibody-binding moiety is bound to a linker moiety. In some embodiments, a is 1. In some embodiments, an agent comprises two or more anti-PLA2R antibody moieties. In some embodiments, two or more anti-PLA2R antibody moieties are bound to a single linker moiety. In some embodiments, a is 2 or more. In some embodiments, one and no more than one cellular receptor-binding moiety is bonded to a linker moiety. In some embodiments, b is 1. In some embodiments, two or more cellular receptor-binding moiety is bonded to a single linker moiety. In some embodiments, b is 2 or more. In some embodiments, an agent comprises one and no more than one cellular receptor-binding moiety. 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, an agent 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.

[0076] In some embodiments, c is 1-20, e.g., 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 the Markush group of size ranges, where c is 1-15, c is 1-10, c is 1-9, c is 1-8, c is 1-7, c is 1-6, c is 1-5, c is 1-4, c is 1-3, and c is 1-2. In some embodiments, c is a size selected from the Markush group of sizes consisting of 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0077] In some embodiments, each cellular receptor-binding moiety in an agent is the same. In some embodiments, each linker moiety connecting a cellular receptor-binding moiety to an antibody moiety is the same. In some embodiments, the TBT in agents is the same. In some embodiments, −L−(TBT)b is the same.

[0078] In some embodiments, b is 1. In some embodiments, c is 1. In some embodiments, c is two or more. In some embodiments, c is 2. Persons having ordinary skill in the biomedical art know that several technologies can conjugate antibody moieties with target-binding moieties, e.g., certain technologies used for preparing antibody-drug conjugates under this specification. In some embodiments, target-binding moieties are connected to antibody moieties through certain types of groups or amino acid residues. In some embodiments, target-binding moieties are connected to lysine residues optionally through linker moieties. In some embodiments, target-binding moieties are connected to cysteine residues optionally through linker moieties. In some embodiments, target-binding moieties are connected to unnatural amino acid residues optionally through linker moieties. In some embodiments, the invention provides technologies for selectively linking target-binding moieties to certain amino acid residues optionally through linker moieties. In some embodiments, provided technologies selectively connect target-binding moieties to certain types of amino acid residues, e.g., lysine residues, optionally through linker moieties. In some embodiments, provided technologies selectively connect target-binding moieties to sites of antibody moieties optionally through linker moieties. In some embodiments, provided technologies selectively connect target-binding moieties to certain types of amino acid residues at sites optionally through linker moieties. In some embodiments, target-binding moieties are connected to K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, target-binding moieties are connected to K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, target-binding moieties are connected to K239 and K241 of an anti-PLA2R antibody heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, a cellular receptor-binding moiety is connected to a particular amino acid residue or site optionally through a linker. In some embodiments, each cellular receptor-binding moiety is independently connected to a particular amino acid residue or site optionally through a linker. As known by persons having ordinary skill in the biomedical art, an antibody agent may comprise more than one sites, e.g., one on each of the more than one chain, e.g., one or each heavy chain. In some embodiments, an antibody moiety comprises two heavy chains and one or both amino acid residues or amino acid residues corresponding thereto are each independently connected to a cellular receptor-binding moiety optionally through a linker. In some embodiments, one and no more than one is connected. In some embodiments, c is 1. In some embodiments, both are connected. In some embodiments, c is 2. In some embodiments, both target-binding moieties or both linker moieties (if any) are the same. The anti-PLA2R antibody-binding moiety

[0079] In some embodiments, the anti-PLA2R antibody-binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues.

[0080] The anti-PLA2R antibody-binding moiety may be or may comprise:[ABT101]or a salt form thereof.

[0081] In some embodiments, the anti-PLA2R antibody-binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues.

[0082] In some embodiments, ABT101 is a universal antibody-binding moiety. In some embodiments, ABT101 is a universal antibody-binding moiety which can bind to anti-PLA2R antibody having different Fab regions. In some embodiments, ABT101 is a universal antibody-binding moiety that binds to a Fc region, e.g., the Fc region that binds to an Fc receptor.

[0083] In some embodiments, the anti-PLA2R antibody-binding moiety comprises a universal antibody-binding moiety which recruit antibodies of diverse specificities, and no more than 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% percent of recruited antibodies are toward the same antigen, protein, lipid, carbohydrate, etc. An advantage of the invention is that provided technologies comprising universal antibody-binding moieties can use diverse pools of antibodies such as those present in serum. In some embodiments, universal antibody-binding moieties of this specification, e.g., those in ARMs, are contacted with a plurality of antibodies, wherein no more than 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% percent of the plurality of antibodies are toward the same antigen, protein, lipid, carbohydrate, etc.

[0084] In some embodiments, the antibody-binding moiety is a universal antibody-binding moiety. In some embodiments, an antibody-binding moiety, e.g., a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, comprises a peptide unit that can be connected to a linker moiety through the C-terminus of the peptide unit. In some embodiments, the antibody-binding moiety is connected to a linker moiety through the N-terminus of the peptide unit. In some embodiments, the antibody-binding moiety is connected to a linker through a side chain group of the peptide unit. In some embodiments, an antibody-binding moiety, e.g., a universal antibody-binding moiety comprises a peptide unit and is connected to an antibody-binding moiety optionally through a linker moiety through the C-terminus of the peptide unit. In some embodiments, the antibody-binding moiety, e.g., a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, comprises a peptide unit connected optionally through a linker moiety through the N-terminus of the peptide unit. In some embodiments, an antibody-binding moiety, e.g., a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, comprises a peptide unit connected optionally through a linker moiety through a side chain of the peptide unit.

[0085] In some embodiments, the antibody-binding moiety comprises a universal antibody-binding moiety which can bind to anti-PLA2R antibody antibodies having different Fab regions. In some embodiments, the universal antibody-binding moiety binds to a Fc region, e.g., the Fc region that binds to an Fc receptor.

[0086] Several antibody-binding moieties, including universal antibody-binding moieties, can be used under the teachings of this specification. Some antibody-binding moieties and technologies for identifying or assessing antibody-binding moieties are described in WO 2019 / 023501 and WO 2019 / 136442, each of which is incorporated in this specification in its entirety by reference. Persons having ordinary skill in the biomedical art know that additional technologies in the biomedical art may be suitable for identifying or assessing antibody-binding moieties under this specification. In some embodiments, an antibody-binding moiety comprises one or more amino acid residues, each independently natural or unnatural.

[0087] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g. a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, has the structure of or a salt form thereof, wherein:each of R7 is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8-membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10-membered bicyclic aromatic carbocyclic ring, a 4-8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6-membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10-membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:an R7 group and the R7’ group attached to the same carbon atom are optionally taken with their intervening carbon atom to form a 3-8-membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring or a 3-8-membered optionally substituted saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each of R7’ is independently hydrogen or optionally substituted C1-3 aliphatic;each of R8 is independently hydrogen, or optionally substituted C1-4 aliphatic, or:an R8 group and its adjacent R7 group are optionally taken with their intervening atoms to form a 4-8-membered optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; andR9 is hydrogen, optionally substituted C1-3 aliphatic, or –C(O)−.

[0088] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g., a universal antibody-binding moiety is or comprises a peptide moiety, e.g., a moiety having the structure of Rc−(Xaa)z− or a salt form thereof, wherein each of Rc, z and Xaa is independently as described in this specification. One or more Xaa may be independently an unnatural amino acid residue. Side chains of two or more amino acid residues may be linked to form bridges. Side chains of two cysteine residues may form a disulfide bridge comprising −S−S−, which, as in many proteins, can be formed by two −SH groups.

[0089] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g. a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, is or comprises a cyclic peptide moiety, e.g., a moiety having the structure of or a salt form thereof, wherein:each Xaa is independently a residue of an amino acid or an amino acid analog;t is 0-50;z is 1-50;each Rc is independently −La−R’;each La is independently a covalent bond, or an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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 bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20-membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20-membered heterocyclyl ring 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 −H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, ortwo R groups are optionally and independently taken together to form a covalent bond, or:two or more R groups on the same atom are optionally and independently taken with the atom to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; ortwo or more R groups on two or more atoms are optionally and independently taken with their intervening atoms to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

[0090] In some embodiments, the heteroatom is independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.

[0091] Several antibody-binding moieties, including universal antibody-binding moieties, can be used under the teachings of this specification. Certain antibody-binding moieties and technologies for identifying or assessing antibody-binding moieties are described in WO2019 / 023501 and WO2019 / 136442, each of which is incorporated in this specification in its entirety by reference. Persons having ordinary skill in the biomedical art know that additional technologies in the biomedical art may be suitable for identifying or assessing antibody-binding moieties under this specification. In some embodiments, an antibody-binding moiety comprises one or more amino acid residues, each independently natural or unnatural.

[0092] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g., a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, has the structure of or a salt form thereof, wherein:each of R1, R3 and R5 is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8-membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10-membered bicyclic aromatic carbocyclic ring, a 4-8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6-membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10-membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:R1 and R1’ are optionally taken with their intervening carbon atom 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;R3 and R3’ are optionally taken with their intervening carbon atom 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;an R5 group and the R5’ group attached to the same carbon atom are optionally taken with their intervening carbon atom 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; ortwo R5 groups are optionally taken with their intervening atoms to form a C1-10 optionally substituted bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-3 methylene units of the chain are independently and optionally replaced with –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 –Cy1–, wherein each –Cy1– is independently a 5-6-membered heteroarylenyl with 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur;each of R1’, R3’ and R5’ is independently hydrogen or optionally substituted C1-3 aliphatic;each of R2, R4 and R6 is independently hydrogen, or optionally substituted C1-4 aliphatic, or:R2 and R1 are optionally taken with their intervening atoms to form a 4-8-membered, optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R4 and R3 are optionally taken with their intervening atoms to form a 4-8-membered optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; oran R6 group and its adjacent R5 group are optionally taken with their intervening atoms to form a 4-8-membered optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;L1 is a trivalent linker moiety; andeach 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.

[0093] In some embodiments, L1 is an optionally substituted trivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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 L1 is –(CH2CH2O)2-4– or –(CH2CH2O)2–.

[0094] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g. a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, has the structure of or a salt form thereof, wherein:each of R7 is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8-membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10-membered bicyclic aromatic carbocyclic ring, a 4-8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6-membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10-membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:an R7 group and the R7’ group attached to the same carbon atom are optionally taken with their intervening carbon atom to form a 3-8-membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring or a 3-8-membered optionally substituted saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each of R7’ is independently hydrogen or optionally substituted C1-3 aliphatic;each of R8 is independently hydrogen, or optionally substituted C1-4 aliphatic, or:an R8 group and its adjacent R7 group are optionally taken with their intervening atoms to form a 4-8-membered optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; andR9 is hydrogen, optionally substituted C1-3 aliphatic, or –C(O)−.

[0095] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g., a universal antibody-binding moiety is or comprises a peptide moiety, e.g., a moiety having the structure of Rc−(Xaa)z− or a salt form thereof, wherein each of Rc, z and Xaa is independently as described in this specification. One or more Xaa may be independently an unnatural amino acid residue. Side chains of two or more amino acid residues may be linked to form bridges. Side chains of two cysteine residues may form a disulfide bridge comprising −S−S− (which, as in many proteins, can be formed by two −SH groups).

[0096] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g. a protein-binding moiety, e.g., an antibody-binding moiety, e.g., a universal antibody-binding moiety, is or comprises a cyclic peptide moiety, e.g., a moiety having the structure of or a salt form thereof, wherein:each Xaa is independently a residue of an amino acid or an amino acid analog;t is 0-50;z is 1-50;each Rc is independently −La−R’;each La is independently a covalent bond, or an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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 bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20-membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20-membered heterocyclyl ring 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 −H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, ortwo R groups are optionally and independently taken together to form a covalent bond, or:two or more R groups on the same atom are optionally and independently taken with the atom to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; ortwo or more R groups on two or more atoms are optionally and independently taken with their intervening atoms to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

[0097] In some embodiments, the heteroatom is independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.

[0098] In some embodiments, an anti-PLA2R antibody-binding moiety is or comprises Rc−(Xaa)z− or a salt form thereof, wherein each variable is as described in this specification.

[0099] In some embodiments, a protein-binding moiety is or comprises Rc−(Xaa)z− or a salt form thereof, wherein each variable is as described in this specification. In some embodiments, a protein-binding moiety is or comprises ABT101 or a salt form thereof, wherein each variable is as described in this specification.

[00100] In some embodiments, an anti-PLA2R antibody-binding moiety, e.g., a universal antibody-binding moiety, is or comprises Rc−(Xaa)z− or a salt form thereof, wherein each variable is as described in this specification. In some embodiments, an anti-PLA2R antibody-binding moiety, e.g., a universal antibody-binding moiety, is or comprises ABT101 or a salt form thereof, wherein each variable is as described in this specification. In some embodiments, an anti-PLA2R antibody-binding moiety, e.g., a universal antibody-binding moiety is Rc−(Xaa)z− or ABT101 , or a salt form thereof, and is or comprises a peptide unit.

[00101] In some embodiments, −(Xaa)z− is or comprises a peptide unit.

[00102] In some embodiments, amino acid residues may form bridges, e.g., connections formed by side chains optionally through linker moieties, e.g., L); for example, as in many polypeptides, cysteine residues may form disulfide bridges.

[00103] In some embodiments, a peptide unit is or comprises a cyclic peptide unit. In some embodiments, a cyclic peptide unit comprises amide group formed by an amino group of a side chain and the C-terminus (−COOH). Persons having ordinary skill in the biomedical art know that in several embodiments, when a peptide unit is connected to another moiety, an amino acid residue of a peptide unit may be connected through several positions, e.g., its backbone, its side chain, etc. In embodiments, an amino acid residue is changed for connection.

[00104] In some embodiments, X5 is XaaA or XaaP. In some embodiments, X5 is XaaA. In some embodiments, X5 is XaaP. In some embodiments, X5 is an amino acid residue whose side chain comprises an optionally substituted saturated, partially saturated, or aromatic ring. In some embodiments, X5 is or . In some embodiments, X6 is selected from the Markush group of amino acids consisting of XaaA, XaaP, and His. In some embodiments, X12 is selected from the Markush group of amino acids consisting of XaaA, XaaP, , and . In some embodiments, X9 is selected from the Markush group of amino acids consisting of Asp and Glu. In some embodiments, each of X7, X10, and X11 is independently an amino acid residue with a hydrophobic side chain (hydrophobic amino acid residue, XaaH). In some embodiments, X7 is selected from the Markush group of amino acids consisting of XaaH, , and Val. In some embodiments, X10 is selected from the Markush group of amino acids consisting of XaaH, Met, and . In some embodiments, X11 is selected from the Markush group of amino acids including XaaH and . In some embodiments, X8 is Gly. In some embodiments, X4 is Pro. In some embodiments, X3 is Lys. In some embodiments, the −COOH of X12 forms an amide bond with the side chain amino group of Lys (X3), and the other amino group of the Lys (X3) is connected to a linker moiety and then an anti-PLA2R antibody-binding moiety.

[00105] In some embodiments, −(Xaa)z− is or comprises −X3X4X5X6X7X8X9X10X11X12−, wherein:each of X3, X4, X5, X6, X7, X8, X9, X10, X11, and X12 is independently an amino acid residue;at least two amino acid residues are connected through one or more linkages Lb;Lb is an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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−, wherein Lb is bonded to a backbone atom of one amino acid residue and a backbone atom of another amino acid residue, and comprises no backbone atoms;X6 is XaaA or XaaP;X9 is XaaN; andX12 is XaaA or XaaP.

[00106] In some embodiments, each of X3, X4, X5, X6, X7, X8, X9, X10, X11, and X12 is independently an amino acid residue of an amino acid of formula LNK101. In some embodiments, two non-neighboring amino acid residues are connected by Lb. In some embodiments, X5 and X10 are connected by Lb. In some embodiments, there is one linkage Lb. In some embodiments, X6 is an amino acid selected from the Markush group of amino acids consisting of XaaA, XaaP, and His. In some embodiments, X9 is Asp or Glu. In some embodiments, X12 is an amino acid selected from the Markush group of amino acids consisting of XaaA, , , and . In some embodiments, each of X4, X7, and X11 is independently XaaH. In some embodiments, X4 is XaaH or Ala. In embodiments, X7 is XaaH. In some embodiments, X7 is . In some embodiments, X11 is XaaH or . In some embodiments, X8 is Gly. In some embodiments, X3 is Lys. In some embodiments, the −COOH of X12 forms an amide bond with the side chain amino group of Lys (X3), and the other amino group of the Lys (X3) is connected to a linker moiety and then an anti-PLA2R antibody-binding moiety. In some embodiments, Lb is . In some embodiments, Lb is . In some embodiments, Lb connects two alpha-carbon atoms of two amino acid residues. In some embodiments, both X5 and X10 are Cys, and the two −SH groups of their side chains form −S−S− (Lb is −CH2−S−S−CH2−).

[00107] In some embodiments, −(Xaa)z− is or comprises −X2X3X4X5X6X7X8X9X10X11X12−, wherein:each of X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, and X12 is independently an amino acid residue;at least two amino acid residues are connected through one or more linkages Lb;Lb is an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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−, wherein Lb is bonded to a backbone atom of one amino acid residue and a backbone atom of another amino acid residue, and comprises no backbone atoms;X4 is XaaA;X5 is XaaA or XaaP;X8 is XaaN; andX11 is XaaA.

[00108] In some embodiments, each of X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, and X12 is independently an amino acid residue of an amino acid of formula LNK101. In some embodiments, two non-neighboring amino acid residues are connected by Lb. In some embodiments, there is one linkage Lb. In some embodiments, X2 and X12 are connected by Lb. In some embodiments, Lb is a linker selected from the Markush group of linkers consisting of −CH2−S−S−CH2−, −CH2−CH2−S−CH2−, , , and −CH2CH2CO−N(R’)−CH2CH2−. In some embodiments, R’ are taken with an R group on the backbone atom that −N(R’)−CH2CH2− is bonded to form a ring, e.g., as in A-34. In some embodiments, a formed ring is 3-, 4-, 5-, 6-, 7- or 8-membered. In some embodiments, a formed ring is monocyclic. In some embodiments, a formed ring is saturated. In some embodiments, Lb is . In some embodiments, Lb connects two alpha-carbon atoms of two amino acid residues. In some embodiments, X4 is XaaA or Tyr. In some embodiments, X5 is an amino acid selected from the Markush group of amino acids consisting of XaaA, XaaP, and His. In some embodiments, X8 is Asp or Glu. X11 is Tyr. In some embodiments, both X2 and X12 are Cys, and the two −SH groups of their side chains form −S−S− (Lb is −CH2−S−S−CH2−). In some embodiments, each of X3, X6, X9, and X10 is independently XaaH. In some embodiments, X3 is XaaH or Ala. In embodiments, X6 is XaaH or Leu. In some embodiments, X9 is an amino acid selected from the Markush group of amino acids consisting of XaaH, Leu, and . In some embodiments, X10 is an amino acid selected from the Markush group of amino acids including XaaH, Val, . In some embodiments, X7 is Gly. In some embodiments, p1 is 1. In some embodiments, X1 is Asp. In some embodiments, p13 is 1. In some embodiments, p14, p15 and p16 are 0. In some embodiments, X13 is an amino acid residue comprising a polar uncharged side chain, e.g., at physiological pH, polar uncharged amino acid residue, XaaL). In some embodiments, X13 is Thr. In some embodiments, X13 is Val. In some embodiments, p13 is 0. In some embodiments, Rc is −NHCH2CH(OH)CH3. In some embodiments, Rc is (R)−NHCH2CH(OH)CH3. In some embodiments, Rc is (S)−NHCH2CH(OH)CH3.

[00109] In some embodiments, −(Xaa)z− is or comprises −X2X3X4X5X6X7X8X9X10X11X12−, wherein:each of X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, and X12 is independently an amino acid residue;at least two amino acid residues are connected through one or more linkages Lb;Lb is an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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−, wherein Lb is bonded to a backbone atom of one amino acid residue and a backbone atom of another amino acid residue, and comprises no backbone atoms;X5 is XaaA or XaaP;X8 is XaaN; andX11 is XaaA.

[00110] Persons having ordinary skill in the biomedical art know that an amino acid residue may be replaced by another amino acid residue having similar properties, e.g., one XaaH, e.g., Val, Leu, etc. may be replaced with another XaaH, e.g., Leu, Ile, Ala, etc. , one XaaA may be replaced with another XaaA, one XaaP may be replaced with another XaaP, one XaaN may be replaced with another XaaN, one XaaL may be replaced with another XaaL, etc.

[00111] In some embodiments, the anti-PLA2R antibody-binding moiety is or comprises optionally substituted moiety disclosed in TABLE 2 of the published patent application WO 2024 / 228935 (Biohaven Therapeutics Ltd.).

[00112] In some embodiments, an antibody-binding moiety is an antibody-binding moiety described in this specification. In some embodiments, a protein-binding moiety is an antibody-binding moiety described in this specification. In some embodiments, −COOH or amino groups of amino acid residues, e.g., those at the C-terminus or N-terminus, is optionally capped. In some embodiments, a −COOH group, e.g., a C-terminus −COOH) is amidated, e.g., converted into −CON(R’)2, e.g., −C(O)NHR, e.g., −C(O)NH2, and in some embodiments, an amino group, e.g. −NH2, e.g., a N-terminus −NH2) is capped with R’− or R’C(O)−, e.g., in some embodiments, by conversion −NH2 into −NHR’, e.g., −NHC(O)R, e.g., −NHC(O)CH3).

[00113] In some embodiments, an antibody-binding moiety is or comprises (X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3), wherein each of X and Xaa is independently an amino acid residue and optionally not a cysteine residue. In some embodiments, Xaa1 is R, L, L, D, E, a 2-amino suberic acid residue, or a diaminopropionic acid residue. In some embodiments, Xaa1 is an arginine residue or a leucine residue. In some embodiments, Xaa2 is L, D, E, N, or Q. In embodiments, Xaa2 is a glutamic acid residue or an aspartic acid residue. In some embodiments, Xaa2 is a lysine residue, a glutamine residue, or an aspartic acid residue. In some embodiments, this antibody-binding moieties are antibody-binding moieties.

[00114] In some embodiments, an antibody-binding moiety is or comprises (X1-3)-C-(Xaa3)-(xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(Xaa5)-(Xaa6)-(Xaa7), wherein each of X and Xaa is independently an amino acid residue and optionally not a cysteine residue. In some embodiments, Xaa3 is an alanine residue or a lysine residue. In some embodiments, Xaa4 is a tryptophan residue or a tyrosine residue. In some embodiments, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-amino suberic acid residue, or a diaminopropionic acid residue. In some embodiments, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. In some embodiments, Xaa5 is a threonine residue or a lysine residue. In some embodiments, Xaa6 is a tyrosine residue, a lysine residue, or absent. In some embodiments, Xaa7 is a histidine residue, a lysine residue, or absent. In some embodiments, this antibody-binding moieties are antibody-binding moieties.

[00115] In some embodiments, an antibody-binding moiety is or comprises D-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(Xaa5)-(Xaa6)-(Xaa7), wherein each of X and Xaa is independently an amino acid residue and optionally not a cysteine residue. In some embodiments, Xaa3 is an alanine residue or a lysine residue. In some embodiments, Xaa4 is a tryptophan residue or a tyrosine residue. In some embodiments, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-amino suberic acid residue, or a diaminopropionic acid residue. In some embodiments, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. In some embodiments, Xaa5 is a threonine residue or a lysine residue. In some embodiments, Xaa6 is a tyrosine residue, a lysine residue, or absent. In some embodiments, Xaa7 is a histidine residue, a lysine residue, or absent. In some embodiments, this antibody-binding moieties are antibody-binding moieties.

[00116] In some embodiments, an antibody-binding moiety is or comprises D-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-T, wherein each of X and Xaa is independently an amino acid residue and optionally not a cysteine residue. In some embodiments, Xaa3 is an alanine residue or a lysine residue. In some embodiments, Xaa4 is a tryptophan residue or a tyrosine residue. In some embodiments, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-amino suberic acid residue, or a diaminopropionic acid residue. In some embodiments, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. In some embodiments, this antibody-binding moieties are antibody-binding moieties.

[00117] In some embodiments, an antibody-binding moiety is or comprises R-G-N-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(Xaa5)- (Xaa6)-(Xaa7), wherein each of X and Xaa is independently an amino acid residue and optionally not a cysteine residue. In some embodiments, Xaa3 is an alanine residue or a lysine residue. In some embodiments, Xaa4 is a tryptophan residue or a tyrosine residue. In some embodiments, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-amino suberic acid residue, or a diaminopropionic acid residue. In some embodiments, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. In some embodiments, Xaa5 is a threonine residue or a lysine residue. In some embodiments, Xaa6 is a tyrosine residue, a lysine residue, or absent. In some embodiments, Xaa7 is a histidine residue, a lysine residue, or absent. In some embodiments, this antibody-binding moieties are antibody-binding moieties.

[00118] In some embodiments, antibody-binding moieties, e.g., several antibody-binding moieties described above, are protein-binding moieties. In some embodiments, antibody-binding moieties are antibody-binding moieties. In some embodiments, LG is or comprises this antibody-binding moiety. In some embodiments, LG is or comprises a protein-binding moiety. In some embodiments, LG is or comprises an antibody-binding moiety.

[00119] In some embodiments, antibody-binding moieties, e.g., antibody-binding moieties, and useful technologies for developing or assessing such moieties are described in, e.g., 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., Bioconjugate Chem., 28(8): 2009-2030 (2017), e.g., Fabsorbent, triazines, etc.; Kruljec et al., Bioconjugate Chem., 29(8), 2763-2775 (2018), WO2012017021A2, etc., the-binding moieties, e.g., antibody-binding moieties) of each of which is incorporated in this specification in its entirety by reference.

[00120] In some embodiments, an antibody-binding moiety, e.g., a protein-binding moiety, e.g., an antibody-binding moiety), is an affinity substance described in AU 2018259856 or WO 2018199337, the affinity substance of each of which is incorporated in this specification by reference.

[00121] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is or comprises an adapter protein agent, e.g., as described in Hui et al., Bioconjugate Chem., 26, 1456−1460 (2015). In some embodiments, when used under this specification, adapter proteins do not require reactive residues, e.g., BPA, to achieve one or more or all advantages.

[00122] In some embodiments, antibody-binding moiety, e.g., an antibody-binding moiety is or comprises a triazine moiety, e.g., one described in US 2009 / 0286693. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety is of such a structure that its corresponding compound is a compound described in US 2009 / 0286693, the compounds of which are independently incorporated in this specification by reference. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is ABT. In some embodiments, ABT is of such a structure that H−ABT is a compound described in US 2009 / 0286693, the compounds of which are independently incorporated in this specification by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to Fc region of an antibody.

[00123] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety is or comprises a triazine moiety, e.g., one described in Teng et al., J. Mol. Recognit., 12, 67–75 (1999). In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety is of such a structure that its corresponding compound is a compound described in Teng, the compounds of which are independently incorporated in this specification by reference. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, ABT is of such a structure that H−ABT is a compound described in Teng, the compounds of which are independently incorporated in this specification by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to Fc region of an antibody.

[00124] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety is a triazine moiety, e.g., one described in Uttamchandani, et al., J. Comb. Chem., 6(6), 862-8 (November-December 2004). In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety is of such a structure that its corresponding compound is a compound described in Uttamchandani, the compounds of which are independently incorporated in this specification by reference. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, ABT is of such a structure that H−ABT is a compound described in Uttamchandani, the compounds of which are independently incorporated in this specification by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to Fc region of an antibody.

[00125] In some embodiments, an antibody-binding moiety binds to one or more-binding sites of a protein selected from the Markush group of proteins consisting of protein A, protein G, protein L, protein Z, protein LG, protein LA, and protein AG. In some embodiments, an antibody-binding moiety is described in Choe, Durgannavar, & Chung, Materials, 9(12) (2016).

[00126] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety can bind to a nucleotide-binding site. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety is a small molecule moiety that can bind to a nucleotide-binding site. In some embodiments, a small molecule is tryptamine. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, ABT is of such a structure that H−ABT is tryptamine. Certain useful technologies were described in Mustafaoglu et al., Analyst, 141(24), 6571–6582 (November 28, 2016).

[00127] Many technologies are available for identifying, assessing, 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 technologies, e.g., those described in Intl. Pat. Publ. WO 2019 / 023501, the technologies of which are incorporated in this specification by reference. In some embodiments, an antibody-binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can selectively bind to IgG, and when used in provided technologies can provide or stimulate ADCC or ADCP. In some embodiments, peptide display technologies, e.g., phase display, non-cellular display, etc., can identify antibody-binding moieties. In some embodiments, an antibody-binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can bind to IgG and optionally can compete with known antibody binders, e.g., protein A, protein G, protein L, etc.

[00128] Persons having ordinary skill in the biomedical art know that antibodies of several properties and activities, e.g., antibodies recognizing different antigens, having optional changes, etc., may be targeted by antibody-binding moieties described in this specification. In some embodiments, such antibodies include antibodies administered to a subject, e.g., for therapeutic purposes. In some embodiments, antibody-binding moieties described in this specification may bind antibodies toward different antigens and are useful for conjugating moieties of interest with several antibodies.

[00129] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is or comprises a meditope agent moiety. In some embodiments, a meditope agent is described in, e.g., US 2019 / 0111149.

[00130] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, can bind to human IgG. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, can bind to an antibody selected from the Markush group of antibodies consisting of rabbit IgG, IgG1, IgG2, IgG3, and IgG4. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, binds to IgG1, IgG2, and IgG4.

[00131] In some embodiments, is used in a reference technology as a non-antibody-binding moiety. In some embodiments, the chemical group used in a reference technology as a non-antibody-binding moiety is selected from the Markush group of chemical groups consisting of CH3−, CH3C(O)−, CH3C(O)NH−, CH3C(O)NHCH2−, CH3CH2−, CH3CH2NH−, and CH3CH2NHC(O)−.

[00132] In some embodiments, antibody-binding moieties, e.g., antibody-binding moieties) bind to targets, e.g., antibody agents for antibody-binding moieties) with a KD that is about 1 mM-1 pM or less. In some embodiments, a KD is about 1 mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, 0.01 mM, 0.005 mM, 0.002 mM, 0.001 mM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or less. In some embodiments, KD is an affinity selected from the Markush group of affinities consisting of about 1 mM or less, about 0.5 mM or less, about 0.1 mM or less, about 0.05 mM or less, about 0.01 mM or less, about 0.005 mM or less, about 0.001 mM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, and about 1 nM or less. In some embodiments, antibody-binding moieties bind to IgG antibody agents with KD described in this specification.

[00133] Persons having ordinary skill in the biomedical art know that antibodies of several properties and activities, e.g., antibodies recognizing different antigens, having optional changes, etc., may be recruited by antibody-binding moieties described in this specification. In some embodiments, such antibodies include antibodies administered to a subject, e.g., for therapeutic purposes. In some embodiments, antibodies recruited by antibody-binding moieties comprise antibodies toward different antigens. In some embodiments, antibodies recruited by antibody-binding moieties comprise antibodies whose antigens are not present on the surface or cell membrane of target cells. In some embodiments, antibodies recruited by antibody-binding moieties comprise antibodies not targeting antigens present on surface or cell membrane of targets. In some embodiments, antigens on surface of target cells may interfere with the structure, conformation, or one or more properties or activities of recruited antibodies which bind such antigens. In some embodiments, recruited antibodies are those in IVIG. In some embodiments, IVIG may be administered before, concurrently with or subsequently to an agent or composition. Antibodies of several types of immunoglobulin structures may be recruited. In some embodiments, one or more subclasses of IgG are recruited. In some embodiments, recruited antibodies are selected from the Markush group of antibody classes consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, recruited antibodies are or comprise IgG1 and IgG2. In some embodiments, recruited antibodies are or comprise IgG1, IgG2 and IgG4. In some embodiments, recruited antibodies are or comprise IgG1, IgG2, IgG3 and IgG4. Recruited antibodies may interact several types of receptors, e.g., those expressed by several types of immune cells. In some embodiments, recruited antibodies can effectively interact several types of Fc receptors and provide desired immune activities. In some embodiments, recruited antibodies can recruit immune cells. In some embodiments, recruited antibodies can effectively interact with hFcγRIIIA. In some embodiments, recruited antibodies can effectively interact with hFcγRIIIA on macrophages. In some embodiments, macrophages are recruited to provide ADCC or ADCP activities toward a virus, e.g., a SARS-CoV-2 virus, or cells infected thereby. In some embodiments, NK cells are recruited to provide immune activities. In some embodiments, recruited antibodies can effectively interact with hFcγRIIA. In some embodiments, recruited antibodies can effectively interact with hFcγRIIA on dendritic cells. In some embodiments, antibody moieties in agents of this specification comprise one or more properties, structures or activities of recruited antibodies described in this specification.

[00134] The ricin domain of PLA2R (rather than the fibronectin type II domain or the C-type lectin domains) has the major epitope on a PLA2R molecule. Peptide screening by competitive ELISA determined two peptides (fragment 1 and fragment 2) of the ricin domain bind the majority of anti-PLA2R antibodies.

[00135] These two fragments were combined into one anti-PLA2R ligand. This 31-mer peptide from the ricin domain inhibits the binding of 80-85% of human anti-PLA2R to PLA2R. Anti-PLA2R antibodies can be effectively targeted by a single peptide. Other fragments (fragment 3, fragment 4, fragment 5, fragment 6, fragment 7) were less effective. One fragment (fragment 8) was ineffective.Fragment 1. WQDKGIFVIQSESLKKC (SEQ ID NO 1)(major epitope).Fragment 2. WSVLTENCK (SEQ ID NO 2) (major epitope).Fragment 3. TREGREDDLLWCATTSR (SEQ ID NO 3).Fragment 4. YLNHIQHEIVEKDAWK (SEQ ID NO 4).Fragment 5. YYATHCEPGWNPYNR (SEQ ID NO 5).Fragment 6. KEEKTWHEARLRSC (SEQ ID NO 6).Fragment 7. AGHVLSOAESGCQEGWER (SEQ ID NO 7).Fragment 8. PRYSGGCVAMRGRHP (SEQ ID NO 8).Synthetic 31-mer. WQDKGIFVIQSESLKKCIQAGKSVLTLENCK (SEQ ID NO 9).

[00136] Most anti-PLA2R antibodies and ~90% of patient anti-PLA2R antibodies bind to the N-C3 domain of the PLA2R. Immunoreactive tryptic fragments of PLA2R were identified by mass spectrometry. Peptide screening by competitive ELISA determined two peptides of the ricin domain of PLA2R bind to most anti-PLA2R antibodies.

[00137] These two fragments were combined into one anti-PLA2R ligand (31-mer). See SEQ ID NO: 9. The synthetic 31-mer binds anti-PLA2R antibodies strongly, KD = 500 pM. TABLE 6Biochemical selectivity. Binding Affinity (nM)#Anti-PLA2RABT3010.3AGN30210±3AGN3043±0.3AGN3037±1AGN30623±1AGN3077 Linker moiety

[00138] In some embodiments, an amino acid has the structure of formula LNK101:NH(Ra1)−La1−C(Ra2)(Ra3)−La2−COOH,[LNK101] or a salt thereof, wherein:each of Ra1, Ra2, Ra3 is independently −La−R’;each of La1 and La2 is independently La;each La is independently a covalent bond, or an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −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 bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20-membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20-membered heterocyclyl ring 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 −H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, ortwo R groups are optionally and independently taken together to form a covalent bond, or:two or more R groups on the same atom are optionally and independently taken with the atom to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; ortwo or more R groups on two or more atoms are optionally and independently taken with their intervening atoms to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

[00139] In some embodiments, an amino acid residue has the structure of −N(Ra1)−La1−C(Ra2)(Ra3)−La2−COO− or a salt form thereof.

[00140] In some embodiments, an amino acid analog is a compound in which the amino group or carboxylic acid group are independently replaced with an optionally substituted aliphatic or heteroaliphatic moiety. As persons having ordinary skill in the biomedical art know, many amino acid analogs, which mimics structures, properties or functions of amino acids, are described in the biomedical art and can be used under this specification, e.g., in several moieties. In some embodiments, one or more peptide groups are optionally and independently replaced with non-peptide groups. In some embodiments, an amino acid moiety in a polypeptide or peptide is replaced with an amino acid analog moiety.

[00141] In some embodiments, the invention provides a derivative of an amino acid of formula LNK101 or a salt thereof. In some embodiments, a derivative is an ester. In some embodiments, the invention composes matter of formula NH(Ra1)−La1−C(Ra2)(Ra3)−La2−COORCT or salt thereof, wherein RCT is R’ and each other variable is independently as described in this specification. In some embodiments, RCT is R. In embodiments, RCT is optionally substituted aliphatic. In some embodiments, RCT is t-butyl.

[00142] In some embodiments, La1 is a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ra1)−C(Ra2)(Ra3)−La2−COOH. In some embodiments, La2 is −CH2SCH2−.

[00143] In some embodiments, La2 is a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ra1)−La1−C(Ra2)(Ra3)−COOH. In some embodiments, an amino acid residue has the structure of −N(Ra1)−La1−C(Ra2)(Ra3)−CO−. In some embodiments, La1 is −CH2CH2S−. In some embodiments, La1 is −CH2CH2S−, wherein the CH2 is bonded to NH(Ra1).

[00144] In some embodiments, La1 is a covalent bond and La2 is a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ra1)−C(Ra2)(Ra3)−COOH. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ra1)−CH(Ra2)−COOH. In some embodiments, a composition of matter of formula LNK101 has a structure selected from the Markush group of peptides consisting of NH(Ra1)−CH(Ra3)−COOH, NH2−CH(Ra2)−COOH, NH2−CH(Ra3)−COOH, −N(Ra1)−C(Ra2)(Ra3)−CO−, −N(Ra1)−CH(Ra2)−CO−, −N(Ra1)−CH(Ra3)−CO−, −NH−CH(Ra2)−CO−, and −NH−CH(Ra3)−CO−.

[00145] In some embodiments, La is a covalent bond. In some embodiments, La is optionally substituted C1-6 bivalent aliphatic. In some embodiments, La is optionally substituted C1-6 alkylene. In some embodiments, La is −CH2−. In some embodiments, La is −CH2CH2−. In some embodiments, La is −CH2CH2CH2−.

[00146] In some embodiments, La is bivalent optionally substituted C1-20 aliphatic, wherein one or more methylene units are independently replaced with −C(O)−, −N(R’)−, −Cy−, or −O−. In some embodiments, La is bivalent optionally substituted C1-20 aliphatic, wherein one or more methylene units are independently replaced with −C(O)N(R’)−, −Cy−, and −O−. In some embodiments, La is bivalent optionally substituted C1-20 aliphatic, wherein two or more methylene units are independently replaced with −C(O)N(R’)−, and −Cy− in addition to other optional replacements. In some embodiments, −Cy− is optionally substituted. In some embodiments, −Cy− is optionally substituted with an electron-withdrawing group as described in this specification. In some embodiments, −Cy− is substituted with one or more −F. In embodiments, −Cy− is optionally substituted 1,3-phenylene. In some embodiments, −Cy− is optionally substituted 1,4-phenylene. In some embodiments, La is or comprises a chemical group selected from the Markush group consisting of , , , , , , , , , , , , , , , , , , and .

[00147] In some embodiments, R’ is R. In embodiments, Ra1 is R, wherein R is as described in this specification. In some embodiments, Ra1 is R, wherein R methyl. In some embodiments, Ra2 is R, wherein R is as described in this specification. In some embodiments, Ra3 is R, wherein R is as described in this specification. In some embodiments, each of Ra1, Ra2, and Ra3 is independently R, wherein R is as described in this specification.

[00148] In some embodiments, Ra1 is hydrogen. In some embodiments, Ra1 is a protective group. In some embodiments, Ra1 is −Fmoc. In some embodiments, Ra1 is −Dde.

[00149] In some embodiments, each of Ra1, Ra2 and Ra3 is independently −La−R’.

[00150] In some embodiments, Ra2 is hydrogen. In some embodiments, Ra3 is hydrogen. In some embodiments, Ra1 is hydrogen, and at least one of Ra2 and Ra3 is hydrogen. In some embodiments, Ra1 is hydrogen, one of Ra2 and Ra3 is hydrogen, and the other is not hydrogen. In some embodiments, Ra2 is −La−R and Ra3 is −H. In embodiments, Ra3 is −La−R and Ra2 is −H. In embodiments, Ra2 is −CH2−R and Ra3 is −H. In embodiments, Ra3 is −CH2−R and Ra2 is −H. In embodiments, Ra2 is R and Ra3 is −H. In embodiments, Ra3 is R and Ra2 is −H.

[00151] In some embodiments, Ra2 is −La−R, wherein R is as described in this specification. In some embodiments, Ra2 is −La−R, wherein R is an optionally substituted group selected from C3-30 cycloaliphatic, C5-30 aryl, 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, Ra2 is −La−R, wherein R is an optionally substituted group selected from C6-30 aryl and 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, Ra2 is a side chain of an amino acid. In some embodiments, Ra2 is a side chain of a standard amino acid.

[00152] In some embodiments, Ra3 is −La−R, wherein R is as described in this specification. In some embodiments, Ra3 is −La−R, wherein R is an optionally substituted group selected from C3-30 cycloaliphatic, C5-30 aryl, 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, Ra3 is −La−R, wherein R is an optionally substituted group selected from C6-30 aryl and 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, Ra3 is a side chain of an amino acid. In some embodiments, Ra3 is a side chain of a standard amino acid.

[00153] In some embodiments, one or Ra2 and Ra3 is −H. In embodiments, one or Ra2 and Ra3 is −La−R, wherein La is as described in this specification. In some embodiments, La is not a covalent bond. In some embodiments, one or more methylene units of La are independently and optionally replaced as described in this specification, e.g., with −C(O)−, −N(R’)−, −O−, −C(O)−N(R’)− or −Cy−, etc. In embodiments, La is or comprises −C(O)−, −N(R’)− and −Cy−. In some embodiments, La is or comprises −C(O)N(R’)− and −Cy−. In some embodiments, −Cy− is substituted and one or more substituents are independently an electron-withdrawing group.

[00154] In some embodiments, an amino acid side chain is Ra2 or Ra3. In some embodiments, an amino acid side chain is or comprises −LLG1−LLG2−LLG3−LLG4−H. In embodiments, an amino acid side chain is or comprises −LLG2−LLG3−LLG4−H. In embodiments, an amino acid side chain is or comprises −LLG3−LLG4−H. In embodiments, an amino acid side chain is or comprises −LLG4−H. In embodiments, this side chain selected from the Markush group consisting of , , , and .

[00155] In some embodiments, R is an optionally a group selected from the Markush group consisting of substituted C1-6 aliphatic, substituted C1-6 alkyl, −CH3, pentyl, and n-pentyl.

[00156] In some embodiments, R is a cyclic group. In some embodiments, R is an optionally substituted C3-30 cycloaliphatic group. In some embodiments, R is cyclopropyl.

[00157] In some embodiments, R is an optionally substituted aromatic group, and an amino acid residue of an amino acid of formula LNK101 is a XaaA. In some embodiments, Ra2 or Ra3 is −CH2−R, wherein R is an optionally substituted aryl or heteroaryl group. In some embodiments, R is an amino acid selected from the Markush group of amino acids consisting of optionally substituted phenyl, phenyl, 4-trifluoromethylphenyl, and 4-phenylphenyl. In some embodiments, R is optionally substituted 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is optionally substituted 5-14-membered heteroaryl having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is . In some embodiments, R is optionally substituted pyridinyl. In some embodiments, R is 1- pyridinyl. In some embodiments, R is 2- pyridinyl. In some embodiments, R is 3- pyridinyl. In some embodiments, R is.

[00158] In some embodiments, R’ is−COOH. In some embodiments, a compound of and an amino acid residue of an amino acid of formula LNK101 is a XaaN.

[00159] In some embodiments, R’ is−NH2. In some embodiments, a compound of an amino acid residue of an amino acid of formula LNK101 is a XaaP.

[00160] In some embodiments, Ra2 or Ra3 is R, wherein R is C1-20 aliphatic. In some embodiments, a compound of an amino acid residue of an amino acid of formula LNK101 is a XaaH. In some embodiments, R is −CH3. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is n-propyl. In some embodiments, R is butyl. In some embodiments, R is n-butyl. In some embodiments, R is pentyl. In some embodiments, R is n-pentyl. In some embodiments, R is cyclopropyl.

[00161] In some embodiments, two or more of Ra1, Ra2, and Ra3 are R and are taken together to form an optionally substituted ring as described in this specification.

[00162] In some embodiments, Ra1 and one of Ra2 and Ra3 are R and are taken together to form an optionally substituted 3-6-membered ring having no additional ring heteroatom other than the nitrogen atom to which Ra1 is bonded to. In some embodiments, a formed ring is a 5-membered ring as in proline.

[00163] In some embodiments, Ra2 and Ra3 are R and are taken together to form an optionally substituted 3-6-membered ring. In some embodiments, Ra2 and Ra3 are R and are taken together to form an optionally substituted 3-6-membered ring having one or more nitrogen ring atom. In some embodiments, Ra2 and Ra3 are R and are taken together to form an optionally substituted 3-6-membered ring having one and no more than one ring heteroatom which is a nitrogen atom. In some embodiments, a ring is a saturated ring.

[00164] In some embodiments, an amino acid is an amino acid selected from the Markush group of amino acids consisting of a natural amino acid, an unnatural amino acid, an alpha-amino acid, and a beta-amino acid. In some embodiments, a composition of matter of formula LNK101 is a natural amino acid or unnatural amino acid.

[00165] In some embodiments, an amino acid comprises a hydrophobic side chain. In some embodiments, an amino acid with a hydrophobic side chain is A, V, I, L, M, F, Y or W. In embodiments, an amino acid with a hydrophobic side chain is A, V, I, L, M, or F. In embodiments, an amino acid with a hydrophobic side chain is A, V, I, L, or M. In embodiments, an amino acid with a hydrophobic side chain is A, V, I, or L. In embodiments, a hydrophobic side chain is R wherein R is C1-10 aliphatic. In some embodiments, R is C1-10 alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is butyl. In some embodiments, R is pentyl. In some embodiments, R is n-pentyl. In some embodiments, an amino acid with a hydrophobic side chain is NH2CH(CH2CH2CH2CH2CH3)COOH. In some embodiments, an amino acid with a hydrophobic side chain is (S)-NH2CH(CH2CH2CH2CH2CH3)COOH. In some embodiments, an amino acid with a hydrophobic side chain is (R)-NH2CH(CH2CH2CH2CH2CH3)COOH. In some embodiments, a hydrophobic side chain is −CH2R wherein R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is phenyl substituted with one or more hydrocarbon group. In some embodiments, R is 4-phenylphenyl. In some embodiments, an amino acid with a hydrophobic side chain is NH2CH(CH2−4-phenylphenyl)COOH. In some embodiments, an amino acid with a hydrophobic side chain is (S)-NH2CH(CH2−4-phenylphenyl)COOH. In some embodiments, an amino acid with a hydrophobic side chain is (R)-NH2CH(CH2−4-phenylphenyl)COOH.

[00166] In some embodiments, an amino acid comprises a positively charged side chain, e.g., at physiological pH. In some embodiments, this amino acid comprises a basic nitrogen in its side chain. In some embodiments, this amino acid is Arg, His or Lys. In some embodiments, this amino acid is Arg. In some embodiments, this amino acid is His. In some embodiments, this amino acid is Lys.

[00167] In some embodiments, an amino acid comprises a negatively charged side chain, e.g., at physiological pH. In some embodiments, this amino acid comprises a −COOH in its side chain. In some embodiments, this amino acid is Asp. In some embodiments, this amino acid is Glu.

[00168] In some embodiments, an amino acid comprises a side chain comprising an aromatic group. In some embodiments, this amino acid is Phe, Tyr, Trp, or His. In some embodiments, this amino acid is Phe. In some embodiments, this amino acid is Tyr. In some embodiments, this amino acid is Trp. In some embodiments, this amino acid is His. In some embodiments, this amino acid is NH2−CH(CH2−4-phenylphenyl)−COOH. In some embodiments, this amino acid is (S)-NH2−CH(CH2−4-phenylphenyl)−COOH. In some embodiments, this amino acid is (R)-NH2−CH(CH2−4-phenylphenyl)−COOH.

[00169] In some embodiments, an amino acid is an amino acid selected from the Markush group of amino acids consisting of or a salt thereof, or a salt thereof, or a salt thereof, or a salt thereof, or a salt thereof, or a salt thereof, or a salt thereof, and or a salt thereof. In some embodiments, a provided compound is . In some embodiments, the invention provides polypeptide agents comprising one or more amino acid residues described in this specification. Amino acids

[00170] In some embodiments, each residue is independently a residue of an amino acid or an amino acid analog, e.g., Xaa, wherein the amino acid or the amino acid analog has the structure of H−La1−La1−C(Ra2)(Ra3)−La2−La2−H or a salt thereof. In some embodiments, an amino acid has the structure of NH(Ra1)−La1−C(Ra2)(Ra3)−La2−COOH or a salt thereof. In some embodiments, an amino acid analog has the structure of H−La1−La1−C(Ra2)(Ra3)−La2−La2−H or a salt thereof. In some embodiments, in this amino acid analog, the first −La1− (bonded to –H in the formula) is not − N(Ra1)−, e.g., is optionally substituted bivalent C1-6 aliphatic. In some embodiments, in H−La1−La1−, −La1−La1− bonds to the –H through an atom that is not nitrogen. In some embodiments, in −La2−La2−H, −La2−La2− is not bonded to the –H through –C(O)O−.

[00171] In some embodiments, each Xaa independently has the structure of −La1−La1−C(Ra2)(Ra3)−La2−La2−. In some embodiments, each Xaa independently has the structure of –LaX1−La1−C(Ra2)(Ra3)−La2−LaX2−, wherein LaX1 is optionally substituted −NH−, optionally substituted −CH2−, − N(Ra1)−, or −S−, LaX2 is optionally substituted −NH−, optionally substituted −CH2−, − N(Ra1)−, or −S−, and each other variable is independently as described in this specification. In some embodiments, LaX1 is optionally substituted −NH−, or − N(Ra1)−. In some embodiments, LaX1 is optionally substituted −CH2−, or −S−. In some embodiments, LaX2 is optionally substituted −NH−, optionally substituted −CH2−, − N(Ra1)−, or −S−. In some embodiments, optionally substituted −CH2− is −C(O)−. In some embodiments, optionally substituted −CH2− is not −C(O)−. In some embodiments, LaX2 is −C(O)−. In some embodiments, each Xaa independently has the structure of −N(Ra1)−La1−C(Ra2)(Ra3)−La2−CO−.

[00172] In some embodiments, two or more residues, e.g., two or more Xaa residues, are linked such that one or more cyclic structures are formed. Residues can be linked, optionally through a linker, e.g., LT) at any suitable positions. A linkage between two residues can connect each residue independently at its N-terminus, C-terminus, a point on the backbone, or a point on a side chain, etc. In embodiments, two or more side chains of residues, e.g., in compounds of formula AGN105, e.g., Ra2 or Ra3 of one amino acid residue with Ra2 or Ra3 of another amino acid residue, are optionally take together to form a bridge, e.g., in some embodiments, two cysteine residues form a −S−S− bridge as typically observed in natural proteins.

[00173] In some embodiments, an amino acid residue has the structure of −N(Ra1)−La1−C(Ra2)(Ra3)−La2−COO− or a salt form thereof.

[00174] In some embodiments, an amino acid analog is a compound in which the amino group or carboxylic acid group are independently replaced with an optionally substituted aliphatic or heteroaliphatic moiety. As persons having ordinary skill in the biomedical art know, many amino acid analogs, which mimics structures, properties or functions of amino acids, are described in the biomedical art and can be used under this specification, e.g., in several moieties. In some embodiments, one or more peptide groups are optionally and independently replaced with non-peptide groups. In some embodiments, an amino acid moiety in a polypeptide or peptide is replaced with an amino acid analog moiety. 

[00175] In some embodiments, moieties are optionally connected to each other through linker moieties. In some embodiments, a reactive group, e.g., RG, is connected to a cellular receptor-binding moiety, e.g., TBT, through a linker, e.g., LRM. In some embodiments, a moiety, e.g., LG, may also comprise one or more linkers, e.g., LLG1, LLG2, LLG3, LLG4, etc., to link several parts. In some embodiments, LLG is a linker moiety described in this specification. In some embodiments, LLG1 is a linker moiety described in this specification. In some embodiments, LLG2 is a linker moiety described in this specification. In some embodiments, LLG3 is a linker moiety described in this specification. In some embodiments, LLG4 is a linker moiety described in this specification. In some embodiments, LRM is a linker moiety described in this specification. In some embodiments, LPM is L. In embodiments, LPM is a linker moiety described in this specification. In some embodiments, LPM is L.

[00176] Linker moieties of several types or for several purposes, e.g., those used in antibody-drug conjugates, etc., may be used under this specification.

[00177] Linker moieties can be bivalent or polyvalent depending on how they are used. In some embodiments, a linker moiety is bivalent. In some embodiments, a linker is polyvalent and connecting over two moieties.

[00178] In some embodiments, L is bivalent. In some embodiments, L is a covalent bond.

[00179] In some embodiments, a linker moiety, or L, is or comprises −(CH2CH2O)n−, wherein each −CH2− is independently and optionally substituted and n is 1-20. In some embodiments, a linker moiety, or L, is or comprises −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently 1-10, and each −CH2− is independently and optionally substituted.

[00180] In some embodiments, a linker moiety is trivalent or polyvalent. In some embodiments, a linker moiety is L, where L is trivalent or polyvalent. In some embodiments, L is trivalent. In some embodiments, L is −CH2−N(−CH2−)−C(O)−.

[00181] In some embodiments, a linker moiety, e.g., L, comprises one or more amino acid residues or analogs thereof.

[00182] In some embodiments, a linker moiety, e.g., L, LRM, etc., is or comprises a reactive group as described in this specification. In some embodiments, an agent (a TRAP) comprises an antibody-binding moiety and a cellular receptor-binding moiety linked through a linker which is or comprises a reactive group. In some embodiments, a reactive group can react with a lysine residue of an antibody in an aqueous buffer. In some embodiments, a reactive group is or comprises −C(O)−O−. In some embodiments, a reactive group is or comprises −C(O)−O−, wherein −O− is bonded to an optionally substituted aryl group. In some embodiments, a reactive group is or comprises −C(O)−O−, wherein −O− is bonded to an aryl group substituted with one or more electron-withdrawing groups. In some embodiments, one or more or each electron-withdrawing group is independently selected from −NO2 and −F. In embodiments, an aryl group has the structure of , wherein Rs is 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, an aryl group has the structure of , wherein each Rs is 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, an aryl group is . In some embodiments, an aryl group is . In some embodiments, C1 is bound to the −O− of −C(O)−O−. In some embodiments, a cellular receptor-binding moiety is at the side of −C(O)− and an antibody-binding moiety is at the side of −O−.

[00183] In some embodiments, a linker moiety, e.g., L, LRM, etc., comprises a reactive group, wherein upon contact with an antibody, the reactive group reacts with a group of the antibody and conjugates a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, to the antibody optionally through a linker. In some embodiments, a reactive group is or comprises , wherein the −C(O)− is connected to a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, optionally through a linker. In some embodiments, a reactive group is or comprises , wherein the −C(O)− is connected to a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, optionally through a linker and the other end of the reactive group is connected to an antibody-binding moiety.

[00184] In some embodiments, a linker moiety, e.g., L, LRM, etc., does not have a reactive group. In some embodiments, a linker moiety, e.g., L, LRM, etc., does not have a reactive group that readily reacts with proteins under aqueous conditions with pH about 6-9, e.g., physiological conditions. In some embodiments, a linker moiety, e.g., L, LRM, etc., does not have a reactive group that readily reacts with natural amino acid residues under aqueous conditions with pH about 6-9, e.g., physiological conditions.

[00185] In some embodiments, a linker moiety, e.g., L, LRM, etc., comprises no −S−, wherein none of the two atoms to which the −S− is bonded to is S. In embodiments, a linker moiety, e.g., L, LRM, etc., comprises no −S−S−. In some embodiments, a linker moiety, e.g., L, LRM, etc., comprises no −S− bonded to a beta carbon of a carbonyl group or a double or triple bond conjugated to a carbonyl group. In some embodiments, a linker moiety, e.g., L, LRM, etc., comprises no . In some embodiments, a linker moiety, e.g., L, LRM, etc., comprises no −S−.

[00186] In some embodiments, an agent (a TRAP) comprises no cleavable groups whose cleavage can release LG except one or more optionally in RG. In some embodiments, an agent comprises no −S−S−, acetal or imine groups except in RG or TBT. In some embodiments, an agent comprises no −S−S−, acetal or imine groups except that the agent may have −S−S− formed by two amino acid residues. In some embodiments, an agent comprises no −S−S−, acetal or imine groups except that the agent may have −S−S− formed by cysteine residues. In some embodiments, an agent comprises no −S−S−, acetal or imine groups.

[00187] In some embodiments, L is a covalent bond. In some embodiments, L is a bivalent optionally substituted, linear or branched C1-100 aliphatic group wherein one or more methylene units of the group are optionally and independently replaced. In some embodiments, L is a bivalent optionally substituted, linear or branched C6-100 arylaliphatic group wherein one or more methylene units of the group are optionally and independently replaced. In some embodiments, L is a bivalent optionally substituted, linear or branched C5-100 heteroarylaliphatic group having 1-20 heteroatoms wherein one or more methylene units of the group are optionally and independently replaced. In some embodiments, L is a bivalent optionally substituted, linear or branched C1-100 heteroaliphatic group having 1-20 heteroatoms wherein one or more methylene units of the group are optionally and independently replaced.

[00188] In some embodiments, a linker moiety, e.g., L) is or comprises 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. In some embodiments, a linker moiety is or comprises −(CH2CH2O)n−, wherein n is as described in this specification. In some embodiments, one or more methylene units of L are independently replaced with −(CH2CH2O)n−.

[00189] 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.

[00190] In some embodiments, a linker moiety, e.g., L) is or comprises 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. As used in this specification, “one or more” can be 1-100, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. In some embodiments, one or more methylene units of L are independently replaced with an amino acid residue. In some embodiments, one or more methylene units of L are independently replaced with an amino acid residue, wherein the amino acid residue is of an amino acid of formula LNK101 or a salt thereof. In some embodiments, one or more methylene units of L are independently replaced with an amino acid residue, wherein each amino acid residue independently has the structure of −N(Ra1)−La1−C(Ra2)(Ra3)−La2−CO− or a salt form thereof.

[00191] In some embodiments, a linker moiety comprises one or more moieties, e.g., amino, carbonyl, etc., that can be used for connection with other moieties. In some embodiments, a linker moiety comprises one or more −NR’−, wherein R’ is as described in this specification. In some embodiments, −NR’− improves solubility. In some embodiments, −NR’− serves as connection points to another moiety. In some embodiments, R’ is −H. In embodiments, one or more methylene units of L are independently replaced with −NR’−, wherein R’ is as described in this specification.

[00192] In some embodiments, a linker moiety, e.g., L, comprises a −C(O)− group, which can be used for connections with a moiety. In some embodiments, one or more methylene units of L are independently replaced with −C(O)−.

[00193] In some embodiments, a linker moiety, e.g., L, comprises a −NR’− group, which can be used for connections with a moiety. In some embodiments, one or more methylene units of L are independently replaced with −N(R’)−.

[00194] In some embodiments, a linker moiety, e.g., L, comprises a −C(O)NR’− group, which can be used for connections with a moiety. In some embodiments, one or more methylene units of L are independently replaced with −C(O)N(R’)−.

[00195] In some embodiments, a linker moiety, e.g., L, comprises a −C(R’)2− group. In some embodiments, one or more methylene units of L are independently replaced with −C(R’)2−. In some embodiments, −C(R’)2− is −CHR’−. In some embodiments, R’ is −(CH2)2C(O)NH(CH2)11COOH. In some embodiments, R’ is −(CH2)2COOH. In some embodiments, R’ is −COOH.

[00196] In some embodiments, a linker moiety is or comprises one or more ring moieties, e.g., one or more methylene units of L are replaced with −Cy−. In some embodiments, a linker moiety, e.g., L, comprises an aryl ring. In some embodiments, a linker moiety, e.g., L, comprises an heteroaryl ring. In some embodiments, a linker moiety, e.g., L, comprises an aliphatic ring. In some embodiments, a linker moiety, e.g., L, comprises an heterocyclyl ring. In some embodiments, a linker moiety, e.g., L, comprises a polycyclic ring. In some embodiments, a ring in a linker moiety, e.g., L, is 3-20-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring in a linker is product of a cycloaddition reaction, e.g., click chemistry, and variants thereof) used to link different moieties.

[00197] In some embodiments, L is L1. In some embodiments, L is Lb.

[00198] In some embodiments, LRM is a covalent bond. In some embodiments, LRM is not a covalent bond. In some embodiments, LRM is or comprises −(CH2CH2O)n−. In some embodiments, LRM is or comprises −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, LRM is −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, LRM is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, wherein n is as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, LRM is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, wherein n is as described in this specification.

[00199] In some embodiments, LPM is a covalent bond. In some embodiments, LPM is not a covalent bond. In some embodiments, LPM is or comprises −(CH2CH2O)n−. In some embodiments, LPM is or comprises −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, LPM is −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, LPM is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, wherein n is as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, LPM is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, wherein n is as described in this specification.

[00200] In some embodiments, LPM, e.g., in a product of a first and a second agents) is or comprises a reaction product moiety formed a first reactive moiety and a second reactive moiety.Cellular receptor-binding moiety

[00201] Several receptor-binding moieties, according to embodiments of present invention, are described in WO2019 / 199621A1 published October 17, 2019, WO2019 / 199634 published October 17, 2019, International Application No. PCT / US2020 / 055053 filed October 9, 2020, and International Application No. PCT / US2020 / 055053 filed October 9, 2020, each of which is incorporated in this specification in its entirety by reference.

[00202] In an embodiment, the cellular receptor-binding moiety may include an asialoglycoprotein receptor (ASGPR) binding group connected through an amine group to the linker moiety.

[00203] The amine group may be a primary alkyl amine group or secondary alkyl amine group, each of which is optionally substituted on the amine group with a C1-C3 alkyl group.

[00204] The cellular receptor-binding moiety may include an ASGPR-binding group according to the chemical structure:, [TBT101] or,[TBT102]wherein X is 1-4 atoms in length and comprises O, S, N(RN1) or C(RN1)(RN1) groups such that:when X is 1 atom in length, X is O, S, N(RN1) or C(RN1)(RN1),when X is 2 atoms in length, no more than 1 atom of X is O, S or N(RN1),when X is 3 or 4 atoms in length, no more than 2 atoms of X are O, S or N(RN1);wherein RN1 is H or a C1-C3 alkyl group optionally substituted with from 1-3 halo groups;R1 and R3 are each independently:H, -(CH2)KOH, -(CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -(CH2)K-vinyl, O-(CH2)K-vinyl, -(CH2)K-alkynyl, -(CH2)K-COOH,-(CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O-C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, orR1 and R3 are each independently a group, which is optionally substituted with up to three halo groups, C1-C4 alkyl groups, each of which alkyl group is optionally substituted with from one to three halo groups or one or two hydroxyl groups, or O-C1-C4 alkyl groups, each of which alkyl groups is optionally substituted with from one to three halo groups or one or two hydroxyl groups; andK is independently an integer of 0 to 4, orR1 and R3 are each independently a group according to the chemical structure:, wherein R7 is O-C1-C4 alkyl, which is optionally substituted with from 1 to 3 halo groups 1 or 2 hydroxy groups, or R7 is a -NRN3RN4 group or a; orR1 and R3 are each independently a group according to the structure:, , , , , or a group according to the chemical structure: , , , , , , , , , , , , , , , , , , , , , , , , , , ,, , , , , or , or R1 and R3 are each independently a group, where , is a C3-C8 saturated carbocyclic group; RC is absent, H, C1-C4 alkyl optionally substituted with from 1-3 halo groups or 1-2 hydroxyl groups, or a group according to the structure:wherein R4, R5 and R6 are each independently, H, halo (F, Cl, Br, I), CN, NRN1RN2, -(CH2)KOH,-(CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C3 alkyl, which is optionally substituted with from 1-3 halo groups, -O-C1-C3-alkyl, which is optionally substituted with from 1-3 halo groups, -(CH2)KCOOH, -(CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O-C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, orRC is a group, a group a group or a group, wherein RN, RN1 and RN2 are each independently H or a C1-C3 alkyl group optionally substituted with from one to three halo groups or one or two hydroxyl groups;K is independently an integer of 0 to 4; K’ is an integer of 1 to 4;RN3 is H, or a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups; andRN4 is H, a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups, or RN4 is a group, where K is preferably 1; is a linker group which comprises at least one anti-PLA2R antibody-binding moiety and links the at least one anti-PLA2R antibody-binding moiety to the cellular receptor-binding moiety through the optional linker moiety, or is a linker group which has at least one or more functional groups which can be used to covalently bond the linker group to at least one anti-PLA2R antibody-binding moiety or optional linker moiety;R2 is a group wherein RN1 and K are the same as above;RAM is H, a C1-C4 alkyl group optionally substituted with up to 3 halo groups and one or two hydroxyl groups, a -(CH2)KCOOH group, a -(CH2)KC(O)O-C1-C4 alkyl group optionally substituted with from 1-3 halo groups, a O-C(O)-C1-C4 alkyl group, which is optionally substituted with from 1-3 halo F groups, a -C(O)-C1-C4 alkyl group, which is optionally substituted with from 1-3 halo groups, a -(CH2)K-NRN3RN4 group where RN3 is H, or a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups; andRN4 is H, a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups, or a group, orR2 is a group, wherein RTA is H, CN, NRN1RN2, -(CH2)KOH, -(CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -(CH2)KCOOH, -(CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O-C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, or RTA is a C3-C10 aryl or a three- to ten-membered heteroaryl group containing up to 5 heteroaryl atoms, each of said aryl or heteroaryl groups being optionally substituted with up to three (preferably 1) CN, NRN1RN2, -(CH2)KOH, -(CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C3 alkyl, which is optionally substituted with from 1-3 halo groups or 1 or 2 hydroxy groups, -O-C1-C3-alkyl, which is optionally substituted with from 1-3 halo groups, -(CH2)KCOOH, -(CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O-C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups or -(CH2)KC(O)-C1-C4 alkyl optionally substituted with from 1-3 halo groups, orRTA is a group, a group, a group a group, a group optionally substituted with up to three C1-C3 alkyl groups optionally substituted with up to three halo groups, orRTA is a group, wherein RN, RN1 and RN2 are each independently H or a C1-C3 alkyl group optionally substituted with from one to three halo groups or one or two hydroxyl groups and each -(CH2)K group is optionally substituted with 1-4, preferably 1 or 2, C1-C3 alkyl groups optionally substituted with from 1-3 fluoro groups or 1-2 hydroxyl groups;and K is independently 0-4.

[00205] In an embodiment, X is -O-C(RN1)(RN1),C(RN1)(RN1)-O-, -S-C(RN1)(RN1), C(RN1)(RN1)-S-, N(RN1)-C(RN1)(RN1),C(RN1)(RN1)-N(RN1) or C(RN1)(RN1)-C(RN1)(RN1) when X is 2 atoms in length,X is -O-C(RN1)(RN1)-C(RN1)(RN1), C(RN1)(RN1)-O-C(RN1)(RN1)-,-O-C(RN1) (RN1)-O-, -O-C(RN1) (RN1)-S-, -O-C(RN1) (RN1)-N(RN1)-,-S-C(RN1)(RN1)-C(RN1)(RN1), C(RN1)(RN1)-S-C(RN1)(RN1)-, C(RN1)(RN1)-C(RN1)(RN1)-S, -S-C(RN1)(RN1)-S-, -S-C(RN1)(RN1)-O-, -S-C(RN1)(RN1)-N(RN1)-, N(RN1)-C(RN1)(RN1)-C(RN1)(RN1), C(RN1)(RN1)-N(RN1)-C(RN1)(RN1), C(RN1)(RN1)-C(RN1)(RN1)- N(RN1), N(RN1)-C(RN1)(RN1)-N(RN1) or C(RN1)(RN1)-C(RN1)(RN1)- C(RN1)(RN1) when X is 3 atoms in length, andX is-O-C(RN1)(RN1)-C(RN1)(RN1)-C(RN1)(RN1), C(RN1)(RN1)-O-C(RN1)(RN1)-(RN1)(RN1)-, -O-C(RN1)(RN1)-O-C(RN1)(RN1)-, -S-C(RN1)(RN1)-C(RN1)(RN1)- C(RN1)(RN1)-, C(RN1)(RN1)-S-C(RN1)(RN1)-C(RN1)(RN1)-, C(RN1)(RN1)-(RN1)(RN1)-S-C(RN1)(RN1)-, -S-C(RN1)(RN1)-S-C(RN1)(RN1)-, N(RN1)-C(RN1)(RN1)-C(RN1)(RN1)- C(RN1)(RN1)-, C(RN1)(RN1)-N(RN1)-C(RN1)(RN1)-C(RN1)(RN1), C(RN1)(RN1)-C(RN1)(RN1)- N(RN1), N(RN1)-C(RN1)(RN1)-N(RN1) or C(RN1)(RN1)-C(RN1)(RN1)- C(RN1)(RN1) when X is 4 atoms in length,wherein RN1 is the same as stated in claim 4 or 6 above.

[00206] In an embodiment, X is OCH2 or CH2O and RN1 is H.

[00207] The cellular receptor-binding moiety may include an ASGPR-binding group according to the chemical structure:, [TBT101] or,[TBT102]where R1, R2 and R3 are the same as in Claim 9, or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof.

[00208] The cellular receptor-binding moiety may have the following structure:or ;where RA is a C1-C3 alkyl group optionally substituted with 1-5 halo (preferably fluoro) groups (preferably RA is a methyl or ethyl group optionally substituted with from 1-3 fluoro groups);ZA is -(CH2)IM, -O-(CH2)IM, S-(CH2)IM, NRM-(CH2)IM, C(O)-(CH2)IM-, a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH2)IMNRM group (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues) whereIM and RM are the same as above; andZB is absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)IM-NRM, whereIM and RM are the same as above.

[00209] In an embodiment, RA may be a methyl or ethyl group optionally substituted with from 1-3 fluoro groups. In an embodiment, ZA may be a PEG group containing from 1 to 4 ethylene glycol residues. In an embodiment, the methyl or ethyl group may be substituted with from 1-3 fluoro groups. In an embodiment, the ASGPR-binding group may be N-acetyl-D-galactosamine. In an embodiment, the cellular receptor-binding moiety may be a low-density lipoprotein receptor-related protein 1 (LRP1), a low-density lipoprotein receptor (LDLR), a FcγRI-binding group, a FcRN-binding group, a transferrin receptor-binding group, or a macrophage scavenger receptor-binding group. Methods of making several agents.

[00210] Agents of this specification may be prepared or isolated by synthetic or semi-synthetic methods or recombinant methods under this specification. In some embodiments, polypeptide agents, e.g., cellular receptor-binding moiety peptide agents, maybe be prepared using biological expression systems. In some embodiments, provided agents are prepared synthetically. In some embodiments, provided agents are prepared using certain technologies described in WO2019 / 023501, which is incorporated in this specification in its entirety by reference.

[00211] Several technologies, e.g., those for preparing antibody-drug conjugates, may be used in preparation of MATE agents. In many such technologies, conjugation is not selective regarding amino acid residue sites, and product compositions usually have several types of agents which may differ from each other regarding number of target-binding moieties conjugated or conjugation sites. In some embodiments, the invention provides technologies that can be used for selective conjugation of target-binding moieties at amino acid residue sites.

[00212] In some embodiments, the invention provides a method of synthesis, comprising the steps of:contacting a first agent comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker with a second agent comprising an antibody moiety linked to a second reactive group optionally through a second linker, wherein the first reactive group reacts with a second reactive group, andforming a product agent comprising a cellular receptor-binding moiety and an antibody-binding moiety optionally through a linker.

[00213] In some embodiments, the invention provides a method of synthesis, comprising the steps of:contacting a first composition comprising a plurality of first agents each independently comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker moiety with a second composition comprising a plurality of second agents each independently comprising an antibody moiety optionally linked to a second reactive group optionally through a second linker moiety,wherein a product composition comprising a plurality of product agents each independently comprising a cellular receptor-binding moiety and an antibody-binding moiety optionally through a linker is formed.MATES

[00214] Persons having ordinary skill in the biomedical art can use MATES materials and methods as guidance to predictable results when making and using the invention.

[00215] In some embodiments, the invention provides an agent comprising:an antibody moiety,a cellular receptor-binding moiety, and a linker moiety (optionally a single peptide linkage) linking an antibody moiety and a cellular receptor-binding moiety.

[00216] This agent may be called a MATE agent or MATE. The MATE agents are described, for example, in International Application No. PCT / US2020 / 061127 filed November 18, 2020, the content of which is incorporated in this specification in its entirety by reference. In some embodiments, an agent comprises an antibody moiety, a cellular receptor-binding moiety, and a linker moiety linking an antibody moiety and a cellular receptor-binding moiety.

[00217] In another embodiment, the anti-PLA2R antibody-binding moiety is an antibody, an antibody variant, or an antigen-binding fragment thereof.

[00218] In another embodiment, the anti-PLA2R antibody-binding moiety is a full IgG antibody or nanobody wherein the heavy chain comprises the three CDR regions of the anti-PLA2R antibody-binding moiety. A full-length human sourced IgG can be native and have variable glycosylation. Plurality of agents. Second agent.

[00219] In another embodiment, the invention provides a composition including the agent and at least one additional agent comprising a moiety capable of-binding to the antibody that forms the antibody moiety of the first compound.

[00220] In some embodiments, a first composition is a composition comprising a first agent as described in this specification. In some embodiments, second agents independently comprise second reactive groups. In some embodiments, a second composition is a composition comprising a plurality of agents as described in this specification, wherein each cellular receptor-binding moiety is independently a reactive group as described in this specification. In some embodiments, a second composition is an antibody composition, wherein antibodies in the composition are not chemically changed. In some embodiments, a second composition is an IVIG preparation. In some embodiments, a product composition is a composition comprising a plurality of agents as described in this specification, wherein each cellular receptor-binding moiety is independently a cellular receptor-binding moiety as described in this specification.

[00221] In some embodiments, a cellular receptor-binding moiety in a product agent is a cellular receptor-binding moiety in a first agent. In some embodiments, an antibody moiety in a product agent is an antibody moiety in a second agent. In some embodiments, a second agent is an antibody agent, e.g., a monoclonal antibody, an antibody in a polyclonal antibody, an antibody in an IVIG preparation, etc. In embodiments, a second reactive group is a function group of an amino acid residue, e.g., −NH2 of Lys, −SH of Cys, etc. In embodiments, a second reactive group is −NH2 of a Lys residue, e.g., of a residue selected from K246 and K248 of IgG1 heavy chain amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of a heavy chain and amino acid residues corresponding thereto. In some embodiments, the invention provides selective reactions at amino acid residues of antibody moieties.

[00222] In some embodiments, a second reactive group is installed to an antibody moiety optionally through a linker. In some embodiments, a second reactive group is installed to an antibody moiety through a linker. In some embodiments, a second reactive group is selectively linked to certain location(s) of an antibody moiety, e.g., certain location(s) selected from K246 and K248 of IgG1 heavy chain amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an anti-PLA2R antibody heavy chain and amino acid residues corresponding thereto. In some embodiments, the invention provides selective reactions at amino acid residues of antibody moieties.

[00223] In some embodiments, the invention provides agents each independently comprising an antibody-binding moiety that binds to an antibody agent, a reactive group, a cellular receptor-binding moiety, and optionally one or more linker moieties linking such groups / moieties. In some embodiments, such agents are useful as reaction partners, e.g., first agents) for conjugating moieties of interest, e.g., target-binding moieties, reactive groups, e.g., second reactive groups) to agents comprising antibody moieties, e.g., second agents). In some embodiments, the invention provides agents for conjugating moieties of interest to antibody moieties in several agents or antibody agents, e.g., monoclonal antibody agents, polyclonal antibody agents, antibody agents of IVIG preparations, etc. In embodiments, provided agents each comprise a cellular receptor-binding moiety, a reactive group, an antibody-binding moiety, and optionally one or more linker moieties (linkers) linking such moieties. In some embodiments, an antibody-binding moiety is part of a leaving group released after contacting this agent, e.g., a first agent, with an antibody moiety, e.g., of a second agent, and reacting a reactive group of this agent, e.g., a first reactive group of a first agent, with a reactive group of an antibody moiety, e.g., a second reactive group of a second agent, such as −NH2 of a Lys residue of an antibody protein. In some embodiments, provided technologies can provide improved conjugation efficiency, high selectivity, or fewer steps (sometimes, single step) to conjugation product agents. In some embodiments, a provided agent, e.g., a first agent, is a composition of matter of formula AGN301 or a salt thereof:LG−RG−LRM−TBT,[AGN301] or a salt thereof, wherein:LG is a group comprising an antibody-binding moiety; RG is a reactive group;LRM is a linker; andTBT is a cellular receptor-binding moiety.

[00224] In some embodiments, LG is or comprises an antibody-binding moiety as described in this specification, and a linker which links an antibody-binding moiety and RG.

[00225] In some embodiments, LG is or comprises RLG−LLG−, wherein RLG is or comprises an antibody-binding moiety, and LLG is a linker moiety as described in this specification. In some embodiments, LG is ABT-LLG−. In some embodiments, LLG is −LLG1−LLG2−, wherein each of LLG1 and LLG2 is independently a linker moiety as described in this specification. In some embodiments, LLG is −LLG1−LLG2−LLG3−, wherein each of LLG1, LLG2 and LLG3 is independently as linker moiety described in this specification. In some embodiments, LLG is −LLG1−LLG2−LLG3−LLG4−, wherein each of LLG1, LLG2, LLG3 and LLG4 is independently a linker moiety as described in this specification. In some embodiments, LLG1 is bonded to RLG. In some embodiments, LLG1 is bonded to cellular receptor-binding moiety. In some embodiments, LLG is −LLG1−, and a reactive group comprises LLG2, LLG3 and LLG4. In some embodiments, LLG is −LLG1−LLG2−, and a reactive group comprises LLG3 and LLG4. In some embodiments, LLG is −LLG1−LLG2−LLG3−, and a reactive group comprises LLG4. In some embodiments, each of LLG1, LLG2, LLG3 and LLG4 is independently L.

[00226] In some embodiments, antibody-binding moieties, LG, etc. are released after reactions, e.g., after first agents, e.g., wherein MOIs are target-binding moieties) react with second agents, e.g., which are antibody agents comprising reactive amino acid residues such as amino groups as second reactive groups or second agents comprising second reactive groups introduced to antibody agents), or after first agents, e.g., wherein MOIs are reactive groups such as second reactive groups) react with second agents which are antibody agents. In some embodiments, an antibody-binding moiety is released after a reaction. In some embodiments, LG is released after a reaction. In some embodiments, a leaving group is released as part of a compound having the structure of LG−H or a salt thereof. In some embodiments, an antibody-binding moiety is released as part of a compound having the structure of LG−H or a salt thereof. In some embodiments, LG is released as part of a compound having the structure of LG−H or a salt thereof. In some embodiments, a released compound has the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof. In some embodiments, an antibody-binding moiety is released as part of a compound having the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof. In some embodiments, an antibody-binding moiety is released as part of a compound having the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof, wherein RLG is or comprises an antibody-binding moiety. In some embodiments, LG is released as part of a compound having the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof, wherein LG is RLG−LLG, and LLG is −LLG1−, −LLG1−LLG2−, −LLG1−LLG2−LLG3−, or −LLG1−LLG2−LLG3−LLG4−. In some embodiments, LG is released as part of a compound having the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof, wherein LG is RLG−LLG1−. In some embodiments, LG is released as part of a compound having the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof, wherein LG is RLG−LLG1−LLG2. In some embodiments, LG is released as part of a compound having the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof, wherein LG is RLG−LLG1−LLG2−LLG3. In some embodiments, LG is released as part of a compound having the structure of RLG−LLG1−LLG2−LLG3−LLG4−H or a salt thereof, wherein LG is RLG−LLG1−LLG2−LLG3−LLG4.

[00227] In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1-100 group comprising one or more aliphatic moieties, aryl moieties, heteroaliphatic moieties each independently having 1-20 heteroatoms, heteroaromatic moieties each independently having 1-20 heteroatoms, or any combinations of any one or more of such moieties, wherein one or more methylene units of the group are optionally and independently replaced with C1-6 alkylene, C1-6 alkenylene, a bivalent C1-6 heteroaliphatic group having 1-5 heteroatoms, , −Cy−, −C(R’)2−, −O−, −S−, −S−S−, −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’)−, an amino acid residue, or −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-20. In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1-100 aliphatic or heteroaliphatic group 1-20 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with , −Cy−, −C(R’)2−, −O−, −S−, −S−S−, −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’)−, or −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-20. In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1, C2, C3, C4, C5, C10, C15, C20, C25, C30, C40, C50, C60, C1-2, C1-5, C1-10, C1-15, C1-20, C1-30, C1-40, C1-50, C1-60, C1-70, C1-80, or C1-90 aliphatic or heteroaliphatic group 1-10 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with , −Cy−, −C(R’)2−, −O−, −S−, −S−S−, −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 residues, or −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-20. In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1, C2, C3, C4, C5, C10, C15, C20, C25, C30, C40, C50, C60, C1-2, C1-5, C1-10, C1-15, C1-20, C1-30, C1-40, C1-50, C1-60, C1-70, C1-80, or C1-90 aliphatic or heteroaliphatic group 1-10 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −C≡C−, −Cy−, −C(R’)2−, −O−, −S−, −S−S−, −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 −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-10. In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1, C2, C3, C4, C5, C10, C15, C20, C25, C30, C40, C50, C60, C1-2, C1-5, C1-10, C1-15, C1-20, C1-30, C1-40, C1-50, C1-60, C1-70, C1-80, or C1-90 aliphatic group, wherein one or more methylene units of the group are optionally and independently replaced with −O−, −N(R’)−, −C(O)−, −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 residues, or −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-10. In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1, C2, C3, C4, C5, C10, C15, C20, C25, C30, C40, C50, C60, C1-2, C1-5, C1-10, C1-15, C1-20, C1-30, C1-40, C1-50, C1-60, C1-70, C1-80, or C1-90 aliphatic group, wherein one or more methylene units of the group are optionally and independently replaced with −O−, −N(R’)−, −C(O)−, −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’)−, or −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-10. In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1-10 aliphatic group, wherein one or more methylene units of the group are optionally and independently replaced with −O−, −N(R’)−, −C(O)−, −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’)−, −Cy−, or −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-10. In some embodiments, L is a covalent bond, or a bivalent optionally substituted, linear or branched C1-10 aliphatic group, wherein one or more methylene units of the group are optionally and independently replaced with −O−, −N(R’)−, −C(O)−, −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’)−, or −[(−O−C(R’)2−C(R’)2−)n]−, wherein n is 1-10. In some embodiments, L comprises no −C(O)O−. In some embodiments, L comprises no −C(O)−N(R’)−. In some embodiments, L comprises no −S−. In some embodiments, L comprises no −S−Cy−. In some embodiments, L comprises no −S−S−. In some embodiments, L does not have one or more or any of −C(O)O−, −C(O)−N(R’)−, −S−, and −S−S−. In some embodiments, L does not have one or more or any of −C(O)O−, −C(O)−N(R’)−, −S−Cy−, and −S−S−. In some embodiments, L does not have one or more or any of −C(O)O−, −S−, and −S−S−. In some embodiments, L does not have one or more or any of −C(O)O−, −S−Cy−, and −S−S−. In some embodiments, L has none of −C(O)O−, −S−, and −S−S−. In some embodiments, L has none of −C(O)O−, −S−Cy−, and −S−S−. In some embodiments, L has none of −C(O)O− and −S−S−.

[00228] In some embodiments, L is a covalent bond. In some embodiments, L is not a covalent bond.

[00229] In some embodiments, LLG1 is a covalent bond. In some embodiments, LLG1 is not a covalent bond. In some embodiments, LLG1 is or comprises −(CH2CH2O)n−. In some embodiments, LLG1 is or comprises a moiety selected from the Markush group of moieties consisting of −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted, −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted, −(CH2)2−O−(CH2CH2O)n−(CH2)2−, wherein n is as described in this specification, and each −CH2− is independently optionally substituted, and −(CH2)2−O−(CH2CH2O)n−(CH2)2−, wherein n is as described in this specification.

[00230] In some embodiments, LLG1 is a moiety selected from the Markush group of moieties consisting of LLG1 is −CH2−, −(CH2)2−, −(CH2)2−C(O)−, −(CH2)2−C(O)−NH−, −(CH2)3−, −(CH2)3NH−, −(CH2)3NH−C(O)−, −C(O)−(CH2)3NH−C(O)−, −C(O)−(CH2)3−, −NH−C(O)−(CH2)3−, and −NHC(O)−(CH2)3NH−C(O)−. In some embodiments, a −CH2− is bonded to an antibody-binding moiety.

[00231] In some embodiments, LLG1 is a moiety selected from the Markush group of moieties consisting of −CH2CH2−O−CH2CH2−O−CH2CH2−, −CH2CH2−O−CH2CH2−O−CH2CH2−C(O)−, −CH2CH2−O−CH2CH2−O−CH2CH2−C(O)NH−, and −CH2CH2−O−CH2CH2−O−CH2CH2−C(O)NH−CH2−. In some embodiments, −CH2CH2− is bonded to an antibody-binding moiety.

[00232] In some embodiments, LLG1 is −(CH2CH2O)n−. In some embodiments, LLG1 is −(CH2CH2O)n−CH2−CH2−. In some embodiments, LLG1 is −(CH2CH2O)n−CH2−CH2−C(O)−. In some embodiments, LLG1 is −(CH2CH2O)2−CH2−CH2−C(O)−. In some embodiments, LLG1 is −(CH2CH2O)4−CH2−CH2−C(O)−. In some embodiments, LLG1 is −(CH2CH2O)8−CH2−CH2−C(O)−. In some embodiments, −C(O)− is bonded to an antibody-binding moiety.

[00233] In some embodiments, LLG1 is −N(R’)−. In some embodiments, LLG1 is −NH−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]n−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]n−CH2CH2−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]n−CH2CH2−NH−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]n−CH2CH2−NH−C(O)−. 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, LLG1 is −NH−CH2CH2−O−. In some embodiments, LLG1 is −NH−CH2CH2−O− CH2CH2−. In some embodiments, LLG1 is −NH−CH2CH2−O− CH2CH2−NH−. In some embodiments, LLG1 is −NH−CH2CH2−O− CH2CH2−NH−C(O)−.

[00234] In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]2−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]2−CH2CH2−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]2−CH2CH2−NH−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]2−CH2CH2−NH−C(O)−.

[00235] In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]3−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]3−CH2CH2−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]3−CH2CH2−NH−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]3−CH2CH2−NH−C(O)−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]4−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]4−CH2CH2−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]4−CH2CH2−NH−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]4−CH2CH2−NH−C(O)−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]5−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]5−CH2CH2−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]5−CH2CH2−NH−. In some embodiments, LLG1 is −NH−[(−CH2CH2−O−)]5−CH2CH2−NH−C(O)−. In some embodiments, −NH− is bonded to an antibody-binding moiety.

[00236] In some embodiments, LLG1 is −CH2−. In some embodiments, LLG1 is −CH2CH2−. In some embodiments, LLG1 is −CH2CH2NH−. In some embodiments, LLG1 is −CH2CH2NH−(CO)−. In some embodiments, −CH2− is bonded to an antibody-binding moiety.

[00237] In some embodiments, LLG1 is −CH2−. In some embodiments, LLG1 is −CH2C(O)−. In some embodiments, LLG1 is −CH2C(O)NH−. In some embodiments, LLG1 is −CH2(CO)NHCH2−. In some embodiments, −CH2−C(O)− is bonded to an antibody-binding moiety at −CH2−.

[00238] In some embodiments, LLG2 is a covalent bond. In some embodiments, LLG2 is not a covalent bond. In some embodiments, LLG2 is −N(R’)C(O)−. In some embodiments, LLG2 is −NHC(O)−. In some embodiments, LLG2 is −(CH2)n−N(R’)C(O)−, wherein −(CH2)n− is optionally substituted. In some embodiments, LLG2 is −(CH2)n−OC(O)−, wherein −(CH2)n− is optionally substituted. In some embodiments, LLG2 is −(CH2)n−OC(O)N(R’)−, wherein −(CH2)n− is optionally substituted. In some embodiments, LLG2 is −(CH2)n−OC(O)NH−, wherein −(CH2)n− is optionally substituted. In some embodiments, n is 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, −(CH2)n− is substituted. In some embodiments, −(CH2)n− is unsubstituted. In some embodiments, LLG2 is −CH2N(CH2CH2CH2S(O)2OH)−C(O)−. In some embodiments, LLG2 is −C(O)−NHCH2−. In some embodiments, LLG2 is −C(O)−NHCH2CH2−. In some embodiments, LLG2 is −C(O)O−CH2−. In some embodiments, LLG2 is −NH−C(O)O−CH2−. In some embodiments, −C(O)− is bonded to LLG3. In some embodiments, −N(R’)−, −NH−, or an optionally substituted −CH2− unit (of optionally substituted −(CH2)n−) is bonded to LLG3. In some embodiments LLG2 is −NH−, −NHC(O)−,−(CH2)n−NHC(O)−, −(CH2)n−OC(O)−, −(CH2)n−OC(O)NH−, −C(O)−NHCH2−, −C(O)−NHCH2CH2−, −C(O)O−CH2−, or −NH−C(O)O−CH2−.

[00239] In some embodiments, LLG2 is −N(R’)−. In some embodiments, LLG2 is −N(R)−. In some embodiments, LLG2 is −NH−.

[00240] In some embodiments, LLG2 is optionally substituted bivalent C1-6 aliphatic. In some embodiments, LLG2 is −CH2−. In some embodiments, LLG2 is −CH2NH−. In some embodiments, LLG2 is −CH2NH−C(O)−. In some embodiments, LLG2 is −CH2NH−C(O)−CH2−.

[00241] In some embodiments, LLG3 is or comprises an optionally substituted aryl ring. In some embodiments, LLG3 is or comprises an optionally substituted phenyl ring. In some embodiments, LLG3 is a phenyl ring substituted with one or more electron-withdrawing groups. As understood by persons having ordinary skill in the biomedical art, several electron-withdrawing groups are known in the biomedical art and may be used under this specification. In some embodiments, an electron-withdrawing group is halogen. In some embodiments, an electron-withdrawing group is −F. In embodiments, it is −Cl. In some embodiments, it is −Br. In embodiments, it is −I. In embodiments, an electron-withdrawing group comprises an X=Y double bond, wherein X is bonded to the group to which the electron-withdrawing group is a substituent, and at least one of X and Y is a heteroatom. In some embodiments, X is a heteroatom. In some embodiments, Y is a heteroatom. In some embodiments, each of X and Y is independently a heteroatom. In some embodiments, Y is O. In embodiments, Y is S. In embodiments, X is C. In embodiments, X is N. In embodiments, X is P. In embodiments, X is S. In embodiments, X=Y is C=O. In embodiments, X=Y is N=O. In embodiments, X=Y is S=O. In embodiments, X=Y is P=O. In embodiments, an electron-withdrawing group is −C(O)−L−R’. In some embodiments, an electron-withdrawing group is −C(O)−R’. In some embodiments, it is −NO2. In some embodiments, it is −S(O)−L−R’. In some embodiments, it is −S(O)−R’. In some embodiments, it is −S(O)2−L−R’. In some embodiments, it is −S(O)2−O−R’. In some embodiments, it is −S(O)2−N(R’)2. In some embodiments, it is −P(O)(−L−R’)2. In some embodiments, it is −P(O)(R’)2. In some embodiments, it is −P(O)(OR’)2. In some embodiments, it is −P(O)[N(R’)2]2.

[00242] In some embodiments, LLG3 is −LLG3a−LLG3b−, wherein LLG3a is a covalent bond or −C(O)O−CH2−, wherein −CH2− is optionally substituted, and LLG3b is an optionally substituted aryl ring. In some embodiments, LLG3a is bonded to LLG2, and LLG3b is bonded to LLG4.

[00243] In some embodiments, LLG3a is a covalent bond. In some embodiments, LLG3a is −C(O)O−CH2−, wherein −CH2− is optionally substituted. In some embodiments, LLG3a is −C(O)O−CH2−, wherein −CH2− is substituted. In some embodiments, LLG3a is −C(O)O−CH2−, wherein −CH2− is unsubstituted.

[00244] In some embodiments, a first group, an antibody-binding moiety, or LG is released as part of a compound having the structure of RLG−LLG1−LLG2−H or a salt thereof.

[00245] In some embodiments, LLG3b is an optionally substituted phenyl ring. In some embodiments, at least one substituent is an electron-withdrawing group as described in this specification.

[00246] In some embodiments, LLG3 is , wherein s is 0-4, each Rs is independently halogen, −NO2, −L−R’, −C(O)−L−R’, −S(O)−L−R’, −S(O)2−L−R’, or −P(O)(−L−R’)2. In some embodiments, C1 is bonded to LLG4. In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is .

[00247] In some embodiments, LLG3b is , wherein s is 0-4, each Rs is independently halogen, −NO2, −L−R’, −C(O)−L−R’, −S(O)−L−R’, −S(O)2−L−R’, or −P(O)(−L−R’)2. In some embodiments, C1 is bonded to LLG4. In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is .

[00248] In some embodiments, s is 0. In some embodiments, s is 1-4. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3. In some embodiments, s is 4.

[00249] In some embodiments, s is 1-4, and at least one Rs is an electron-withdrawing group, e.g., an electron-withdrawing group described above. In some embodiments, at least one Rs is −NO2. In some embodiments, at least one Rs is −F. In embodiments, each Rs is independently an electron-withdrawing group. In some embodiments, each Rs is −NO2. In some embodiments, each Rs is −F.

[00250] In some embodiments, an electron-withdrawing group or Rs is at C2. In some embodiments, an electron-withdrawing group or Rs is at C3. In some embodiments, an electron-withdrawing group or Rs is at C4. In some embodiments, an electron-withdrawing group or Rs is at C2 and C5.

[00251] In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is . In some embodiments, LLG3 is .

[00252] In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is . In some embodiments, LLG3b is .

[00253] In some embodiments, LLG3b is optionally substituted . In some embodiments, the nitrogen atom is bound to LLG4 which is −O−. In some embodiments, the nitrogen atom is bound to LLG4 which is −O−, and −LRG1−LRG2− is −C(O)−.

[00254] In some embodiments, −LLG4−LRG1−LRG2− is −O−C(O)−. In some embodiments, −LLG4−LRG1−LRG2− is −S−C(O)−.

[00255] In some embodiments, LLG4 is a covalent bond. In some embodiments, LLG4 is not a covalent bond. In some embodiments, LLG4 is −O−. In some embodiments, LLG4 is −N(R’)−. In some embodiments, LLG4 is −NH−. In some embodiments, LLG4 is −N(CH3)−. In some embodiments, LLG4 is −N(R’)−, and LLG3 is −O−. In some embodiments, R’ is optionally substituted C1-6 alkyl. In some embodiments, LLG4 is −S−.

[00256] In some embodiments, RLG is or comprises an antibody-binding moiety. In some embodiments, RLG is or comprises a protein-binding moiety. In some embodiments, RLG is or comprises an antibody-binding moiety. In some embodiments, RLG is an antibody-binding moiety. In some embodiments, RLG is a protein-binding moiety. In some embodiments, RLG is an antibody-binding moiety.

[00257] In some embodiments, RLG is ABT101, Rc−(Xaa)z−, a nucleic acid moiety, or a small molecule moiety. In some embodiments, RLG is or comprises ABT101. In some embodiments, RLG is or comprises Rc−(Xaa)z−. In some embodiments, RLG is or comprises a small molecule moiety. In some embodiments, RLG is or comprises a peptide agent. In some embodiments, RLG is or comprises a nucleic acid agent. In some embodiments, RLG is or comprises an aptamer agent. In some embodiments, an antibody-binding moiety is or comprises ABT101. In some embodiments, a protein-binding moiety is or comprises ABT101. In some embodiments, an antibody-binding moiety is or comprises ABT101. In some embodiments, an antibody-binding moiety is or comprises Rc−(Xaa)z−. In some embodiments, a protein-binding moiety is or comprises Rc−(Xaa)z−. In some embodiments, an antibody-binding moiety is or comprises Rc−(Xaa)z−.

[00258] In some embodiments, target-binding moieties may be conjugated to antibody moieties optionally through linker moieties using technologies described in US 2020 / 0190165.

[00259] In some embodiments, where a particular protecting group (PG), leaving group (LG), or transformation condition is depicted, persons having ordinary skill in the biomedical art know that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated. Such groups and transformations are described in Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (John Wiley & Sons, 2001), Larock, Comprehensive Organic Transformations, 2nd edition (John Wiley & Sons, 1999), and Greene & Wuts, Protecting Groups in Organic Synthesis, 3rd edition (John Wiley & Sons, 1999), the entirety of each of which is incorporated in this specification by reference.

[00260] In some embodiments, leaving groups include but are not limited to, halogens, e.g. fluoride, chloride, bromide, iodide, sulfonates, e.g. mesylate, tosylate, benzenesulfonate, brosylate, nosylate, triflate), diazonium.

[00261] In some embodiments, an oxygen protecting group includes carbonyl protecting groups, hydroxyl protecting groups, etc. Hydroxyl protecting groups are well known in the biomedical art and include those described in Greene & Wuts, Protecting Groups in Organic Synthesis, 3rd edition (John Wiley & Sons, 1999), the entirety of which is incorporated in this specification by reference. Examples of suitable hydroxyl protecting groups include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, phalobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, and 2- and 4-picolyl.

[00262] Amino protecting groups are well known in the biomedical art and include those described in Greene & Wuts, Protecting Groups in Organic Synthesis, 3rd edition (John Wiley & Sons, 1999), the entirety of which is incorporated in this specification by reference. Suitable amino protecting groups include, but are not limited to, aralkylamines, carbamates, cyclic imides, allyl amines, amides. Examples of such groups include t-butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl (CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl.

[00263] Persons having ordinary skill in the biomedical art know that provided agents may have one or more stereocenters and may be present as a racemic or diastereomeric mixture. Persons having ordinary skill in the biomedical art know there are many methods known in the biomedical art for the separation of isomers to obtain stereoenriched or stereopure isomers of those compounds, including but not limited to HPLC, chiral HPLC, fractional crystallization of diastereomeric salts, kinetic enzymatic resolution, e.g. by fungal- derived, bacterial- derived, or animal-derived lipases or esterases, and formation of covalent diastereomeric derivatives using an enantioenriched reagent.

[00264] persons having ordinary skill in the biomedical art know that several functional groups present in compounds of this specification such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles can be interconverted by techniques well known in the biomedical art including, but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (John Wiley & Sons, 2001), the entirety of which is incorporated in this specification by reference. Such interconversions may require one or more techniques, and certain methods for synthesizing compounds of this specification are described below in the Exemplification.

[00265] As known by persons having ordinary skill in the biomedical art, reaction partners are generally contacted with each other under conditions and for a time sufficient for production of the desired results, e.g., formation of product agents and compositions thereof to desired extents. Many reaction conditions / reaction times may be assessed and used if they are suitable for desired purposes under this specification; certain such conditions, reaction times, assessment, etc. are described in the Examples.

[00266] In some embodiments, an agent formed, e.g., a product MATE agent, has the structure of formula AGN101 or AGN102, or a salt thereof. In some embodiments, a cellular receptor-binding moiety in a product agent, e.g., a MATE agent, is the same as a cellular receptor-binding moiety in a reaction partner, e.g., a first agent comprising a cellular receptor-binding moiety) used to prepare a product agent. In some embodiments, an antibody moiety in a product agent, e.g., a MATE agent, is the same as an antibody moiety in a reaction partner, e.g., a second agent comprising an antibody moiety) used to prepare a product agent.

[00267] In some embodiments, linker moieties or a part connected to target-binding moieties or antibody moieties may be transferred from reaction partners, e.g., LRM of formula AGN301 or a salt thereof. In some embodiments, a linker moiety in a product agent (may be called LPM; e.g., L in formula AGN101 or AGN102, is or comprises a linker moiety in a reaction partner, e.g., one between a reactive group and a cellular receptor-binding moiety, e.g., LRM. In some embodiments, LPM is or comprises LRM. In some embodiments, LPM is −LRM−LRG2−. In some embodiments, LRG2 is −C(O)−. In some embodiments, LRG2 is −C(O)−, and is bonded to −NH− of a target agent moiety, e.g., −NH− in a side chain of a lysine residue of a protein moiety, which in some embodiments, is an antibody moiety.

[00268] Reaction partners, e.g., compounds of formula AGN301 or salts thereof, rarely have moieties that can react with reactive groups under conditions under which reactive groups react with target agents. In some embodiments, to the extent that some moieties in reaction partners may react with reactive groups under conditions under which reactive groups react with target agents, reactions between such moieties and reactive groups are significantly slower or less efficient compared to reactions between reactive groups and target agents. In some embodiments, reactions between such moieties and reactive groups do not significantly reduce, e.g., no more than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. of reduction, efficiencies, yields, rates, or conversions, etc., of reactions between reactive groups and target agents. In some embodiments, reactive groups, e.g., ester groups, activated carboxylic acid derivatives, etc. react with amino groups, e.g., −NH2 groups, of target agents, e.g., protein agents such as antibody agents. In some embodiments, reaction partners, e.g., compounds of formula AGN301 or salts thereof, do not have amine groups. In some embodiments, compounds of formula AGN301 or salts thereof (or parts of it, such as RLG, LLG, LLG1, LLG2, LLG3, LLG4, LRG1, LRG2, LRM, or MOI, do not have amine groups. In some embodiments, they do not have primary amine groups (−NH2). In some embodiments, they do not have −CH2NH2. In some embodiments, they do not have −CH2CH2NH2. In some embodiments, they do not have −CH2CH2CH2NH2. In some embodiments, they do not have −CH2CH2CH2CH2NH2. In some embodiments, amine groups, e.g., primary amine groups, are capped, e.g., by introduction of acyl groups, e.g., R−C(O)−, e.g., acetyl to form amide groups) to prevent or reduce undesired reactions.

[00269] In some embodiments, reactions are performed in buffer systems. In some embodiments, buffer systems of present disclosure maintain structures or functions of target agents, cellular receptor-binding moiety, etc. In embodiments, a buffer is a phosphate buffer. In some embodiments, a buffer is a phosphate-buffered saline (PBS) buffer. In some embodiments, a buffer is a borate buffer. In some embodiments, buffers of this specification provide and optionally maintain certain pH value or range. In some embodiments, a useful pH is about 7-9, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 9.0, etc. In embodiments, a pH is 7.4. In some embodiments, a pH is 7.5. In some embodiments, a pH is 7.8. In some embodiments, a pH is 8.0. In some embodiments, a pH is 8.2. In some embodiments, a pH is 8.3.

[00270] Provided technologies can provide several advantages. In some embodiments, connection of a cellular receptor-binding moiety in a reaction partner, e.g., a compound comprising a reactive group between an antibody-binding moiety and a cellular receptor-binding moiety, e.g., a composition of matter of formula AGN301 or a salt thereof to an agent comprising an antibody moiety, e.g., a second agent such as an antibody agent, and release of an antibody-binding moiety in a provided reaction partner can be achieved in one reaction or in one pot. In many embodiments, no separate reactions / steps are performed to remove antibody-binding moieties. As known by persons having ordinary skill in the biomedical art, by performing connection of cellular receptor-binding moiety and release of antibody-binding moiety in a single reaction / operation, provided technologies can avoid separate steps for antibody-binding moiety removal and can improve overall efficiency, e.g., by simplify operations, increasing overall yield, etc. , reduce manufacturing cost, improve product purity, e.g., by avoiding exposure to antibody-binding moiety removal conditions, which typically involve one or more of reduction, oxidation, hydrolysis, e.g., of ester groups), etc., conditions and may damage target agent moieties, e.g., for protein agent moieties, protein amino acid residues, overall structures, or post-translational changes, e.g., glycans of antibodies) thereof. In some embodiments, provided technologies can provided improved efficiency, e.g., in terms of reaction rates or conversion percentages), increased yield, increased purity / homogeneity, or enhanced selectivity, particularly compared to reference technologies wherein a reaction partner containing no antibody-binding moieties is used, without introducing step(s) for antibody-binding moiety removal, e.g., antibody-binding moiety is removed in the same step as cellular receptor-binding moiety conjugation.

[00271] In some embodiments, the invention provides products of provided processes, which have low levels of damage to antibody moieties compared to processes comprising steps performed for antibody-binding moiety removal but not for substantial conjugation of moieties of interest, e.g. target-binding moieties. In some embodiments, provided product agent compositions have high homogeneity, e.g., regarding the number of cellular receptor-binding moiety per antibody moiety, or positions of amino acid residues in antibody moieties conjugated to moieties of interest) compared to reference product compositions, e.g., those from technologies without using antibody-binding moieties, or using extra step(s) for antibody-binding moiety removal, e.g., not using reaction partners described in this specification which comprise a reactive group between an antibody-binding moiety and a cellular receptor-binding moiety.

[00272] In some embodiments, the invention provides a product agent which is an agent comprising an antibody moiety, a cellular receptor-binding moiety and optionally a linker moiety linking an antibody-binding moiety and a cellular receptor-binding moiety. In some embodiments, the invention composes such agents.

[00273] In some embodiments, the invention provides a composition comprising a plurality of agents, wherein each agent independently comprises:an antibody moiety,a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody-binding moiety and a cellular receptor-binding moiety.

[00274] In some embodiments, product agents are MATE agents. In some embodiments, an antibody agent moiety comprises IgG Fc region. In some embodiments, an antibody moiety is connected to a cellular receptor-binding moiety through an amino group optionally through a linker. In some embodiments, it is through a lysine residue wherein the amino group of the side chain is connected to a cellular receptor-binding moiety optionally through a linker, e.g., forming −NH−C(O)− as part of an amide group, a carbamate group, etc.

[00275] In some embodiments, selected locations of antibody moieties are used for conjugation. In some embodiments, K246 or K248 of an antibody agent (EU numbering, or corresponding residues) are conjugation locations. In some embodiments, a conjugation location is K246 of heavy chain (unless otherwise specified, locations in this specification include corresponding residues in, e.g., changed sequence, e.g., longer, shorter, rearranged, etc., sequences. In some embodiments, a location is K248 of heavy chain. In some embodiments, a location is K288 or K290 of heavy chain. In some embodiments, a location is K288 of heavy chain. In some embodiments, a location is K290 of heavy chain. In some embodiments, a location is K317. In some embodiments, an antibody moiety is a moiety of an IgG1 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG2 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an antibody or a fragment thereof. In some embodiments, a composition comprises a plurality of MATE agents, wherein antibody moieties of the plurality of MATE agents are independently an antibody moiety of an IgG1, IgG2, or IgG4 antibody, or a fragment thereof.

[00276] In some embodiments, antibody heavy chains are selectively conjugated / labeled over light chains.

[00277] In some embodiments, in provided agents, e.g., agents of formula AGN101 or AGN102, or a salt thereof) substantially all conjugation sites of antibody moieties have the same changes, e.g., all share the same moieties of interest optionally connected through the same linker moieties. In some embodiments, no conjugation sites bear different changes, e.g., different moieties of interest or no moieties of interest or different linker moieties.

[00278] In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target-binding moieties, conjugated to antibody moieties of a particular type of antibodies, e.g., IgG1, or fragments thereof are conjugated to one or more particularly sites, typically one or two particularly sites, e.g., K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target-binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target-binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K239 and K241 of a heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., for a plurality of agents, for a composition, etc. are conjugated to antibody moieties of IgG1, IgG2, or IgG4 antibodies, or fragments thereof, e.g., for conjugation products with IgG1 antibodies or fragments thereof (antibody moieties being of IgG1 antibodies or fragments thereof), IgG2 antibodies or fragments thereof (antibody moieties being of IgG2 antibodies or fragments thereof), IgG4 antibodies or fragments thereof (antibody moieties being of IgG4 antibodies or fragments thereof), or for conjugation products with IVIG (when certain provided technologies described in this specification are used, selective conjugation with IgG1, IgG2 and IgG4). In some embodiments, a percentage is about 10% or more. In some embodiments, a percentage is about 20% or more. In some embodiments, a percentage is about 25% or more. In some embodiments, a percentage is about 30% or more. In some embodiments, a percentage is about 40% or more. In some embodiments, a percentage is about 50% or more. In some embodiments, a percentage is about 60% or more. In some embodiments, a percentage is about 65% or more. In some embodiments, a percentage is about 70% or more. In some embodiments, a percentage is about 75% or more. In some embodiments, a percentage is about 80% or more. In some embodiments, a percentage is about 85% or more. In some embodiments, a percentage is about 90% or more. In some embodiments, a percentage is about 95% or more. In some embodiments, a percentage is about 100%.

[00279] In some embodiments, a composition comprises a plurality of agents, e.g., MATE agents, agents of formula AGN101 or AGN102, or a salt thereof, each independent comprising a cellular receptor-binding moiety, an antibody moiety, and optionally a linker moiety linking a cellular receptor-binding moiety and an antibody moiety. In some embodiments, substantially all target-binding moieties of a plurality of agents are the same. In some embodiments, substantially all target-binding moieties of a plurality of agents comprise peptide moieties of a common amino acid sequence. In some embodiments, substantially all target-binding moieties of a plurality of agents are peptide moieties of a common amino acid sequence. In some embodiments, substantially all conjugation sites of antibody moieties in a plurality of agents have the same changes, e.g., all share the same moieties of interest optionally connected through the same linker moieties. In some embodiments, no conjugation sites of a plurality of agents bear different changes, e.g., different moieties of interest or no moieties of interest or different linker moieties. In some embodiments, a plurality of agents does not have agents that share the same (or substantially the same) antibody moieties but different changes, e.g., different moieties of interest or no moieties of interest or different linker moieties. In some embodiments, agents that share the same (or substantially the same) antibody moieties but different changes, e.g., different moieties of interest or no moieties of interest or different linker moieties) are intermediates of multiple-step preparations, e.g., comprising steps for removal of antibody-binding moieties in addition to steps for cellular receptor-binding moiety conjugation) of final product agents.

[00280] In some embodiments, the invention provides a composition comprising a plurality of agents each of which independently comprising:an antibody moiety,a cellular receptor-binding moiety, andoptionally a linker moiety linking the antibody moiety and the cellular receptor-binding moiety;wherein antibody moieties of agents of the plurality comprise a common amino acid sequence, and agents of the plurality share a common cellular receptor-binding moiety independently at least one common amino acid residue of the common amino acid sequence; andwherein about 1%-100% of all agents that comprise an antibody moiety that comprise the common amino acid sequence and the cellular receptor-binding moiety are agents of the plurality.

[00281] In some embodiments, the invention provides a composition comprising a plurality of agents each of which independently comprising:an antibody moiety,a cellular receptor-binding moiety, andoptionally a linker moiety linking an antibody moiety and a cellular receptor-binding moiety;wherein agents of the plurality share the same or substantially the same antibody moiety, and a cellular receptor-binding moiety at least one common location; andwherein about 1%-100% of all agents that comprise the antibody moiety and the cellular receptor-binding moiety are agents of the plurality.

[00282] In some embodiments, an antibody moiety is a moiety of an IgG1 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG2 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG3 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an antibody or a fragment thereof. In some embodiments, about 1-100% of all moieties of interest are at common locations. In some embodiments, a cellular receptor-binding moiety is a cellular receptor-binding moiety as described in this specification. In some embodiments, agents of a plurality are each independently of formula AGN101 or AGN102, or a salt thereof.

[00283] In some embodiments, antibody moieties of agents of a plurality comprise a common amino acid sequence. In some embodiments, antibody moieties of agents of a plurality comprise a common amino acid sequence in a Fc region. In some embodiments, antibody moieties of agents of a plurality comprise a common Fc region. In some embodiments, antibody moieties of agents of a plurality can bind a common antigen specifically. In some embodiments, antibody moieties are monoclonal antibody moieties. In some embodiments, antibody moieties are polyclonal antibody moieties. In some embodiments, antibody moieties bind to two or more different antigens. In some embodiments, antibody moieties bind to two or more different proteins. In some embodiments, antibody moieties are IVIG moieties.

[00284] In some embodiments, a cellular receptor-binding moiety in an agent of a plurality is a cellular receptor-binding moiety. In some embodiments, each cellular receptor-binding moiety is independently a cellular receptor-binding moiety. In some embodiments, a composition comprises a plurality of agents, antibody moieties of agents of the plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently and optionally through a linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a common cellular receptor-binding moiety independently comprise a common cellular receptor-binding moiety linked to a common amino acid residue independently and optionally through a linker. In some embodiments, a composition comprises a plurality of agents, antibody moieties of agents of a plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently through a common linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a common cellular receptor-binding moiety independently comprise a common cellular receptor-binding moiety linked to a common amino acid residue independently and through a common linker. In some embodiments, a composition comprises a plurality of agents, antibody moieties of agents of a plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently and optionally through a linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a common cellular receptor-binding moiety are agents of a plurality. In some embodiments, a composition comprises a plurality of agents, wherein antibody moieties of agents of a plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently through a common linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence, a common cellular receptor-binding moiety, and a common linker are agents of a plurality.

[00285] In some embodiments, “at least one” or “one or more” is 1-1000, 1-500, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 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 one. In some embodiments, it is two 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.

[00286] In some embodiments, a common amino acid sequence comprises 1-1000, 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 10-1000, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 20-1000, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 50-1000, 50-500, 50-400, 50-300, 50-200, 50-100, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 400, 500, 600 or more amino acid residues. In some embodiments, a length is at least 5 amino acid residues. In some embodiments, a length is at least 10 amino acid residues. In some embodiments, a length is at least 50 amino acid residues. In some embodiments, a length is at least 100 amino acid residues. In some embodiments, a length is at least 150 amino acid residues. In some embodiments, a length is at least 200 amino acid residues. In some embodiments, a length is at least 300 amino acid residues. In some embodiments, a length is at least 400 amino acid residues. In some embodiments, a length is at least 500 amino acid residues. In some embodiments, a length is at least 600 amino acid residues.

[00287] In some embodiments, a common amino acid sequence is at least 10%-100%, 50%-100%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence of an antibody moiety, a protein agent moiety, etc. In embodiments, it is 10% or more. In some embodiments, it is 20% or more. In some embodiments, it is 30% or more. In some embodiments, it is 40% or more. In some embodiments, it is 50% or more. In some embodiments, it is 60% or more. In some embodiments, it is 70% or more. In some embodiments, it is 80% or more. In some embodiments, it is 90% or more. In some embodiments, it is 100%.

[00288] In some embodiments, in a common amino acid sequence, one and only one amino acid residue is linked to a common cellular receptor-binding moiety, e.g., a common cellular receptor-binding moiety. In some embodiments, in a common amino acid sequence, two and only two amino acid residues are linked to a common cellular receptor-binding moiety, e.g., a common cellular receptor-binding moiety. In some embodiments, in a common amino acid sequence, two or more amino acid residues are linked to a common cellular receptor-binding moiety, e.g., a common cellular receptor-binding moiety. In some embodiments, each common cellular receptor-binding moiety, e.g., a common cellular receptor-binding moiety, is independently linked to an amino acid residue in a common amino acid sequence.

[00289] In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an antibody heavy chain and amino acid residues corresponding thereto. In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto. In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto. In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K239 and K241 of an antibody heavy chain and amino acid residues corresponding thereto. In some embodiments, a cellular receptor-binding moiety is connected to this amino acid residue (unless explicitly noted, optionally through a linker moiety). In some embodiments, each cellular receptor-binding moiety is connected to this amino acid residue each optionally and independently through a linker moiety.

[00290] In some embodiments, antibody moieties share a high percentage of amino acid sequence homology. In some embodiments, the amino acid sequence homology is about 50%-100%. In some embodiments, the amino acid sequence homology is a percentage selected from the Markush group of percentages consisting of 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

[00291] In some embodiments, a percentage used in this specification is a percentage selected from the Markush group of percentages consisting of about 1%-100%, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100%.

[00292] In some embodiments, antibody moiety of agents of a plurality comprises a common Fc region or a fragment thereof.

[00293] In some embodiments, moieties of interest of agents of a plurality are at specific locations. In some embodiments, all moieties of interest are at amino acid residues of a common amino acid sequence. In some embodiments, all moieties of interest are at common locations of amino acid residues of a common amino acid sequence. In some embodiments, the number of common locations is 1. In some embodiments, it is 2. In some embodiments, it is 3. In some embodiments, it is 4. In some embodiments, antibody moieties comprise two heavy chains or fragments thereof, and the number of common locations is 2 (one on each chain). In some embodiments, common locations are selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an antibody heavy chain and amino acid residues corresponding thereto.

[00294] In some embodiments, agents of a plurality share a common cellular receptor-binding moiety independently at least one location. In some embodiments, agents of a plurality share a common cellular receptor-binding moiety and linker independently at least one location. In some embodiments, moieties of interest at two or more or all locations comprise a common cellular receptor-binding moiety. In some embodiments, moieties of interest are the same.

[00295] In some embodiments, agents share common changes at least one common amino acid residue. In some embodiments, agents of a plurality share common changes at each location connected to a cellular receptor-binding moiety and optionally a linker. In some embodiments, agents of a plurality the same −LPM−TBT at each location connected to a linker moiety.

[00296] In some embodiments, a location is selected from K246, K248, K288, K290, K317 of antibody agents and locations corresponding thereto. In some embodiments, a location is selected from K246 and K248, and locations corresponding thereto. In some embodiments, a location is selected from K288 and K290, and locations corresponding thereto. In some embodiments, a location is K246 or a location corresponding thereto. In some embodiments, a location is K248 or a location corresponding thereto. In some embodiments, a location is K288 or a location corresponding thereto. In some embodiments, a location is K290 or a location corresponding thereto. In some embodiments, a location is K317 or a location corresponding thereto. In some embodiments, a location is K185 of light chain or a location corresponding thereto. In some embodiments, a location is K187 of light chain or a location corresponding thereto. In some embodiments, a location is K133 of heavy chain or a location corresponding thereto. In some embodiments, a location is K246 or K248 of heavy chain or a location corresponding thereto. In some embodiments, a location is K414 of heavy chain or a location corresponding thereto. In some embodiments, a common sequence is a sequence that is about or at least about 10-100, 20-50, e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, amino acid residues in length, and comprises one or more of such residues or residues corresponding thereto. In some embodiments, a common sequence is a sequence that is about or at least about 10-100, 20-50, e.g., about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, amino acid residues in length, and comprises one, two or more residues selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an antibody heavy chain and amino acid residues corresponding thereto.

[00297] In some embodiments, about 1%-100% of all agents that comprise an antibody moiety and a cellular receptor-binding moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a cellular receptor-binding moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety that comprise a common amino acid sequence or can bind to a common antigen and a cellular receptor-binding moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety that comprise the common amino acid sequence are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise a protein agent moiety that comprise the common amino acid sequence are agents of a plurality. In some embodiments, a range of all agent agents that comprise an antibody agent moiety that comprise the common amino acid sequence or can bind to the common antigen are selected from a Markush group of ranges where the agents are of a plurality consisting of about 1%-100%, about 5%-100%, 10%-100%, about 20%-100%, about 25%-100%, about 30%-100%, and about 40%-100%, about 50%-100%. In some embodiments, a range of all agent agents that comprise an antibody agent moiety that comprise the common amino acid sequence or can bind to the common antigen are selected from a Markush group of ranges where the agents are of a plurality consisting of about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100%. In some embodiments, a range of all agent agents that comprise an antibody agent moiety that comprise the common amino acid sequence or can bind to the common antigen are selected from a Markush group of ranges where the agents are of a plurality consisting of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50 at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99%.

[00298] In some embodiments, each agent of the plurality does not have −S−Cy−, wherein −Cy− is optionally substituted 5-membered monocyclic ring, does not have −S−S− which is not formed by cysteine residues and does not have −SH or salt form thereof that is not of a cysteine residue. In some embodiments, each agent of the plurality does not have −S−CH2−CH2−. In some embodiments, each agent of the plurality does not have a moiety that can specifically bind to an antibody agent. In some embodiments, a composition is substantially free from a moiety that can specifically bind to an antibody agent.

[00299] In some embodiments, the invention provides product agent compositions comprising product agents, e.g., agents of formula AGN101 or AGN102, or a salt thereof. In some embodiments, a product agent composition, e.g., aa agent composition formed from certain methods, comprises a product agent comprising an antibody moiety and a cellular receptor-binding moiety and optionally a linker, e.g., an agent of formula AGN101 or AGN102, or a salt thereof, a released antibody-binding moiety, e.g., a compound comprising RLG−(LLG1)0-1−(LLG2)0-1−(LLG3)0-1−(LLG4)0-1−) or a compound comprising a released antibody-binding moiety, e.g., a compound having the structure of RLG−(LLG1)0-1−(LLG2)0-1−(LLG3)0-1−(LLG4)0-1−H or a salt thereof, and a reaction partner, e.g., a composition of matter of formula AGN301 or a salt thereof. In some embodiments, released antibody-binding moieties may bind to antibody moieties in target agents or formed product agents. Several technologies are available to separate released antibody-binding moieties from antibody moieties under this specification, for example, in some embodiments, contacting a composition with a composition comprising glycine at certain pH. In some embodiments, each agent of a plurality is independently this product agent.Reactive Group

[00300] In some embodiments, provided agents, compounds, e.g., those useful as reaction partners such as first agents, comprise reactive groups, e.g., RG. In some embodiments, reactive groups, e.g., RG) are between antibody-binding moieties, e.g., ABT) and moieties of interest, e.g., MOI), and are optionally and independently linked to antibody-binding moieties and moieties of interest via linkers. In some embodiments, RG is a reaction group as described in this specification.

[00301] In some embodiments, reactive groups when used in agents that comprise no antibody-binding moieties react slowly and provide low level of, in some embodiments, substantially no conjugation of moieties of interest with target agents. As shown in this specification, combination of reactive groups with antibody-binding moieties in the same agents, e.g., as in compounds of formula AGN301 or salts thereof, can promote reactions between reactive groups and target agents, enhance reaction efficiency, reduce side reactions, or improve reaction selectivity, e.g., in terms of target sites wherein conjugation of moieties of interest with target agents occurs.

[00302] Reactive groups in agents can react with several types of groups in target agents. In some embodiments, reactive groups in agents selectively react with amino groups of target agents, e.g., −NH2 groups on side chains of lysine residues of proteins. In some embodiments, reactive groups when used in agents, e.g., those of formula AGN301 or salts thereof, selectively react with particular sites of target agents, e.g., as shown in examples in this specification, one or more of K246, K248, K288, K290, K317, etc. of IgG1, K251, K253, etc. for IgG2, K239, K241 for IgG4 , etc. In embodiments, a site is K246 or K248 of an antibody heavy chain. In some embodiments, sites are K246 or K248 of an antibody heavy chain. In some embodiments, a site is K246 of an antibody heavy chain. In some embodiments, a site is K248 of an antibody heavy chain. In some embodiments, a site is K288 or K290 of an antibody heavy chain. In some embodiments, a site is K288 of an antibody heavy chain. In some embodiments, a site is K290 of an antibody heavy chain. In some embodiments, a site is K317. In some embodiments, a site is K414 of an antibody heavy chain. In some embodiments, a site is K185 of an antibody light chain. In some embodiments, a site is K187 of an antibody light chain. In some embodiments, sites are K251 or K253 of an IgG2 heavy chain. In some embodiments, a site is K251 of an IgG2 heavy chain. In some embodiments, a site is K253 of an IgG2 heavy chain. In some embodiments, sites are K239 or K241 of an antibody heavy chain. In some embodiments, a site is K239 of an antibody heavy chain. In some embodiments, a site is K241 of an antibody heavy chain. In some embodiments, conjugation selectively occurs at one or more heavy chain sites over light chain sites. In some embodiments, for technologies without antibody-binding moieties, conjugation occurs at light chain sites more than heavy chain sites.

[00303] In some embodiments, a reactive group, e.g., RG, is or comprises an ester group. In some embodiments, a reactive group, e.g., RG, is or comprises an electrophilic group, e.g., a Michael acceptor.

[00304] In some embodiments, a reactive group, e.g., RG, is or comprises −LRG1−LRG2−, wherein each of LRG1 and LRG2 is independently L. In embodiments, a reactive group, e.g., RG, is or comprises −LLG4−LRG1−LRG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −LLG3−LLG4−LRG1−LRG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −LLG2−LLG3−LLG4−LRG1−LRG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −LLG4−LRG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −LLG3−LLG4−LRG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −LLG2−LLG3−LLG4−LRG2−, wherein each variable is as described in this specification.

[00305] In some embodiments, LLG4 is −O−. In some embodiments, LLG4 is −N(R)−. In some embodiments, LLG4 is −NH−.

[00306] In some embodiments, LLG3 is or comprises an optionally substituted aryl ring. In some embodiments, LLG3 is or comprises a phenyl ring. In some embodiments, an aryl or phenyl ring is substituted. In some embodiments, a substituent is an electron-withdrawing group as described in this specification, e.g., −NO2, −F, etc.

[00307] In some embodiments, LRG1 is a covalent bond. In some embodiments, LRG1 is not a covalent bond. In some embodiments, LRG1 is −S(O)2−.

[00308] In some embodiments, LRG2 is −C(O)−. In some embodiments, a reactive group is or comprises −LLG4−C(O)−, wherein each variable is as described in this specification. In some embodiments, a reactive group is or comprises −LLG3−LLG4−C(O)−, wherein each variable is as described in this specification. In some embodiments, a reactive group is or comprises −LLG2−LLG3−LLG4−C(O)−, wherein each variable is as described in this specification.

[00309] In some embodiments, LRG2 is −LRG3−C(=CRRG1RRG2)−CRRG3RRG4−, wherein each of RRG1, RRG2, RRG3 and RRG4 is independently −L−R’, and LRG3 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, each of RRG1, RRG2, RRG3 and RRG4 is independently R’. In some embodiments, one or more of RRG1, RRG2, RRG3 and RRG4 is independently −H. In embodiments, LRG3 is −C(O)−. In some embodiments, LRG3 is −C(O)O−. In some embodiments, −O−, −N(R’)−, etc. of LRG3 is bonded to LPM.

[00310] In some embodiments, RRG1 is −H. In embodiments, RRG3 is −H.

[00311] In some embodiments, LRG2 is optionally substituted −LRG3−C(=CHRRG2)−CHRRG4−, wherein each variable is as described in this specification.

[00312] In some embodiments, RRG2 and RRG4 are taken with their intervening atoms to form an optionally substituted ring as described in this specification. In some embodiments, a formed ring is an optionally substituted 3-10-membered monocyclic or bicyclic ring having 0-5 heteroatoms. In some embodiments, a formed ring is an optionally substituted 3-10-membered cycloaliphatic ring. In some embodiments, a formed ring is selected from the Markush group consisting of optionally substituted cycloaliphatic rings consisting of a 3-8-membered cycloaliphatic ring, a 5-8-membered cycloaliphatic ring., a 5-membered cycloaliphatic ring, a 6-membered cycloaliphatic ring, and a 7-membered cycloaliphatic ring. In some embodiments, a formed ring is substituted. In some embodiments, a formed ring is not substituted. In some embodiments, a formed ring has no additional unsaturation in addition to the double bond in C(=CHRRG2) or C(=CRRG1RRG2).

[00313] In some embodiments, −C(=CHRRG2)−CHRRG4 or −C(=CRRG1RRG2)−CRRG3RRG4 is optionally substituted or is . In some embodiments, −[C(=CHRRG2)−CHRRG4]−LRG3− or −[C(=CRRG1RRG2)−CRRG3RRG4]−LRG3− is optionally substituted or is . In some embodiments, −LRG1−[C(=CHRRG2)−CHRRG4]−LRG3− or −LRG1−[C(=CRRG1RRG2)−CRRG3RRG4]−LRG3− is optionally substituted . In some embodiments, −LRG1−[C(=CHRRG2)−CHRRG4]−LRG3− or −LRG1−[C(=CRRG1RRG2)−CRRG3RRG4]−LRG3− is optionally substituted.

[00314] In some embodiments, the reactive group has a structure disclosed in TABLE 3 of the published International patent application WO 2024 / 228935 (Biohaven Therapeutics Ltd.).

[00315] In some embodiments, −LLG4−LRG2− is −O−C(O)− or −S−C(O)−. In some embodiments, −LLG4−LRG1−LRG2− is −S−C(O)−.

[00316] In some embodiments, −LLG4−LRG2− is −N(−)−C(O)−, wherein N is a ring atom of an optionally substituted heteroaryl ring. In some embodiments, −LLG4−LRG2− is −N(−)−C(O)−, wherein N is a ring atom of LLG4 which is or comprises an optionally substituted heteroaryl ring. In some embodiments, −LLG4−LRG2− is −N(−)−C(O)−O−, wherein N is a ring atom of LLG4 which is or comprises an optionally substituted heteroaryl ring.

[00317] In some embodiments, LRG2 is optionally substituted −CH2−C(O)−, wherein −CH2− is bonded to an electron-withdrawing group comprising or connected to an antibody-binding moiety. In some embodiments, LRG2 is optionally substituted −CH2− bonded to an electron-withdrawing group comprising or connected to an antibody-binding moiety. In some embodiments, LRG1 is an electron-withdrawing group. In some embodiments, LRG1 is selected 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)−.

[00318] In some embodiments, LRG2 is optionally substituted −CH2−C(O)−, wherein −CH2− is bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, LRG2 is optionally substituted −CH2− bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, LRG1 is selected from the Markush group consisting of −O−C(O)−, −OS(O)2−, −OP(O(OR)−, −OP(O(SR)−, and −OP(O(N(R)2)−.

[00319] In some embodiments, a reactive group reacts with an amino group of a target agent. In some embodiments, an amino group is −NH2 of the side chain of a lysine residue.

[00320] In some embodiments, a target agent is a protein agent. In some embodiments, a target agent is an antibody agent. In some embodiments, a reactive group reacts with an amino acid residue of this protein or antibody agent. In some embodiments, an amino acid residue is a lysine residue. In some embodiments, a reactive group reacts with −NH2 of the side chain of a lysine residue. In some embodiments, a reactive group is or comprises −C(O)−O−, it reacts with −NH2, e.g., of the side chain of a lysine residue), and forms an amide group −C(O)−O− with the −NH2.

[00321] In some embodiments, reactive groups, e.g., a first reactive group, a second reactive group, etc., are at terminal locations. In some embodiments, agents such as first agents comprise first reactive groups linked to target-binding moieties optionally through linker moieties, and do not have antibody-binding moieties.

[00322] In some embodiments, the invention provides methods for preparing a composition comprising a plurality of agents, wherein each agent independently comprises:an antibody moiety,a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody moiety and a cellular receptor-binding moiety;which method comprise:contacting a plurality of agents each of which independently comprises a reactive group with a plurality of antibody agents.

[00323] In some embodiments, an agent comprising a reactive group comprises an antibody-binding moiety, a cellular receptor-binding moiety and optionally a linker. In some embodiments, agents comprising a reactive group share the same cellular receptor-binding moiety. In some embodiments, agents comprising a reactive group share the same structure. In some embodiments, antibody molecules are of such structures, properties or activities to provide antibody moieties in agents described in this specification. In some embodiments, a plurality of antibody molecules comprises two or more IgG subclasses. In some embodiments, a plurality of antibody molecules comprises IgG1. In some embodiments, a plurality of antibody molecules comprises IgG2. In some embodiments, a plurality of antibody molecules comprises IgG4. In some embodiments, a plurality of antibody molecules comprises IgG1 and IgG2. In some embodiments, a plurality of antibody molecules comprises IgG1, IgG2 and IgG4. In some embodiments, a plurality of antibody molecules comprises IgG1, IgG2, IgG3 and IgG4. In some embodiments, a plurality of antibody molecules is IVIG antibody molecules.

[00324] In some embodiments, provided agents comprise a reactive group, e.g., . In some embodiments, −C(O)− is connected to a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, optionally through a linker and the other end is connected to an antibody-binding moiety. In some embodiments, reacts with an amino group of another moiety, e.g., an antibody moiety, forming an amide group with the moiety and releasing a moiety which is or comprises antibody-binding moiety. In some embodiments, an amino group is −NH2 of a lysine side chain. In some embodiments, −C(O)− is connected to a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, optionally through a linker and the other end is connected to R’ or an optional substituent. In some embodiments, provided agents comprise optionally substituted . Such reactive groups may be useful for conjugation with detection, diagnosis, or therapeutic agents. Persons having ordinary skill in the biomedical art know that many agents, and many technologies, e.g., click chemistry, reactions based on functional groups such as amino groups, e.g., amide formation), hydroxyl groups, carboxyl groups, etc. can be used for conjugation under this specification.

[00325] In some embodiments, antibody-binding moieties bind to Fc regions of antibodies. In some embodiments, reactions occur at residues at Fc regions. In some embodiments, target-binding moieties are conjugated to residues of Fc regions, optionally through linker moieties. In some embodiments, a residue is a Lys residue. In some embodiments, an antibody is or comprises IgG1. In some embodiments, an antibody is or comprises IgG2. In some embodiments, an antibody is or comprises IgG4. In some embodiments, an antibody composition used in a method comprises IgG1 and IgG2. In some embodiments, an antibody composition used in a method comprises IgG1, IgG2 and IgG4. In some embodiments, an antibody composition used in a method comprises IgG1, IgG2, IgG3 and IgG4.

[00326] In some embodiments, a product is or comprises IgG1. In some embodiments, a product is or comprises IgG2. In some embodiments, a product is or comprises IgG4. In some embodiments, a product composition comprises IgG1 and IgG2. In some embodiments, a product composition comprises IgG1, IgG2 and IgG4. In some embodiments, a product composition comprises IgG1, IgG2, IgG3 and IgG4.

[00327] In some embodiments, provided agents comprising antibody moieties provide one or more or substantially all antibody immune activities, e.g. for recruiting one or more types of immune cells or provide short-term and long-term immune activities. In some embodiments, provided agents comprising antibody moieties do not significantly reduce one or more or substantially all relevant antibody immune activities. In some embodiments, provided agents comprising antibody moieties improve one or more or substantially all relevant antibody immune activities, e.g., compared to antibody moieties by themselves. In some embodiments, provided agents provides comparable or better stability compared to antibody moieties by themselves, e.g., home time in blood. In some embodiments, antibody moieties in provided agents can bind to FcRy of immune cells, e.g., several FcRy of immune effector cells for desired immune activities, typically, at comparable or better levels. In some embodiments, antibody moieties in provided agents have comparable Fab / antigen-binding capabilities. In some embodiments, antibody moieties in provided agents have comparable Fab / antigen-binding capabilities. In some embodiments, antibody moieties in provided agents provide FcRn-binding. In some embodiments, antibody moieties in provided agents provide FcRn-binding, e.g., for antibody recycle or prolonged half-life. In some embodiments, provided technologies are useful for changing blood-derived IgG products as provided technologies are suitable for and can use all IgG subclasses.

[00328] In some embodiments, a provided method comprises one step described below. In some embodiments, reacts with an amino group of a lysine side chain to form an amide bond with an antibody molecule, and releases or a salt form thereof. Pharmaceutically acceptable excipients.

[00329] Formulations suitable for parenteral administration, such as by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can have antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[00330] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include these components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the change in tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Methods of assessing the chemical structure and function of the agent.

[00331] HDX-MS measures the exposure of hydrogen molecules on the surface of a protein or protein complex. The first step causes exposed hydrogen molecules to be readily exchanged with deuterium. Hydrogen molecules buried within the structure, for example at the binding site of an antibody-antigen complex, are not exchanged as readily.

[00332] Comparing the deuterium uptake between the free and the complex states of proteins via mass spectrometry provides valuable information about the binding regions.

[00333] Protein sequencing. In one method of protein sequencing, eight digestions are prepared using five enzymes (Pepsin, Lys C, Trypsin, Chymotrypsin, Asp N). The digestions for the sample are processed with disulfide reduction, cysteine blocking, and then enzyme digestion. Digestions were analyzed by LC-MS / MS using a Thermo-Fisher Orbitrap fusion™ mass spectrometer. Peptides are characterized from LC -MS / MS data using de nova peptide sequencing and then assembled into antibody sequences.

[00334] Competition ELISA assay protocol.

[00335] (1) Dilute patient plasma samples at 1:100 in the provided ELISA sample buffer.

[00336] (2) Make the working stock solution for the degraders for a final concentration of 400 nM in 115 µl total reaction volume per well.

[00337] (3) Incubate 100 ul patient plasma sample with 15 ul of degrader for 2 hours at room temperature.

[00338] (4) Transfer 100 ul from this reaction to the EuroImmun anti-PLA2R ELISA plate.

[00339] (5) Also transfer the calibrators, and positive and negative controls to the plate.

[00340] (6) Incubate for 30 minutes at room temperature.

[00341] (7) Empty the wells and wash thrice with 300 ul / well of wash buffer.

[00342] (8) Pipette 100 ul of enzyme conjugate into each well of the ELISA plate.

[00343] (9) Incubate for 30 minutes at room temperature.

[00344] (10) Empty the wells and wash thrice with 300 ul / well of wash buffer.

[00345] (11) Pipette 100 ul of substrate solution into each well of the ELISA plate.

[00346] (12) Incubate for 15 minutes at room temperature.

[00347] (13) Pipette 100 ul of stop solution into each well of the ELISA plate.

[00348] (14) Measure absorbance at 450 nm within 30 minutes of adding the stop solution.

[00349] ASGPR-mediated endocytosis of anti-PLA2R antibody.

[00350] For routine culture of HEK-ASGPR cells, use DMEM high glucose with phenol + 10% FBS + 2 mM glutamine + 1% Pen Strep + 200 ug / ml geneticin.

[00351] For seeding of HEK-ASGPR cells in step 2, use DMEM high glucose without phenol + 10% FBS + 2 mM glutamine + 1% Pen Strep + 200 ug / ml geneticin.

[00352] (1) Harvest HEK-ASGPR1 cells using Accutase

[00353] (2) Seed 30,000 cells / well in 100 ul media / well in poly-D-lysine-coated black TC-treated 96-well plates

[00354] (3) The next day, label rabbit anti-PLA2R with anti-rabbit-F(ab)2-AF488 as follows (amounts for 4:1 molar ratio of anti-PLA2R:anti-rabbit-F(ab)2-AF488, with anti-PLA2R at 12.5 nM, for one 96-well plate):

[00355] (a) Add 11.6 ul 6B3-1 anti-PLA2R (@1.63 ug / ul) and 2.3 ul anti-rabbit-F(ab)2-Alexa Fluor488 (@ 1.5 ug / ul) to 10.4 ml Opti-MEM.

[00356] (b) Invert a couple of times to mix.

[00357] (c) Incubate for 30 min at 37 C in the dark, inverting to mix every 10-15 min.

[00358] (4) Prepare a 11-point titration series for the degraders using PBS, starting at 20 uM top concentration with three-fold serial dilutions

[00359] (5) Remove media from the plate by flicking and dispense 90 ul / well of the anti-PLA2R + anti-rabbit-F(ab)2-AlexaFluor488 mix at the slowest dispense speed

[00360] (6) Add 10 ul / well of the degrader titration series to achieve a final top concentration of 2 uM

[00361] (7) Place plates in the IncuCyte and scan at 0, 3, 6, 9, 12, 15, 18, 21 and 24 hours

[00362] (8) Analyze the images to obtain green fluorescent area per image using the following algorithm parameters: Surface fit segmentation, 0.75 threshold, edge split off, 0.5 µm2 hole fill.TABLE 7ReagentsVendorCat # DMEM high glucose with phenolGibco11960044DMEM high glucose without phenolGibco31053028Opti-MEM Reduced serum mediumThermo Fisher (Gibco)31985062Geneticin 50mg / mlGibco10131-035 FBS heat inactivatedR&D systems (Optima)S12450HGlutamineCorning25-005-CI Penicillin Streptomycin Corning30-002-CI AccutaseStemcell technologies07920 96-well black, flat clear bottom, poly-D lysine coatedCorning3842Anti-PLA2R antibodyGenScript  Alexa Fluor® 488 AffiniPure™ Goat Anti-Rabbit IgG, F(ab')₂ fragment specificJackson ImmunoResearch111-545-006 Immunoglobulin Sequence Analysis

[00363] Sequence Analysis Tools. The following free online tools for sequence analysis of immunoglobulin variable regions: (1) NCBI Nucleotide BLAST, (2) IMGT / V Quest program, and (3) NCBI IgBLAST.

[00364] Sequence Analysis Materials. B cells prepared by GenScript; RNA-easy Isolation Reagent (Vazyme, Cat. No. : R701-01-AA); PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Cat. No.: 6110A).

[00365] Sequence Analysis Methods. Total RNA was isolated from the B cells following the technical manual of RNA-easy Isolation Reagent. Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti-sense primers or universal primers following the technical manual of PrimeScript™ 1st Strand cDNA Synthesis Kit. Antibody fragments of heavy chain and light chain were amplified using specific primers according to the standard operating procedure (SOP) of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. The consensus sequence was provided.

[00366] Phospholipase A2 receptor (PLA2R) antibody, IgG with reflex to titer. This commercially-available assay aids in the differential diagnosis of membranous glomerulonephritis (MGN) or nephrotic syndrome of unknown etiology. This assay is commercially-available from ARUP Laboratories. Component Test Code*2011829. Component Chart Name, Phospholipase A2 Receptor, IgG. LOINC. 82991-1. This assay is New York State approved.

[00367] Patient Preparation. Instructions patient must follow before / during specimen collection.

[00368] Collect. Specimen type to collect. May include collection media, tubes, kits, etc.

[00369] Serum Separator Tube. Specimen preparation, instructions for specimen prep before / after collection and prior to transport. Separate serum from cells ASAP or within two hours of collection. Transfer 1 mL serum to an ARUP Standard Transport Tube. (minimum 0.2 mL).

[00370] Processes used to perform the test. Semi-Quantitative Cell-Based Indirect Fluorescent Antibody.

[00371] Additional information related to the test. If phospholipase A2 receptor antibody, IgG is positive, then a phospholipase receptor A2 antibody, IgG titer can be added.  Statistical analyses.

[00372] Normal distribution quantitative variables can be expressed as means and standard deviations and compared by an independent-samples t test. For non-normally distributed variables, we used median and interquartile range and analyzed with the Mann-Whitney U test. Categorical data is summarized by percentages. A two-sided p value <0.05 is statistically significant. All statistical tests are performed using SPSS version 16.0. EXAMPLES

[00373] The invention is further illustrated by non-limiting EXAMPLES. EXAMPLE 1Bifunctional degrader directed to anti-PLA2R Abs in idiopathic membranous nephropathy.

[00374] Anti-PLA2R degraders were constructed using the reported 31-mer peptide ligand as the antibody-binding moiety. Depleting anti-PLA2R antibodies with a degrader should result in remission of idiopathic membranous nephropathy faster and safer than immunosuppressive therapy. Depleting anti-PLA2R antibodies could treat patients unresponsive to immunosuppressants.Material generation.

[00375] The inventors constructed PLA2R bifunctional degraders. TABLE 8MoietyaPLA2R-binding moietyASGPR-binding moietyKD (nM) as assayed by surface plasmon resonance ABT30531-merN / A7AGN30631-merβGN323 ± 1ABT30131-mer (bicyclic)N / A0.3 ± 2AGN30231-mer (bicyclic)βGN310 ± 3 AGN30431-mer (bicyclic)CF3 pyrazine3 ± 0.3 AGN30331-mer (bicyclic)⍺GN37 ± 1AGN30831-merCF3 pyrazine9 ± 2Measure uptake in vitro.

[00376] The inventors have successfully measured the uptake of anti-PLA2R antibodies and biodegraders in HepG2 assays. The inventors now have an assay to measure antibody degradation in vitro. The inventors assaying and analyzing patient plasma in this assay.

[00377] The inventors are demonstrating the biophysical characterization of peptide alone and degrader to monoclonal anti-PLA2R antibody and patient anti-PLA2R antibody. The inventors are proceeding to measure competition of idiopathic membranous nephropathy anti-PLA2R to PLA2R by Meso Scale Discovery. In vitro toxicity assays.

[00378] The inventors successfully completed a hemagglutination assay using bifunctional degraders. TABLE 9PLA2R degrader hemagglutinin activity assayAgentPLA2R-binding moietyASGPR ligand50 µM500 µMAGN306WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K (31-mer)βGN+-AGN307WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K (31-mer)⍺GN3+-AGN308WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K (31-mer)CF3 pyrazine-+ABT305WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K (31-mer)None-+AGN302[WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K](bic 31-mer)βGN--AGN303[WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K](bic 31-mer)⍺GN3--AGN304[WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K](bic 31-mer)CF3 pyrazine-+ABT301[WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K](bic 31-mer)None--ABT309VIQSESLKK(CIQAGKSVLTLENKC)KNone-- 

[00379] Both N-C cyclization and shortened peptide (ABT309) show no hemagglutinin activity at 50 / 500 µM.

[00380] AGN306 (βGN3) shows hemagglutinin activity in mice, rats, and humans. AGN302 (βGN3) only shows moderate hemagglutinin activity in rats.TABLE 10AGN303 and peptide alone show HA activity at 5 mMCompoundPLA2R ligandASGPR ligand5 mM500 µMAGN303[WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K](bic 31-mer)⍺GN3+-ABT301[WQDKGIFVIQSESLKK(CIQAGKSVLTLENKC)K](bic 31-mer)None+-ABT309**VIQSESLKK(CIQAGKSVLTLENKC)KNone+-ABT310**VIQSES [SEQ ID NO: 10] None+-ABT311**SVLTENCK [SEQ ID NO: 11]None+-** Insoluble at 5 mM 

[00381] The inventors successfully completed a proliferation and cytotoxicity assay using bifunctional degraders.

[00382] The inventors will be performing assays of PBMC cytokine release using standard assays known to persons having ordinary skill in the art.  EXAMPLE 2Depletion of anti-PLA2R antibody in vivo.

[00383] The inventors are demonstrating the ability of the degraders to deplete anti-PLA2R antibody in vivo using the in vitro-validated anti-PLA2R degraders.

[00384] The inventors measured depletion of anti-PLA2R antibody in mice using in vitro-validated anti-PLA2R bifunctional degraders. The inventors have successfully completed assays of murine tolerability of the degraders.

[00385] The inventors are proceeding with assays of mouse pharmacokinetics / pharmacodynamics. Mouse pharmacokinetics / pharmacodynamics studies were run using AGN303 (⍺GN3) and AGN302 (βGN3). Mice were dosed at 0.1 mg / kg rabbit anti-PLA2R antibody (commercially available antibody that binds to the 31-mer). Degraders were dosed at 0.3 or 1 mg / kg, intravenous or subcutaneous. Degraders rapidly depleted anti-PLA2R from plasma for all doses tested. An antibody rebound effect was observed at four-eight hours. EXAMPLE 3Mouse maximum tolerated dose (MTD).

[00386] AGN303 (⍺GN3) was better tolerated intravenously than AGN302 (βGN3). Clinical observations at 25 and 50 milligram / kilogram were mild and transient. Mice were fully recovered by end of the assay.

[00387] AGN303 (⍺GN3)-treated mice showed severe toxic clinical signs at 100 milligram / kilogram (+++) when dosed intravenously: low motility, hunched posture, cyanosis, and coldness.

[00388] AGN303 (⍺GN3)-treated mice showed no clinical signs of toxicity up to 100 milligram / kilogram when dosed subcutaneously. EXAMPLE 4Assays to characterize activities of anti-PLA2R bifunctional degraders.

[00377] GN3 sortase reagent. A basic protocol for sortase conjugation including the ASPGR binder for the sortase. C-terminal sortase tag (LPETGG) for conjugation (GN3 / linker).

[00378] Surface plasmon resonance. Direct binding of anti-PLA2R antibody binding moiety to anti-PLA2R antibody was determined using Biacore™ instruments and IBIS-MX96 systems. Anti-PLA2R antibody was immobilized by amine coupling to the surface of a sensor chip as the ligand. Anti-PLA2R antibody binding moiety is flowed over in solution as analyte. Binding affinity was determined using single-cycle kinetics, e.g., by bio-layer interferometry (ForteBio™oCtet™ systems).TABLE 11Compound aPLA2R SPR SCK KD (nM)Theoretical binding ratioImmobili-zation (RU)Experimental Rmax (RU)Theoretical Rmax (RU)Active SurfacePeptidesABT3056.90.96ABT3010.260.92ABT309No bindingDegradersAGN30622.70.80AGN30632.70.40AGN30623.50.75AGN30851.60.27AGN3086.720.81AGN3089.450.86AGN30815.70.26AGN3089.640.41AGN30814.40.28AGN30727.80.37AGN30741.80.27AGN30917.80.26AGN31045.50.27AGN30211.90.81AGN3027.860.43AGN3042.75AGN3043.17AGN3037.69AGN3036.34AGN364*1362:1416.838.5124.70.31AGN364*2192:145437.2135.80.27AGN365*9592:1416.839.6249.60.16AGN365*6612:145449271.90.18AGN579*4511:138255.53580.16AGN579*12401:142936.84020.09AGN579*11501:141841.13920.10*Ligand: Boster Bio pAb 

[00379] GN3-maleimide reagent (maleimide-GN3 / linker), with a C-terminal Cys for conjugation.

[00380] Biophysical / biochemical potency assay to measure target-engagement. A biomolecule coupled to surface of sensor chip as ligand. As analyte is flowed in solution over immobilized ligand, binding to the sensor chip surface induces a change in refractive index proportional to bound mass.

[00381] ASGPR-dependent uptake assay. An HEK293 fluorescent cell-based assay was used to assess antibody uptake by cells in vitro. This on-mechanism endocytosis assay measures the anti-PLA2R antibody accumulation in HEK293 cells.

[00382] Other off-target assays include measurement of cytotoxicity through In vitro toxicity with HEPG2 cells, e.g., in a CellTiter-Glo assay, measurement of hemagglutination by red blood cell interactions, measurement of PMBC, and measurement of off-target bindings in a house C-type lectin panel. Signal is boosted in the HEK293 versus HepG2 cells when measuring signal accumulation not degradation. Western assays can measure the degradation in HEK cells and other cell lines.

[00383] Ternary complex formation. Formation of a ternary complex of anti-PLA2R antibody and ASGPR mediated by anti-PLA2R bifunctional molecules was determined.TABLE 12DegraderTernary Complex HTRF (max signal:noise)Ternary Complex [degrader] at max signal (nM)Ternary Complex EC50 (nM)AGN3069.2785.6AGN3066.9392.6AGN3065782.3AGN30833.4192.8AGN30827.6192.3AGN30713.5191.3AGN30712390.9AGN30917.139 AGN30915.1781.3AGN3103.7156 AGN3103.51565.2AGN3027.52.5 AGN3026.192AGN30435.350.9AGN3043091AGN30316.950.7AGN30314.3191.3

[00384] Animal models. Testing for antidrug antibodies (ADA) can be done in mouse models. See Meyer-Schwesinger et al., Kidney International, 97(5), 913-919 (2020). Many of the ADAs are IgG4 , such as those in Factor 8 hemophilia.  EXAMPLE 5Preparation of TBT307 - APreparation of Building block #1:Preparation of Intermediate-26:

[00385] A mixture of Intermediate-25 (75.0 g, 64.4 mmol) in HBr / water (40% HBr, 1000 mL in total) was stirred at 140°C for sixteen hours. The solvent was removed at 70°C under reduced pressure. The residue was triturated in MeCN (50 mL) for ten minutes. After filtration, the solid was dried under lyophilization to afford Intermediate-26 (100.0 g, 427.9 mmol, 88.4% yield, HBr salt) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.03 (s, 1 H) 8.20 (s, 3 H) 7.32 (dd, J = 12.17, 1.88 Hz, 1 H) 7.11 (dd, J = 8.28, 1.51 Hz, 1 H) 6.95-7.03 (m, 1 H) 3.93 (q, J = 5.27 Hz, 2 H).Preparation of Intermediate-27:

[00386] To a mixture of Intermediate-26 (60.0 g, 270.2 mmol, 1.00 equivalent, HBr salt), Reactant26a (111.1 g, 270.2 mmol, 1.00 equivalent), DIEA (34.9 g, 270.2 mmol, 47.0 mL, 1.00 equivalent) and HOBt (54.7 g, 405.3 mmol, 1.50 equivalents) in DMF (1 L) was added EDCI (56.7 g, 297.2 mmol, 1.10 equivalents) at 20oC. The mixture was stirred at 20°C for three hours. The mixture was precipitated with 0.5 M HCl (cold, 10 L). After filtration, the solid was dissolved in DCM (2 L), washed with 0.5 M HCl (800 mL), water (800 mL), brine (800 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column (DCM / methanol = from 1 / 0 to 20 / 1) to afford Intermediate-27 (120.0 g, 90% purity, containing a little DMF, 83.3% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.70 (s, 1 H) 8.34 (t, J = 5.77 Hz, 1 H) 7.90 (d, J = 7.53 Hz, 2 H) 7.71 (d, J = 7.53 Hz, 2 H) 7.61 (d, J = 8.28 Hz, 1 H) 7.39-7.47 (m, 2 H) 7.29-7.36 (m, 2 H) 7.02 (d, J = 12.30 Hz, 1 H) 6.85-6.92 (m, 2 H) 4.20-4.39 (m, 4 H) 4.11-4.19 (m, 2 H) 1.36 (s, 9 H).Preparation of Building block #1:

[00387] A mixture of Intermediate-27 (120.0 g, 224.4 mmol, 1.00 equivalent) in trifluoroacetic acid (600 mL) and DCM (600 mL) was stirred at 20°C for one half hour. The solvent was removed under reduced pressure. The residue was purified by silica gel column (DCM / methanol = from 1 / 0 to 10 / 1) to afford Building block #1 (100.0 g, 93.5% purity, 93.1% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.69 (s, 1 H) 8.34 (t, J = 5.90 Hz, 1 H) 7.90 (d, J = 7.28 Hz, 2 H) 7.71 (d, J = 7.53 Hz, 2 H) 7.54 (d, J = 6.53 Hz, 1 H) 7.42 (t, J = 7.40 Hz, 2 H) 7.27-7.37 (m, 1 H) 7.27-7.37 (m, 1 H) 7.02 (d, J = 12.05 Hz, 1 H) 6.82-6.93 (m, 2 H) 4.35-4.43 (m, 1 H) 4.20-4.31 (m, 3 H) 4.13-4.19 (m, 2 H). EXAMPLE 6Preparation of TBT307 – BPreparation of Intermediate-30:

[00388] The peptide was synthesized using standard Fmoc chemistry (CTC resin).

[00389] (1) Resin preparation: To the vessel containing CTC resin (2.0 g, 2.0 mmol, 1.00 mmol / g) and Fmoc-Thr(tBu)-OH (795 mg, 2.0 mmol, 1.00 equivalent) in DCM (50 mL) was added DIEA (4.00 equivalents) dropwise and mixed for two hours with nitrogen gas bubbling at 25°C. Then methanol (2 mL) was added and bubbled with nitrogen gas for another thirty minutes. The resin was washed with DMF (100 mL) * 5, followed by the addition of 20% piperidine in DMF (100 mL) and bubbled with nitrogen gas for thirty minutes at 25°C for Fmoc deprotection. The mixture was filtered. The resin was washed with DMF (100 mL) * 5 before proceeding to next step.

[00390] (2) Coupling: A solution of Fmoc-Cys(Trt)-OH (3.51 g, 6.0 mmol, 3.00 equivalents), HBTU (2.16 g, 5.7 mmol, 2.85 equivalents) in DMF (50 mL) was added to the resin with nitrogen gas bubbling. Then DIEA (6.00 equivalents) was added to the mixture dropwise and bubbled with nitrogen gas for thirty minutes at 25°C. The coupling reaction was monitored by ninhydrin test, if it showed colorless, the coupling was completed. The resin was then washed with DMF (100 mL) * 5.

[00391] (3) Deprotection: 20% piperidine in DMF (100 mL) was added to the resin and the mixture was bubbled with nitrogen gas for thirty minutes at 25°C. The resin was then washed with DMF (100 mL) * 5.

[00392] (4) Steps 2 and 3 were repeated for the following amino acids elongation: Number # 3-15, TABLE 13.

[00393] (5) After all the steps were completed, the resin was washed with DMF (100 mL) * 5, methanol (100 mL) * 5, then dried under reduced pressure to afford resin-bound peptide Intermediate-28 (CTC resin, 2.0 mmol).TABLE 13List of amino acids and the corresponding reagents used on solid-phase peptide synthesis#MaterialsCoupling reagents1Fmoc-Thr(tBu)-OH (3.00 equivalents)DIEA (4.00 equivalents)2Fmoc-Cys(Trt)-OH (3.00 equivalents)HBTU (2.85 equiv.) and DIEA (6.00 equiv.)3Fmoc-Trp-OH (3.00 equivalents)HBTU (2.85 equiv.) and DIEA (6.00 equiv.)4Fmoc-Val-OH (3.00 equivalents)HBTU (2.85 equiv.) and DIEA (6.00 equiv.)5Fmoc-Leu-OH (3.00 equivalents)HBTU (2.85 equiv.) and DIEA (6.00 equiv.)6Fmoc-Glu(OtBu)-OH (3.00 equivalents)HBTU (2.85 equiv.) and DIEA (6.00 equiv.)7Fmoc-Gly-OH (3.00 equivalents)HBTU (2.85 equiv.) and DIEA (6.00 equiv.)8Fmoc-Leu-OH (3.00 equivalents)HBTU (2.85 equiv.) and DIEA (6.00 equiv.)9Building block #1 (2.00 equivalents)DIC (2.00 equiv.) and HOBt (2.00 equiv.)10Fmoc-Trp-OH (3.00 equivalents)DIC (3.00 equiv.) and HOBt (3.00 equiv.)11Fmoc-Ala-OH (3.00 equivalents)DIC (3.00 equiv.) and HOBt (3.00 equiv.)12Fmoc-Cys(Trt)-OH (3.00 equivalents)DIC (3.00 equiv.) and HOBt (3.00 equiv.)13Fmoc-Asp(OtBu)-OH (3.00 equivalents)DIC (3.00 equiv.) and HOBt (3.00 equiv.)14ACAc2O:NMM:DMF=10:5:8515Boc-PEG2-OH (3.00 equivalents)DIC:HOBt:DMAP=3:3:3Peptide cleavage and cyclization:

[00394] (1) Cleavage solution (trifluoroacetic acid / TIS / water, 95 / 2.5 / 2.5, v / v / v, 500 mL) was added to the flask containing the side-chain protected resin-bound peptide (CTC resin, 5 g, 2.0 mmol) at 25oC and stirred for two hours.

[00395] (2) After filtration, the filtrate was collected.

[00396] (3) The filtrate was precipitated with cold isopropyl ether (2.5 L). After filtration, the solid was washed with isopropyl ether (2.5 L) twice, and the crude peptide was dried under reduced pressure for two hours to afford Intermediate-29 (3.0 g, crude) as a white solid.

[00397] To the mixture of Intermediate-29 (3.0 g, crude) in HOAc / MeCN / water (4 / 3 / 3, v / v / v, 2 L) was added 0.1 M I2 / AcOH dropwise until a yellow color persisted, then the mixture was stirred at 25oC for five minutes. The mixture was quenched by addition of 0.1 M aq. Na2S2O3 dropwise until the yellow color disappeared. After filtration, the filtrate was purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: MeCN), followed by lyophilization to afford Intermediate-30 (360 mg, 90.0% purity, 50.8% yield) as a white solid. LCMS: retention time = 0.907=0.960 minutes, MS calculated: Mav = 1833.02, mass observed: [M + 2H]2+ =917.00, [M + H]+ =1832.91.Preparation of Intermediate-31:

[00398] To a solution of Bis-PEG4-TFP (2.53 g, 5.00 equivalents) in DMF (25 mL) was added a mixture of Target A001 (1.2 g, 1.00 equivalent) and DIEA (332.9 mg, 449.9 μL, 3 equivalents) in 10 mL DMF at 0°C. The resulting reaction was stirred for five minutes at 0°C. After completion monitored by LC-MS, the mixture was directly injected into the reverse column, purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: MeCN), followed by lyophilization to afford Intermediate-31 (700 mg, 95.0% purity, 57.5% yield) as colorless oil. LCMS: retention time = 0.709 minutes, MS calculated: Mav = 1821.81, mass observed: [M+16 + 3H]3+ =613.10.Preparation of TBT307:

[00399] To a solution of Intermediate-30 (140.86 mg, 76.85 μmol, 1.00 equivalent) and Intermediate-31 (140 mg, 76.85 μmol, 1.00 equivalent) in DMF (2 mL) was added DIEA (29.80 mg, 230.54 μmol, 40.16 μL, 3.00 equivalents). The mixture was stirred at 20°C for one hour.After completion monitored by LC-MS, the mixture was acidified by 1 M HCl to pH = 5. The mixture was purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: MeCN) directly, followed by lyophilization to afford TBT307(103 mg, 92.8% purity, 35.6% yield.) as a white solid. LCMS: retention time = 1.391 minutes, MS calculated: Mav = 3488.76, mass observed: [M + 3H]3+ =1163.90. EXAMPLE 7Procedure for Preparation of Intermediate-27:Preparation of Intermediate-2:

[00400] Pd / C (4.00 g, 10% purity) in reaction bottle (purged with argon gas for three times) was added THF (40 mL) slowly, then a solution of trifluoroacetic acid (7.44g, 65.2 mmol, 4.86 mL, one equivalent) and Intermediate-1 (40.0 g, 65.2 mmol, 1.00 equivalent) in THF (360 mL) was added to the reaction slowly under nitrogen gas. The reaction was degassed and purged with argon gas and hydrogen gas for three times, then stirred at 25°C for three hours under hydrogen gas atmosphere (40 psi). TLC (DCM: methanol = 10: 1, Rf = 0.20) indicated Intermediate-1 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was dissolved in THF (100 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (100 mL * 2), and the filtrate was concentrated under reduced pressure to get Intermediate-2 (38.7 g, 38.5 mmol, 59.0% yield, 59.0% purity, trifluoroacetic acid salt) as a white solid. LCMS: retention time = 0.428 minutes, MS calculated: 478.22, mass observed: [M + Na]+ = 501.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.79-7.91 (m, 3 H), 5.16-5.28 (m, 1 H), 4.97 (br dd, J = 11.07, 2.81 Hz, 1 H), 4.54 (br d, J = 8.50 Hz, 1 H), 4.03 (s, 2 H), 3.77-3.93 (m, 2 H), 3.53-3.62 (m, 8 H), 2.98 (br d, J = 5.00 Hz, 2 H), 2.10 (s, 3 H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.78 (s, 3 H).Preparation of Intermediate-4:

[00401] To a stirring solution of Intermediate-3 (8.50 g, 16.0 mmol, 1.00 equivalent) and Intermediate-2 (38.1 g, 64.3 mmol, 4.00 equivalents, trifluoroacetic acid salt) in DMF (17 mL) and DCM (340 mL) was added HOBT (8.69 g, 64.3 mmol, 4.00 equivalents), EDCI (12.3 g, 64.3 mmol, 4.00 equivalents) and DIEA (9.35 g, 72.3 mmol, 4.50 equivalents) successively. The reaction was stirred at 25°C for two hours. TLC (DCM: methanol = 10: 1, Rf = 0.5) indicated Intermediate-3 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was slowly poured into a stirring cold 1.0 mol / L HCl solution (350 mL) and stirred for ten minutes. A white precipitate formed and was filtered. The aqueous phase was extracted with DCM (350 mL* 2) twice. The combined organic layers were washed with sat. NaHCO3 (350 mL), dried over Na2SO4, and concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiO2, DCM:methanol = 100: 1 to 15: 1) to afford Intermediate-4 (22.0 g, 10.6 mmol, 66.0% yield, 92.2% purity) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.91 (br t, J = 5.32 Hz, 3 H), 7.81 (d, J = 9.26 Hz, 3 H), 7.26-7.49 (m, 6 H), 7.12 (s, 1 H), 5.21 (d, J = 3.25 Hz, 3 H), 5.02 (s, 2 H), 4.97 (dd, J = 11.13, 3.25 Hz, 3 H), 4.55 (d, J = 8.38 Hz, 3 H), 4.03 (s, 9 H), 3.84-3.92 (m, 3 H), 3.74-3.82 (m, 3 H), 3.47-3.65 (m, 38 H), 3.39 (br s, 3 H), 3.20 (q, J = 5.42 Hz, 6 H), 2.30 (br t, J = 6.13 Hz, 6 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H). LCMS: retention time = 0.403 minutes, MS calculated: 1908.81, mass observed: [M + 2H]2+ = 1910.2.Preparation of Intermediate-5:

[00402] The 500 mL round-bottom flask was purged with argon gas for three times and added dry Pd / C (1.60 g, 1.50 mmol, 10% purity, 0.17 equivalents) carefully. Then THF (60.0 mL) was added to infiltrate the Pd / C completely, followed by the solution of Intermediate-4 (16.4 g, 8.59 mmol, 1.00 equivalent) and trifluoroacetic acid (979 mg, 8.59 mmol, 637.8 μL, 1.00 equivalent) in THF (100 mL) slowly under argon gas atmosphere. The resulting mixture was degassed and purged with hydrogen gas for three times, and then the mixture was stirred at 25°C for three hours under hydrogen gas atmosphere (15 psi). TLC (DCM:methanol = 10: 1, Rf = 0.6) indicated Intermediate-9 was consumed completely, and one major new spot was detected. The reaction mixture was filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (50 mL*2). The filter cake was added water immediately. The organic layer concentrated under reduced pressure to afford Intermediate-5 (15.7 g, 6.78 mmol, 78.9% yield, 81.6% purity, trifluoroacetic acid salt) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.91-7.98 (m, 5 H), 7.82 (br d, J = 9.13 Hz, 3 H), 7.74 (s, 1 H), 7.12-7.28 (m, 1 H), 5.22 (br d, J = 2.63 Hz, 3 H), 4.97 (br dd, J = 11.13, 2.75 Hz, 3 H), 4.54 (d, J = 8.51 Hz, 3 H), 3.84-3.93 (m, 3 H), 3.78 (br dd, J = 10.13, 4.88 Hz, 3 H), 3.53-3.62 (m, 22 H), 3.33-3.45 (m, 22 H), 3.18-3.25 (m, 6 H), 2.31 (br t, J = 6.13 Hz, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.73-1.81 (m, 14 H). LCMS: retention time = 0.341 minutes, MS calculated: 1774.7, found: [M + 2H]2+ = 1776.9.Preparation of Target-A001A:

[00403] To a solution of Intermediate-5 (5.5 g, 2.91 mmol, 1.00 equivalent, trifluoroacetic acid) in methanol (50.0 mL) was added NaOMe (707 mg, 13.1 mmol, 4.50 equivalents) at 0°C. The mixture was stirred at 0°C for 0.5 hour. The reaction was monitored by LCMS, which showed the desired mass (one main peak with desired was detected.). The reaction mixture was added with 1.0 M HCl solution till the pH = 6. The mixture was diluted with water (75.0 mL) and extracted with DCM (120 mL * 3). The mixture was freeze-dried to afford Target-A001A(4.1 g, 2.74 mmol, 94.1% yield, 93.4% purity, HCl) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.94 (br t, J = 5.50 Hz, 3 H), 7.62 (d, J = 9.01 Hz, 3 H), 7.54 (s, 1 H), 4.28 (d, J = 8.50 Hz, 3 H), 3.70-3.88 (m, 6 H), 3.61-3.70 (m, 6 H), 3.49-3.57 (m, 31 H), 3.38-3.43 (m, 12 H), 3.31 (br d, J = 6.25 Hz, 4 H), 3.17 (s, 6 H), 3.09 (s, 2 H), 2.30 (br t, J = 6.32 Hz, 6 H), 1.80 (s, 9 H). LCMS: retention time = 0.22 minutes, MS calculated: 1396.6, found: [M + H]+= 1397.8.Preparation of Intermediate-27:

[00404] To a solution of Target-A001A (2.00 g, 1.43 mmol, 1.00 equivalent) and Azido-PEG3-CH2CO2-NHS (567 mg, 1.71 mmol, 1.20 equivalents) in DMF (20 mL) was added DIEA (554 mg, 4.29 mmol, 709 μL, 3.00 equivalents) at 0°C. The mixture was stirred at 0°C for two hours under nitrogen gas atmosphere. LCMS indicated Target-A001A was consumed completely, one main peak with desired MS was detected. The residue was purified by prep-high performance liquid chromatography (AcOH condition) directly to afford Intermediate-27 (1.75 g, 1.04 mmol, 73.2% yield, 96.5% purity) as a white solid. LCMS: retention time = 0.286 minutes, MS calculated: Mav = 1612.68, [M + 2H]2+ = 807.1, [M-sugar + 2H]2+ = 705.6. EXAMPLE 8Procedure for Preparation of TBT544:Preparation ofIntermediate-37:

[00405] Peptide was synthesized using standard Fmoc chemistry (C resin).

[00406] (1) Resin preparation: DMF (100 mL) was added to the vessel containing Rink MBHA-Amide Resin (5.00 mmol, 16.7 g, 0.30 mmol / g) with nitrogen gas bubbling for thirty minutes. The resin was washed with DMF (100 mL * 5), followed by adding 20% piperidine in DMF (50 mL) and bubbled with nitrogen gas for thirty minutes at 25°C for Fmoc deprotection. The mixture was filtered. The resin was washed with DMF (100 mL * 5) before proceeding to next step.

[00407] (2) Coupling: A solution of Fmoc-Pra-OH (3.00 equivalents), HATU (2.85 equivalents) in DMF (50 mL) was added to the resin with nitrogen gas bubbling. Then DIEA (6.00 equivalents) was added to the mixture dropwise and bubbled with nitrogen gas for thirty minutes at 25°C. The coupling reaction was monitored by ninhydrin test, if it showed colorless, the coupling was completed. The resin was then washed with DMF (100 mL) * 5.

[00408] (3) Deprotection: 20% piperidine in DMF (50 mL) was added to the resin and the mixture was bubbled with nitrogen gas for thirty minutes at 25°C. The resin was then washed with DMF (100 mL) * 5. The deprotection reaction was monitored by ninhydrin test. If it showed blue or brownish red, the reaction was completed.

[00409] (4) Steps 2 and 3 were repeated for the following amino acids elongation: Number # 2-3, in TABLE 14.

[00410] (5) After the last position completed, the resin was then washed with DMF (100 mL) * 5, methanol (100 mL) * 5, and dried under reduced pressure to afford peptide-bound-resin (5 mmol).TABLE 14The list of amino acids and the corresponding reagents used on solid-phase peptide synthesis#MaterialsCoupling reagents1Fmoc-Pra-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)2Fmoc-Gly-Gly-Gly-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)3Fmoc-Gly-Gly-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)Peptide TFA deprotection:

[00411] Trifluoroacetic acid deprotection: peptide-bound-resin was stirred in a solution of trifluoroacetic acid / 3-MPA / Tis / water (92.5 / 2.5 / 2.5 / 2.5, v / v / v / v, 200 mL) at 25°C for two hours. The mixture was precipitated with isopropyl ether (cold, 2 L). After filtration, the solid was washed with isopropyl ether (cold, 2 L) for two additional times, and dried under reduced pressure for two hours to afford Intermediate-37 (620 mg, crude) as a white solid. The crude was purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: MeCN) directly to afford Intermediate-37 (1.59 g, 3.77 mmol, 75.5% yield, 94.3% purity) as a white solid. LCMS: retention time = 0.071 minutes, MS calculated: Mav = 397.39, mass observed: [M + H]+ = 398.1.Preparation of TBT544:

[00412] To a solution of Intermediate-37 (500 mg, 1.25 mmol, 1.20 equivalents) and Intermediate-27 (1.69 g, 1.04 mmol, 1.00 equivalent) in DMF (10 mL) was added a solution of CuSO4 (0.4 M, 2.62 mL, 1.00 equivalent), sodium L-ascorbate (0.4 M, 10.4 mL, 4.00 equivalents) at 0°C. The mixture was stirred at 0°C for two hours under nitrogen gas atmosphere. LCMS indicated Intermediate-27 was consumed completely, one main peak with desired MS was detected. The residue was purified by prep-high performance liquid chromatography (AcOH condition) directly to afford TBT544 (1.377 g, 666 μmol, 63.5% yield, 97.3% purity) as a white solid. LCMS: retention time = 1.372 minutes, MS calculated: Mav = 2010.06, [M + 2H]2+ = 1005.7, [M + 3H]3+ = 671.0, [M -sugar + 2H]2+ = 904.2, [M -2sugar + 2H]2+ = 802.6. EXAMPLE 9Procedure for Preparation of Target-A001A:Preparation of Intermediate-2:

[00413] To a solution of 1a (60.0 g, 400 mmol, 2.00 equivalents) in 2-Methyltetrahydrofuran (450 mL) was added 1 (34.2 g, 200 mmol, 1.00 equivalent) in 2-Methyltetrahydrofuran (160 mL) at 0°C. The mixture was stirred at 25°C for two hours. TLC (DCM:methanol = 20: 1, Rf = 0.70) showed the reaction was completed, one major new spot with lower polarity was detected. The reaction mixture was added HCl / EA (1 N, 27.0 mL) and stirred for thirty minutes, and the white precipitate was removed by filtration, the filtrate was concentrated under reduced pressure to afford Intermediate-2 (crude, 105.0 g, 370.6 mmol) as yellow oil. LCMS: retention time = 0.797 minutes, MS calculated: 283.14, mass observed: [M + Na]+ = 306.1. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.23-7.41 (m, 5 H), 5.01 (s, 2 H), 4.60 (br s, 1 H), 3.45-3.52 (m, 6 H), 3.38-3.43 (m, 5 H), 3.14 (q, J = 5.94 Hz, 2 H), 2.53-2.55 (m, 1 H).Preparation of Intermediate-3:

[00414] To a solution of Intermediate-2a (100.0 g, 257 mmol, 1.00 equivalent) in DCE (500 mL) was added TMSOTf (85.6 g, 385 mmol, 1.50 equivalents) and stirred at 60°C for two hours. The reaction was then cooled to room temperature (25°C) and stirred for another one hour. A mixture of Intermediate-2 (80.0 g, 282 mmol, 1.10 equivalents) and 4 Å powder molecular sieves (50.0 g) in DCE (500 mL) was added to the reaction. The resulting mixture was stirred for thirty minutes under nitrogen gas atmosphere. Then a solution of Intermediate-2a (100.0 g, 257 mmol, 1.00 equivalent) in DCE was added dropwise to the mixture at 0°C. The mixture was stirred for sixteen hours at 25°C under nitrogen gas atmosphere. TLC (DCM:methanol = 10: 1, Rf = 0.42) indicated Intermediate-2a was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was filtered and washed with sat. NaHCO3 (500 mL), water (500 mL) and brine (500 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE: EA = 3: 1 to 1: 6, then DCM:methanol = 20: 1) to afford Intermediate-3 (90.0 g, 146.9 mmol, 91.6% purity, 57.2% yield) as yellow oil. LCMS: retention time = 0.860 minutes, MS calculated: 612.25, mass observed: [M + H]+ = 613.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.80 (d, J = 9.03 Hz, 1 H), 7.24-7.39 (m, 6 H), 5.22 (d, J = 3.51 Hz, 1 H), 4.95-5.05 (m, 3 H), 4.53-4.59 (m, 1 H), 3.99-4.06 (m, 3 H), 3.84-3.92 (m, 1 H), 3.73-3.82 (m, 1 H), 3.55-3.61 (m, 1 H), 3.45-3.53 (m, 7 H), 3.41 (t, J = 5.90 Hz, 2 H), 3.11-3.18 (m, 3 H), 2.10 (s, 3 H), 1.99 (s, 3 H), 1.89 (s, 3 H), 1.77 (s, 3 H).Preparation of Intermediate-4:

[00415] Pd / C (9.00 g, 10% purity) in reaction bottle (purged with argon gas three times) was added THF (180 mL) slowly, then a solution of trifluoroacetic acid (16.7 g, 147 mmol, 1.00 equivalent) and Intermediate-3 (90.0 g, 147.0 mmol, 1.00 equivalent) in THF (720 mL) was added to the reaction slowly under nitrogen gas. The reaction was degassed and purged with nitrogen gas and hydrogen gas for three times, then stirred at 25°C for three hours under hydrogen gas atmosphere (40 psi). TLC (DCM:methanol = 10: 1, Rf = 0.20) indicated Intermediate-3 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was dissolved in THF (100 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (100 mL * 2), and the filtrate was concentrated under reduced pressure to get the residue. The residue was diluted with water (1000 mL), washed with DCM (300 mL * 3), the aqueous layer was lyophilized to afford Intermediate-4 (80.0 g, 139.0 mmol, 95.1% purity, 91.8% yield, trifluoroacetic acid salt) as a white solid. LCMS: retention time = 0.484 minutes, MS calculated: 478.22, mass observed: [M + H]+ = 478.9. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.91 (br t, J = 9.03 Hz, 4 H), 5.21 (d, J = 3.26 Hz, 1 H), 4.96 (dd, J = 11.17, 3.39 Hz, 1 H), 4.54 (d, J = 8.53 Hz, 1 H), 3.98-4.08 (m, 3 H), 3.85-3.93 (m, 1 H), 3.75-3.84 (m, 1 H), 3.59 (br t, J = 5.14 Hz, 3 H), 3.50-3.56 (m, 6 H), 2.98 (br s, 2 H), 2.10 (s, 3 H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.78 (s, 3 H).Preparation of Intermediate-6:

[00416] To a mixture of 5 (60.0 g, 495.0 mmol, 1.00 equivalent) in DMSO (166 mL) was added aqueous NaOH (5.0 M, 9.91 mL, 0.10 equivalents) dropwise at 0-15°C for over five minutes. After addition, the mixture was stirred at 0-15°C for five minutes, then 5a (254.0 g, 1.98 mol, 287 mL, 4.00 equivalents) was added to the reaction mixture dropwise at 20°C. The resulting mixture was stirred at 25°C for sixteen hours. TLC (DCM:methanol = 10: 1, Rf = 0.7) indicated Intermediate-5 was consumed completely, and one major new spot with lower polarity was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate (400 mL), quenched by addition of water (400 mL), and extracted with ethyl acetate (400 mL * 3). The combined organic layers were washed with brine (300 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to afford Intermediate-6 (100.0 g, 197.8 mmol, 96.0% purity, 40.0% yield) as colorless oil. 1H NMR (400 MHz, DMSO-d6) δ ppm 3.51-3.61 (m, 7 H), 3.17 (s, 5 H), 2.39 (t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H).Preparation of Intermediate-7:

[00417] To a solution of Intermediate-6 (40.0 g, 79.1 mmol, 1.00 equivalent) in MeCN (400 mL) was added HOBt (10.7 g, 79.1 mmol, 1.00 equivalent). Then 6a (16.5 g, 79.1 mmol, 1.00 equivalent) and DCC (16.3 g, 79.1 mmol, 1.00 equivalent) were added. The reaction was stirred at 25°C for sixteen hours. TLC (PE: EA = 1: 1, Rf = 0.80) indicated Intermediate-6 was consumed completely, and one major new spot with lower polarity was detected. MeCN was evaporated to get the residue. The residue was purified by column chromatography (SiO2, PE: EA = 10: 1 to 1: 1) to afford Intermediate-7 (40.0 g, 57.4 mmol, 82.9% purity, 72.5% yield) as a white solid. LCMS: retention time = 1.151 minutes, MS calculated: 696.38, mass observed: [M + H]+ = 697.3, [M + Na]+ = 719.3. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.26-7.40 (m, 6 H), 7.06 (s, 1 H), 5.03 (s, 2 H), 3.49-3.61 (m, 14 H), 2.39 (br t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H).Preparation of Intermediate-8:

[00418] A solution of Intermediate-7 (30.0 g, 43.0 mmol, 1.00 equivalent) in HCOOH (300 mL) was stirred at 25°C for sixteen hours. TLC (PE: EA = 1: 1, Rf = 0.04) indicated Intermediate-7was consumed completely, and one major new spot with larger polarity was detected. Solvent was evaporated under reduced pressure, then co-evaporated with toluene (50 mL * 3) under reduced pressure and dried under reduced pressure to get the residue. The residue was purified by prep-high performance liquid chromatography (A: 0.1% FA condition / water, B: MeCN) to afford Intermediate-8 (20.0 g, 37.8 mmol, 98.2% purity, 87.9% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.17 (br s, 3 H), 7.26-7.43 (m, 6 H), 7.06 (s, 1 H), 5.02 (s, 2 H), 3.49-3.65 (m, 14 H), 2.42 (br t, J = 6.27 Hz, 6 H). LCMS: retention time = 0.790 minutes, MS calculated: 528.20, mass observed: [M + H]+ = 529.2.Preparation of Intermediate-9:

[00419] To a stirring solution of Intermediate-8 (20.0 g, 37.8 mmol, 1.00 equivalent) and Intermediate-4 (78.5 g, 132 mmol, 3.50 equivalents, trifluoroacetic acid salt) in DMF (400 mL) was added HOBT (20.4 g, 151 mmol, 4.00 equivalents), EDCI (29.0 g, 151 mmol, 4.00 equivalents) and DIEA (22.0 g, 170 mmol, 4.50 equivalents) successively. The reaction was stirred at 25°C for two hours. TLC (DCM:methanol = 10: 1, Rf = 0.4) indicated Intermediate-8 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was slowly poured into a stirring cold 0.5 mol / L HCl solution (900 mL) and stirred for ten minutes. White precipitate formed and was filtered. The aqueous phase was extracted with DCM (600 mL* 2) twice. The combined organic layers were washed with 5% NaHCO3 (450 mL), dried over Na2SO4, and concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiO2, DCM:methanol = 100: 1 to 5: 1) to afford Intermediate-9 (58.0 g, 30.4 mmol, 82.7% purity, 80.3% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.92 (br t, J = 5.14 Hz, 3 H), 7.81 (d, J = 9.03 Hz, 3 H), 7.28-7.39 (m, 6 H), 7.13 (s, 1 H), 5.21 (d, J = 3.26 Hz, 3 H), 5.02 (s, 2 H), 4.97 (dd, J = 11.17, 3.39 Hz, 3 H), 4.54 (d, J = 8.53 Hz, 3 H), 4.03 (s, 9 H), 3.84-3.92 (m, 3 H), 3.75-3.81 (m, 3 H), 3.45-3.61 (m, 37 H), 3.39 (br s, 3 H), 3.18-3.23 (m, 6 H), 2.30 (br t, J=6.15 Hz, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H). LCMS: retention time = 3.455 minutes, MS calculated: 1908.81, mass observed: [M + 2H]2+ = 955.7.Preparation of Intermediate-10:

[00420] The 500 mL round-bottom flask was purged with argon gas three times and added dry Pd / C (1.50 g, 1.41 mmol, 10% purity, 1.00 equivalent) carefully. Then THF (150.0 mL) was added to infiltrate the Pd / C completely, followed by the solution of Intermediate-9 (15.0 g, 7.85 mmol, 1.00 equivalent) and trifluoroacetic acid (895 mg, 7.85 mmol, 583 μL, 1.00 equivalent) in tetrahydrofuran (75 mL) slowly under argon gas atmosphere. The resulting mixture was degassed and purged with hydrogen gas for three times, and then the mixture was stirred at 25°C for three hours under hydrogen gas atmosphere (15 psi). The reaction was monitored by LCMS. LCMS showed the desired mass. One main peak with desired was detected. The reaction mixture was filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (100 mL*2). The filter cake was added water immediately. The organic layer concentrated under reduced pressure to afford Intermediate-10 (13.0 g, 6.54 mmol, 83.2% yield, 99.7% purity, trifluoroacetic acid salt) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.89-7.99 (m, 5 H), 7.82 (d, J = 9.3 Hz, 3 H), 7.74 (s, 1 H), 7.14-7.28 (m, 1 H), 5.22 (d, J = 3.3 Hz, 3 H), 4.97 (dd, J = 11.3, 3.4 Hz, 3 H), 4.54 (d, J = 8.5 Hz, 3 H), 3.84-3.93 (m, 3 H), 3.76-3.82 (m, 3 H), 3.47-3.61 (m, 43 H), 3.21 (q, J = 5.8 Hz, 6 H), 2.29-2.34 (m, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 8 H), 1.77 (s, 9 H). LCMS: retention time = 1.333 minutes, MS calculated: 1774.7, found: [M + 2H]2+ = 888.6.Preparation of Target-A001A:

[00421] To a solution of Intermediate-10 (12.0 g, 6.35 mmol, 1.00 equivalent, trifluoroacetic acid) in methanol (120.0 mL) was added NaOMe (5.4 M, 5.01 mL, 4.26 equivalents) at 0°C. The mixture was stirred at 0°C for 0.5 h. The reaction was monitored by LCMS, LCMS showed the desired mass (one main peak with desired was detected.). The reaction mixture was added with 1.0 M HCl solution (10.0 mL) till the pH = 6. The mixture was diluted with water (75.0 mL) and extracted with DCM (120 mL * 3). The mixture was freeze-dried to afford Target-A001A(9.0 g, 5.96 mmol, 93.9% yield, >95% purity, HCl) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.96 (br t, J = 5.4 Hz, 3 H), 7.67 (d, J = 8.9 Hz, 3 H), 7.51 (s, 1 H), 4.27 (d, J = 8.4 Hz, 3 H), 3.75-3.80 (m, 6 H), 3.70 (br d, J = 10.0 Hz, 6 H), 3.49 (br d, J = 4.0 Hz, 31 H), 3.37-3.42 (m, 12 H), 3.30 (br d, J = 6.1 Hz, 4 H), 3.20 (br d, J = 5.8 Hz, 6 H), 2.99 (s, 2 H), 2.30 (br t, J = 6.4 Hz, 6 H), 1.80 (s, 9 H). LCMS: retention time = 0.966 minutes, MS calculated: 1396.6, found: [M + 2H]2+= 699.1. EXAMPLE 10Procedure for Preparation of AGN306:Preparation of Intermediate-17:

[00422] The peptide was synthesized using standard Fmoc chemistry (CTC resin).

[00423] (1) Resin preparation: To the vessel containing CTC Resin (4.00 mmol, 20.0 g, 1.00 mmol / g) and Fmoc-Lys(Boc)-OH (1.87 g, 4.00 mmol, 1.00 equivalent) in DCM (100 mL) was added DIEA (4.00 equivalents) dropwise and mix for two hours with nitrogen gas bubbling at 25°C. Methanol (20 mL) was added. The mixture was bubbled with nitrogen gas for another thirty minutes. The resin was washed with DMF (100 mL) * 5. Then 20% piperidine in DMF (50 mL) was added and the mixture was bubbled with nitrogen gas for thirty minutes at 25°C. The mixture was filtered to obtain the resin. The resin was washed with DMF (100 mL) * 5 before proceeding to next step.

[00424] (2) Coupling: A solution of Fmoc-Cys(Trt)-OH (7.02 g, 12.0 mmol, 3.00 equivalents), HATU (4.33 g, 2.85 equivalents) in DMF (50 mL) was added to the resin with nitrogen gas bubbling. DIEA (6.00 equivalents) was added to the mixture dropwise. The mixture was bubbled with nitrogen gas for thirty minutes at 25°C. The coupling reaction was monitored by ninhydrin test, if it showed colorless, the coupling was completed. The resin was washed with DMF (100 mL) * 5.

[00425] (3) Deprotection: 20% piperidine in DMF (50 mL) was added to the resin and the mixture was bubbled with nitrogen gas for thirty minutes at 25°C. The resin was washed with DMF (100 mL) * 5. The deprotection reaction was monitored by ninhydrin test, if it showed blue or brownish red, the reaction was completed.

[00426] (4) Steps 2 and 3 were repeated for the following amino acids elongation: Number # 3-31, TABLE 15.

[00427] (5) After the last position completed, the resin was washed with DMF (100 mL) * 5, methanol (100 mL) * 5, and dried under reduced pressure to afford Intermediate-11 (peptide-bound-resin, 4.00 mmol, 47.0 g).TABLE 15The list of amino acids and the corresponding reagents used on solid-phase peptide synthesis#MaterialsCoupling reagents1Fmoc-Lys(Boc)-OH (1.00 equivalent) DIEA (4.00 equivalents)2Fmoc-Cys(Trt)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)3Fmoc-Asn(Trt)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)4Fmoc-Glu(OtBu)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)5Fmoc-Leu-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)6Fmoc-Thr(Trt)-OH (3.00 equivalents)DIC (3.00 equiv.) and HOAt (3.00 equiv.)7Fmoc-Leu-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)8Fmoc-Val-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)9Fmoc-Lys(Boc)-Ser(psi(Me,Me)pro)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)10Fmoc-Gly-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)11Fmoc-Ala-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)12Fmoc-Gln(Trt)-OH (3.00 equivalents)DIC (3.00 equiv.) and HOAt (3.00 equiv.)13Fmoc-Ile-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)14Fmoc-Cys(Trt)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)15Fmoc-Lys(Boc)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)16Fmoc-Lys(Boc)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)17Fmoc-Leu-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)18Fmoc-Ser(tBu)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)19Fmoc-Glu(OtBu)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)20Fmoc-Ser(tBu)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)21Fmoc-Gln(Trt)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)22Fmoc-Ile-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)23Fmoc-Val-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)24Fmoc-Phe-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)25Fmoc-Ile-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)26Fmoc-Gly-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)27Fmoc-Lys(Boc)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)28Fmoc-Asp(OtBu)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)29Fmoc-Gln(Trt)-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)30Fmoc-Trp-OH (3.00 equivalents)HATU (2.85 equiv.) and DIEA (6.00 equiv.)31Fmoc-Dab(N3)-OH (1.50 equivalents)DIC (1.50 equiv.) and HOAt (1.50 equiv.)Peptide cleavage and disulfide formation:

[00428] 1)(1) TFA deprotection: Intermediate-11 (47.0 g, resin) was stirred in a solution of trifluoroacetic acid / 3-MPA / Tis / water (92.5 / 2.5 / 2.5 / 2.5, v / v / v / v, 500 mL) at 25°C for two hours. After filtration, the filtrate was precipitated with isopropyl ether (cold, 5 L). After filtration, the solid was washed with isopropyl ether (cold, 5 L) for two additional times, and dried under reduced pressure for two hours to afford Intermediate-12 (14.0 g, crude) as a white solid. LCMS: retention time = 0.474 minutes, MS calculated: Mav = 3621.19, mass observed: [M + 2H]2+ = 1811.0 [M + 3H]3+ = 1207.7, [M + 4H]4+ = 906.1, [M + 5H]5+ = 725.0, [M + 6H]6+ = 604.4, [M + 7H]7+ = 518.3.

[00429] 2)(2) Disulfide formation: Intermediate-12 (14.0 g, crude) was dissolved in water (2.8 L) and MeCN (1.2 L) at 25°C. To the mixture was added 0.1 M I2 / AcOH dropwise until a yellow color persisted. Then the mixture was stirred at 25°C for five minutes. After filtration, the filtrate was purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: MeCN) directly to afford Intermediate-17 (1.75 g, 438 μmol, 10.95% yield, 90.26% purity) as a white solid. LCMS: retention time = 0.439 minutes, MS calculated: Mav = 3619.18, mass observed: [M + 2H]2+ = 1810.5 [M + 3H]3+ = 1207.1, [M + 4H]4+ = 905.6, [M + 5H]5+ = 724.6.Preparation of Intermediate-14:

[00430] To a solution of Intermediate-13 (600 mg, 2.31 mmol, 1.00 equivalent), Intermediate-13a (2.30 g, 13.8 mmol, 6.00 equivalents) in DMF (6 mL) was added EDCI (1.33 g, 6.92 mmol, 3.00 equivalents) at 0°C. The mixture was stirred at 0°C for one hour. The mixture was purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: MeCN) directly to afford Intermediate-14 (800 mg, 83.4% yield, 98.2 % purity) as yellow oil. LCMS: retention time = 1.4 thirty minutes, MS calculated: Mav = 408.34, mass observed: [M + Na]+ = 431.0. Preparation of Target-A001A-PEG4-Alkyne:

[00431] To a solution of Intermediate-14 (276 mg, 676 μmol, 1.05 equivalents) and Target-A001A (1.00 g, 644 μmol, 1.00 equivalent) in DMF (0.25 mL) was added DIEA (166 mg, 230 µl, 2.00 equivalents). The mixture was stirred at 25°C for twelve hours. The mixture was diluted with MeCN / water (cold, v / v, 3 / 3, 10 mL), then dried by lyophilization to remove DMF. The residue was purified by prep-high performance liquid chromatography (AcOH condition) directly to afford Target-A001A-PEG4-Alkyne (0.60 g, 56.8% yield, 93.5% purity) as colorless oil. LCMS: retention time = 0.368 minutes, MS calculated: Mav = 1639.74, mass observed: [M + H]+ = 1639.8, [M-sugar + H]+ = 1426.6, [M – 2*sugar + H]+ = 1233.6, [M – 3*sugar + H]+ = 1030.3, [M + 2H]2+ = 820.6. Preparation of AGN306:

[00432] To a solution of Intermediate-17 (370 mg, 102 μmol, 1.00 equivalent) and Target-A001A-PEG4-Alkyne (201 mg, 122 μmol, 1.20 equivalents) in DMF (2 mL) and water (2 mL) was added a fresh solution of CuSO4 (0.4 M, 255 μL, 1.00 equivalent), sodium L-ascorbate (0.4 M, 1.02 mL, 4.00 equivalents) and THPTA (44.4 mg, 102 μmol, 1.00 equivalent) at 0°C. The mixture was stirred at 0°C for one hour under nitrogen gas atmosphere. The residue was purified by prep-high performance liquid chromatography (ACOH condition) directly to afford AGN306 (144 mg, 26.6 μmol, 26.08% yield, 96.91% purity) as a white solid. LCMS: retention time = 1.639 minutes, MS calculated: Mav = 5258.92, [M + 3H]3+ = 1754.2, [M + 4H]4+ = 1315.9, [M + 5H]5+ = 1052.7. EXAMPLE 11Maleimide-beta-GN3(TBT506)Procedure for Preparation of Target-A001A.See EXAMPLE 9 above.Preparation of Intermediate-2:

[00433] To a solution of 1a (60.0 g, 400 mmol, 2.00 equivalents) in 2-Methyltetrahydrofuran (450 mL) was added 1 (34.2 g, 200 mmol, 1.00 equivalent) in 2-methyltetrahydrofuran (160 mL) at 0°C. The mixture was stirred at 25°C for two hours. TLC (DCM: MeOH = 20: 1, Rf = 0.70) showed the reaction was completed, one major new spot with lower polarity was detected. The reaction mixture was added HCl / EA (1 N, 27.0 mL) and stirred for thirty minutes, and the white precipitate was removed by filtration, the filtrate was concentrated under reduced pressure to afford Intermediate-2 (crude, 105.0 g, 370.6 mmol) as yellow oil. LCMS: retention time = 0.797 minutes, MS cal.: 283.14, mass observed: [M + Na]+ = 306.1. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.23-7.41 (m, 5 H), 5.01 (s, 2 H), 4.60 (br s, 1 H), 3.45-3.52 (m, 6 H), 3.38-3.43 (m, 5 H), 3.14 (q, J = 5.94 Hz, 2 H), 2.53-2.55 (m, 1 H).Preparation of Intermediate-3:

[00434] To a solution of Intermediate-2a (100.0 g, 257 mmol, 1.00 equivalent) in DCE (500 mL) was added TMSOTf (85.6 g, 385 mmol, 1.50 equivalents) and stirred at 60°C for 2 h. The reaction was then cooled to room temperature (25°C) and stirred for another one hour. A mixture of Intermediate-2 (80.0 g, 282 mmol, 1.10 equivalents) and 4 Å powder molecular sieves (50.0 g) in DCE (500 mL) was added to the reaction. The resulting mixture was stirred for thirty minutes under nitrogen gas atmosphere. Then a solution of Intermediate-2a (100.0 g, 257 mmol, 1.00 equivalent) in DCE was added dropwise to the mixture at 0°C. The mixture was stirred for sixteen hours at 25°C under nitrogen gas atmosphere. Thin-layer chromatography (DCM: MeOH = 10: 1, Rf = 0.42) indicated Intermediate-2a was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was filtered and washed with sat. NaHCO3 (500 mL), water (500 mL) and brine (500 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE: EA = 3: 1 to 1: 6, then DCM: MeOH = 20: 1) to afford Intermediate-3 (90.0 g, 146.9 mmol, 91.6% purity, 57.2% yield) as yellow oil. LCMS: retention time = 0.860 minutes, MS cal.: 612.25, mass observed: [M + H]+ = 613.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.80 (d, J = 9.03 Hz, 1 H), 7.24-7.39 (m, 6 H), 5.22 (d, J = 3.51 Hz, 1 H), 4.95-5.05 (m, 3 H), 4.53-4.59 (m, 1 H), 3.99-4.06 (m, 3 H), 3.84-3.92 (m, 1 H), 3.73-3.82 (m, 1 H), 3.55-3.61 (m, 1 H), 3.45-3.53 (m, 7 H), 3.41 (t, J = 5.90 Hz, 2 H), 3.11-3.18 (m, 3 H), 2.10 (s, 3 H), 1.99 (s, 3 H), 1.89 (s, 3 H), 1.77 (s, 3 H).Preparation of Intermediate-4:

[00435] Pd / C (9.00 g, 10% purity) in reaction bottle (purged with Ar for three times) was added THF (180 mL) slowly, then a solution of trifluoroacetic acid (16.7 g, 147 mmol, 1.00 equivalent) and Intermediate-3 (90.0 g, 147.0 mmol, 1.00 equivalent) in THF (720 mL) was added to the reaction slowly under nitrogen gas. The reaction was degassed and purged with nitrogen gas and hydrogen gas for three times, then stirred at 25°C for three hours under H2 atmosphere (40 psi). TLC (DCM:methanol = 10: 1, Rf = 0.20) indicated Intermediate-3 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was dissolved in THF (100 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (100 mL * 2), and the filtrate was concentrated under reduced pressure to get the residue. The residue was diluted with water (1000 mL), washed with DCM (300 mL * 3), the aqueous layer was lyophilized to afford Intermediate-4 (80.0 g, 139.0 mmol, 95.1% purity, 91.8% yield, trifluoroacetic acid salt) as a white solid. LCMS: retention time = 0.484 minutes, MS cal.: 478.22, mass observed: [M + H]+ = 478.9. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.91 (br t, J = 9.03 Hz, 4 H), 5.21 (d, J = 3.26 Hz, 1 H), 4.96 (dd, J = 11.17, 3.39 Hz, 1 H), 4.54 (d, J = 8.53 Hz, 1 H), 3.98-4.08 (m, 3 H), 3.85-3.93 (m, 1 H), 3.75-3.84 (m, 1 H), 3.59 (br t, J = 5.14 Hz, 3 H), 3.50-3.56 (m, 6 H), 2.98 (br s, 2 H), 2.10 (s, 3 H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.78 (s, 3 H).Preparation of Intermediate-6:

[00436] To a mixture of Intermediate-5 (60.0 g, 495.0 mmol, 1.00 equivalent) in DMSO (166 mL) was added aqueous NaOH (5.0 M, 9.91 mL, 0.10 equivalents) dropwise at 0-15°C for over five minutes . After addition, the mixture was stirred at 0-15°C for five minutes, then 5a (254.0 g, 1.98 mol, 287 mL, 4.00 equivalents) was added to the reaction mixture dropwise at 20°C. The resulting mixture was stirred at 25°C for sixteen hours. TLC (DCM: MeOH = 10: 1, Rf = 0.7) indicated Intermediate-5 was consumed completely, and one major new spot with lower polarity was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate (400 mL), quenched by addition of water (400 mL), and extracted with ethyl acetate (400 mL * 3). The combined organic layers were washed with brine (300 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to afford Intermediate-6 (100.0 g, 197.8 mmol, 96.0% purity, 40.0% yield) as colorless oil. 1H NMR (400 MHz, DMSO-d6) δ ppm 3.51-3.61 (m, 7 H), 3.17 (s, 5 H), 2.39 (t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H).Preparation of Intermediate-7:

[00437] To a solution of Intermediate-6 (40.0 g, 79.1 mmol, 1.00 equivalent) in MeCN (400 mL) was added HOBt (10.7 g, 79.1 mmol, 1.00 equivalent). Then 6a (16.5 g, 79.1 mmol, 1.00 equivalent) and DCC (16.3 g, 79.1 mmol, 1.00 equivalent) were added. The reaction was stirred at 25°C for sixteen hours. TLC (PE: EA = 1: 1, Rf = 0.80) indicated Intermediate-6 was consumed completely, and one major new spot with lower polarity was detected. MeCN was evaporated to get the residue. The residue was purified by column chromatography (SiO2, PE: EA = 10: 1 to 1: 1) to afford Intermediate-7 (40.0 g, 57.4 mmol, 82.9% purity, 72.5% yield) as a white solid. LCMS: retention time = 1.151 minutes, MS cal.: 696.38, mass observed: [M + H]+ = 697.3, [M + Na]+ = 719.3. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.26-7.40 (m, 6 H), 7.06 (s, 1 H), 5.03 (s, 2 H), 3.49-3.61 (m, 14 H), 2.39 (br t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H).Preparation of Intermediate 8:

[00438] A solution of Intermediate-7 (30.0 g, 43.0 mmol, 1.00 equivalent) in HCOOH (300 mL) was stirred at 25°C for sixteen hours. TLC (PE: EA = 1: 1, Rf = 0.04) indicated Intermediate-7was consumed completely, and one major new spot with larger polarity was detected. Solvent was evaporated under reduced pressure, then co-evaporated with toluene (50 mL * 3) under reduced pressure and dried under reduced pressure to get the residue. The residue was purified by prep-high performance liquid chromatography (A: 0.1% FA condition / water, B: MeCN) to afford Intermediate-8 (20.0 g, 37.8 mmol, 98.2% purity, 87.9% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.17 (br s, 3 H), 7.26-7.43 (m, 6 H), 7.06 (s, 1 H), 5.02 (s, 2 H), 3.49-3.65 (m, 14 H), 2.42 (br t, J = 6.27 Hz, 6 H). LCMS: retention time = 0.790 minutes, MS cal.: 528.20, mass observed: [M + H]+ = 529.2.Preparation of Intermediate-9:

[00439] To a stirring solution of Intermediate-8 (20.0 g, 37.8 mmol, 1.00 equivalent) and Intermediate-4 (78.5 g, 132 mmol, 3.50 equivalents, trifluoroacetic acid salt) in DMF (400 mL) was added HOBT (20.4 g, 151 mmol, 4.00 equivalents), EDCI (29.0 g, 151 mmol, 4.00 equivalents) and DIEA (22.0 g, 170 mmol, 4.50 equivalents) successively. The reaction was stirred at 25°C for 2 h. TLC (DCM: MeOH = 10: 1, Rf = 0.4) indicated Intermediate-8 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was slowly poured into a stirring cold 0.5 mol / L HCl solution (900 mL) and stirred for ten minutes. White precipitate was formed and filtered. The aqueous phase was extracted with DCM (600 mL* 2) twice. The combined organic layers were washed with 5% NaHCO3 (450 mL), dried over Na2SO4, and concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiO2, DCM: MeOH = 100: 1 to 5: 1) to afford Intermediate-9 (58.0 g, 30.4 mmol, 82.7% purity, 80.3% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.92 (br t, J = 5.14 Hz, 3 H), 7.81 (d, J = 9.03 Hz, 3 H), 7.28-7.39 (m, 6 H), 7.13 (s, 1 H), 5.21 (d, J = 3.26 Hz, 3 H), 5.02 (s, 2 H), 4.97 (dd, J = 11.17, 3.39 Hz, 3 H), 4.54 (d, J = 8.53 Hz, 3 H), 4.03 (s, 9 H), 3.84-3.92 (m, 3 H), 3.75-3.81 (m, 3 H), 3.45-3.61 (m, 37 H), 3.39 (br s, 3 H), 3.18-3.23 (m, 6 H), 2.30 (br t, J=6.15 Hz, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H). LCMS: retention time = 3.455 minutes, MS cal.: 1908.81, mass observed: [M + 2H]2+ = 955.7.Preparation of Intermediate 10:

[00440] The 500 mL round-bottom flask was purged with Ar gas for 3 times and added dry Pd / C (1.50 g, 1.41 mmol, 10% purity, 1.00 equivalent) carefully. Then THF (150.0 mL) was added to infiltrate the Pd / C completely, followed by the solution of Intermediate-9 (15.0 g, 7.85 mmol, 1.00 equivalent) and trifluoroacetic acid (895 mg, 7.85 mmol, 583 μL, 1.00 equivalent) in THF (75 mL) slowly under Ar atmosphere. The resulting mixture was degassed and purged with H2 for 3 times, and then the mixture was stirred at 25°C for three hours under H2 atmosphere (15 psi). The reaction was monitored by LCMS, LCMS showed the desired mass (one main peak with desired was detected.). The reaction mixture was filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (100 mL*2). Then, the filter cake was added water immediately. The organic layer concentrated under reduced pressure to afford Intermediate-10 (13.0 g, 6.54 mmol, 83.2% yield, 99.7% purity, trifluoroacetic acid salt) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.89-7.99 (m, 5 H), 7.82 (d, J = 9.3 Hz, 3 H), 7.74 (s, 1 H), 7.14-7.28 (m, 1 H), 5.22 (d, J = 3.3 Hz, 3 H), 4.97 (dd, J = 11.3, 3.4 Hz, 3 H), 4.54 (d, J = 8.5 Hz, 3 H), 3.84-3.93 (m, 3 H), 3.76-3.82 (m, 3 H), 3.47-3.61 (m, 43 H), 3.21 (q, J = 5.8 Hz, 6 H), 2.29-2.34 (m, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 8 H), 1.77 (s, 9 H). LCMS: retention time = 1.333 minutes, MS cal.: 1774.7, found: [M + 2H]2+ = 888.6.Preparation of Intermediate Target-A001A:

[00441] To a solution of Intermediate-10 (12.0 g, 6.35 mmol, 1.00 equivalent, trifluoroacetic acid) in MeOH (120.0 mL) was added NaOMe (5.4 M, 5.01 mL, 4.26 equivalents) at 0°C. The mixture was stirred at 0°C for 0.5 h. The reaction was monitored by LCMS, LCMS showed the desired mass (one main peak with desired was detected.). The reaction mixture was added with 1.0 M HCl solution (10.0 mL) till the pH = 6. The mixture was diluted with water (75.0 mL) and extracted with DCM (120 mL * 3). The mixture was freeze-dried to afford Target-A001A(9.0 g, 5.96 mmol, 93.9% yield, >95% purity, HCl) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.96 (br t, J = 5.4 Hz, 3 H), 7.67 (d, J = 8.9 Hz, 3 H), 7.51 (s, 1 H), 4.27 (d, J = 8.4 Hz, 3 H), 3.75-3.80 (m, 6 H), 3.70 (br d, J = 10.0 Hz, 6 H), 3.49 (br d, J = 4.0 Hz, 31 H), 3.37-3.42 (m, 12 H), 3.30 (br d, J = 6.1 Hz, 4 H), 3.20 (br d, J = 5.8 Hz, 6 H), 2.99 (s, 2 H), 2.30 (br t, J = 6.4 Hz, 6 H), 1.80 (s, 9 H). LCMS: retention time = 0.966 minutes, MS cal.: 1396.6, found: [M + 2H]2+= 699.1.Procedure for Preparation of TBT506:Preparation of TBT506:

[00442] To a solution of Target-A001A (500 mg, 357 μmol, 1.00 equivalent) and 3-maleimidopropionic acid N-hydroxysuccinimide ester (142.869 mg, 536 μmol, 1.50 equivalents) in DMF (5 mL) was added DIEA (46.2 mg, 357 μmol, 59.1 μL, 1.00 equivalent) degassed and purged with nitrogen gas for three times. The mixture was stirred at 0°C for five minutes under nitrogen gas atmosphere. LCMS showed the desired mass (one main peak with desired was detected.). The reaction mixture was purified by prep-high performance liquid chromatography (AcOH condition) directly to afford TBT506 (251.2 mg, 156 μmol, 43.7% yield, 96.5% purity) as a colorless solid. LCMS: retention time = 1.382 minutes, MS cal.: Mav = 1548.59, [M + H]+ = 1548.9, [M + 2H]2+ = 775.1. EXAMPLE 12Preparation of TBT644 - AProcedure for Preparation of Intermediate-27:Preparation of Intermediate 2:

[00443] Pd / C (4.00 g, 10% purity) in reaction bottle (purged with Ar for three times) was added THF (40 mL) slowly, then a solution of trifluoroacetic acid (7.44g, 65.2 mmol, 4.86 mL, one equivalent) and Intermediate-1 (40.0 g, 65.2 mmol, 1.00 equivalent) in THF (360 mL) was added to the reaction slowly under nitrogen gas. The reaction was degassed and purged with argon gas and hydrogen gas for three times, then stirred at 25°C for under hydrogen gas atmosphere (40 psi). TLC (DCM: MeOH = 10: 1, Rf = 0.20) indicated Intermediate-1 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was dissolved in THF (100 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (100 mL * 2), and the filtrate was concentrated under reduced pre...

Claims

1. A composition of matter comprising:an anti-PLA2R antibody-binding moiety, anda cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on surface degrading cells,wherein the anti-PLA2R antibody-binding moiety and the cellular receptor-binding moiety are covalently connected,optionally through a linker moiety connecting the anti-PLA2R antibody-binding moiety and the cellular receptor-binding moiety.

2. The composition of matter of Claim 1, having a structure of:RCN−(Xaa)y−RCC,[AGN101],[AGN102],[AGN103], [AGN104]or a salt thereof.

3. The composition of matter of claim 1, wherein the composition of matter is selected from the group consisting of AGN302, AGN303, AGN304, AGN306, AGN307, AGN308, AGN309, AGN310, AGN362, AGN363, AGN364, AGN365, AGN579, AGN580, and AGN167.

4. The composition of matter of claim 1, wherein the anti-PLA2R antibody-binding moiety is selected from the group of compounds listed in TABLE 72.5 The composition of matter of claim 1, wherein the anti-PLA2R antibody-binding moiety is a peptide selected from the groups of secretory phospholipase A2 receptor peptides in SEQ ID NO: 60-145.6 The composition of matter of claim 1, wherein the anti-PLA2R-autoantibody-binding moiety is selected from the group consisting of ABT301, ABT305, ABT309, ABT310, ABT311, ABT312, ABT407, ABT408, ABT409, ABT427, ABT530, ABT603, ABT606, ABT816, ABT817, ABT818, ABT819, ABT976, ABT983, and ABT985.

7. The composition of matter of claim 1, wherein the cellular receptor-binding moiety has the structure:or ;where RA is a C1-C3 alkyl group optionally substituted with 1-5 halo, preferably fluoro, groups, preferably RA is a methyl or ethyl group optionally substituted with from 1-3 fluoro groups;ZA is -(CH2)IM, -O-(CH2)IM, S-(CH2)IM, NRM-(CH2)IM, C(O)-(CH2)IM-, a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH2)IMNRM group (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues); andZB is absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)IM-NRM.

8. The composition of matter of claim 1, wherein the cellular receptor-binding moiety is selected from the group consisting of TBT103, TBT104, TBT105, TBT307, TBT506, and TBT544.

9. The composition of matter of claim 1, for use as a medicament.

10. The composition of matter of claim 1, for use in removing anti-PLA2R-autoantibody from a subject.

11. The composition of matter of claim 1, for use in treating a disease state or condition associated with the upregulation of anti-PLA2R-autoantibody in a subject or patient.

12. The composition of matter of claim 1, for use in treating membranous nephropathy.

13. A method of making the composition of matter of claims 1-8, wherein the method comprises a conjugation step to link an anti-PLA2R-autoantibody-binding moiety to a cellular receptor-binding moiety through a linker moiety, wherein the conjugation step is selected from the group consisting of a MATE conjugation step, a maleimide conjugation step, and a sortase conjugation step.

14. A pharmaceutical composition comprising the composition of matter of claims 1-8 and a pharmaceutically acceptable excipient.

15. A method of removing anti-PLA2R-autoantibody from a subject or patient comprising administering to the subject or patient the composition of matter of any of claims 1-8.

16. A method of treating a disease state or condition associated with the upregulation of anti-PLA2R-autoantibody in a subject or patient comprising administering to the subject or patient an effective amount of the composition of matter of any of claims 1-8.

17. A method of treating membranous nephropathy in a subject or patient comprising administering to the subject or patient an effective amount of the composition of matter of any of claims 1-8.

18. A composition comprising:a first composition of matter comprising:an anti-PLA2R-autoantibody-binding moiety,a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors which are on surface degrading cells, and a linker moiety connecting the anti-PLA2R-autoantibody-binding moiety and the cellular receptor-binding moiety, andat least one additional composition of matter comprising a moiety comprising:a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors which are on surface degrading cells, and a linker moiety connecting the anti-PLA2R-autoantibody-binding moiety and the cellular receptor-binding moiety.

19. The composition of claim 18, wherein the additional agent has a structure selected from the group consisting of formula LG−RG−LRM(−TBT)b,, LG−RG−H, and a combination thereof. 

Images