Bifunctional degraders for the treatment of graves' disease

Abstract

A composition of matter including an anti-TSH receptor antibody-binding moiety, a cellular receptor-binding moiety that 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-TSH receptor antibody-binding moiety and the cellular receptor-binding moiety, wherein the composition of matter is useful for removing anti-TSH receptor autoantibody from a patient or subject, such as a Graves' disease or thyroid eye disease patient.

Description

BIFUNCTIONAL DEGRADERS FOR THE TREATMENT OF GRAVES' DISEASEFIELD OF THE INVENTION

[0001] 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 ceil surface antigen or a ceil surface determinant, and particularly to bifunctional molecules which contain a circulating protein-binding moiety linked through a linker group to a cellular receptorbinding moiety for the treatment of Graves' disease, thyroid eye disease, and other thyroid stimulating hormone receptor (TSHR)-related diseases.BACKGROUND OF THE INVENTION

[0002] Graves' disease (GD) is an autoimmune condition and the most common cause of hyperthyroidism. In Graves' disease, the feedback control mechanism of thyroid function is no longer effective in the presence of thyroid stimulating autoantibodies. Graves' disease patients present with clinical symptoms of a hyperactive thyroid characterized by excess of thyroid hormones in serum and their metabolic consequences. Extrathyroidal manifestations include Graves orbitopathy (GO) or thyroid eye disease (TED).

[0003] Thyroid stimulating hormone (TSH) receptor autoantibodies (TRAbs) are responsible for the pathology of Graves' disease. There are two main types of TSH receptor autoantibodies, the stimulating type and the blocking type. See Rees Smith et al., Thyroid, 17, 923-938 (2007) and Rees Smith et al., Hormone and Metabolic Research, 41, 448-455 (2009). Thyroid-stimulating autoantibodies bind to the TSH receptor and mimic the actions of TSH, thereby stimulating the thyroid to produce high levels of hormones triiodothyronine (T3) and thyroxine (T4). Thyroid-stimulating autoantibodies have TSH receptor agonist activity. Rees Smith et al., Thyroid, 17, 923-938 (2007). The detection of high anti-TSH receptor levels establishes a Graves' disease diagnosis. See Davies et al., Nature Reviews Disease Primers, 6(1), 52 (July 2, 2020). Stimulating anti-TSH receptor antibodies are correlated with clinical activity score. See Lytton et al. J. Clin. Endocrinol. Metab. (2010).

[0004] The standard of care for treatment for Graves' disease and other TSH receptor-related diseases centers on antithyroid drugs, radioiodine therapy and surgery, with no significant changes in treatment for many years. There remains a need in the biomedical art for new medicines capable of treating, preventing, or slowing down the progression of Graves' disease and other TSH receptor-related diseases.SUMMARY OF THE INVENTION

[0005] The invention provides anti-TSH receptor selective compositions of matter for the targeted degradation of anti-TSH receptor antibodies.

[0006] In one embodiment, the invention provides a composition of matter (agent, TRAP™) comprising: a binding moiety that can bind to anti-TSH receptor antibody, wherein the binding moiety comprises at least one leucine rich domain (LRD) of human TSH receptor or a variant or fragment thereof, 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 of hepatocytes or other degrading cells in a patient or subject, and a linker moiety connecting the anti-TSH receptor antibody-binding moiety and the cellular receptor-binding moiety.

[0007] In another embodiment, the leucine rich domain comprises amino acids 22-260 of the human TSH receptor.

[0008] In some embodiments, the leucine rich domain variant comprises amino acids 22-260 of the human TSH receptor, as modified to make the leucine rich domain thermostable. In a specific embodiment comprises the amino acid sequence of TSHR260™, described by International Patent Publication WO 2015 / 189543 (RSR Ltd.). See SEQ ID NO: 6. Thermostable TSH receptors are commercially available. In some commercially available thermostable TSH receptors, the protein is rendered stable by the introduction of six mutations.

[0009] In some embodiments, the binding moiety that can bind to anti-TSH receptor antibody comprises two leucine rich domains of human TSH receptor or variants thereof.

[0010] In some embodiment, the leucine rich domain variant has 95% sequence identity to the human TSH receptor.

[0011] In some embodiment, the leucine rich domain variant is leucine rich domain from a mammalian homologue to the human TSH receptor or a variant thereof. In another embodiment, the leucine rich domain variant is selected from the Markush group consisting of a rat TSH receptor, a mouse TSH receptor, and a cynomolgus monkey receptor. Human TSH receptor (SEQ ID NO: 14), rat TSH receptor (SEQ ID NO: 15), mouse TSH receptor (SEQ ID NO: 16), and cynomolgus monkey TSH receptor (SEQ ID NO: 17) have a high sequence consensus among the species, with at least 86.3% sequence identity with full-length TSH receptor and 87.9% sequence identity with the leucine-rich domain (amino acids 22-260).

[0012] In some embodiments, the binding moiety further comprises an Fc (e.g., human IgGl Fc with LALA / PA mutations) or VHH moiety conjugated to the linker moiety, wherein one or two leucine rich domains or variants thereof are conjugated to one or each of the protein chains of the VHH moiety.

[0013] In some embodiments, the composition of matter (agent, TRAP™) has a structure of:RCN-(Xaa)y-Rcc,[AGN102], or a salt thereof.

[0014] In some embodiments, the composition of matter (agent, TRAP™) has a structure of:[AGN103] or a pharmaceutically acceptable salt thereof, wherein: is a moiety comprising at least one leucine rich domain (LRD) of human TSH receptor or a variant thereof;connected tvia a side chain amino group of a lysine residue of to formeachis an asialoglycoprotein receptor ("ASGPR") binding moiety that is either present or absent, provided that at least oneis present.

[0015] In some embodiments,may be present.

[0016] In some other embodiments,may be absent.

[0017] In some embodiments, the composition of matter may have a structure of:pharmaceutically acceptable salt thereof.

[0018] In some embodiments, the composition of matter may have a structure of:( A J wherein each may be the same or different and may be a moiety comprising one leucine rich domain (LRD) of human TSH receptor or a variant thereof.

[0019] For the compositions of matter AGN332 and AGN333 described in the specification below, the compositions of matter are variants of AGN103 having the structure 6x E groups-D-B-A-A.

[0020] In some embodiments, the composition of matter (agent, TRAP™) has a structure:Formula (I) wherein,each is a moiety comprising a leucine rich domain (LRD) of human TSH receptor or a modified variant thereof;(B) (A) ( c ) each is a means for connecting and ;( C ) each is a carrier polypeptide moiety, which may be a human Fc polypeptide or a derivative thereof having a structure similar to the constant region of SEQ ID NO: 26(amino acids 120-447) (SEQ ID NO: 49) or a sequence having 95% sequence identity therewith, wherein( C ) the moieties are linked together via two disulfide bridgesas shown in Formula (II), wherein one disulfide bridge links cysteine residues located in a position corresponding to position 11 of SEQ ID NO: 26 of each moiety, and wherein the other disulfide bridge links cysteine residues located in a position corresponding to position 14 of a peptide moiety having a structure similar to SEQ ID NO: 26 ofa lysine residue of to formeachis an asialoglycoprotein receptor ("ASGPR") binding moiety, which may be present or absent. In an embodiment, at least onemay include an N-acetyl-D-galactosamine("GalNAc") group having the structureFormula (II)

[0021] In a particular embodiment, the composition of matter is AGN301 , which comprises a thermostable leucine rich domain linked to a Fc-fusion region (thermostable LRD hlgGl Fc-fusion protein) conjugated to 0GN3. This protein corresponds to amino acids 22-260 of the TSH receptor protein. Relative to wild-type human TSH receptor protein, this protein contains mutations LALA / PA, H63C, R112P, D143P, D151E, V169R, and I253R. The conjugation can be accomplished using the MATE®technology. The binding moiety is ABT301 (SEQ ID NO: 24). The cell binding moiety (0GN3) is TBT301. The target BAR is 2. The moiety is 476 amino acids, Relative to wild-type human TSHR receptor, the amino acid mutations are H63C, R112P, D143P, D151E, V169R, I253R, LALA / PA, and C->A. According to the full-length IgGl Heavy Chain Sequence from Uniprot PODOX5 (SEQ ID NO: 26), these mutations are L236A, L237A, and P331A. LALA / PA mutations. The C / A mutation numbering according to the same Uniprot entry PODOX5 is C222A. The molecular weight with 0GN3 and other modifications is about 160 kDa. AGN301 has in vivo activity as low as 0.1 mpk in mouse. AGN301 spontaneously forms and is used as a dimer.

[0022] In some embodiments, the composition of matter (agent, TRAP™) has a structure that is a variant of Formula I above, where the C region comprises a portion of an Fc.

[0023] In some embodiments, the composition of matter (agent, TRAP™) has a structure that is a variant of Formula I above, where the C region is missing or substantially missing.

[0024] In some embodiments, the composition of matter (agent, TRAP™) has a structure that is a variant of Formula I above, where the C region is mutated so that the C-regions do not spontaneously dimerize.

[0025] In a particular embodiment, the composition of matter is AGN302, which comprises a thermostable leucine rich domain conjugated to 0GN3. The conjugation can be accomplished using a sortase enzyme. The anti-TSH receptor antibody binding moiety is ABT302. This protein corresponds to amino acids 22-260 of the TSH receptor protein, also containing a C-terminal sortase tag and aHis6 tag. Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, and I253R, also containing a C-terminal sortase site-His6. (256 amino acids. The cell binding moiety (0GN3) is TBT302. The molecular weight with 0GN3 and other modifications is about 42 kDa. The target BAR is 1.

[0026] In a related aspect, the invention provides AGN303, which comprises a thermostable leucine rich domain conjugated to biotin by sortase linkage. The conjugation can be accomplished using a sortase enzyme. The binding moiety is ABT302. The cell binding moiety (biotin) is TBT303. The target BAR is about 1, e.g., 0.94. AGN303 can be used as a reagent in composition of matter characterization assays.

[0027] In a particular embodiment, the binding moiety is ABT301 (SEQ ID NO: 24), a thermostable leucine rich domain Fc-fusion protein. Of the 476 amino acids, the mutated amino acids are C222A, L236A, L237A, and P331A , and LALA / PA. The molecular weight is approximately 150000 g / mol.

[0028] In a particular embodiment, the binding moiety is ABT302 (SEQ ID NO: 23), a thermostable leucine rich domain protein with a C-terminal sortase site and a His6 tag. Of the 256 amino acids, , the mutated amino acids are H63C, R112P, D143P, D151E, V169R, and I253R. The molecular weight is approximately 40000 g / mol.

[0029] In a particular embodiment, the binding moiety is ABT303, a leucine rich domain protein with a C-terminal sortase site and a His6 tag. Of the 256 amino acids, none are mutated. ABT303 contains a wild-type leucine rich domain region. The molecular weight is about 40000 Daltons.

[0030] In other embodiments, the composition of matter (agent, TRAP™) is selected from the Markush group of the following:

[0031] AGN304 contains a wild-type LRD Fc-fusion protein region (LRD hlgGl Fc-fusion). This protein corresponds to amino acids 22-260 of the TSH receptor protein. This protein contains mutationsLALA / PA.

[0032] AGN305 contains an ectodomain Fc-fusion region.

[0033] AGN306 contains an ectodomain with a sortase tag.

[0034] AGN307 protein contains a thermostable leucine rich domain region (untagged- thrombin). This protein corresponds to amino acids 22-260 of the TSH receptor protein, also containing an N-term His6 tag, GS tag, and thrombin tag. Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, and I253R, with an N-term His6, GS, thrombin tag. (256 amino acids). The molecular weight is 54583.

[0035] AGN308 contains an extended thermostable leucine rich domain region (22-281). Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, and I253R, also containing a C-terminal sortase site-His6 tag. The composition has 271 amino acids and a molecular weight of about 43912 Daltons.

[0036] AGN309 contains an extended thermostable leucine rich domain region (22-289). Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, C283S, and C284S, also containing a C-terminal His6 tag. The composition has 279 amino acids and a molecular weight of about 44812 Daltons.

[0037] AGN310 contains an extended thermostable leucine rich domain region (22-300). Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, C283S, and C284S, also containing a C-terminal His6 tag. The composition has 290 amino acids, and a molecular weight of about 47028 Daltons.

[0038] AGN311 contains an extended thermostable leucine rich domain region (22-316). Relative to wild-type human TSH receptor protein, this ABT418 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, C283S, C284S, and C301S, also containing a C-terminal His6 tag. The composition has 306 amino acids and a molecular weight of about 49520 Daltons.

[0039] AGN312 contains an extended thermostable TSH receptor region (22-302-GSPG-388-402) (22-302, 388-402). Relative to wild-type human TSH receptor protein, this ABT419 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has 312 amino acids, and a molecular weight of about 47360 Daltons.

[0040] AGN313 contains an extended thermostable TSH receptor region (22-302-GGGS-388-402) (22-302, 388-402). Relative to wild-type human TSH receptor protein, this ABT420 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has 312 amino acids, and a molecular weight of about 48266 Daltons.

[0041] AGN314 contains an extended thermostable TSH receptor region (22-289-GSPG-396-402) (22-289, 396-402). Relative to wild-type human TSH receptor protein, this ABT421 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has 290 amino acids, and a molecular weight of about 44631 Daltons.

[0042] AGN316 contains an extended thermostable TSH receptor region (22-289-GGGS-396-402) (22-289, 396-402). Relative to wild-type human TSH receptor protein, this ABT422 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has 290 amino acids and a molecular weight of about 44883 Daltons.

[0043] AGN317 contains an extended thermostable TSH receptor region (22-402). Relative to wildtype human TSH receptor protein, this ABT423 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has 392 amino acids and a molecular weight of possibly 59500 Daltons.

[0044] AGN318 contains a thermostable LRD Fc-fusion region with a C-terminal sortase tag (22-260, Human IgGl). Relative to wild-type human TSH receptor protein, this ABT506 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R; LALA / PA; C->A, The LALA / PA; C->A mutations are C222A, L236A, L237A, and P331A. The composition has 487 amino acids, and a molecular weight of about 130000 Daltons.

[0045] AGN319 contains a dimeric thermo-LRD (GGGGS)x2 region with a sortase region (22-260).Relative to wild-type human TSH receptor protein, this protein contains mutations C222A, L236A, L237A, and P331A, also containing a C-terminal His6 tag. The composition has 510 amino acids and a molecularweight of about 74000 Daltons. ABT111 has the structure of LRD-(GGGGS)2-LRD-sortase site-His6 tag.The inventors have conjugated ABT111 to a cellular binding moiety.

[0046] AGN320 contains a dimeric thermo-LRD (GGGGS)x4 region with a sortase region (22-260). Relative to wild-type human TSH receptor protein, this ABT112 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, also containing a C-terminal His6 tag. The composition has 510 amino acids, and a molecular weight of about 74000 Daltons ABT112 has the structure of LRD-(GGGGS)4-LRD- sortase site-His6 tag. The inventors have conjugated ABT112 to a cellular binding moiety.

[0047] AGN322 contains a thermostable TSH receptor (22-289-GSPG-396-402) region with a sortase region (22-289, 396-402). Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has 301 amino acids, and a molecular weight of about 45792 Daltons ABT113 has the structure of TSH receptor amino acids 22-289-GSPG-396-402-sortase. The inventors have conjugated ABT113 to a cellular binding moiety.

[0048] AGN323 contains a thermostable TSH receptor (22-289-GGGS-396-402) region with a sortase region (22-289, 396-402). Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has 301 amino acids, and a molecular weight of about 45751 Daltons. ABT114 has the structure of TSH receptor amino acids 22-289-GGGS-396-402-sortase. The inventors have conjugated ABT114 to a cellular binding moiety.

[0049] AGN324 contains a thermostable LRD-Fc-fusion minus CH3 sortase (22-260, human IgGl (without CH3)). Relative to wild-type human TSH receptor protein, this protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C284S, also containing a C-terminal His6 tag. The composition has about 386 amino acids.

[0050] AGN325 contains a thermostable LRD short hinge region (22-260, Human IgGl Hinge). Relative to wild-type human TSH receptor protein, this ABT428 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C222A mutation of human IgGl hinge region, also containing a C- terminal His6 tag. The composition has about 281 amino acids.

[0051] AGN326 contains a thermostable LRD full hinge region (22-260, human IgGl hinge). Relative to wild-type human TSH receptor protein, this ABT429 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, C222A, L236A, and L237A mutations of human IgGl hinge region, also containing a C-terminal His6 tag. The composition has about 284 amino acids.

[0052] AGN327 contains a thermostable LRD-hinge-CH3 region (22-260, human IgGl hinge and CHS). Relative to wild-type human TSH receptor protein, this ABT446 protein contains mutations H63C, R112P, D143P, D151E, V169R, I253R, and C222A mutation of human IgGl hinge region. The composition has about 388 amino acids. Of the human IgGl Fc region, ABT546 contains a CH3 region only, with the structure of LRD-hinge-CH3-sortase. The inventors are conjugating ABT546 to a cellular binding moiety.

[0053] AGN328 has an extended leucine rich domain with a GSPG linker.

[0054] AGN329 has an extended leucine rich domain with a GGGS linker.

[0055] AGN330 comprises a leucine rich domain dimer with a (GGGGS)x2 linker, GN3.

[0056] AGN331 comprises a leucine rich domain dimer with a (GGGGS)x4 linker, GN3.

[0057] AGN332 comprises a leucine rich domain dimer with a (GGGGS)x2 linker, dimeric GN3.

[0058] AGN333 comprises a leucine rich domain dimer with a (GGGGS)x4 linker, dimeric GN3. SeeSEQ ID NO: 25. AGN333 is similar to AGN301 (dimer) but with only hinge and CH3 domains of Fc.

[0059] AGN334 comprises a leucine rich domain linked to truncated Fc (hlgGl hinge-CH3 domains only), with a GN3 cellular binding moiety. AGN334 is similar to AGN301 (dimer) but with only hinge and CH3 domains of Fc.

[0060] In another embodiment, the cellular receptor-binding moiety comprises an ASGPR binding group according to the chemical structure:XR1^O^ R3OHwherein the cellular receptor-binding moiety has additional elements described in this specification. Groups Ri, R2, R3, and X may be the same as described in International Patent Publication WO 2019 / 199634 and International Patent Publication WO 2019 / 199621.

[0061] In a specific embodiment, the cellular receptor-binding moiety is TBT301 or a conjugation reaction derivative thereof. See FIG. 4.

[0062] In another embodiment, the invention provides a method of making the composition of matter (agent, TRAP™).

[0063] In a specific embodiment, the method of making the composition of matter comprises the step of conjugating ABT301 (thermostable leucine rich domain (LRD)-Fc) and TBT301 to make the AGN301 composition of matter.

[0064] In another embodiment, the method of making the composition of matter comprises the step of conjugating ABT302 (thermostable leucine rich domain (LRD)) and TBT302 to make the AGN302 composition of matter.

[0065] In some embodiments, anti-TSH receptor antibody-binding moieties are connected to antibody moieties through certain types of groups or amino acid residues. In some embodiments, anti- TSH receptor antibody-binding moieties are connected to lysine residues optionally through linker moieties. In some embodiments, anti-TSH receptor antibody-binding moieties are connected to cysteine residues optionally through linker moieties. In some embodiments, anti-TSH receptor antibody-binding moieties are connected to unnatural amino acid residues optionally through linker moieties. In some embodiments, the invention provides technologies for selectively linking anti-TSH receptor antibodybinding moieties to certain amino acid residues optionally through linker moieties. In some embodiments, provided technologies selectively connect anti-TSH receptor antibody-binding moieties to certain types of amino acid residues, e.g., lysine residues, optionally through linker moieties. In some embodiments, provided technologies selectively connect anti-TSH receptor antibody-binding moieties to sites of antibody moieties optionally through linker moieties. In some embodiments, provided technologies selectively connect anti-TSH receptor antibody-binding moieties to certain types of amino acid residues at sites optionally through linker moieties. In some embodiments, anti-TSH receptor antibody-binding moieties are connected to K246 or K248 or both of an IgGl heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, anti-TSH receptor antibody-binding moieties are connected to K251 and K253 of an lgG2 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, anti-TSH receptor antibody-binding moieties are connected to K239 and K241 of a heavy chain and amino acidresidues 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.

[0066] In another embodiment, the invention provides a method of removing anti-TSH receptor antibodies from a subject by administering the composition of matter (agent, TRAP™) to the subject. In yet another embodiment, the composition of matter is administered so that in vivo activity is as low as 0.1 mpk, based upon results shown in this specification.

[0067] In another embodiment, the invention provides a method of treating a disease state or condition associated with the upregulation of anti-TSH receptor antibody, such as Graves' disease, Graves orbitopathy, or thyroid eye disease, in a patient by administering an effective amount of the agent to the patient.

[0068] In another embodiment, the invention provides a method of treating Graves' disease, Graves orbitopathy, or thyroid eye disease, in a patient by administering an effective amount of the composition of matter (agent, TRAP™) to the patient.

[0069] In another embodiment, the invention provides a pharmaceutical composition including the agent and at least one pharmaceutically acceptable excipient.

[0070] 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 accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0071] 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.

[0072] FIG. 1 is a diagram showing that the leucine-rich domain (LRD) of the thyroid stimulating hormone receptor (TSHR) binds agonistic autoantibodies (TRAbs) according to an embodiment of the invention. Anti-TSH receptor autoantibodies bind the extracellular leucine-rich domain of TSH receptor. The antibody binding overlaps with that of the thyroid stimulating hormone (TSH), the native ligand. See Faust et al., Nature (2022). The inventors selected the leucine-rich domain as bait for bifunctional degrader development.

[0073] FIG. 2 is a diagram showing a concept of incorporating the leucine-rich domain (LRD) domain of the thyroid stimulating hormone receptor into a degrader molecule according to an embodiment of the invention.

[0074] FIG. 3 is a diagram showing site-specific conjugation of the degrader portion to the Fc domain of the protein using MATE® technology according to embodiments of the invention.

[0075] FIG. 4 shows the structures of reagents TBT301 and TBT302 according to two embodiments of the invention.

[0076] FIG. 5 shows the results of AGN301 and AGN302 binding to patient-derived anti-TSHR antibody M22 in a surface plasmon resonance assay. Surface plasmon resonance (SPR) assays assessed the binding of bifunctional degraders to anti-TSH receptor tool antibody.

[0077] FIG. 6 shows the formation of the ternary complex between ASGPR and anti-TSHR antibody M22 for TSHR degraders AGN301 and AGN302 in the TR-FRET assay. Ternary complex formation assays showed bifunctional degrader-induced ternary complex of anti-TSH receptor tool antibody (M22) and ASGPR. This TR-FRET, ternary complex formation assay uses 12.5 nM mouse anti-TSH receptor antibody (M22 antibody) and 50 nM ASGPR1 (biotinylated ASGPR CRD). The reported EC5o correspond to complex formation. The bifunctional degraders and baits (ABT301 and ABT302) tested from 1 pM, three-fold dilutions, 16 points. This FIG. 6 shows that these bifunctional degraders mediate concentrationdependent ternary complex between ASGPR and anti-TSH receptor antibody (M22 antibody. The resulting bell-shaped curve is typical for bifunctional ligand interactions.

[0078] FIG. 7 shows the capture of anti-TSHR IgG from patient samples by THSR-leucine rich domain protein baits ABT301 and ABT302 according to two embodiments of the invention. In the Meso Scale Discovery assay, immobilized ABT301 (Fc-fusion) and ABT302 (sortase) TSHR-leucine rich domain protein baits capture IgG. The anti-TSHR status was determined by clinical-grade ELISA using a TSHR- leucine rich domain protein bait. This FIG. 7 shows that the baits of bifunctional degraders bind anti-TSH receptor autoantibodies from Graves' disease patient samples.

[0079] FIG. 8 is a diagram showing that the leucine rich domain protein bait selectively engages patient IgG by competitive Meso Scale Discovery. The binding of serum IgG to a TSH receptor-leucine rich domain bait (ABT301, Fc-fusion) was effectively competed by a soluble TSH receptor-leucine rich domain protein in Graves patient samples but not a control. The Meso Scale Discovery assay included immobilized leucine rich domain protein bait (Fc-fusion). The assay detected IgG (% depletion). The competition was with leucine rich domain protein bait (sortase).

[0080] FIG. 9 shows results of the cellular internalization of the agonistic, patient-derived anti-TSH receptor antibody upon addition of AGN301 or AGN302, in a concentration-response manner.Internalization assays showed bifunctional degrader-induced internalization of anti-TSH receptor tool antibody (M22) via ASGPR-mediated uptake. AGN301 and AGN302 internalize M22 antibody in cells. The endocytosis cellular assay uses HEK293-ASGPR1 cell line. The anti-TSH receptor antibody M22 is directly conjugated to AlexaFluor-647. FA = fluorescence area. The EC5o fitting was limited to [Ligand] < 34 nM. The data points are mean ± SEM from two technical replicates. This FIG. 9 shows that these bifunctional degraders mediate the uptake of patient-derived anti-TSH receptor antibody (M22 antibody) into ASGPR-expressing cells.

[0081] FIG. 10 shows results of the in vivo pharmacokinetic / pharmacodynamic study with AGN301 Fc-fusion bifunctional degrader in mouse, dosed intravenously (IV). M22 antibody was dosed at 0.016 mpk to match reported Graves' disease patient concentration (~200 ng / ml). The top line graph shows the results of the AGN301 pharmacokinetic (IV) assay. The bottom line graph shows the results of the AGN301 pharmacodynamic (IV) assay. The 8 mpk dose achieved higher Cmax than 3 mpk. AGN301 (3 mpk intravenous) achieved ~95% depletion of added M22 antibody within two hours. Both doses achieved similar reduction of added M22 antibody. 8 mpk = '“500:1 D:T. 3 mpk = ~200:l D:T. Dosed intravenously, AGN301 is rapidly cleared to about l / 1000ththe concentration, stabilizing at 100 ng / ml. No rebound of M22 antibody was observed.

[0082] FIG. 11 shows results of the in vivo pharmacokinetic / pharmacodynamic study with AGN301 in mouse, dosed subcutaneously (SC). The top line graph shows the results of the AGN301 pharmacokinetic (SC) assay. The bottom line graph shows the results of the AGN301 pharmacodynamic (SC) assay. Both doses of AGN301 (3 mpk and 8 mpk) achieved robust depletion of the added M22 antibody, ~95%. Both doses achieved similar M22 reduction. No rebound of M22 antibody was observed. The in vivo pharmacodynamic results of FIG. 10 and FIG. 11 show that AGN301 rapidly depletes patient-derived anti-TSH receptor (M22 antibody) in mice both intravenously and subcutaneously.

[0083] FIG.12 shows results of the in vivo pharmacodynamic dose-titration of AGN301 in mouse, dosed subcutaneously (SC). All dose levels tested (3 mpk, 0.3 mpk, and 0.1 mpk), successfully depleted >90% of the added M22 antibody (0.4 pg / mouse). AGN301 maintains potent activity at a dose level of 0.1 mpk.

[0084] FIG. 13 shows the in vivo pharmacokinetic / pharmacodynamic study with AGN302 sortase- conjugated bifunctional degrader in mouse, dosed intravenously (IV). M22 antibody was dosed at 200 ng / mL (0.2 mpk) to match average reported anti-TSH receptor autoantibodies (TRAbs) in Graves' disease patients. See Nakatake et al., Thyroid, 16(11), 1077-84 (2006). AGN302 (8 mpk intravenous) achieved ~80% depletion of added M22 antibody within two hours. The 0.8 mpk dose was less effective. For this AGN302 assay, some rebound of M22 antibody occurs between two to seven hours.

[0085] FIG. 14 shows the in vivo pharmacokinetic / pharmacodynamic study with AGN302 in mouse, dosed subcutaneously (SC). AGN302 (8 mpk subcutaneously) rapidly depleted ~98% of the added M22 antibody.

[0086] FIG. 15 is a table (TABLE 1) of R-Groups for MATE® reagents and bifunctional MoDE final compounds.

[0087] FIG. 16 is a sequence comparison of the constant heavy chains of human IgGl, lgG2, and lgG4, which can be therapeutic antibodies. Sequence differences in lgG2 and lgG4 from IgGl are noted. The hinge and lower hinge regions, with the N-glycosylation site, N297, are noted. Numbering is according to the EU numbering scheme. Sequences important for FcgR binding, Clq, and FcRn are noted by shading. Some increased ADCC mutants are shown above or below the sequences, including Xencor's S239D, A330L, I332E; Genentech's S298A, E333A, K334A; and Eli Lilly / AM E's P247I, A339D / Q. Examples of FcRn-binding mutants for prolongation of half-life are also shown, including Medlmmune's YTE mutant (M252Y, S254T, T256E), PDL's T250Q, M428L mutant, Sally Ward's H433K mutant, N434Y mutant, and Genentech's N434A mutant are shown.Y1

[0088] FIG. 17 is a set of diagrams showing several configurations of alternative formats for the compositions of matter (agent, TRAP™).

[0089] FIG. 18 is a line graph showing representative EC5o values from an endocytosis degradation assay for several LRD constructs.

[0090] FIG. 19 Is a dot graph showing the results of the in vivo efficacy, on thyroids, of AGN301 or AGN302 dosed subcutaneously (SC) following intravenous administration of randomly biotinylated M22 antibody (bt-M22).DETAILED DESCRIPTION OF THE INVENTION

[0091] The following detailed description is provided to aid persons having ordinary skill in the biomedical art. Exemplary embodiments are described, but 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

[0092] The invention provides a medically useful composition of matter (agent, TRAP™) for treating or slowing down the progression of Graves' disease, Graves orbitopathy (GO), thyroid eye disease (TED), or other diseases associated with autoantibodies that bind the TSH receptor (TRAbs). Autoimmune thyroid disease is one of the most common autoimmune conditions. See Pokhrel & Bhusal, Graves' Disease. In StatPearls [Internet] (Treasure Island (FL), StatPearls Publishing, January 2024). Early diagnosis and management of Graves' disease can also prevent severe cardiac complications such as atrial flutter, atrial fibrillation, and high-output cardiac failure.

[0093] Anti-TSH receptor antibodies are agonistic and pathogenic in Graves' disease.

[0094] For the pathogenesis of Graves' disease, anti-TSH receptor autoantibodies act as TSH receptor agonists to induce thyroid cells to secrete excess thyroid hormones.

[0095] For Graves' disease diagnosis, the gold standard is a positive TSH receptor autoantibody test. The amount of stimulating anti-TSH receptor antibodies correlate with the clinical activity score.Definitions

[0096] 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.

[0097] As used in this application, except as otherwise expressly provided in this specification, each of these terms 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.

[0098] The articles "a" and "an" have the plain meaning of one or 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.

[0099] 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.

[0100] The term "agent" has the biomedical art-recognized meaning of a composition of matter useful for performing a function. Several biomedically useful functions are described in this specification.

[0101] 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 decreased. 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.

[0102] The term "asialoglycoprotein receptor (ASGPR) binding group" has the biomedical art- recognized meaning of a binding group that binds to a hepatocyte asialoglycoprotein receptor. The ASGPR binding group selectively binds to hepatocyte asialoglycoprotein receptors on the surface of hepatocytes. In several embodiments of this specification, an ASGPR binding group is a component of a bifunctional agent as a cellular receptor-binding moiety covalently bound to the antibody-binding moiety through a linker group or directly. Bifunctional agents complexed with a circulating protein, e.g., bind to hepatocytes, through this ASGPR moiety. After the bifunctional agent complexed with a circulating protein is bound to a hepatocyte or other cell, the circulating protein is taken into thehepatocyte or other cell via an endocytosis mechanism, wherein the circulating protein is degraded through lysosomal degradation.

[0103] 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.

[0104] 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.

[0105] The term "AT" or "ABT" means a binding moiety that binds to or is bound by an anti-TSH receptor antibody.

[0106] The term "autoantibody" has the biomedical art-recognized meaning. TSH receptor autoantibodies (TRAbs) bind to the TSH receptor. TSH receptor autoantibodies with stimulating activity are responsible for hyperthyroidism in Graves' disease. TSH receptor autoantibodies with blocking activity may cause hypothyroidism. Rees Smith, McLachlan, & Furmaniak, Endocrine Reviews, 9, 106- 121 (1988); Rapoport et al., Endocrine Reviews. 19, 673-716 (1988); Furmaniak et al., Clinical Endocrinology, 96, 878-887 (2022). In a first example of an autoantibody, Kl-70™ IgGl lambda is a blocking type human monoclonal autoantibody to the TSH receptor obtained from the peripheral blood lymphocytes of a patient with hypothyroidism. See Inti. Pat. Publ. WO 2010 / 073012; Evans et al, Clinical Endocrinology 73, 404-412 (2010). Kl-70™ is a powerful inhibitor of TSH action. Nunez Miguel et al., Structure of full-length TSH receptor in complex with antibody Kl-70™. Journal of Molecular Endocrinology, 70, e220120 (2022). Kl-70™ IgG can be purified from heterohybridoma culture supernatants. Fab can be manufactured using mercuripapain. Sanders et al., Journal of Molecular Endocrinology, 46, 81-99 (2011). The Kl-70™ Fab structure consists of Kl-70™ heavy chain (HC) residues Glnl to Ser229 and light chain (LC) residues Ser2 to Ala212 and is the structure of a typical Fab fragment. Kl-70™ IgGl lambda is commercially available from RSR limited (Order Code K170 / FR / 1.0). In a second example of an autoantibody, human monoclonal TSH receptor autoantibody M22™ IgGl lambda is a powerful stimulator of the TSH receptor. See European Pat. No. EP1565493B1. M22™ is commercially available from RSR Limited (Order Code M22 / FR / 1.0). In a third example of an autoantibody, mouse monoclonal TSH receptor antibody 14C4 binds to a conformational epitope within amino acids 22-260 of the TSH receptor distinct from the Kl-70™ binding site. A fourth example is Kl-18™ IgGl kappa, which is commercially available from RSR limited (Order Code K118 / FR / 1.0).

[0107] The term "autoimmunity" has the biomedical art-recognized meaning of the system of immune responses of an organism against its own healthy cells, tissues and other normal body constituents. Autoimmunity means the presence of antibodies or T cells that react with self-protein. Self-reactivity can lead to tissue damage. A disease resulting from this type of immune response is an "autoimmune disease".

[0108] The term "binder to antibody ratio" (BAR) has biomedical art-recognized meaning when used in making antibody conjugates. In this specification, the terms DAR and BAR are interchangeable.

[0109] The term "binding moiety" has the biomedical art-recognized meaning of a moiety on a binding protein, e.g., an antibody, an antibody variant, or an antigen-binding fragment thereof, that binds to the anti-TSH receptor antibody.

[0110] The term "cellular receptor-binding moiety" has the biomedical art-recognized meaning. In several embodiments of this specification, the cellular receptor-binding moiety is an asialoglycoprotein receptor (ASGPR) binding group.

[0111] 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.

[0112] The term "chimerized" has a 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. Through these biotechnical manipulations, chimeric antibodies retain the foreign antibody's antigen specificity and affinity.

[0113] The term "drug to antibody ratio" (DAR) has biomedical art-recognized meaning when used in making antibody conjugates. In this specification, the terms DAR and BAR are interchangeable.

[0114] The term "conservative substitution" has the biomedical art-recognized meaning. For example, Sequence identity can be measured using sequence analysis software, for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, or other modifications. Conservative substitutions include substitutions within these groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. A residue having similarphysiochemical characteristics can replace an amino acid, e.g., substituting one aliphatic residue for another, such as He, Vai, Leu, or Ala for one another,, or substitution of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn. Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known.Polypeptides comprising conservative amino acid substitutions can be tested in any of the assays to confirm that the desired activity, e.g., the ability to bind to anti-TSH receptor antibodies.

[0115] The term "epitope of a TSH receptor" has the biomedical art-recognized meaning and includes epitopes that are recognized by anti-TSH receptor antibodies. A first epitope (Kl-70™) binds to the TSH receptor well clear of the lipid bilayer. Kl-70™ Fab binding is principally to the concave surface of the TSH receptor leucine rich domain (LRD) which has no glycans attached. Kl-70™ Fab HC and LC bind to the N-terminal leucine-rich repeats (LRR) of the TSH receptor from amino acid Glu35 in the TSH receptor N-cap to Lysl83 in the 7th LRR. There is a mixture of an extensive hydrogen bonding and salt bridge network (twenty hydrogen bonds and salt bridges), five ion pairs, thirteen polar interactions, and thirteen hydrophobic / aromatic contacts. Differences in some of the interactions seen in the cryo-EM structure of the full-length TSH receptor bound to Kl-70 Fab compared to the crystal structure of the TSH receptor leucine rich domain in complex with Kl-70 Fab are most likely due to side chain flexibility in the interacting amino acids. There are no interactions between the Kl-70™ Fab and either the HR or the TMD. A second epitope binds to the TSH receptor at a region where it would clash with the bilayer unless the TSH receptor HR rotates upwards as part of the M22™ binding process. M22™ interacts with TSH receptor amino acids more C-terminal than those that interact with Kl-70™. Sanders et al., Journal of Molecular Endocrinology, 46, 81-99 (2011). This 'push' process may explain the observations of Chazenbalk et al., Journal of Clinical Investigation 110 209-217 (2002), who noted that thyroid- stimulating antibodies, but not thyroid-blocking antibodies were partially sterically hindered when binding to the full-length TSH receptor compared to the TSH receptor ECD alone. A partial obstruction of the thyroid-stimulating autoantibody binding site may lead to a torsion effect on the TSH receptor ECD, on antibody binding, which may explain how thyroid-stimulating autoantibodies were able to activate the TSH receptor. Nunez Miguel et al., Journal of Molecular Endocrinology, 70, e220120 (2022).

[0116] The term "Graves' disease," also known as toxic diffuse goiter or Basedow's disease, has the biomedical art-recognized meaning of an autoimmune disease characterized by the presence of anti- TSHR auto antibodies that lead to a generalized overactivity of the thyroid gland (hyperthyroidism). Classic symptoms of Graves' disease include exophthalmos, eyelid retraction, insomnia, muscle weakness, tremor, oligomenorrhea, dyspnea, arrhythmia, and fatigue. Persons having ordinary skill inthe biomedical art diagnose Graves' disease using methods such as blood tests assaying body levels of anti-TSHR antibodies, thyroid-stimulating hormone (TSH), thyroid hormones, radioactive iodine uptake assays.

[0117] The term "Graves' ophthalmopathy" (GO) has the biomedical art-recognized meaning of an autoimmune disorder affecting the tissues around the eyes.

[0118] The term "thyroid eye disease" (TED) has the biomedical art-recognized meaning of an autoimmune disorder affecting the tissues around the eyes.

[0119] The term "Hashimoto thyroiditis" has the biomedical art-related meaning of an autoimmune thyroiditis when the autoantibodies that attack the thyroid cells. Many Hashimoto thyroiditis patients have hypothyroidism. See Choe, Durgannavar, & Chung, Materials (Basel), 9(12), 994 (December 8, 2016). While Hashimoto's thyroiditis and Graves' disease can coexist as comorbidities, Hashimoto's thyroiditis is linked to anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin autoantibodies, not anti- TSHR.

[0120] 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.

[0121] The term "humanized" has the biomedical art-recognized meaning that a protein, e.g., an antibody, is genetically engineered to closely resemble 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, thus retaining the specificity of the rodent antibody. The human origin domain need not originate directly from a human when it is first synthesized in a human. Human domains can be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially or completely humanized. In one approach, four general steps are used to humanize a monoclonal antibody. These steps 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.

[0122] The term "IC5o" 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.

[0123] The term "IgG" antibody has the biomedical art-recognized meaning. Each IgG molecule consists of the basic four-chain immunoglobulin structure— two y (gamma) heavy chains and twoidentical 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.

[0124] The term "IgGl" antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-1 chain C region is a protein encoded by the IGHG1 gene in humans.

[0125] The term "lgG2" antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-2 chain C region is a protein encoded by the IGHG2 gene in humans.

[0126] The term "lgG4" antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-4 chain C region is a protein encoded by the IGHG4 gene in humans. lgG4 has little effector function. lgG4 cannot fix complement.

[0127] The term "I VIG" has the biomedical art-recognized meaning of administering intravenous immunoglobulin (I VIG).

[0128] 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.

[0129] The term "leucine rich domain" (LRD) has biomedical art-recognized meaning of the extracellular portion of the TSH receptor.

[0130] 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.

[0131] The term "MoDE" has the proprietary meaning of molecular degraders. See International Pat. Publ. WO 2019 / 199634 (Yale University) and International Pat. Publ. WO 2019 / 199621 (Yale University).

[0132] 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. In some embodiments, a binding moiety maintains one or more desired structural features, properties, functions, or properties, e.g., 3-dimension structure, antigen specificity, antigen-binding capacity, or immunological functions, etc., comparable to its corresponding binding protein, e.g., an antibody. In some embodiments, a moiety is monovalent. In some embodiments, a moiety is bivalent. In other embodiments, a moiety is polyvalent.

[0133] The term "monotherapy" is a biomedical art-recognized term for administering a single active or therapeutic compound to a subject or patient in need. Monotherapy usually involves administering a therapeutically effective amount of an active composition.

[0134] The term "Multimodal Antibody Therapy Enhancers (MATE®)" has the proprietary meaning. See International Pat. Publ. WO 2021 / 102052 (Kleo Pharmaceuticals). This antibody conjugation technology enables site-directed pairing with therapeutic monoclonal antibodies (mAbs), or therapeutic immunoglobulin (IG) pooled from donors. Persons having ordinary skill in the biomedical art can use MATE® materials and methods as guidance to predictable results when making and using the invention.

[0135] The term "on" has the plain meaning. When an element is referred to as being on another element, it can be directly in contact with the other element, or intervening elements may be present therebetween. When an element is referred to as being "directly on" another element, no intervening elements are present.

[0136] 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.

[0137] The term "other degrading cells" has the biomedical art-recognized meaning. Asialoglycoprotein receptors (ASGPRs) are found on hepatocytes and the glandular cells of the gallbladder and stomach.

[0138] The term "partially humanized" has the biomedical art-recognized meaning that a protein, e.g., an antibody, is genetically engineered to resemble the polypeptide structure of the human homologue more closely. A variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, thus retaining 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. Human domains can be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially or completely humanized.

[0139] 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).

[0140] The term "pharmaceutically acceptable" has the biomedical art-recognized meaning of those compounds, anions, cations, materials, compositions, carriers, or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animalswithout excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0141] 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.

[0142] The term "ROC" has the biomedical art-recognized meaning of the receiver operating characteristic curve.

[0143] The term "TRAP™" has the proprietary meaning of an agent for the targeted removal of aberrant protein. A TRAP is a bifunctional degrader.

[0144] The term "TBT" has the biomedical art-recognized meaning of a cellular receptor-binding moiety. In some embodiments of this specification, the TBT binds to ASGPR.

[0145] The term "thyroglobulin" (Tg) has the biomedical art-recognized meaning. Thyroglobulin autoantibodies (TgAbs) are serological markers of thyroid autoimmunity disease, including Hashimoto's thyroiditis, Graves' disease, a post-partum thyroiditis, and other autoimmune diseases. Rees Smith et al., Thyroid, 17, 923-938 (2007); Choe, Durgannavar, & Chung, Materials (Basel), 9(12), 994 (December 8, 2016).

[0146] The term "thyroid peroxidase" (TPO) has the biomedical art-recognized meaning. Thyroid peroxidase autoantibodies are serological markers of thyroid autoimmunity disease, includingHashimoto thyroiditis, Graves' disease, a post-partum thyroiditis, and other autoimmune diseases. Rees Smith et al., Thyroid, 17, 923-938 (2007); Choe, Durgannavar, & Chung, Materials (Basel), 9(12), 994 (December 8, 2016).

[0147] The term "thyroid stimulating hormone receptor" (TSH receptor, TSHR) has the biomedical art-recognized meaning of a G-protein coupled receptor (GPCR) that regulates thyroid function. TSH receptors are present on the basal membrane of thyroid follicular epithelial cells. Binding of thyroid stimulating hormone (TSH) to the TSH receptor starts the activation of the TSH receptor signaling cascade which involves binding of G-proteins to the TSH receptor followed by stimulation of the cyclic AMP pathway. Sanders et al., Ball iere's Clinical Endocrinology and Metabolism, 11, 451-479 (London, Ball iere Tindall, 1997), Latif et al., Endocrinology and Metabolism Clinics of North America, 38, 319-341 (2009). The TSH receptor can be a human wild-type TSH receptor having the sequence shown in SEQ ID NO: 4 or a functional fragment thereof. In one embodiment, the TSH receptor is a mutated human TSH receptor or a functional fragment thereof. International Pat. Publ. WO 2015 / 189543 describes mutated human TSH receptor functional fragments of the extracellular domain of the TSH receptor (see SEQ IDNO: 5), wherein the mutant or fragment thereof comprises one, two, three, four, five or six mutations within residues 22-260 selected from P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D143P, D151E, S166T, I167F, P168Y, V169R, N170W, T179C, I253R and R255Y, wherein the mutant TSH receptor fragment thereof has increased thermostability with respect to the equivalent wild type TSH receptor. A thyroid- stimulating hormone receptor protein has a leucine-rich domain (LRD), a hinge region (HR), and a transmembrane domain (TMD). The TSH receptor extracellular domain (ECD) is composed of the leucine rich domain and hinge region. The structure of the ECD, formed by the leucine-rich domain and hinge region, is that of a typical leucine-rich-repeat (LRR) structure with eleven repeats in the leucine-rich domain and one repeat in the hinge region. See FIG. 1. Each leucine-rich-repeat has a parallel 0-strand on its concave surface while the N-terminal cap (N-cap) has an additional 0-strand antiparallel to the 0- strand of the first repeat. The leucine-rich domain and the HR form a continuum structure with the N- cap, with two disulfide bonds, and a C-terminal cap (C-cap) with three disulfide bonds and an alpha helix. One of the two N-terminal disulfide bonds between Cys31 and Cys41 is visible in the structure while the other one between Cys24 and Cys29 is not. All three intra-domain disulfide bonds which form the C-cap are visible in the structure between Cys283 and Cys398, Cys284 and Cys408, and Cys301 and Cys390. The first twenty-one amino acids of the TSH receptor make up the cleaved signal peptide. See also Choe, Durgannavar, & Chung, Materials (Basel), 9(12), 994 (December 8, 2016).

[0148] The term "thyroid-stimulating hormone" (TSH, thyrotropin, thyrotropic hormone) has biomedical art-recognized meaning of the pituitary hormone that stimulates the thyroid gland to produce thyroxine (T4), and then triiodothyronine (T3) which affects almost every tissue in the body.

[0149] The term "TSHR260" has the biomedical art-recognized meaning of a subdomain of TSH receptor consisting of residues 22-260 and encompassing most of the leucine-rich domain (LRD). Sanders et al., Thyroid, 17, 395-410 (2007). TSHR260 shows similar binding to TR antibodies as the full- length TSH receptor. See Rees Smith et al., Hormone and Metabolic Research, 41, 448-455 (2009) and International Patent Publication WO 2010 / 073012 (RSR Limited). The structure of TSHR260 remained essentially unchanged upon ligand binding. Miller-Gallacher et al., Journal of Molecular Endocrinology, 62, 117-128 (2019). TSHR260 is commercially available from RSR Limited (TSHR260 STABL™, Order Code STABL / FR / 1.0). TSHR260-JMG55™ maintains select properties of the native domain.

[0150] 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.

[0151] The term "variant" has the biomedical art-recognized meaning of an alteration in a polynucleotide sequence or a polypeptide sequence. The term variant can be used to describe an alteration that may be significant or of unknown significance. The term variant is often used for the term mutation. See NCI Dictionary of Genetics Terms.

[0152] The term "VHH" has the biomedical art-recognized meaning. VHH has nine 0-sheets forming a cylindric structure.

[0153] The terms "an effective amount" and "a therapeutically effective amount" have the biomedical art-recognized meaning of an amount effective to achieve its intended purpose. The effect can be detected by any assay method known in the art. The precise effective amount for a subject depends 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 given 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.

[0154] The terms "combination therapy" and "co-therapy" have 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 coaction of these therapeutic agents. The beneficial effect of the combination may include but is not limited to pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. These therapeutic agents are typically administered in combination 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 described above in 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 combination of the therapeutic agents 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.

[0155] The terms "comprises," "comprising," "includes," and "including" specify the presence of stated features, regions, integers, steps, operations, elements, or components but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, or groups thereof.

[0156] The terms "first," "second," "third," etc., have the plain meaning of describing several elements, components, regions, layers, or sections. These terms should not limit these elements, components, regions, layers, or sections. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. 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.

[0157] The terms "subject" and "patient" have the biomedical art-recognized meanings. The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0158] The terms "treating" and "treat" have the biomedical art-recognized meaning of managing and caring for a patient to combat a disease, condition, or disorder. Treating includes administering 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.

[0159] Some embodiments are described below by referring to structures and schemes to explain aspects of the description.

[0160] 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.

[0161] This specification does not concern a process for cloning humans, methods for modifying the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or procedures for modifying the genetic identity of animals likely to cause them suffering with no substantial medical benefit to humans or animals resulting from these processes.Methods of removal of autoantibodies from a subject or patient.

[0162] How to administer. The best mode of administration depends on where treatment is taking place, whether a hospital or outpatient. In one embodiment, the method of administration is by subcutaneous administration to a patient or subject of 50 mg / ml of the composition of matter (agent, TRAP™).

[0163] After the bifunctional degrader and the bound anti-TSH receptor autoantibody protein are endocytosed, they are released from the ASGPR through calcium depletion from the endosome and changes in binding site amino acid protonation due to decreased pH. The ASGPR is recycled back to the hepatocyte surface. Endocytosed proteins are trafficked to late endosomes, which are fused withlysosomes. Lysosomal proteases then degrade endocytosed proteins, permanently removing them from circulation.

[0164] The confirmation of anti-TSH receptor autoantibody presence in a subject or patient can be measured by methods known to persons having ordinary skill in the art. The inventors tested samples tested in the KRONUS TRAb ELISA. Standard curve of calibrators from 1 to 40 U / L was made. Samples were marked as positive when above the suggested diagnostic threshold of 1 U / L.

[0165] Several types of assays are used by persons having ordinary skill in the biomedical art to measure TSH receptor autoantibodies. See European Pat. No EP1021721B1 and Inti. Pat. Publ. WO 2015 / 189543. For example, Elecsys® Anti-TSHR (TRAK) (Roche Diagnostics) is a fully automated test for detection of autoantibodies to the TSH receptor.

[0166] The 4E31 antibody is a mouse monoclonal antibody to residues 603-764 of the C-terminus of the TSH receptor (C-TSHR), which can be used to immobilize the full-length TSH receptor onto ELISA plate wells. See EP1021721B1 (RSR Ltd.) and Bolton et al., Clin Chem., 45,2285-2287 (1999). The 4E31 antibody can be prepared by immunization with electroeluted C-TSHR / GST fusion protein. See Inti. Pat. Publ. WO 2015 / 189543. 4E31 is available for purchase from RSR Ltd, Cambridge, UK.The chemical structure of the composition of matter (agent, TRAP™).

[0167] In one embodiment, the invention provides a composition of matter (agent, TRAP™) comprising: a binding moiety that can bind to anti-TSH receptor antibody, a cellular receptor-binding moiety that binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface of degrading cells in a patient or subject, and a linker moiety linking the antibody moiety and the cellular receptor-binding moiety.

[0168] In some embodiments, the invention provides a composition of matter (agent, TRAP™) having a structure selected from the Markush group of structures consisting of:RCN-(Xaa)y-Rcc,[AGN102], or a pharmaceutically acceptable 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.

[0169] In some embodiments, the agent (TRAP™) has the structure of formula AGN101:[AGN101], or a pharmaceutically acceptable salt thereof, wherein: each of a, b, and c is independently 1-200; each AT is independently a binding moiety;L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety, wherein AT is an anti-TSH receptor antibody-binding moiety, an anti-TSH receptor antibodybinding variant, or an anti-TSH receptor antibody-binding fragment thereof.

[0170] In some embodiments, the agent (TRAP™) has the structure of formula AGN102:[AGN102] or a pharmaceutically acceptable salt thereof, wherein: each of a and b is independently 1-200; each AT is independently a binding moiety; L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety, wherein AT is an anti-TSH receptor antibody-binding moiety, an anti-TSH receptor antibodybinding variant, or an anti-TSH receptor antibody-binding fragment thereof.

[0171] In some embodiments, an agent (TRAP™) comprises one and no more than one binding moiety. In some embodiments, one or no more than one binding moiety is bound to a linker moiety. In some embodiments, a is 1. 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 moieties are bonded to a single linker moiety. In some embodiments, b is 2 or more. In some embodiments, an agent (TRAP™) 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 (TRAP™) comprises two or more anti-TSH receptor antibody-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.

[0172] 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.

[0173] In some embodiments, each cellular receptor-binding moiety in an agent (TRAP™) 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, the -L-(TBT)b are the same.

[0174] As known by persons having ordinary skill in the biomedical art, an antibody may comprise more than one site, e.g., one on each of the more than one chain, e.g., one or each heavy chain. In someembodiments, an antibody moiety comprises two heavy chains. One or both amino acid residues or amino acid residues corresponding thereto are each independently connected to a cellular receptorbinding 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 anti-TSH receptor antibody-binding moieties or both linker moieties (if any) are the same.

[0175] In some embodiments, an agent (TRAP™) comprises one and no more than one binding moiety. In some embodiments, one or no more than one binding moiety is bound to a linker moiety. In some embodiments, a is 1. In some embodiments, an agent comprises two or more moieties. In some embodiments, two or more 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 receptorbinding moieties are 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 anti-TSH receptor antibody-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.

[0176] 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.

[0177] 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.

[0178] As known by persons having ordinary skill in the biomedical art, an antibody agent may comprise more than one site, 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. 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 someembodiments, c is 2. In some embodiments, both anti-TSH receptor antibody-binding moieties or both linker moieties (if any) are the same.

[0179] In some embodiments, the composition of matter has the structure of:[AGN103] or a pharmaceutically acceptable salt thereof, wherein: is a moiety comprising at least one leucine rich domain (LRD) of human TSH receptor or a variant thereof;connected tvia a side chain amino group of a lysine residue of to formeachis an asialoglycoprotein receptor ("ASGPR") binding moiety, which is either present or absent, provided that at least oneis present.

[0180] In some embodiments,may be present.

[0181] In some other embodiments,may be absent.

[0182] In some embodiments, the composition of matter may have a structure of:pharmaceutically acceptable salt thereof.

[0183] In some embodiments, the composition of matter may have a structure of:, or a pharmaceutically acceptable salt thereof, wherein eachmay be the same or different and may be a moiety comprising one leucine rich domain (LRD) of human TSH receptor or a variant thereof.

[0184] In some embodiments, the composition of matter has the structure of:Formula (I), wherein,each is a moiety having the peptide sequence of SEQ ID NO: TBD or a sequence having 95% sequence identity therewith;(B) (A) (each is a means for connecting and each ICJ is a carrier polypeptide moiety, which may be a human Fc polypeptide or a derivative thereof having SEQ ID NO: TBD or a sequence having 95% sequence identitytherewith, wherein the moieties are linked together via two disulfidebridges ’ as shown in Formula (II), wherein one disulfide bridge links cysteine residues located in position 11 of SEQ ID NO: TBD of each moiety (CJ , and wherein the other disulfide bridge links cysteine residues located in position 14 of SEQ ID NO: TBD ofeach moietya lysine residue of to formeachis an asialoglycoprotein receptor ("ASGPR") binding moiety which may be present or absent. In an embodiment, at least onemay include an N-acetyl-D-galactosamine("GalNAc") group having Formula (II):Formula (II).

[0185] The following embodiments apply to any of the above chemical structures.(j\)

[0186] In an embodiment, each has the peptide sequence of SEQ ID NO: TBD.

[0187] In an embodiment, is a single bond or a peptide connecting moiety comprising an amino acid selected from the group consisting of glycine (G), glutamate (E), leucine (L), proline (P), glutamine (Q), serine (S), and threonine (T).®

[0188] In an embodiment, each has the peptide sequence of SEQ ID NO: 9.

[0189] In an embodiment, eachcomprises a moiety selected from the Markush group consisting of:wherein,X2are independently CH2, O, S, NR4, C(O), S(O), S(O)2, S(O)2O, OS(O)2, or OS(O)2O;X3are independently O, S, NR4, wherein R4is H or a C1-C3 alkyl; and k and n are independently 1 to 25.

[0190] In an embodiment, eachfurther comprises a moiety selected from the group consisting of:wherein,X2are independently CH2, O, S, NR4, C(O), S(O), S(O)2, S(O)2O, OS(O)2, or OS(O)2O;X3are independently O, S, NR4, wherein R4is H or a C1-C3 alkyl; k and n are independently 1 to 25.

[0191] In an embodiment, eachcomprises one or more -(O)C-[(CH2)nO]m(CH2)nNH-,-[(CH2)nO]m(CH2)nNHC(O)[(CH2)nO]m-, and -[(CH2)nO]m(CH2)n{NHC(O)[(CH2)nO]m}p(CH2)nC(O)NH-, wherein each m and n are independently 1 to 10.

[0192] In an embodiment, eachcomprises one or more -[(CH2)n-O]m-, wherein each m and n are independently 1 to 10.

[0193] In an embodiment, eachcomprises -(CH2)n-O-(CH2CH2O)n-(CH2)n-, wherein each n is independently 1 to 10.

[0194] In an embodiment, each

[0195] (HI):Formula (V)

[0197] In an embodiment, the lysine residue of is located in position 31 or position 33 of SEQ ID. NO: TBD.

[0198] In an embodiment, the cysteine residues in positions 46 and 106 of SEQ ID NO: TBD are connected to form a disulfide bridge, and wherein the cysteine residues in positions 152 and 210 of SEQ ID NO: TBD are connected to form a disulfide bridge.

[0199] In a specific embodiment, the composition of matter is AGN301.Anti-TSH receptor antibody binding moiety

[0200] In an embodiment, the anti-TSH receptor binding moiety may be a moiety derived from the TSH receptor. The TSH receptor is involved in the control of thyroid function and is a major autoantigen in autoimmune thyroid diseases. The TSHR is a member of the G-protein coupled receptor (GPCR) family and consists of three domains, (1) the extracellular leucine-rich repeat domain (LRD), (2) the hinge region and (3) a transmembrane domain (TMD) with an intracellular C-terminus. Nunez Miguel et al., Thyroid, 14, 991-1011 (2004). TSHR260 is a subdomain of TSHR consisting of residues 22-260 and encompassing most of the leucine rich domain. Sanders et al., Thyroid, 17, 395-410 (2007). The TSHR260 shows similar binding to TRAbs as the full-length TSHR. See Rees Smith, Hormone and Metabolic Research, 41, 448-455 (2009) and International Patent Publication WO 2010 / 073012.

[0201] Proteins such as TSH receptor and TSHR260 have poor stability and are denatured during purification. Accordingly, more thermostable proteins, for example more thermostable TSHR260 and full length TSHR have been created and described. See, for example, EP3155011, which is incorporated herein in its entirety by reference.

[0202] In an embodiment, a mutant TSH receptor or fragment thereof may include a single point mutation. Such mutation may be P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D143P, D151E, S166T, I167F, P168Y, V169R, N170W, T179C, I253R, or R255Y.

[0203] In another embodiment,may be a mutant TSH receptor or fragment thereof containing two point mutations. One of these mutations may be I253R, and the second mutation may be P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D143P, D151 E, S166T, I167F, P168Y, V169R, N170W, T179C, or R255Y. In another aspect, a mutant TSHR or fragment thereof may contain three point mutations. One of these mutations may be I253R, the second mutation may be D143P, and the third mutation may be one of P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D151 E, S166T, P168Y, V169R, andN170W.

[0204] In another embodiment, ) may be a mutant TSH receptor or fragment thereof containing four point mutations. One of these mutations may be I253R, the second mutation may be D143P, the third mutation may be R112P, and the fourth mutation may be one of L59F, H63C, D151 E, S166T, V169R, and N170W.

[0205] In another embodiment, Z) may be a mutant TSH receptor or fragment thereof containing five point mutations. One of these mutations may be I253R, the second mutation may be D143P, the third mutation may be R112P, the fourth mutation may be D151 E or H63C, and a fifth mutation may be one of L59F, (H63C or D151 E), S166T and V169R.(A)

[0206] In another embodiment, may be a mutant TSH receptor or fragment thereof containing six mutations. One of these mutations may be I253R, the second mutation may be D143P, the third mutation may be R112P, the fourth mutation may be D151 E, the fifth mutation may be H63C, and the sixth mutation may be either S166T or V169R.(A)

[0207] In another embodiment, may be a mutant TSH receptor or fragment thereof containing from one, two, three, four, five, or six point mutations selected from P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D143P, D151 E, S166T, I167F, P168Y, V169R, N170W, T179C, I253R, and R255Y.

[0208] In another embodiment, (H) may be a mutant TSH receptor or fragment thereof including the subdomain TSHR260 of the TSHR receptor and preferably may also include one of the following set of mutations:(1) I253R;(2) I253R and one of the following: P28E, L59F, H63C, L64Y, R112P, D143P, D151E, S166T, P168Y, V169R, or N170W;(3) I253R and D143P and one of the following: L59F, H63C, R112P, D151E, S166T, or V169R;(4) I253R and D143P and R112P and one of the following: L59F, H63C, D151E, S166T, or V169R;(5) I253R and D143P and R112P and D151E and one of the following: L59F, H63C, S166T, orV169R;(6) I253R and D143P and R112P and D151E and H63C and one of the following: S166T orV169R; and(7) I253R and D143P and R112P and H63C and one of the following: S166T or V169R.( )

[0209] In another embodiment, may be the mutant TSH receptor or fragment thereof including the subdomain TSHR260 of the TSHR receptor and preferably may also include one of the following set of mutations:(1) I253R;(2) D143P + I253R;(3) R112P + D143P + I253R;(4) R112P + O143P + D151 E + I253R;(5) R112P + D143P + D151 E + V169R + I253R;(6) H63C + R112P + D143P + D151 E + V169R + I253R;(7) H63C + R112P + D143P + V169R + I253R; or(8) H63C + R112P + D143P + S166T + I253R.

[0210] In another embodiment, ) may be the TSHR260 mutant or fragment thereof that is stabilized by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55™, which is approximately 900 times more thermostable than wild-type TSHR260. Such TSHR260 mutant was described by Miller-Gallacher et al., Journal of Molecular Endocrinology, 62, 117- 128 (2019), which is incorporated herein in its entirety by reference.

[0211] In an embodiment, Q may be the anti-TSH receptor antibody-binding moiety ABT301( )(SEQ ID NO: 24) or a conjugation reaction derivative thereof. In another embodiment, may be the anti-TSH receptor antibody-binding moiety including a sequence having 80% or greater sequence identity with SEQ ID NO: TBD. For example, the sequence identity with SEQ ID NO: TBD may be 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater

[0000] Linking moiety serves as a spacer structure connecting moieties and( C ). A spacer is a structure that is located between different structural modules and can spatially separate the structural modules. The definition of spacer is not limited by whether it has a certain function or whether it can be cleaved or degraded in vivo. Examples of spacers include but are not limited to amino acids and non-amino acid structures, wherein non-amino acid structures can be, but are not limited to, amino acid derivatives or analogues. In an embodiment a spacer structure may include a spacer sequence. As used herein, "spacer sequence" refers to an amino acid sequence serving as a spacer, and examples thereof include but are not limited to a single amino acid, a sequence containing a plurality of amino acids, for example, a sequence containing two amino acids such as GA, etc., or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc.

[0213] In Formula (I), ( ' —H ') may be a single chemical bond or a polypeptide that providesseparation between the TSHR binding moiety and Fc polypeptide moiety. In an embodiment,may be a peptide connecting moiety comprising an amino acid selected from the group consisting of G, E, L, P, Q, S, and T.

[0214] In an embodiment,may include a (G4S)4 linker. For example,may include a G4S polypeptide having sequence GGGGS. In another example,may include a 2XG4S polypeptide having sequence GGGGSGGGGS. In another example,may include a 3XG4S polypeptide having sequence GGGGSGGGGSGGGGS. In another example,may include a 4XG4S polypeptide having sequence GGGGSGGGGSGGGGSGGGGS. In another example,may include a 5XG4S polypeptide having sequence GGGGSGGGGSGGGGSGGGGSGGGGS.

[0215] In an embodiment, ® may include a G4S polypeptide and further include one or more of the amino acids selected from the group consisting of E, P, K, and A. For example,may be a polypeptide having the sequence GGGGSEPKSA.

[0216] Methods of connecting or fusing the TSHR binding moiety and Fc polypeptidemoiety are known in the art. Either one or both moieties may have a recognition sequence Gnof a ligase acceptor substrate, which facilitates enzyme-catalyzed coupling of compound of formula (I) with the targeting molecule under the catalysis of the ligase. The targeting molecule optionally modified and comprises the corresponding recognition sequence of a ligase acceptor substrate.

[0217] In an embodiment, the ligase may be a transpeptidase. In an embodiment, the ligase may be selected from the group consisting of a natural transpeptidase, an unnatural transpeptidase, variants thereof, and the combination thereof. Unnatural transpeptidase enzymes can be, but are not limited to, those obtained by engineering of natural transpeptidase. In an embodiment, the ligase may be selected from the group consisting of a natural sortase, an unnatural sortase, and the combination thereof. The species of natural Sortase include Sortase A, Sortase B, Sortase C, Sortase D, Sortase L. plantarum, etc. A description can be found in U.S. Pat. Publ. 2011 / 0321183, which is incorporated herein by reference.The type of ligase corresponds to the ligase recognition sequence and is thereby used to achieve specific conjugation between different molecules or structural fragments.

[0218] In some embodiments, the ligase may be a Sortase selected from Sortase A, Sortase B, Sortase C, Sortase D and Sortase L. plantarum. In these embodiments, the recognition sequence of the ligase acceptor substrate may be selected from the group consisting of oligomeric Glycine, oligomeric alanine, and a mixture of oligomeric Glycine / alanine having a degree of polymerization of 3-10. In a particular embodiment, the recognition sequence of the ligase acceptor substrate may be Gn, wherein G is glycine (Gly) , and n is an integer of 2 to 10.

[0219] In another embodiment, the ligase may be Sortase A from Staphylococcus aureus. The ligase recognition sequence may be typical recognition sequence of the enzyme Sortase A from Staphylococcus aureus.

[0220] In an embodiment, the ligase may be Sortase B from Staphylococcus aureus, and the corresponding donor substrate recognition sequence may be sequence may be the typical recognition sequence of the enzyme Sortase B from Staphylococcus aureus. In another embodiment, the ligase may be Sortase B from Bacillus anthracis, and the corresponding donor substrate recognition sequence may be the typical recognition sequence of the enzyme Sortase B from Bacillus anthracis.

[0221] In yet another embodiment, the ligase may be Sortase A from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be typical recognition sequence of the ligase enzyme Streptococcus pyogenes. In another embodiment, the ligase may be Sortase subfamily 5 from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence may be typical recognition sequence of the ligase enzyme Streptomyces coelicolor.

[0222] In yet another embodiment, the ligase may be Sortase A from Lactobacillus plantarum. The corresponding donor substrate recognition sequence may be the typical recognition sequence of the enzyme Sortase A from Lactobacillus plantarum.

[0223] The ligase recognition sequence can also be other totally new recognition sequence for transpeptidase optimized by manual screening.( C )

[0224] In an embodiment, the polypeptide moiety may be a Fc fragment generated from the heavy chain constant region of an immunoglobulin, an Fc monomer, an Fc dimer, or fragments thereof (e.g., synthetic peptides). For example,may be an Fc- monomer. In some embodiments, the Fc fragment may be conjugated to a single variable domain on a heavy chain (VHH) antibody, a nanobody, a domain, or a fragment thereof, to form an VHH antigen fusion. In some embodiments, the Fc polypeptide may be conjugated to an antigen to form an Fc- antigen fusion. In various embodiments, the Fc fragment may be conjugated to the TSHR bonding moiety through the linking polypeptide.( C 1

[0225] In an embodiment, the polypeptide moiety may have SEQ ID NO:( . C . )TBD. In another embodiment, the polypeptide moiety may have a sequencehaving 80% or greater sequence identity with SEQ ID NO: TBD. For example, the sequence identity with SEQ ID NO: TBD may be 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater.

[0226] Various linkers described in the art may be used as the linking groupsinFormula (I) according to embodiments of the invention.

[0227] In an embodiment, each linking groupin Formula (I) may include any of the linkers [LINKERS] and [CON] disclosed in International Patent Publication WO 2019 / 199634, which is incorporated herein in its entirety by reference.

[0228] In another embodiment, each linking groupin Formula (I) may include any of the linkers [LinkerA], [LinkerB], [LinkerC], and [LinkerD] disclosed in International Patent Publication WO 2021 / 155317, which is incorporated herein in its entirety by reference.

[0229] In another embodiment, each linking groupin Formula (I) may include any of the linkers L disclosed in International Patent Publication WO 2024155750 , which is incorporated herein in its entirety by reference.

[0230] In an embodiment, eachin Formula (I) may include one or more-(O)C-[(CH2)nO]m(CH2)nNH-, -[(CH2)nO]m(CH2)nNHC(O)[(CH2)nO]m-, and -[(CH2)nO]m(CH2)n{NHC(O)[(CH2)nO]m}p(CH2)nC(O)NH-, wherein each m and n are independently 1 to 10.

[0231] In another embodiment, eachin Formula (I) may include one or more- [(CH2)n- O]m- , wherein each m and n are independently 1 to 10.

[0232] In another embodiment, eachin Formula (I) may include-(CH2)n-O-(CH2CH2O)n-(CH2)n-, wherein each n is independently 1 to 10.D)

[0233] In an embodiment, each - in Formula (I) may include a moiety selected from the group consisting of:wherein,X2are independently CH2, O, S, NR4, C(O), S(O), S(O)2, S(O)2O, OS(O)2, or OS(O)2O;X3are independently O, S, NR4, wherein R4is H or a C1-C3 alkyl; and k and n are independently 1 to 25.

[0234] Various ASGPR binding moieties described in the art may be used as the ASGPR binding moietiesin Formula (I) according to embodiments of the invention.

[0235] In an embodiment, each ASGPR binding moietyin Formula (I) may include any of the moieties [CRBM] and [ASGPRBM] disclosed in International Patent Publication WO 2019 / 199634, which is incorporated herein in its entirety by reference.

[0236] In another embodiment, each ASGPR binding moietyin Formula (I) may include any of the galactose and mannose ASGPR binding moieties disclosed in International Patent Publication WO2021 / 155317, which is incorporated herein in its entirety by reference.

[0237] In another embodiment, each ASGPR binding moietyin Formula (I) may include any of the lysosomal targeting moieties X disclosed in International Patent Publication WO 2024 / 155750 , which is incorporated herein in its entirety by reference.

[0238] in Formula (I) may include a group having the structure:Formula (Ila)

[0239] may further include a moiety selected from the group consisting of:wherein,X2are independently CH2, O, S, NR4, C(O), S(O), S(O)2, S(O)2O, OS(O)2, or OS(O)2O;X3are independently O, S, NR4, wherein R4is H or a C1-C3 alkyl; k and n are independently 1 to 25.

[0240] Formula (III):wherein,ZBis absent, -(CH2)IM-, -C(=O)-(CH2)IM-, or -C(=O)-(CH2)IM-NRM-;RM is H or Ci-C3alkyl; and each occurrence of IM is independently 1, 2, or 3.

[0241] Formula (IV) or Formula (V):Formula (V)Antibody Fc binding moiety.

[0242] The composition of matter according to embodiments of the invention may be prepared by site-specific (MATE®) conjugation of a reagent having binding specificity to the antibody Fc region as described in International Patent Publication WO 2021 / 102052, which is incorporated herein in its entirety by reference. This process provides attachment of the linking moietyto Fc moiety of the described composition of matter.

[0243] In some embodiments, the Fc-binding moiety comprises a moiety selected from theMarkush 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.

[0244] In some embodiments, each binding moiety in an agent (TRAP™) is of the same binding moiety or a pharmaceutically acceptable salt thereof.

[0245] In some embodiments, the Fc-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.

[0246] The Fc-binding moiety may comprise:[ABT101] or a pharmaceutically acceptable salt form thereof.

[0247] In some embodiments, a 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 binding moiety comprises ABT101 or a salt form thereof, wherein each variable is as described in this specification.

[0248] 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 proteinbinding moiety is or comprises ABT101 or a salt form thereof, wherein each variable is as described in this specification.

[0249] In some embodiments, a 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, a 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, a 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.

[0250] In some embodiments, -(Xaa)z- is or comprises a peptide unit.

[0251] 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.

[0252] In some embodiments, a peptide unit comprises an amino acid residue, e.g., at physiological pH about 7.4, positively charged amino acid residue, Xaap), e.g., a residue of an amino acid of formula LNK101 with a positively charged side chain. In some embodiments, a peptide unit comprises R. In some embodiments, at least one Xaa is R.

[0253] In some embodiments, ABT101 is a universal antibody-binding moiety. In some embodiments, ABT101 is a universal antibody-binding moiety that can bind to different Fab regions. In some embodiments, ABT101 is a universal antibody-binding moiety that binds to an Fc region, e.g., the Fc region that binds to an Fc receptor.

[0254] Several antibody-binding moieties, including universal antibody-binding moieties, can be used following the teachings of this specification. Certain antibody-binding moieties and technologies for identifying or assessing antibody-binding moieties are described in W02019 / 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 in accordance with this specification. In some embodiments, an antibody-binding moiety comprises one or more amino acid residues, each independently natural or unnatural.

[0255] In some embodiments, a binding moiety, e.g., a protein-binding moiety, e.g., an antibodybinding moiety, e.g., a universal antibody-binding moiety, has the structure of ABT101 or a salt form thereof, wherein: each of R1, R3and R5is independently hydrogen or an optionally substituted group selected from Ci-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:R1and R1are optionally taken together 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;R3and R3are optionally taken together 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 R5group and the R5group attached to the same carbon atom are optionally taken together 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; or two R5groups are optionally taken together with their intervening atoms to form a Ci-io 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 Ci-3aliphatic; each of R2, R4and R6is independently hydrogen, or optionally substituted C1-4 aliphatic, or:R2and R1are optionally taken together 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;R4and R3are optionally taken together 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; or an R6group and its adjacent R5group are optionally taken together 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;L1is a trivalent linker moiety; and each of m and n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0256] In some embodiments, L1is 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')z-, -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(0)-, -S(0)2-, -S(O)2N(R')-, -C(O)S- or - C(O)O- . In some embodiments L1is -(CH2CH2O)2.4- or -(CH2CH2O)2-

[0257] In some embodiments, a binding moiety, e.g., a protein-binding moiety, e.g., an antibodybinding moiety, e.g., a universal antibody-binding moiety, has the structure of ABT101 or a salt form thereof, wherein: each of R7is independently hydrogen or an optionally substituted group selected from Ci_6aliphatic, 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 R7group and the R7group attached to the same carbon atom are optionally taken together 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 Ci_3aliphatic; each of R8is independently hydrogen, or optionally substituted Ci-4aliphatic, or: an R8group and its adjacent R7group are optionally taken together 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; andR9is hydrogen, optionally substituted Ci_3aliphatic, or -C(O)-.

[0258] In some embodiments, a 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 together to form bridges. Side chains of two cysteine residues may form a disulfide bridge comprising -S-S-.

[0259] In some embodiments, a binding moiety, e.g., a protein-binding moiety, e.g., an antibodybinding moiety, e.g., a universal antibody-binding moiety, is or comprises a cyclic peptide moiety, e.g., a moiety having the structure of ABT101 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 Rcis independently -La-R'; each Lais 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')z-, -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 C6-2o 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, heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, Ce-3o aryl, Ce-3o arylaliphatic, Ce-3o 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, or two 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 together 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; or two or more R groups on two or more atoms are optionally and independently taken together 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.

[0260] In some embodiments, a peptide unit comprises a functional group in an amino acid residue that can react with a functional group of another amino acid residue. In some embodiments, a peptide unit comprises an amino acid residue with a side chain which comprises a functional group that can react with another functional group of the side chain of another amino acid residue to form a linkage, e.g., see moieties described in TABLE 2 of International Patent Publication WO 2024 / 228935 (Biohaven Therapeutics, Ltd.). In some embodiments, one functional group of one amino acid residue is connected to a functional group of another amino acid residue to form a linkage (or bridge). Linkages are bonded to backbone atoms of peptide units and comprise no backbone atoms. In some embodiments, a peptide unit comprises a linkage formed by two side chains of non-neighboring amino acid residues. In some embodiments, a linkage is bonded to two backbone atoms of two non-neighboring amino acid residues. In some embodiments, both backbone atoms bonded to a linkage are carbon atoms.

[0261] 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., Vai, Leu, etc., may be replaced with another XaaH, e.g., Leu, He, Ala, etc., one XaaAmay be replaced with another XaaA, one Xaapmay be replaced with another Xaap, one XaaNmay be replaced with another XaaN, one XaaLmay be replaced with another XaaL, etc.

[0262] In some embodiments, antibody-binding moieties, e.g., antibody-binding moieties. Useful technologies for developing or assessing these moieties are described in, e.g., Alves, Langmuir, 28, 9640-9648 (2012), Choe et al., Materials, 9 (20), 994 (2016), Gupta et al., Nature Biomedical Engineering, 3, 917-929 (2019), Muguruma et al., ACS Omega, 4, 14390-14397 (2019), Yamada et al., Angewandte Chemie 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), International Patent Publication WO 2012 / 017021, etc., the binding moieties, e.g., antibody-binding moieties of each of which is incorporated in this specification in its entirety by reference.

[0263] 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 International Patent Publication WO 2018199337, the affinity substance of each of which is incorporated in this specification by reference.

[0264] In some embodiments, an antibody-binding moiety comprises an adapter, e.g., as described by Hui et al., Bioconjugate Chem., 26, 1456-1460 (2015). In some embodiments, when used inaccordance with this specification, adapter proteins do not require reactive residues, e.g., BPA, to achieve one or more advantages.

[0265] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is or comprises a triazine moiety, e.g., one described in U.S. Pat. Publ. 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 U.S. Pat. Publ. 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 U.S. Pat. Publ. 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 the Fc region of an antibody.

[0266] 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. Recognition, 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 antibodybinding moiety, e.g., an antibody-binding moiety, 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 the Fc region of an antibody.

[0267] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is a triazine moiety, e.g., one described by 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, is of such a structure that H-ABT is a compound described by Uttamchandani, which is 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 the Fc region of an antibody.

[0268] 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).

[0269] Other useful technologies are described in Mustafaoglu et al., Analyst, 141(24), 6571-6582 (November 28, 2016).

[0270] Several antibody-binding moieties, including universal antibody-binding moieties, can be used in accordance with the teachings of this specification. Some antibody-binding moieties and technologies for identifying or assessing antibody-binding moieties are described in International Patent Publications 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 in accordance with this specification. In some embodiments, an antibody-binding moiety comprises one or more amino acid residues, each independently natural or unnatural.Linker moiety.

[0271] In some embodiments, the agent (TRAP™) may comprise one or more amino acid moieties, e.g., antibody-binding moieties, linker moieties, etc. Amino acid moieties can either be those of natural amino acids or unnatural amino acids. In some embodiments, an amino acid has the structure of the formula LNK101:NH(Ral)-Lal-C(Ra2)(Ra3)-La2-COOH,[LNK101] or a salt thereof, wherein: each of Ral, Ra2, and Ra3is independently -La-R' or an amino acid side chain; each of Laland La2is independently La; each Lais 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')z-, -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 C6-2o 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 Ci-30aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, C6-3o aryl, C6-3o arylaliphatic, C6-3o 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, or two 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 together 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; or two or more R groups on two or more atoms are optionally and independently taken together 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.In some embodiments, an amino acid residue, e.g., of an amino acid having the structure of formula LNK101, has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-La2-CO-. In some embodiments, each amino acid residue in a peptide independently has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-La2-CO-.

[0272] 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 provides a composition of matter of formula NH(Ral)-Lal-C(Ra2)(Ra3)-La2-COORCTor salt thereof, wherein RCTis R' and each other variable is independently as described in this specification. In some embodiments, RCTis R. In some embodiments, RCTis optionally substituted aliphatic. In some embodiments, RCTis t-butyl.

[0273] In some embodiments, Lalis a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-C(Ra2)(Ra3)-La2-COOH. In some embodiments, La2is -CH2SCH2-

[0274] In some embodiments, La2is a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-Lal-C(Ra2)(Ra3)-COOH. In some embodiments, anamino acid residue has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-CO-. In some embodiments, Lalis -CH2CH2S-. In some embodiments, Lalis -CH2CH2S-, wherein the CH2is bonded to NH(Ral).

[0275] In some embodiments, Lalis a covalent bond, and La2is a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-C(Ra2)(Ra3)-COOH. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-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(Ral)-CH(Ra3)-COOH, NH2-CH(Ra2)-COOH, NH2-CH(Ra3)-COOH, -N(Ral)-C(Ra2)(Ra3)-CO-, -N(Ral)-CH(Ra2)-CO- -N(Ral)-CH(Ra3)-CO-, -NH-CH(Ra2)-CO-, and -NH-CH(Ra3)-CO-

[0276] In some embodiments, Lais a covalent bond. In some embodiments, Lais optionally substituted by Ci-6bivalent aliphatic. In some embodiments, Lais optionally substituted for Ci_6alkylene. In some embodiments, Lais -CH2-. In some embodiments, Lais -CH2CH2-. In some embodiments, Lais -CH2CH2CH2-.

[0277] In some embodiments, Lais 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, Lais 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, Lais 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 electronwithdrawing group as described in this specification. In some embodiments, -Cy- is substituted with one or more -F. In some embodiments, -Cy- is optionally substituted 1,3-phenylene. In some embodiments, -Cy- is optionally substituted 1,4-phenylene. In some embodiments, Lais or comprises a chemical group disclosed in International Patent Publication WO 2024 / 228935 (Biohaven Therapeutics, Ltd.), the contents of which are incorporated herein in their entireties by reference.

[0278] In some embodiments, moieties are optionally connected 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 portions. In some embodiments, LLGis a linker moiety described in this specification. In some embodiments, LLG1is a linker moiety described in this specification. In some embodiments, LLG2is a linker moiety described in this specification. In some embodiments, LLG3is a linker moiety described in this specification. In some embodiments, LLG4is a linkermoiety described in this specification. In some embodiments, LRMis a linker moiety described in this specification. In some embodiments, LPMis L. In some embodiments, LPMis a linker moiety described in this specification. In some embodiments, LPMis L.

[0279] Linker moieties of several types or for several purposes, e.g., those used in antibody-drug conjugates, etc., may be used in accordance with this specification.

[0280] 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 polyvalent linker connects more than two moieties.

[0281] In some embodiments, a linker moiety, e.g., Lz, e.g., LPM, LRM, LLG, LLG1, etc., is or comprises L.

[0282] In some embodiments, L is a covalent bond, or a bivalent or polyvalent optionally substituted, linear or branched Ci-ioo group comprising one or more aliphatic, aryl, heteroaliphatic having 1-20 heteroatoms, heteroaromatic having 1-20 heteroatoms, or any combinations thereof, wherein one or more methylene units of the group are optionally and independently replaced with Ci-6alkylene, Ci-6alkenylene, a bivalent Ci-6heteroaliphatic group having 1-5 heteroatoms, 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-, -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. The linker optionally contains a cyclic group, Cy, defined below, and a reactive group, RG, as defined below. In some embodiments, each amino acid residue is independently a residue of an amino acid having the structure of formula LNK101 or a salt thereof. In some embodiments, each amino acid residue independently has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-La2-CO- or a salt form thereof.

[0283] In some embodiments, L is bivalent. In some embodiments, L is a covalent bond.

[0284] In some embodiments, L is a bivalent or optionally substituted linear or branched group, selected from Ci-oo aliphatic and Ci-ioo heteroaliphatic having 1-50 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with Ci-6alkylene, Ci-6alkenylene, a bivalent Ci-6 heteroaliphatic group having 1-5 heteroatoms, 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-, -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]- . In some embodiments, L is a bivalent or optionally substituted linear or branched group selected from Ci-20aliphatic and Ci-20heteroaliphatic having 1-10heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with Ci-6alkylene, Ci-6alkenylene, a bivalent Ci-6heteroaliphatic group having 1-5 heteroatoms, — CEC— , -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]-. In some embodiments, L is a bivalent or optionally substituted linear or branched group selected from Ci-20aliphatic, wherein one or more methylene units of the group are optionally and independently replaced with — CEC— , -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]-. In some embodiments, L is a bivalent or optionally substituted, linear or branched Ci-20aliphatic wherein one or more methylene units of the group are optionally and independently replaced with CECt-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-, an amino acid residue or - [(- O- C(R')2- C(R')2- )n]— • In some embodiments, L is a bivalent or optionally substituted linear or branched group Ci-100 aliphatic, wherein one or more methylene units of the group are optionally and independently replaced with CEC , -Cy-, -C(R')2-, -O-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, an amino acid residue or -[(-O-C(R')2-C(R')2-)n]-. In some embodiments, L is a bivalent or optionally substituted linear or branched group C1-50 aliphatic, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear, or branched group C1-40 aliphatic, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear, or branched group Ci-20 aliphatic, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted linear or branched group C1-10 aliphatic, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted linear or branched group Ci-100 alkylene, wherein one or more methylene units of the group are optionally and independently replaced as described in thisspecification. In some embodiments, L is a bivalent or optionally substituted linear or branched group Ci- so alkylene, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted linear or branched group C1-40 alkylene, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted linear or branched group C1-20 alkylene, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted linear or branched group Ci- 10 alkylene, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification.

[0285] In some embodiments, a linker moiety, e.g., L, LPM, LRM, etc., comprises an acidic group, e.g., -S(O)2OH.

[0286] In some embodiments, L is or comprises -[(-O-C(R')2-C(R')2-)n]-. In some embodiments, L is or comprises -[(-O-CH2-CH2-)n]-. In some embodiments, L is -[(-CH2-CH2-O)6]-CH2-CH2-. In some embodiments, L is -[(-CH2-CH2-O)8]-CH2-CH2-. In some embodiments, -CH2-CH2-O- is bonded to an antibody-binding moiety at a -CH2-. In some embodiments, -CH2-CH2-O- is bonded to a cellular receptor-binding moiety at a -CH2-. In some embodiments, LPMis such L. In some embodiments, LRMis such L.

[0287] In some embodiments, a linker moiety comprises one or more -(CH2)n-O-, wherein each n is independently 1-20. In some embodiments, it is or comprises one or more -[(CH2)n-O]m-, wherein each n is independently 1-20, and m is 1-100. In some embodiments, it comprises two or more -[(CH2)n-O]m-, wherein each n is independently 1-20, and each m is 1-100. In some embodiments, it is or comprises one or more -(O)C-[(CH2)nO]m(CH2)nNH-, -[(CH2)nO]mNHC(O)[(CH2)nO]mNH-, -[(CH2)nO]m{NHC(O)[(CH2)nO]m}pNH— wherein each n is independently 1-20, and each m is independently 1-100, and where each p is independently 1 to 10. In some embodiments, n is selected from the Markush group of numbers consisting of 1-10, 1-5, and 2. In some embodiments, m is 1-50. In some embodiments, m is 1-40. In some embodiments, m is selected from the Markush group of numbers consisting of 1-30, 1-20, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0288] In some embodiments, a linker moiety, or L, is or comprises -(CFhCl-hOJn-, 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.

[0289] 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)-.

[0290] In some embodiments, a linker moiety, e.g., L, comprises one or more amino acid residues or analogues.

[0291] 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 comprises an antibody-binding moiety and a cellular receptor-binding moiety linked through a linker comprising 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 comprises -C(O)-O-. In some embodiments, a reactive group comprises -C(O)-O-, wherein -O- is bonded to an optionally substituted aryl group. In some embodiments, a reactive group 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 of each electron-withdrawing group is independently selected from -NO2and -F. In some embodiments, an aryl group has the structure of, wherein Rsis 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 ofwherein each Rsis 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 groupsome embodiments, Cl 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-.

[0292] In some embodiments, a linker moiety, e.g., LRM, comprises a reactive group, wherein upon contact with an antibody, the reactive group reacts with a group of the antibody and conjugates acellular 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 compriseswherein 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.

[0293] 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 comprises -(CH2CH2O)n-, wherein n is described in this specification. In some embodiments, one or more methylene units of L are independently replaced with -(CH2CH2O)n-.

[0294] 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.

[0295] 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(Ral)-Lal-C(Ra2)(Ra3)-La2-CO- or a salt form thereof.

[0296] 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 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 some embodiments, one or more methylene units of L are independently replaced with -NR'-, wherein R' is as described in this specification.

[0297] 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)-.

[0298] 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')-.

[0299] 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')-.

[0300] 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)uCOOH. In some embodiments, R' is -(CH2)2COOH. In some embodiments, R' is -COOH.

[0301] 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 a 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 a 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 the product of a cycloaddition reaction, e.g., click chemistry, and variants thereof used to link different moieties together.N=N

[0302] In some embodiments, a linker moiety, e.g., L, is or comprises. In some N=N embodiments, a methylene unit of L is replaced with. in some embodiments, a methylene unit of L is replaced with -Cy-. In some embodiments, -Cy- is.

[0303] In some embodiments, a linker moiety, e.g., L) is or comprises -CO)y-. In some embodiments, L is or comprises -[(CH2)nO]mCy[(CH2)nO]mNH, or L is - [(CH2)nO]mCy[(CH2)nO]mNHC(O)[(CH2)nO]mNH-, or L is - [(CH2)nO]mCy[(CH2)nO]m{NHC(O)[(CH2)nO]m}pNH-, where n, m, and p are independently chosen at each occurrence from 1-20, from 1-12, or 2-10. In some embodiments, each n is 2, and m isindependently chosen at each occurrence from an integer from 2-10. In some embodiments, m isN=N independently chosen from an integer from 2-6, and Cy is. In some embodiments, a methylene unit of L is replaced with -Cy-. In some embodiments, -Cy- is -. In some embodiments, -Cy- is

[0304] In some embodiments, LRMis a covalent bond. In some embodiments, LRMis not a covalent bond. In some embodiments, LRMis or comprises -(CFhCFhOJn-. In some embodiments, LRMis 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, LRMis-(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, LRMis -(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, LRMis -(CH2)2-O-(CH2CH2O)n-(CH2)2-, wherein n is as described in this specification.

[0305] In some embodiments, LPMis a covalent bond. In some embodiments, LPMis not a covalent bond. In some embodiments, LPMis or comprises -(Cl-bCI-hOJn-. In some embodiments, LPMis 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, LPMis -(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, LPMis -(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, LPMis -(CH2)2-O-(CH2CH2O)n-(CH2)2-, wherein n is as described in this specification.

[0306] In some embodiments, LPM, e.g., in a product of a first and a second agent, is or comprises a reaction product moiety formed by a first and second reactive moiety.

[0307] In some embodiments, a linker moiety, e.g., LPMin a product of a first and a second agent, is or comprises. In some embodiments, a methylene unit of a linker moiety, e.g., a linker moiety that can be L, e.g., LRMor LPM, is replaced with -Cy-. In some embodiments, -Cy- is optionally substitutedn some em o men s, y s In some embodiments L is -[(CH2)nO]mCH2Cy[(CH2)nO]m- or In some embodiments, -Cy- is. In some embodiments,Cellular receptor-binding moiety.

[0308] According to several embodiments of the invention, several receptor-binding moieties are described in International Patent Publications WO 2019 / 199621 (Yale University), WO2019 / 199634 (Yale University), and WO 2021 / 072246 (Yale University), each incorporated in this specification by reference.

[0309] 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.

[0310] The amine group may be a primary alkyl amine group or a secondary alkyl amine group, each of which is optionally substituted on the amine group with a C1-C3 alkyl group.

[0311] The cellular receptor-binding moiety may include an ASGPR binding group according to the chemical structure disclosed in International Patent Publication WO 2019 / 199621 (Yale University).

[0312] The cellular receptor-binding moiety may have the following structure:where RAis a C1-C3 alkyl group optionally substituted with 1-5 halo groups (preferably RAis a methyl or ethyl group optionally substituted with from 1-3 fluoro groups);ZAis -(CH2)IM, -O-(CH2)IM, S-(CH2)IM, N RM-(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)|MNRMgroup (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues) where IM and RMare the same as above; andZBis absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)|M-NRM, where IM and RMare the same as above.

[0314] In an embodiment, RAmay be a methyl or ethyl group optionally substituted with one to three fluoro groups.

[0315] In an embodiment, ZAis a PEG group containing 1 to 4 ethylene glycol residues.

[0316] In an embodiment, the methyl or ethyl group may be substituted with from 1-3 fluoro groups.

[0317] In an embodiment, the ASGPR binding group may be N-acetyl-D-galactosamine.

[0318] 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 FcyRI binding group, a FcRn binding group, a transferrin receptor-binding group, or a macrophage scavenger receptor-binding group.MATE®

[0319] The MATE® antibody conjugation technology enables site-directed pairing with therapeutic monoclonal antibodies (mAbs), or therapeutic immunoglobulin (IG) pooled from donors. Persons having ordinary skill in the biomedical art can use MATE® materials and methods as guidance to predictable results when making and using the invention.

[0320] This reagent may be referred to as a MATE® agent or MATE®. The MATE® agents are described, for example, in International Patent Publication WO 2021 / 102052, 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.

[0321] In a specific embodiment, the cellular receptor-binding moiety is TBT301 or a conjugation reaction derivative thereof. See FIG. 16.Second agent.

[0322] In another embodiment, the invention provides a composition including the agent (TRAP™) and at least one additional agent comprising a moiety capable of binding to the antibody that forms the antibody moiety of the first compound.

[0323] In some embodiments, a first composition comprises a first agent (TRAP™) described in this specification. In some embodiments, second agents independently comprise second reactive groups. In some embodiments, a second composition comprises 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 modified. In some embodiments, a second composition is an IVIG preparation. In some embodiments, a product composition comprises 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.

[0324] In some embodiments, a cellular receptor-binding moiety 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 some embodiments, a second reactive group is a function group of an amino acid residue, e.g., -NH2 of lysine, -SH of cysteine, etc. In some embodiments, a second reactive group is -NH2 of a lysine residue, e.g., of a residue selected from K246 and K248 of IgGl heavy chain amino acid residues corresponding thereto, K251 and K253 of an lgG2 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.

[0325] 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 IgGl heavy chain amino acid residues corresponding thereto, K251 and K253 of an lgG2 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.

[0326] 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 these groups / moieties. In some embodiments, these agents are useful as reaction partners, e.g., first agents for conjugating moieties of interest, e.g., anti-TSH receptor antibody-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 I VIG preparations, etc. In some 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 these moieties. In some embodiments, an antibody-binding moiety is part of a leaving group that is 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 (in some cases, single steps) to conjugation product agents.

[0327] In some embodiments, anti-TSH receptor antibody-binding moieties may be conjugated to antibody moieties optionally through linker moieties using technologies described in U.S. Patent Publication 2020 / 0190165.

[0328] 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. These groups and transformations are described in Greene's Protecting Groups in Organic Synthesis, Wuts editor (John Wiley & Sons, 2014), the entirety of each of which is incorporated in this specification by reference.

[0329] In some embodiments, an oxygen-protecting group includes carbonyl and hydroxylprotecting groups, etc. Hydroxyl-protecting groups and amino-protecting groups are well known in the biomedical art and include those described in Greene's Protecting Groups in Organic Synthesis, Wuts, editor (John Wiley & Sons, 2014), the entirety of which is incorporated in this specification by reference.

[0330] Persons having ordinary skill in the biomedical art know that provided agents may contain one or more stereocenters and may be present as a racemic or diastereomeric mixture. They know thatthere 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 high performance liquid chromatography, chiral high performance liquid chromatography, 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.

[0331] 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, 5thedition (John Wiley & Sons, 2001), which is incorporated in this specification by reference.

[0332] 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 producing 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 in accordance with this specification.

[0333] 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., anti- TSH receptor antibody-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 antibodybinding 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 located between an antibodybinding moiety and a cellular receptor-binding moiety.

[0334] 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 provides compositions of such agents.Plurality of agents.

[0335] 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.

[0336] In some embodiments, product agents are MATE® agents. In some embodiments, an antibody agent moiety comprises the 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.

[0337] 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 the heavy chain (unless otherwise specified, locations in this specification include corresponding residues in, e.g., modified sequence, e.g., longer, shorter, rearranged, etc., sequences. In some embodiments, a location is K248 of the heavy chain. In some embodiments, a location is K288 or K290 of heavy chain. In some embodiments, a location is K288 of the 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 IgGl antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an lgG2 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 IgGl, lgG2, or lgG4 antibody, or a fragment thereof.

[0338] In some embodiments, antibody heavy chains are selectively conjugated / labeled over light chains.

[0339] In some embodiments, the invention provides a composition comprising a plurality of agents, each of which independently comprises an antibody moiety, a cellular receptor-binding moiety, and optionally 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 of at least onecommon amino acid residue of the common amino acid sequence; and wherein about l%-100% of all agents that comprise an antibody moiety that comprises the common amino acid sequence and the cellular receptor-binding moiety are agents of the plurality.

[0340] In some embodiments, the invention provides a composition comprising a plurality of agents, each of which 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; 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; and wherein about l%-100% of all agents that comprise the antibody moiety and the cellular receptor-binding moiety are agents of the plurality.Reactive Group

[0341] 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 are located between antibody-binding moieties and moieties of interest. Reactive groups 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.

[0342] In some embodiments, reactive groups, when used in agents that comprise no antibodybinding moieties, react slowly and provide a low level of, in some embodiments, substantially no conjugation of moieties of interest with target agents. As shown in this specification, a combination of reactive groups with antibody-binding moieties in the same agents, 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.

[0343] Reactive groups in agents can react with several groups in target agents. In some embodiments, reactive groups in agents selectively react with amino groups of target agents, e.g., -NH2groups on side chains of lysine residues of proteins. In some embodiments, reactive groups when used in agents, selectively react with sites of target agents, e.g., as shown in examples in this specification, one or more of K246, K248, K288, K290, K317, etc. for IgGl, one or more of K251, K253, etc. for lgG2, and one or more of K239, K241 for lgG4. In some 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 someembodiments, 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 lgG2 heavy chain. In some embodiments, a site is K251 of an lgG2 heavy chain. In some embodiments, a site is K253 of an lgG2 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.

[0344] 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.

[0345] In some embodiments, a reactive group, e.g., RG, is or comprises — LRG1— 1_RG2— , wherein each of LRG1and LRG2is independently L. In some 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.

[0346] In some embodiments, LLG4is -O-. In some embodiments, LLG4is -N(R)-. In some embodiments, LLG4is -NH-.

[0347] In some embodiments, LLG3is or comprises an optionally substituted aryl ring. In some embodiments, LLG3is 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.

[0348] In some embodiments, LRG1is a covalent bond. In some embodiments, LRG1is not a covalent bond. In some embodiments, LRG1is -S(O)2-.

[0349] In some embodiments, LRG2is -C(O)-. In some embodiments, a reactive group comprises LLG4-C(0), wherein each variable is described in this specification. In some embodiments, a reactive group comprises -LLG3-LLG4-C(O)-, wherein each variable is as described in this specification. In some embodiments, a reactive group comprises -LLG2-LLG3-LLG4-C(O)-, wherein each variable is as described in this specification.

[0350] In some embodiments, LRG2is -LRG3-C(=CRRG1RRG2)-CRRG3RRG4-, wherein each of RRG1, RRG2, RRG3and RRG4is independently -L-R', and LRG3is -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 RRG4is independently R'. In some embodiments, one or more of RRG1, RRG2, RRG3and RRG4is independently -H. In some embodiments, LRG3is -C(O)-. In some embodiments, LRG3is -C(O)O-. In some embodiments, -O-, -N(R')-, etc. of LRG3is bonded to LPM.

[0351] In some embodiments, RRG1is -H. In some embodiments, RRG3is -H.

[0352] In some embodiments, LRG2is optionally substituted -LRG3-C(=CHRRG2)-CHRRG4-, wherein each variable is as described in this specification.

[0353] In some embodiments, RRG2and RRG4are taken together 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 contains no additional unsaturation besides the double bond in C(=CHRRG2) or C(=CRRG1RRG2).

[0354] In some embodiments, -C(=CHRRG2)-CHRRG4or -C(=CRRG1RRG2)-CRRG3RRG4is optionally substituted 'Q^ or isIn some embodiments, -[C(=CHRRG2)-CHRRG4]-LRG3- or -[C(=CRRG1RRG2)-CRRG3RRG4]-LRG3- is optionally substituted 'Q^ or isIn some embodiments, -LRG1-[C(=CHRRG2)-CHRRG4]-LRG3- or -LRG1-[C(=CRRG1RRG2)-CRRG3RRG4]-LRG3- is optionally substitutedIn some embodiments, -LRG1-[C(=CHRRG2)-CHRRG4]-LRG3- or -LRG1-[C(=CRRG1RRG2)-CRRG3RRG4]-LRG3- is optionally substituted.

[0355] In some embodiments, a reactive group, e.g., -LLG2-LLG3-LLG4-LRG1-LRG2- or _LLG2_|_LG3_LLG4_|_RGI_is a structureselected from TABLE 3 of International Patent Publication WO 2024 / 228935 (Biohaven Therapeutics, Ltd.).

[0356] In some embodiments, -LLG4-LRG2- is -O-C(O)- or -S-C(O)-. In some embodiments, -LLG4_LRG1_|_RG2_is_S-C(0)_

[0357] 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.

[0358] In some embodiments, LRG2is 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, LRG2is optionally substituted -CH2- bonded to an electron-withdrawing group comprising or connected to an antibody-binding moiety. In some embodiments, LRG1is an electron-withdrawing group. In some embodiments, LRG1is 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)-.

[0359] In some embodiments, LRG2is optionally substituted -CH2-C(O)-, wherein -CH2- is bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, LRG2is optionally substituted -CH2- bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, LRG1is 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)-.

[0360] In some embodiments, a reactive group reacts with an amino group of a target agent. In some embodiments, an amino group is -NH2of the side chain of a lysine residue.

[0361] 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- reacts with -NH2, e.g., of the side chain of a lysine residue) and forms an amide group -C(O)-O- with the -NH2.

[0362] In some embodiments, reactive groups, e.g., a first reactive group, a second reactive group, etc., are located at terminal locations. In some embodiments, the first agents comprise the first reactivegroups linked to anti-TSH receptor antibody-binding moieties optionally through linker moieties. They do not contain antibody-binding moieties.

[0363] 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 comprises contacting a plurality of agents, each independently comprising a reactive group with a plurality of antibody agents.

[0364] 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 IgGl. In some embodiments, a plurality of antibody molecules comprises lgG2. In some embodiments, a plurality of antibody molecules comprises lgG4. In some embodiments, a plurality of antibody molecules comprises IgGl and lgG2. In some embodiments, a plurality of antibody molecules comprises IgGl, lgG2, and lgG4. In some embodiments, a plurality of antibody molecules comprises IgGl, lgG2, lgG3 and lgG4. In some embodiments, a plurality of antibody molecules is I VIG antibody molecules.°FW

[0365] In some embodiments, provided agents comprise a reactive group, e.g., O UIn 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 a °FW some embodiments, 0 reacts with an amino group of another moiety, e.g., an antibody moiety, forming an amide group with the moiety and releasing a moiety comprising an antibody-binding moiety. In some embodiments, an amino group is -NH2of 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 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 in accordance with this specification.

[0366] 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, anti-TSH receptor antibody-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 IgGl. In some embodiments, an antibody is or comprises lgG2. In some embodiments, an antibody is or comprises lgG4. In some embodiments, an antibody composition used in a method comprises IgGl and lgG2. In some embodiments, an antibody composition used in a method comprises IgGl, lgG2, and lgG4. In some embodiments, an antibody composition used in a method comprises IgGl, lgG2, lgG3, and lgG4.

[0367] In some embodiments, a product is or comprises IgGl. In some embodiments, a product is or comprises lgG2. In some embodiments, a product is or comprises lgG4. In some embodiments, a product composition comprises IgGl and lgG2. In some embodiments, a product composition comprises IgGl, lgG2, and lgG4. In some embodiments, a product composition comprises IgGl, lgG2, lgG3, and lgG4.

[0368] In some embodiments, provided agents comprising antibody moieties provide one or more or substantially all antibody immune activities, e.g., recruiting one or more types of immune cells or providing 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 provide comparable or better stability than antibody moieties by themselves, e.g., residence time in blood. In some embodiments, antibody moieties in provided agents can bind to the 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 bindingcapabilities. 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 recycling or prolonged half-life. In some embodiments, provided technologies are useful for modifying blood-derived IgG products as provided technologies are suitable for and can use all IgG subclasses.

[0369] In some embodiments, a provided method comprises one of the steps described below. In some embodiments, a 0 J reacts with an amino group of a lysine side chain to form an amide bond with an antibody molecule and release it.Pharmaceutically acceptable excipients.

[0370] 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 contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral, oral, and intravenous are the preferred administration methods. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0371] 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 adjustment of tonicities 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.

[0372] Protein sequencing. In one protein sequencing method, eight digestions are prepared using five different 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 liquid chromatography-mass spectroscopy (LC-MS) / mass spectroscopy (MS) using a Thermo-Fisher Orbitrap fusion™ mass spectrometer. Peptides are characterized from liquid chromatography-mass spectroscopy / mass spectroscopy data using de novo peptide sequencing and assembled into antibody sequences.

[0373] Total protein concentration can be determined by Bradford assay (Biorad). Nunez Miguel et al., Journal of Molecular Endocrinology, 70, e220120 (2022).Statistical analyses.

[0374] Normal distribution quantitative variables can be expressed as means and SDs and compared by an independent-sample t-test. The inventors used median and interquartile ranges for non-normally distributed variables and analyzed them with the Mann-Whitney U test. Categorical data was summarized by percentages. A two-sided p-value <0.05 was considered statistically significant. All statistical tests were performed using SPSS version 16.0.

[0375] GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA) can be used to fit a doseresponse curve using non-linear regression and calculate the EC5o of each agonist. See Miller-Gallacher et al., Journal of Molecular Endocrinology, 62, 117-128 (2019).Methods to detect TSH receptor antibodies.

[0376] Measurements of TSH receptor autoantibodies are important in diagnosing and managing Graves' disease and other thyroid disorders. It is well documented in the biomedical art that patient TSH receptor autoantibodies bind to regions on the TSHR leucine-rich domain overlapping with each other and the TSH binding site. There are differences in the TSH receptor residues in contact with autoantibodies present in different sera. Persons having ordinary skill in the art can use any of four types of assay are used to measure TSH receptor autoantibodies described by International Patent Publication WO 2015 / 189543 (RSR Ltd.). (1) Competitive binding assays which measure the ability of patient serum TSH receptor autoantibodies to inhibit the binding of TSH or human monoclonal TSH receptor autoantibodies to preparations of TSH receptor. (2) Bioassays which measure the ability of TSH receptor autoantibodies to stimulate cells expressing the TSH receptor in culture. (3)Immunoprecipitation of labeled TSH receptor preparations with TSH receptor autoantibodies. (4) Bridgetype assays in which divalent TSH receptor autoantibodies bind to TSHR coated onto ELISA plate wells with one arm and to liquid phase TSHR260-alkaline phosphatase fusion protein with the other arm to form a bridge. An ELISA described in International Patent Publication WO 2010 / 07301 is based on the ability of divalent TSHR antibodies to bind with one antigen binding site to TSHR coated onto an ELISA plate well and with the other antigen binding site to TSHR260-AP in liquid phase, i.e., forming a bridge. The assay relies on the bivalent properties of human monoclonal stimulating type TSHR autoantibodies and the human monoclonal blocking type TSHR autoantibody to form a bridge between immobilized full-length wild type TSHR and alkaline phosphatase labeled TSHR260 mutants.

[0377] A suitable binding assay may comprise a plate having bound thereto a TSHR fragment to be tested and a labeled antibody or autoantibody to TSHR. The TSHR to be tested is suitably bound to the plate in such a way so as not to interfere with the binding of the antibody to the TSHR protein. The TSHR may be bound to the plate using any suitable antibody. One such antibody is mouse monoclonal TSH receptor antibody 14C4. See International Patent Publication WO 2015 / 189543 (RSR Ltd.). The amount of labeled antibody bound can be used to indicate the amount of active mutant protein.

[0378] The activity in the TSHR activity assay may be expressed in any suitable way, for example as the amount of activity per volume of sample, i.e., units per ml. The activity is the specific activity as measured in units of activity per quantity of protein— for example, units per mg. See International Patent Publication WO 2015 / 189543 (RSR Ltd.).Characterizing antibody-binding moieties.

[0379] Many technologies are available for identifying, assessing, or characterizing antibodybinding 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 International Patent Publication 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., a small molecule, peptide, nucleic acid, etc., that can selectively bind to IgG and 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.

[0380] Persons having ordinary skill in the biomedical art know that antibody-binding moieties described in this specification target antibodies with several properties and activities, e.g., antibodies recognizing different antigens, having optional modifications, etc. In some embodiments, these 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.

[0381] In some embodiments, an antibody-binding moiety comprises a meditope agent moiety. A meditope agent is described in, e.g., Patent Publication US 2019 / 0111149.

[0382] 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, IgGl, lgG2, lgG3, and lgG4. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, binds to IgGl, lgG2, and lgG4.Specific mutation strategies to reduce FcyR and cig binding and effector functions and to enhance FcRn binding and exposure of antibodies.Fc mutation strategies to reduce FcyR and cig binding.

[0383] The inventors mutate an antibody, an antibody variant, or an antigen-binding fragment thereof in the Fc region to insert a LALA mutation using biomedical art-recognized methods first described by the Winter group in the 1990s. In this EXAMPLE, LALA = L234A / L235A.

[0384] The inventors mutate an antibody, an antibody variant, or an antigen-binding fragment thereof in the Fc region to insert a LALA mutation using biomedical-art recognized methods first described by the Winter group in the 1990s, then inserted P mutations, such as by the biomedical-art recognized methods introduced by Roche team in 2016, such as the technology for adding P329G and P329G combined with LALA: In this EXAMPLE, LALA / PA = L234A / L235A / P329A. LALA / PG = L234A / L235A / P329G. See Tilman et al., Protein Engineering, Design and Selection, Volume 29, Issue 10, pages 457-466 (October 2016), which shows that LALA abolishes clq binding. P329A alone was tested. P329A abolishes clq binding and reduces FcgR binding. They show that P329G / LALA further reduces FcyR binding beyond LALA alone.

[0385] Persons having ordinary skill in the biomedical art can also mutate an antibody, an antibody variant, or an antigen-binding fragment thereof in other regions. N297A / Q removes the natural N-linked glycosylation site in the hinge region.Fc mutation strategies to enhance FcRn binding to prolong exposure.

[0386] There are many publicly available approaches and mutation sets to increase the binding of an Fc to FcRn. See TABLE 4. Strohl, Current Opinion in Biotechnology, 20(6), 685-691 (2009), which provided an older summary.

[0387] For an example of introducing a YTE region in a protein (M252Y / S254T / T256E), see Acqua et al., The Journal of Immunology 169(9), 5171-5180 (2002). For a recent example combining LALA and YTE, see Cobb et al., bioRxiv, 2021-09 (2021). Additional references with details of engineering are also available.

[0388] For an example of introducing an LS region in a protein (M428L / N434S), see Zalevsky et al., Nature Biotechnology, 28(2), 157-159 (2010).Structure of recombinont ligond constructs.

[0389] GN3 sortose reogent. Persons having ordinary skill in the biomedical art have access to commercially available protocols for sortase conjugation using the ASPGR binder for the C-terminal sortase tag (LPETGG) for conjugation (GN3 / linker).

[0390] GN3-mgleimide reggent (maleimide-GN3 / linker), with a C-terminal cysteine for conjugation.Methods of ma kina agents.

[0391] Agents of this specification may be prepared or isolated by synthetic or semi-synthetic methods or recombinant methods in accordance with this specification. In some embodiments, polypeptide agents, e.g., cellular receptor-binding moiety peptide agents, may 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 International Patent Publication WO 2019 / 023501, which is incorporated in this specification in its entirety by reference.

[0392] Several technologies, e.g., those for preparing antibody-drug conjugates, may be used in preparing MATE® agents. In some embodiments, the invention provides technologies that can be used for selective conjugation of anti-TSH receptor antibody-binding moieties at amino acid residue sites.

[0393] 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, and forming a product agent comprising a cellular receptor-binding moiety and an antibodybinding moiety optionally through a linker.

[0394] In some embodiments, the invention provides a method of synthesis comprising the steps: 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.Commercially available reagents useful for making or using agents.

[0395] Anti-TSH receptor antibody M22™ (Fab), stimulating antibody (Cell Sciences, catalogue # FHD18110B), which binds to the leucine-rich domain of TSHR. The antibody binding overlaps with that of the native ligand, TSH.

[0396] Anti-human TSHR antibody Kl-70 (Dima Biotech, BME100080).

[0397] Recombinant human TSHR ectodomain Fc- fusion chimera (Biotechne, catalogue # 8950-TR).

[0398] Recombinant human TSHR extracellular domain, with C-terminal histidine (SinoBiological, catalogue # 17299-H08B).

[0399] Recombinant Human TSHR leucine-rich domain, amino acids 22-260 with histidine tag (MyBioSource, catalogue # MBS355800).

[0400] Thyrotropic hormone from human pituitary (Millipore Sigma, catalogue # T9265).Method of gssessing ggent for gdministrgtion

[0401] Persons having ordinary skill in the biomedical art can analyze an agent formulation for appearance, color, and achromicity using the criteria described by the United States Pharmacopeia- National Formulary in USP <631> or by the European Pharmacopoeia in Ph. Eur. 2.2.2.

[0402] Persons having ordinary skill in the biomedical art can analyze an agent formulation for appearance and clarity using the criteria described by the United States Pharmacopeia-National Formulary in USP <1061> or by the European Pharmacopoeia in Ph. Eur. 2.2.2.

[0403] Persons having ordinary skill in the biomedical art can analyze an agent formulation for pH using the criteria described by the United States Pharmacopeia-National Formulary in USP < <791> or by the European Pharmacopoeia in Ph. Eur. 2.2.3. A generally accepted criterion is ±0.5 pH of the targeted pH.

[0404] Persons having ordinary skill in the biomedical art can analyze the drug product for osmolality using the criteria described by the United States Pharmacopeia-National Formulary in USP <785> or by the European Pharmacopoeia in Ph. Eur. 2.2.35.

[0405] Persons having ordinary skill in the biomedical art can quantify the concentration of an agent by UV-VIS spectroscopy, e.g., at A280 (280 nm). A generally accepted criterion is ±10% of the targeted concentration.

[0406] Persons having ordinary skill in the biomedical art can confirm the Identity of an agent by the charge variant peak retention time or pl as measured by imaged capillary isoelectric focusing (iCIEF), a high-resolution technique that separates proteins into groups based on their isoelectric point (pl).

[0407] Persons having ordinary skill in the biomedical art can confirm the purity of the agent by the HIC-HPLC method. For example, the persons having ordinary skill in the biomedical art can confirm the DAR of the agent. A generally accepted criterion is ±10% of the targeted concentration.

[0408] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the SE-HPLC method. For example, the persons having ordinary skill in the biomedical art can confirm the percentage of a monomer, high molecular weight, and low molecular weight. A generally accepted criterion is a result of >90% or <5%.

[0409] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the CE-SDS-NR method. For example, the persons having ordinary skill in the biomedical art can confirm the percentage of a main peak and fragment peaks. A generally accepted criterion is a result of >85%.

[0410] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the CE-SDS-R method. For example, the persons having ordinary skill in the biomedical art can confirm the percentage of monomer and Minor species peaks. A generally accepted criterion is a result of >85%.

[0411] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the iCIEF method. For example, the persons having ordinary skill in the biomedical art can confirm the percentage of charge variants.

[0412] Persons having ordinary skill in the biomedical art can confirm the potency of an agent for binding by the ELISA method. A generally accepted criterion is ± a defined percentage as compared to a reference standard.

[0413] Persons having ordinary skill in the biomedical art can detect a process impurity (HCP) in an agent formulation by the ELISA method. A generally accepted criterion is < 100 ng / mg.

[0414] Persons having ordinary skill in the biomedical art can detect a process impurity (rProtein A) in an agent formulation by the ELISA method. A generally accepted criterion is < 50 ng / mg.

[0415] Persons having ordinary skill in the biomedical art can detect an rDNA impurity in an agent formulation by the rtPCR method. A generally accepted criterion is < 10 ng per dose.

[0416] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the RP-HPLC method. For example, the persons having ordinary skill in the biomedical art can confirm the presence of residual free drug & free protein intermediates.

[0417] Persons having ordinary skill in the biomedical art can analyze an agent formulation for the presence of endotoxins using the criteria described by the United States Pharmacopeia-NationalFormulary in USP <85> or by the European Pharmacopoeia in Ph. 2.6.14. A generally accepted criterion is dose dependent based on < 5 EU / kg.

[0418] Persons having ordinary skill in the biomedical art can analyze a drug substance for its bioburden using the criteria described by the United States Pharmacopeia-National Formulary in USP <61> or by the European Pharmacopoeia in Ph. 2.6.12. A generally accepted criterion is < 1 CFU / 10 mL.

[0419] Persons having ordinary skill in the biomedical art can analyze a drug product for its sterility using the criteria described by the United States Pharmacopeia-National Formulary in USP <71> or by the European Pharmacopoeia in Ph. 2.6.1. A generally accepted criterion is no growth.

[0420] Persons having ordinary skill in the biomedical art can analyze a drug product for the presence of excipients using the HPLC-ELSD method For example, the persons having ordinary skill in the biomedical art can measure the percentage of surfactants. A generally accepted criterion is ±50% of the targeted value.

[0421] Persons having ordinary skill in the biomedical art can analyze a drug product for the subvisible particles in a container using the criteria described by the United States Pharmacopeia- National Formulary in USP <787> or by the European Pharmacopoeia in Ph. 2.6.19. Several accepted criterion are known to persons having ordinary skill in the biomedical art.

[0422] Persons having ordinary skill in the biomedical art can analyze a drug product for the extractable volume using the criteria described by the United States Pharmacopeia-National Formulary in USP <697> or by the European Pharmacopoeia in Ph. 2.6.17.

[0423] Persons having ordinary skill in the biomedical art can analyze a drug product for Container Closure Integrity Test (CCIT), using the criteria described by the United States Pharmacopeia-National Formulary in USP <1207>. A bioburden test is usually performed at the final stability time only. A CCIT test is usually performed at repeated time intervals, e.g., annually.EXAMPLES

[0424] The invention is further illustrated by non-limited EXAMPLES.EXAMPLE 1Procedure for Preparation of lnt-00027

[0425] Preparation of Intermediate 2

[0426] Pd / C (4.00 g, 10% purity) in reaction bottle (purged with argon gas for three times) was added tetrahydrofuran (40 mL) slowly, then a solution of trifluoroacetic acid (7.44 g, 65.2 mmol, 4.86 mL, one equivalent) and Intermediate 1 (40.0 g, 65.2 mmol, 1.00 equivalent) in tetrahydrofuran (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). Thin-layer chromatography (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 tetra hydrofuran (100 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with tetrahydrofuran (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, liquid chromatography-mass spectroscopy: retention time is 0.428 minutes, MS calculated: 478.22, mass observed: [M + Na]+= 501.2.1H NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6) <5 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).

[0427] Preparation of Intermediate 4

[0428] To a stirring solution of Intermediate 3 (8.50 g, 16.0 mmol, 1.00 equivalent) andIntermediate 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. Thin-layer chromatography (DCM: methanol = 10: 1, Rf = 0.5) indicated Intermediate 3 was consumed completely. One major new spot with larger polarity was detected. The reaction mixture was slowly poured into a stirring cold 1.0 mol / L HCI solution (350 mL) and stirred for ten minutes. White precipitate was formed and filtered. The aqueous phase was extracted with DCM (350 mL* 2) twice. The combined organic layers were washed with saturated 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 make Intermediate 4 (22.0 g, 10.6 mmol, 66.0% yield, 92.2% purity) as a white solid.1H NMR (400 MHz, DMSO-d6) <5 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). liquid chromatography-mass spectroscopy: retention time is = 0.403 minutes, MS calculated: 1908.81, mass observed: [M + 2H]2+= 1910.2.

[0429] Preparation of Intermediate 5

[0430] 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. Tetrahydrofuran (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 pL, 1.00 equivalent) in tetra hydrofuran (100 mL) slowly under argon gas atmosphere. The resulting mixture was degassed and purged with hydrogen gas for three times. The mixture was then stirred at 25°C for three hours under hydrogen gas atmosphere (15 psi). Thin-layer chromatography (DCM: methanol = 10: 1, Rf = 0.6) indicated Intermediate 9 was consumed completely. One major new spot was detected. The reaction mixture was filtered carefully through siliceous earth under nitrogen gas atmosphere. The cake was washed with tetra hydrofuran (50 mL*2). Then, the filter cake was added water immediately. The organic layer concentrated under reduced pressure to make 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-cfe) 6 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). liquid chromatography-mass spectroscopy: retention time is 0.341 minutes, MS calculated: TI J , found: [M + 2H]2+= 1776.9.

[0431] Preparation of Intermediate Target A001A

[0432] 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 equivalent) at 0°C. The mixture was stirred at 0°C for 0.5 hours. The reaction was monitored by liquid chromatography-mass spectroscopy, which showed the desired mass (one main peak with desired was detected.). The reaction mixture was added with 1.0 M HCI 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 make Target A001A (4.1 g, 2.74 mmol, 94.1% yield, 93.4% purity, HCI) as a white solid.XH NMR (400 MHz, DMSO-d6) 6 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). liquid chromatography-mass spectroscopy: retention time is 0.22 minutes, MS calculated: 1396.6, found: [M + H]+= 1397.8.

[0433] Preparation of Intermediate lnt-00027

[0434] 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 pL, 3.00 equivalents) at 0°C. The mixture was stirred at 0°C for two hours under nitrogen gas atmosphere, liquid chromatography-mass spectroscopy 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 make lnt-00027 (1.75 g, 1.04 mmol,73.2% yield, 96.5% purity) as a white solid, liquid chromatography-mass spectroscopy: Retention time is0.286 minutes, MS calculated: Mo„ = 1612.68, [M + 2H]2+= 807.1, [M - sugar + 2H]2+= 705.6.EXAMPLE 1AProcedure for Preparation of TBT302lnt-00037

[0436] Peptide was synthesized using standard Fmoc chemistry (C resin).

[0437] (1) Resin preparation: DMF (100 mL) was added to the vessel containing Rink MBHA-AmideResin (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.

[0438] (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 reactionwas monitored by ninhydrin test, if it showed colorless, the coupling was completed. The resin was then washed with DMF (100 mL) * 5.

[0439] (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.

[0440] (4) Steps 2 and 3 were repeated for the following amino acids elongation: Number # 2-3,TABLE 2A.

[0441] (5) After the last position completed, the resin was then washed with DMF (100 mL) * 5,MeOH (100 mL) * 5, then dried under reduced pressure to afford peptide-bound-resin (5 mmol).

[0442] Peptide TFA de-protection-. The peptide-bound-resin was stirred in a solution of TFA / 3-MPA / Tis / H2O (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, then dried under reduced pressure for two hours to afford lnt-00037 (620 mg, crude) as a white solid. The crude was purified by prep-HPLC (A: 0.075% TFA / H2O, B: MeCN) directly to afford lnt-00037 (1.59 g, 3.77 mmol, 75.5% yield, 94.3% purity) as a white solid. LCMS: retention time = 0.071 min, MS calculated: Mo„ = 397.39, mass observed: [M + H]+= 398.1.

[0443] Preparation of TBT302 :

[0444] To a solution of lnt-00037 (500 mg, 1.25 mmol, 1.20 equiv.) and lnt-00027 (1.69 g, 1.04 mmol, 1.00 equiv.) in DMF (10 mL) was added a solution of CuSO4(0.4 M, 2.62 mL, 1.00 equiv.), sodiumL-ascorbate (0.4 M, 10.4 mL, 4.00 equiv.) at 0 °C. The mixture was stirred at 0 °C for 2 h under N2atmosphere. LCMS indicated lnt-00027 was consumed completely, one main peak with desired MS was detected. The residue was purified by prep-HPLC (AcOH condition) directly to afford TBT302 (1.377 g, 666 pmol, 63.5% yield, 97.3% purity) as a white solid. LCMS: RT = 1.372 min, MS cal.: MOT= 2010.06, [M + 2 H]2+= 1005.7, [M + 3 H]3+= 671.0, [M - sugar + 2H]2+= 904.2, [M - 2sugar + 2 H]2+= 802.6.EXAMPLE 2Preparation of Building block #1

[0445] Preparation of Intermediate 2:25 26

[0446] 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 16 h. The solvent was removed at 70°C under reduced pressure. The residue was triturated in acetonitrile (50 mL) for ten minutes. After filtration, the solid was dried under lyophilization to make Intermediate 26 (100.0 g, 427.9 mmol, 88.4% yield, HBr salt) as a brown solid.TH NMR (400 MHz, DMSO-d6) 6 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).

[0447] Preparation of Intermediate 27:2627

[0448] To a mixture of Intermediate 26 (60.0 g, 270.2 mmol, 1.00 equivalent, HBr salt), Reactant 26a (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 20°C. The mixture was stirred at 20°C for three hours. The mixture was precipitated with 0.5 M HCI (cold, 10 L). After filtration, the solid was dissolved in DCM (2 L), washed with 0.5 M HCI (800 mL), water (800 mL), brine (800 mL), dried over anhydrous Na2SO4and concentrated under reduced pressure. The residue was purified by silica gel column (DCM / methanol = from 1 / 0 to 20 / 1) to make Intermediate 27 (120.0 g, 90% purity, containing a little DMF, 83.3% yield) as a white solid.TH NMR (400MHz, DMSO-dg) 6 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).

[0449] Preparation of Building block #1:O NHFmoc O NHFmocMolecular Weight: 478.4727 Building block #1

[0450] 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 0.5 h. The solvent was removed under reduced pressure. The residue was purified by silica gel column (DCM / methanol = from 1 / 0 to 10 / 1) to make Building block #1 (100.0 g, 93.5% purity, 93.1% yield) as a white solid.XH NMR (400 MHz, DMSO-dg) 6 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 3Procedure for preparation of TBT301

[0451] Preparation of Intermediate 30: Peptide was synthesized using standard Fmoc chemistry(CTC resin).

[0452] (1) Resin preparation: To the vessel containing CTC resin (2.0 g, 2.0 mmol, 1.00 mmol / g) andFmoc-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.

[0453] (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.

[0454] (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.

[0455] (4) Steps 2 and 3 were repeated for the following amino acids elongation: Number # 3-15,TABLE 3 below.

[0456] (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 make resin-bound peptide Intermediate 28 (CTC resin, 2.0 mmol).

[0457] Peptide cleavage and cyclization.

[0458] (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 25°C and stirred for two hours.

[0459] (2) After filtration, the filtrate was collected.

[0460] (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. The crude peptide was dried under reduced pressure for two hours to make Intermediate 29 (3.0 g, crude) as a white solid.

[0461] To the mixture of Intermediate 29 (3.0 g, crude) in HOAc / acetonitrile / 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 25°C for five minutes. The mixture was quenched by addition of 0.1 M aqueous Na2S2O3dropwise until the yellow color disappeared. After filtration, the filtrate was purified by prep-high performance liquid chromatograph (A: 0.075% trifluoroacetic acid / water, B: acetonitrile), followed by lyophilization to make Intermediate 30 (360 mg, 90.0% purity, 50.8% yield) as a white solid, liquid chromatography-mass spectroscopy: retention time is 0.907=0.960 minutes, MS calculated: Mo„ = 1833.02, mass observed: [M + 2H]2+=917.00, [M + H]+=1832.91.

[0463] 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 pL, three equivalents) in 10 mL DMF at 0°C. The resulting reaction was stirred for five minutes at 0°C. After completion monitored by liquid chromatography-mass spectroscopy, the mixture was directly injected into the reverse column, purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: acetonitrile), followed by lyophilization to make Intermediate 31 (700 mg, 95.0% purity, 57.5% yield) as colorless oil. liquid chromatography-mass spectroscopy: retention time is 0.709 minutes, MS calculated: Mo„ = 1821.81, mass observed: [M+16 + 3H]3+=613.10.

[0464] Preparation of TBT301:

[0465] To a solution of Intermediate 30 (140.86 mg, 76.85 pmol, 1.00 equivalent) and Intermediate 31 (140 mg, 76.85 pmol, 1.00 equivalent) in DMF (2 mL) was added DIEA (29.80 mg, 230.54 pmol, 40.16 pL, 3.00 equivalents). The mixture was stirred at 20°C for one hour. After completion monitored by liquid chromatography-mass spectroscopy, the mixture was acidified by 1 M HCI to pH = 5. The mixture was purified by prep-high performance liquid chromatography (A: 0.075% trifluoroacetic acid / water, B: acetonitrile) directly. This step was followed by lyophilization to make TBT301 (103 mg, 92.8% purity, 35.6% yield.) as a white solid, liquid chromatography-mass spectroscopy: retention time is 1.391 minutes, MS calculated: MOT= 3488.76, mass observed: [M + 3H]3+=1163.90.EXAMPLE 4ABT301 Transient Expression

[0466] (1) The inventors seeded and passaged CHO KI host cells in advance of performing the assay.

[0467] (2) On the day of transfection, the inventors centrifuged host cells to specific cell density for transfection.

[0468] (3) Added DNA and transfection reagent (PEI) into host cell for transfection.

[0469] (4) Incubated the transfected culture by shaking at 150 rpm.

[0470] (5) Shifted temperature from 36.5 degree to 33 degree at 24 hours post transfection.

[0471] (6) Performed feeding on day 0 and day 4, then harvested cell culture on day 7 or whenVIA<60%.Harvest and clarification

[0472] Subjected the cell culture fluid to centrifugation at 10000 g for thirty minutes and then pass through a 0.22 pm sterile filter.Affinity Capture by MabSelect SuRe Resin for ABT301

[0473] (1) Purified at room temperature.

[0474] (2) Chose the column based on titer, recommended load density (5-10 mg / ml resin).

[0475] (3) Equilibrated, five column volumes, with the buffer of 25 mM Tris-HCI, 150 mM sodium chloride, 5 mM EDTA, pH7.5.

[0476] (4) Sampled load.

[0477] (5) EQ wash. Washed with the buffer of 25 mM Tris-HCI, 150 mM sodium chloride, 5 mMEDTA, pH7.5, for five column volumes.

[0478] (6) Triton wash. Washed with the buffer of 25 mM Tris-HCI, 150 mM sodium chloride, 5 mMEDTA, 0.05% Triton 114, 0.05% Triton 100, pH 7.5, for sixty minutes.

[0479] (7) EQ wash. Washed with the buffer of 25 mM Tris-HCI, 150 mM sodium chloride, 5 mMEDTA, pH7.5, for ten column volumes.

[0480] (8) Eluted with elution buffer 0.1M HAC, for five column volumes (neutralize immediately with neutralization buffer to make a final pH around 5.5).

[0481] (9) Tested for affinity chromatography eluates: SDS-PAGE gel non-reducing and reducing, size exclusion chromatography-HPLC.Size Exclusive Chromatography forABT301

[0482] (1) Purified at room temperature.

[0483] (2) Chose the column based on protein amount and molecule weight, e.g., Superdex 200 pg.

[0484] (3) Equilibrated with a buffer of phosphate-buffered saline, pH7.4 for ABT301-01, 200 mMArginine, 137 mM succinic acid, pH 5.0 for ABT301-02 for one column volume.

[0485] (4) Sampled load.

[0486] (5) Elution with a buffer of phosphate-buffered saline, pH7.4 for ABT301-01, 200 mMArginine, 137 mM succinic acid, pH 5.0 for ABT301-02 for one column volume.

[0487] (6) Collected target protein with desirable purity. Testing for size exclusion chromatography eluates: SDS-PAGE gel nonreducing and reducing, size exclusion chromatography-HPLC.Buffer Exchange for ABT301

[0488] The size exclusion chromatography pool of ABT301-02 was in a buffer containing 200 mM arginine, 137 mM succinic acid, pH 5.0. The sample was then concentrated and dialyzed into a formulation buffer of phosphate-buffered saline at pH 7.4.Size exclusion chromatography-HPLC

[0489] (1) Perform size exclusion chromatography-HPLC analysis on a Vanquish Flex Duo liquid chromatography instrument using a TSKgel G3000SWxl stainless steel column (7.8 x 300 mm, particle size 5pm).

[0490] (2) The mobile phase consists of 50 mM sodium phosphate and 300 mM sodium chloride at pH 6.8.

[0491] (3) Inject 25 pg of sample per run. Perform isocratic elution for fifteen minutes at a flow rate of 1.0 mL / min. Monitor protein elution by ultraviolet light absorbance at 280 nm. Integrate the peaks corresponding to aggregates, monomers and LMW species, and calculate the percentage of each species.SDS-PAGE

[0492] (1) Perform Non-reducing and reducing SDS-PAGE analysis using precast NuPAGE™ 4-12%Bis-Tris Gel from Thermo Scientific. Sample loading buffer (4X LDS) is from Invitrogen. Gel running buffer (20X MES) is from GenScript.

[0493] (2) Treat non-reducing samples with 30 mM iodoacetamide and heat at 95 °C for 5 min before analysis. Treat reducing samples with 50 mM DTT and heat at 95 °C for 5 min before analysis. Carry out electrophoresis at a constant voltage of 180 V for 40 min. Stain gels using Coomassie blue for 30 min and destain with water for one hour.Endotoxin Level Testing

[0494] (1) Dilute purified proteins based on maximum valid dilution (MVD) in LAL reagent water(Charles River / WllO).

[0495] (2) Load samples into Charles River Laboratories EndoSafe LAL Cartridges (Charles River / PTS11F) and detect them using Charles River Laboratories EndoSafe NexGen-MCS™ instrument.Molecular Mass Analysis by LC-MS

[0496] (1) Conduct LC-MS analysis by using Agilent 6230 AdvanceBio LC / TOF system with a PL1912-1502, PLRP-S 1000A, 2.1 x 50 mm, 5 pm column (Agilent / PL1912-1502).

[0497] (2) For the Non-reduced intact mass analysis, dilute 200 pmol sample by ddH2O. The diluted sample was subjected to LC / MS. For the reduced mass analysis, denature and reduce 100 pmol sample using lOmM Urea (Sigma / 15568), 30 mM Tris-HCI (lnvitrogen / 15568), and DTT (Sigma / D0632) in a total volume of 50 pL. Incubate samples at 37°C for twenty minutes and then subjected them to LC / MS.

[0498] (3) Analyze the mass spectroscopy data using Bioconfirm 10.0 software and identify molecules based on molecular masses.EXAMPLE 5AGN301-001

[0499] The composition of matter AGN301-001 is a lysine-based antibody conjugate. The molecule consists of two same single chains covalently linked by disulfide bonds (ABT301, Fc-fusion). The linker payload TBT301 is conjugated to lysine amines in the antibody. The leaving group will be eliminated when the payload is conjugated to amines. The targeting BAR is 2.

[0500] Purification method for antibody conjugate by UF / DF. An ultrafiltration membrane (Pellicon3 0.11m2Cassette, Ultracel 30 kDa) having a molecular weight of 30 kDa is preferable to concentrate the sample. The UF / DF was preprocessed according to the following procedure:

[0501] (1) The inventors assembled the UF / DF system and install the cassette (0.11 m2membrane area, 30 kDa MWCO).

[0502] (2) The inventors flushed the system with water, cleaned with the 0.1 M sodium hydroxide for thirty minutes, and flushed with water again.

[0503] (3) The inventors added phosphate-buffered saline, pH 7.4 to the feed tank. Start the feed pump. Verify that the pH and conductivity in the system have been equilibrated to the level of the phosphate-buffered saline.

[0504] (4) The inventors added reaction mixture solution to the feed tank. Start the feed pump by partially closing the retentate valve and adjusting the pump speed. Diafilter the product with the phosphate-buffered saline, pH7.4, 10% dimethyl sulfoxide (Sigma-Aldrich, BCCL4606) for 40 DV, followed by phosphate-buffered saline, pH7.4 for twenty DV.

[0505] (5) Open the retentate valve fully and collect all the product. Filter the product by 0.22 pm membrane.

[0506] (6) Test the quality and concentration of product. Ultraviolet-visible spectroscopy platform by Nanodrop to determine protein concentration of in-process sample and final composition of matter. 750 nm was set up as baseline. The ultraviolet absorption at 280 nm and 220 nm were measured respectively. The calculation method based on Beer-Lambert Law A=E*C*I.A280= EmAt>280 *C[mAb] * I

[0507] Where E = molar extinction coefficient, C = molar concentration, and I = light path (Nanodrop: 0.1 cm).

[0508] BAR determination by liquid chromatography-mass spectroscopy. LC-MS was performed using a combination of Agilent 1260 series high performance liquid chromatography system and time-of- flight mass spectrometry. BAR was calculated based on the peak abundance of the deconvoluted mass.

[0509] (1) Preparation of sample used in liquid chromatography-mass spectroscopy analysis(deglycosylation)

[0510] (a) Denaturation. Transfer sample into 1.5 mL tube and incubate at 75°C for five minutes.

[0511] (b) Deglycosylation. Transfer 15 pg of denatured samples into a 1.5 mL tube, add 3 pL RapidPNGase F (non-reducing format, 5X) enzyme buffer and 0.5 pL Rapid PNGase F (non-reducing format) enzyme for de-glycosylation and some water to ensuring the total volume is 15 pL, then incubate this solution at 50°C for fifteen minutes. The obtained sample was used in a liquid chromatography-mass spectroscopy analysis.

[0512] A high performance liquid chromatography analysis was carried out under the following measurement conditions.

[0513] Aggregation determination by size exclusion chromatography-high performance liquid chromatograph. Size-exclusion chromatography was performed using an Agilent 1260 series HPLC system with the TSK gel G3000SWXL Size-exclusion chromatography column (7.8x300 mm, 5 pm) at 25°C. The mobile phase was consisted of 78 mM KH2PO4, 122 mM K2HPO4, 250 mM potassium chloride, 15% isopropanol at pH 7.0±0.1. The flow rate was set at 0.75 mL / min. Sample loading was 40~50 pg per injection. Samples were detected at 280 nm and 370 nm with a UV detector. The retention time of the aggregation peak was recorded based on its relative molecular weight. And the aggregation level was determined by the relative area of the peak at 280 nm.

[0514] Residual free drug determined by reverse phase-UPLC. The residual free drug level was determined by reverse phase UPLC. After protein precipitation, supernatant was loaded to Luna Omega 1.6 pm Polar C18 100A column and eluted by a gradient of increasing the organic mobile phase. Thepercentage of residual free drug was quantified via peak area by comparing it to external standard curve.Solvent preparation

[0515] Solvent I (for protein precipitation). Weighed 10 g NaCI to the pre-mixed organic solvent of 30 mL methanol and 50 mL acetonitrile, mixed and stirred one hour at least, allowed the solution to stand for at least one hour before use. The supernatant was the saturated sodium chloride solution.

[0516] Dilution Buffer I. Mix 250 pL of dimethyl sulfoxide, 700 pL of phosphate-buffered saline, lOOOpL of 6 mg / mL Herceptin® in phosphate-buffered saline as Buffer I.

[0517] Dilution Buffer II. Mix 300 pL of dimethyl sulfoxide, 700 pL of phosphate-buffered saline, lOOOpL of 6 mg / mL Herceptin® in phosphate-buffered saline as Buffer II.

[0518] Preparation of standard curve. (uABT (cpdl724) / linker payload (TBT301)). The stock standard uABT (cpdl724) / linker payload (TBT301) solution was firstly diluted to 1000 pM with DMSO for standard curve preparation. Add 10 pL of the 1000 pM uABT to 390 pL of dilution buffer I for a final concentration of 25 pM. Then the standard curve and sample were prepared. Take 160 pL of each standard samples and add 240 pl solvent buffer I as final standard curve (10 pM, 5 pM, 2 pM, 1 pM, 0.5 pM, 0.2 pM, 0.1 pM). Vortex solution for ten minutes at room temperature. Centrifuge solution for ten minutes at 16,000 ref at room temperature. Remove the supernatant immediately into a glass vial for analysis.

[0519] Preparation of sample. The inventors added phosphate-buffered saline and dimethyl sulfoxide (Sigma-Aldrich, BCCL4606) to sample to 6 mg / mL with 15% (v / v) dimethyl sulfoxide. Then they mixed 30 pL 6 mg / mL sample with 45 pL Solvent I (sample: precipitant l:1.5v / v). Vortex solution for ten minutes at room temperature. Centrifuge solution for ten minutes at 16,000 ref at room temperature. Remove the supernatant immediately into a glass vial for analysis.Data analysis (linker payload)

[0520] Integrated the stand BAR curve injections. Plotted the peak area (Y) as a function of concentration (X), Y=Kx + b. Calculated the slope (k) and intercept (b).

[0521] Integrated the related drug peak in the sample. Recorded the sum peak area (Y) and interpolated against the standard curve. Calculated the concentration obtained.

[0522] Where Cnnker payload—concentration of linker payload by C18-UPLC, Y — sum peak areas of drug related impurities, k = slope of standard curve, and b = intercept of standard curve.

[0523] Where C linker payload — concentration of linker payload by C18-UPLC (mg / mL), C protein — concentration of protein (mg / mL), BAR = drug to antibody ratio by liquid chromatography-mass spectroscopy, Mw Linker payload = molecular weight of drug, and Mwprotein = molecular weight of monoclonal antibody.Data analysis (uABT).

[0524] Integrated the standard BAR curve injections. Plotted the peak area (Y) as a function of concentration (X), Y=Kx + b. Calculated the slope (k) and intercept (b).

[0525] Integrated the related drug peak in the sample. Recorded the sum peak area (Y) and interpolated against the standard curve. Calculated the concentration obtained.

[0526] Where CUBT = concentration of uABT by C18-UPLC, Y = sum peak areas of drug related impurities, k = slope of standard curve, and b = intercept of standard curve.

[0527] Where C UABT concentration of uABT by C18-UPLC (mg / mL), C protein- concentration of protein (mg / mL), BAR = drug to antibody ratio by liquid chromatography-mass spectroscopy, MwUABT = molecular weight of uABT, and Mwprotein = molecular weight of monoclonal antibody.Residual Payload determination by reverse phase-UPLC.

[0528] The residual free drug level was determined by reverse phase UPLC. After protein precipitation, supernatant was loaded to Luna Omega 1.6 pm Polar C18 100A column and eluted by a gradient of increasing the organic mobile phase. The percentage of residual free drug was quantified via peak area by comparing it to external standard curve.

[0529] Preparation of standard curve (pay load cpdl777). The stock standard payload cpdl777 solution was firstly diluted to 1000 pM with formulation buffer for standard curve preparation. Add 10 pL of the 1000 pM payload cpdl777 to 90 pL of solvent II for a final concentration of 100 pM. Then the standard curve and sample were prepared. Vortex solution for ten minutes at room temperature. Centrifuge solution for ten minutes at 16,000 ref at room temperature. Remove the supernatant immediately into a glass vial for analysis.

[0530] Preparation of sample. Mix 30 pL sample with 60 pL Solvent I (sample: precipitant 1:2 v / v).Vortex solution for ten minutes at room temperature. Centrifuge solution for ten minutes at 16,000 ref at room temperature. Remove the supernatant immediately into a glass vial for analysis.

[0531] Integrated the standard BAR curve injections. Plotted the peak area (Y) as a function of concentration (X), Y=Kx + b. Calculated the slope (k) and intercept (b).

[0532] Integrated the related drug peak in the sample. Recorded the sum peak area (Y) and interpolated against the standard curve. Calculated the concentration obtained.

[0533] Where Cpayioaci = concentration of payload by C18-UPLC, Y = sum peak areas of drug related impurities, k = slope of standard curve, and b = intercept of standard curve.

[0534] Report the relative amount of residual payload in antibody conjugate composition of matter (% mol / mol) with two decimal place.

[0535] Where C payload — concentration of payload by C18-UPLC (mg / mL), C protein- concentration of protein (mg / mL), BAR = drug to antibody ratio by liquid chromatography-mass spectroscopy, MWpayioad = molecular weight of payload, and MWprotein = molecular weight of monoclonal antibody.

[0536] Hydrophobicity determination by hydrophobic interaction chromatography-high performgnce liguid chromgtogrgphy. Hydrophobic interaction chromatography was performed using an Agilent 1260 series HPLC system with the TSK gel Butyl-NPR Hydrophobic Interaction chromatography column (4.6x35 mm, 5 pm) at 25°C. The mobile phase A was consisted of 1.5 M (NH^zSC and 50 mM K2HPO43H2O, at pH 7.2±0.2, Cond. 190,0001200 us / cm. The mobile phase B was consisted of 50 mM Potassium Phosphate and 25% 2-propanol, at pH 7.410.2, Cond. 34001200 us / cm. The flow rate was set at 0.6 mL / min. Sample loading was 8 pL per injection. Samples were detected at 280 nm with an ultraviolet light detector.

[0537] Endotoxin determination. The endotoxin level was determined by kinetic turbidimetric assay.

[0538] Preparation of endotoxin standards and quality control. The Control Standard Endotoxin (CSE) (10 EU / vial, lyophilized was reconstituted with 1 mL of water for BET to yield a 10 EU / mL standard solution. Made ten-fold dilution using the water for BET, prepare standard solutions with four concentrations (Sl_10 EU / mL, S2_l EU / mL, S3_0.1 EU / mL, S4_0.01 EU / mL). Diluted Control Standard Endotoxin with water for BET to prepare quality control (0.05 EU / mL).

[0539] Preparation of Tachypleus Amebocyte Lysate (TAL) reagents. The TAL reagent is stable when stored at 2~8°C before expiration. Allow reagents to equilibrate to room temperature before use. Added 1.25 mL Water for BET to dissolve one bottle of TAL reagent.

[0540] Preparation of samples. The inventors diluted all the sample X / 2 folds with water for BET. Taken half volume of X / 2 folds sample and added equal volume of water for BET to make the finally tested sample with X folds dilution. Added equal volume of S3 in the remaining half of volume of X / 2 folds sample to make its PPC sample with endotoxin concentration to quality control.

[0541] Add 100 pL of the standard curve, quality control, sample, PPC and NTC into wells. Add 100 pL of TAL reagent into the 96-well plat wells (Greiner-655185). All standards control and samples need duplicate. Read microplate with a plate reader.

[0542] OD405 nm was read during incubating at 37°C for one hundred minutes in a microplate reader.

[0543] A standard curve was prepared by plotting the average 405 nm measurement for each standard compared to its endotoxin level in EU / mL.

[0544] Standard curve was used to determine the endotoxin level of samples.Conjugation buffer and formulation buffer.

[0545] (1) ABT301 in original buffer (phosphate-buffered saline, pH7.4) was buffer exchanged to 20 mM phosphate-buffered saline, pH 6.5 by Amicon® Ultra Centrifugal Filter Unit. Concentration was tested with 10.71 mg / mL by Nanodrop.

[0546] (2) 77 mg of antibody was transferred to 50mL tube (Greiner, GN227270) and placed in ice bath; then added with conjugation buffer (20 mM PB, pH 6.5) and 10 mM TBT301 solution (4.77 equivalents). The total dimethyl sulfoxide concentration in the reaction mixture was 3.05%; the concentration of the reaction is 10 mg / mL. The reaction vessel was allowed to approach ambient temperature and then incubated at 25°C for twenty-four hours.

[0547] (3) The crude conjugation was purified with UF / DF (Pellicon 3, 30 kDa, 0.11m2) with phosphate-buffered saline, pH7.4 (Gibco, 10010031), 10% dimethyl sulfoxide (Sigma, D4540) for 40 DV, followed by phosphate-buffered saline, pH7.4 for twenty DV.

[0548] (4) The purified sample was filtered with 0.22 pm filter.

[0549] (5) Performed the quality control tests including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography- high performance liquid chromatography, liquid chromatography-mass spectroscopy, and endotoxin level.Results of bulk conjugation.

[0550] After conjugation, the reaction mixture was purified with UF / DF (Pellicon 3, 30 kDa, 0.11m2) with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) contain 10% dimethyl sulfoxide (Sigma- Aldrich) for 40 DV and followed by phosphate-buffered saline, pH7.4 for twenty DV. Then filter with 0.22 pM membrane.

[0551] Reaction conditions and results for AGN301-001 bulk conjugation. TBT301 to ABT301 ratio was 4. Conjugation Concentration was 10 mg / mL. Conjugation temperature and time was 4-25°C fortwenty-four hours. Theoretical mass was 109683.17 Daltons. Measured mass was 109700.63 Da Amount was 54.71 mg. MS-BAR was 2.00 Size exclusion chromatography-purity was 97.44% Yield was 71.06 %.>>EXAMPLE 6Surface plasmon resonance binding

[0552] Binding confirmation by surface plasmon resonance. Surface plasmon resonance (SPR) assays were performed on a Cytiva Biacore S200 and Cytiva Biacore 8k+ instrument. Anti-TSH receptor antibody (M22™) was diluted in 10 mM sodium acetate buffer pH 4.5 to 10 pg / mL. The antibody ligand was immobilized to the surface of a CM5 chip via amine-coupling, targeting 300 RU. AGN301 was prepared in_HBS-P+ running buffer in concentrations ranging from 1-1000 nM. AGN301 was then injected over M22™ antibody as analyte using single-cycle kinetics method. As analyte is flowed in solution over an immobilized ligand, binding events between analyte and ligand induce a change in the refractive index proportional to bound mass. Data were analyzed using the Biacore Insight Evaluation Software to determine binding affinity.

[0553] The surface plasmon resonance results for AGN301 were KD = 123 ± 2.8 nM. AGN301 binds to M22™ antibody by surface plasmon resonance (KD~100 nM), similar to unfunctionalized LRD-Fc- fusion (ABT301, KD~265 nM).

[0554] AGN301 binds to human TSH in the surface plasmon resonance binding assay with less affinity to than to the M22™ antibody. In this surface plasmon resonance assay setup, AGN301 is immobilized as a ligand to the surface of a CM5 chip via amine-coupling and human TSH or anti-TSHR antibody (M22™) are flowed over as analyte. For human TSH KD = 497 ± 118 nM. For M22™ (positive control), KD= 11.2 ± 0.5 nM.

[0555] FIG. 5 shows the results of AGN301 binding to patient-derived anti-TSH receptor antibody (M22™) in the surface plasmon resonance assay.

[0556] Conclusion. Degraders bind to patient-derived anti-TSH receptor antibody (M22™) by surface plasmon resonance.EXAMPLE 7Ternary complex formation assay.

[0557] The ability of degraders to induce a ternary complex between anti-TSH receptor (M22™) and ASGPR can be evaluated using a TR-FRET assay.

[0558] Method for the ternary complex assay (HTRF). Fresh assay buffer was prepared immediately prior to assay (25 mM HEPES pH 7.5, 150 mM NaCI, 5 mM CaCI2, 0.01% Tween-20, 1 mM DTT). 20x degrader or bait protein dilutions were prepared in assay buffer (final top concentration at 1000 nM). A2x mix of anti-TSH receptor antibody (M22™) and biotinylated ASGPR CRD was prepared in assay buffer (final assay concentrations of 12.5 nM and 50 nM, respectively). The anti-TSHR antibody and ASGPR mix were aliquoted into an assay plate along with dilutions of bifunctional molecules and their unconjugated bait counterparts. Negative control wells with assay buffer were also prepared. The plate was sealed and incubated at room temperature for one hour. A 2.22x mix of HTRF fluorophore pair, comprising of polyclonal anti-mouse IgG d2-conjugate (Revvity) and streptavidin-europium cryptate conjugate (Revvity), was prepared in assay buffer (final assay concentrations of 15 nM and 2.5 nM, respectively). The HTRF fluorophore pair mix was aliquoted into the assay plate, including negative control wells. The plate was sealed and incubated at room temperature for one hour. The FRET ratio was read using an Envision plate reader and data were analyzed using GraphPad Prism 10. EC5o reported correspond to complex formation.

[0559] The results were AGN301 EC5o = 0.01 nM.

[0560] Degraders AGN301 and AGN302 mediate concentration-dependent ternary complex between ASGPR and anti-TSH receptor antibody (M22™). Their unconjugated bait counterparts ABT301 and ABT302 do not.

[0561] FIG. 6 shows formation of the ternary complex between ASGPR and M22™ for TSHR degrader AGN301 in the TR-FRET assay.EXAMPLE 8Meso Scale Discovery (MSP)

[0562] Immobilized protein bait is a leucine-rich domain (LRD) protein bait (Fc-fusion), ABT301 or an LRD bait without Fc, ABT302. IgG (% depletion) is detected.

[0563] TSHR-leucine rich domain protein baits capture IgG from patient samples.

[0564] A patient's anti-TSHR autoantibody status determined by clinical-grade ELISA using a TSHR- leucine rich domain protein bait.

[0565] LRD protein bait (ABT302, SEQ ID NO: 23) selectively engages patient IgG by competitive Meso Scale Discovery.

[0566] Binding of serum IgG to a TSHR-leucine rich domain bait (ABT301, Fc-fusion) is effectively competed in Graves' patient samples but not in control.

[0567] FIG. 7 illustrates capture of IgG from patient samples by THSR-LRD protein baits ABT301 andABT302 according to an embodiment of the present invention.

[0568] FIG. 8 is a diagram showing that the LRD protein bait selectively engages patient IgG by competitive Meso Scale Discovery.EXAMPLE 9 cAMP assay for functional validation of degrader activity

[0569] The goal of the assay is measuring the activation of TSHR by stimulating auto-antibodies. In combination with internalization / degradation experiments can then measure correlated reduced activation in response to compound.

[0570] An alternative goal is to classify a polyclonal auto-antibody pool against TSH receptor as stimulating or blocking. Can perform assay with or without TSH to measure blocking vs stimulating activity.

[0571] See HitHunter® cAMP Assay for Biologies (Eurofins DiscoverX), Catalog #: 90-0075LM2.EXAMPLE 10 cAMP conditioned media assay

[0572] The inventors developed a successful cAMP functional assay using conditioned media post- endocytosis. Compounds + M22™ antibody was added first to HEK293-ASGPR1 cell line. Twenty-four hour-conditioned media was applied to CHOK1-TSHR cell line. A cAMP-Glo Max assay (Promega) was used to measure TSHR stimulation by M22™. Compounds representing the LRD-bait successfully functioned as decoys in this cAMP functional assay.

[0573] Increasing doses of TSHR-LRD-Fc fusion protein (ABT301) effectively eliminated in vitro stimulation of TSHR by patient-derived anti-TSHR antibody M22™.

[0574] Cyclic AMP concentration in lysates can be assayed using direct cAMP ELISA kits (Enzo Life Sciences, Exeter, UK). See Miller-Gallacher et al., Journal of Molecular Endocrinology, 62, 117-128 (2019).EXAMPLE 11Endocytosis cellular assay (HEK293-ASGPR mediated endocytosis method)

[0575] ASGPR-dependent uptake assay. An on-mechanism endocytosis assay indirectly measures the ternary complex formed by a fluorescently labeled tool anti-TSHR antibody (M22™), a degrader compound, and ASGPR. The assay directly measures degrader-dependent antibody internalization in HEK293 cells.

[0576] HEK293-ASGPR-mediated endocytosis assay methods. HEK293-ASGPR1 cell line cells were cultured in DMEM media containing 10% Hl-Fetal Bovine Serum, 1% penicillin / streptomycin, and 200 pg / ml G418 (complete HEK293 media) maintaining a density at or below 90% confluency. Cells were harvested by washing twice with phosphate-buffered saline followed by detachment with Accutase. Cells were strained with a 40 pm strainer into a fresh centrifuge tube then centrifuged for five minutes at 500 x g. Supernatant was poured off. The pellet was resuspended in fresh complete HEK293 media. Viable cell density was measured using a Countess II cell counter and trypan blue dye. Viable cells were diluted with complete HEK293 media with 2 pg / mL PDL to a concentration 3.0 x 105cells / mL to achieve 3.0 x 104cells / well in a 96-well assay plate. Assay plate was incubated overnight 37QC and 5% CO2to allow for cell attachment and monolayer establishment.

[0577] The next day, M22™ antibody was labeled with Alexa Fluor 647 using an Alexa Fluor antibody labeling kit to make M22™ directly conjugated to AlexaFluor-647. Alternatively, LysoLight-Deep Red (LLDR, Invitrogen) can be used as the fluorophore. LysoLight-Deep Red dye visible after cleavage with cathepsin B (Thermo L36004). Briefly, M22™ antibody was buffer exchanged into phosphate- buffered saline using a Zeba desalting column. The concentration of the antibody was verified using a Nanodrop and 100 pg was mixed with 0.1 volumes IM sodium bicarbonate provided in the kit and added to a vial of reactive AF-647 dye. The reaction was allowed to incubate for one hour in the dark. Following conjugation, residual unreacted dye was removed through a Zeba desalting column. The final product was measured for IgG and AF-647 concentration via Nanodrop.

[0578] A 2.2 nM solution of M22-647 conjugate was prepared in OptiMEM for the HEK293 endocytosis assay. AGN301 was serial diluted in OptiMEM with a starting concentration of 20 nM down to 30 pM in three-fold increments to create a lOx dilution series for dosing. A vehicle control was also included each compound. The assay plate containing cells was removed from the incubator and media was gently removed by aspiration. 90 pL of 2.2 nM M22-647 was added to each well of the assay plate. Ten pL of the AGN301 dilution series or vehicle control was added to select wells in duplicate allowing for a 2 nM final concentration of M22-647. The final concentration of AGN301 from 2 nM to 3 pM with a vehicle control.

[0579] The assay plate was incubated at 37°C and 5% CO2in the Incucyte instrument, collecting three phase and near-infrared images per well for twenty-four hours after AGN301 dosing. Incucyte analysis software was used to process individual images and subtract background fluorescent signal. For each independent assay, the total area of near-infrared signal (pm2 / image) per well was measured from two replicate wells and concentration response curves were analyzed in GraphPad Prism 10. Peakfluorescent area and EC50 values were determined by fitting the data to a four-parameter non-linear regression model.

[0580] The results for [Ligand] < 34 nM were fitted to obtain an EC50. Data points were mean ± SEM from two technical replicates. Peak fluorescent area follows the 100 nM AGN301 dose over time. Cellular degradation of patient-derived anti-TSHR antibody M22™ was visualized in vitro after lysosomal breakdown of cathepsin B cleavage.

[0581] There was successful cellular internalization of a patient-derived monoclonal anti-TSHR antibody M22™ with lead degraders. The degraders mediate uptake of patient-derived anti-TSHR (M22™) into ASGPR-expressing cells.

[0582] FIG. 9 shows the results of the cellular internalization of the agonistic, patient-derived anti- TSHR antibody M22™ by AGN301 and AGN302.EXAMPLE 12In vivo pharmacodynamics (biotinylated M22 method)

[0583] AGN301 rapidly depletes patient-derived anti-TSHR antibody M22™ added to mice (0.4 pg / mouse) from the mice both intravenously and subcutaneously using an in-house assay to measure added M22 antibody levels.

[0584] Pharmacodynamic biotinylated M22 method. Biotinylated M22 levels were checked using an Meso Scale Discovery assay. Briefly, multi array 96-well streptavidin coated plates were blocked with 150 pL / well Meso Scale Discovery Blocker A for at least one hour at room temperature with shaking at 700 rpm. During the block incubation, a standard curve of biotinylated-M22 was prepared in diluent-100 starting at 500 ng / mL with five-fold dilutions for eight total concentrations. Samples were diluted in diluent-100 accordingly for each assay. The goal was for the initial dose of antibody to be approximately 100 ng / mL. After the blocking incubation, the plates were washed three times with phosphate-buffered saline with Tween 20 at 150 pL / well using a plate-washer. The M22 dilution curve and each sample were added 25 pL / well to the appropriate wells in duplicate and incubated for two hours at room temperature with shaking. After incubation, the plates were washed with phosphate-buffered saline with Tween 20 three times. The plates were then incubated with a sulfo-tagged anti-human IgG antibody that detects the Fab region of the IgG at 0.8 pg / mL. The plates were incubated for 1.5 hours at room temperature with shaking then finally washed three times with phosphate-buffered saline with Tween 20. Read Buffer A was added to each well. The plates were immediately read on the Meso Scale Discovery instrument.

[0585] Clearance results. For AGN301 subcutaneously (SC), all dose levels tested, 3 mpk, 0.3 mpk, and 0.1 mpk, successfully depleted >90% added M22 antibody.

[0586] For AGN301 intravenous (IV), dosing, the 8 mpk dose achieved higher Cmax than 3 mpk.

[0587] The 3 mpk intravenous achieved ~95% depletion of M22 antibody. No rebound of M22 antibody effect was observed. TSH was not depleted by AGN301 dosed intravenously.

[0588] For the in vivo proof-of-concept, degraders rapidly remove patient-derived anti-TSH receptor antibody M22 in mouse. FIG. 10 shows results of the in vivo PK / PD study with AGN301 in mouse, dosed intravenously. FIG. 11 shows results of the in vivo PK / PD with AGN301 in mouse, dosed subcutaneously. FIG.12 shows results of the in vivo PD dose-titration of AGN301 in mouse, dosed subcutaneously.EXAMPLE 13Pharmacokinetics assaysPharmacokinetics (AGN301)

[0589] Multi array 96-well streptavidin plates were coated with 25 pL / well biotinylated soybean lectin (SBA) for 1 hour at room temperature with shaking at 700 rpm. The solution was dumped and 150 pL Meso Scale Discovery Blocker A was added to each well. The plates were incubated at room temperature for one hour with shaking. During the block incubation, a standard curve of AGN301 was prepared in diluent-100 starting at 1000 ng / mL with five-fold dilutions for eight total concentrations. Samples were diluted in diluent-100 accordingly for each assay with a minimum twenty-fold dilution to prevent background plasma interference. After the blocking incubation, the plates were washed three times with phosphate-buffered saline with Tween 20 at 150 pL / well using a plate-washer. The AGN301 dilution curve and each sample were added 25 pL / well to the appropriate wells in duplicate, then incubated for two hours at room temperature with shaking. After incubation, the plates were washed with phosphate-buffered saline with Tween 20 three times and then incubated with a sulfo-tagged M22 antibody at 4 pg / mL. The plates were incubated for 1.5 hours at room temperature with shaking then finally washed three times with phosphate-buffered saline with Tween 20. Read Buffer A was added to each well and the plates were immediately read on the Meso Scale Discovery instrument.Pharmacokinetics (AGN302)

[0590] Multi array 96-well streptavidin plates were coated with 25 mL / well biotinylated M22 antibody for one hour at room temperature with shaking at 700 rpm. The solution was dumped. Then,150 mL Meso Scale Discovery Blocker A was added to each well. The plates were incubated at roomtemperature for one hour with shaking. During the block incubation, a standard curve of AGN302 was prepared in diluent-100, starting at 1000 ng / mL with five-fold dilutions for eight total concentrations. Samples were diluted in diluent-100 accordingly for each assay with a minimum twenty-fold dilution to prevent background plasma interference. After the blocking incubation, the plates were washed three times with phosphate-buffered saline with Tween 20 at 150 mL / well using a plate-washer. The AGN302 dilution curve and each sample were added 25 mL / well to the appropriate wells in duplicate and incubated for two hours at room temperature with shaking. After incubation, the plates were washed with phosphate-buffered saline with Tween 20 three times, then incubated with a mixture of 1 mg / mL mouse CS-17 anti-human TSHR antibody and 1 mg / mL sulfo-tagged goat anti-mouse antibody. The plates were incubated for 1.5 hours at room temperature with shaking then finally washed three times with phosphate-buffered saline with Tween 20. Read buffer A was added to each well. The plates were immediately read on the Meso Scale Discovery instrument.EXAMPLE 14Patient sample screening (UPLEX)

[0591] Each protein bait was assigned a spot for a UPLEX Meso Scale Discovery sector plate each with a spot-specific linker that enables multiplexed loading. Loading complexes were prepared by adding 400 pL 420 nM of biotinylated ABT301 to 600 pL of the assigned linker, mixing well by pipette, then incubation at room temperature for thirty minutes. The reaction was stopped by adding 400 pL of Stop solution and incubated for thirty minutes at room temperature before being combined into a single loading solution. The loading solution was added 50 pL / well to two 6-spot UPLEX Meso Scale Discovery plates and incubated at room temperature for one hour with shaking at 700 rpm. The solution was dumped and 150 pL Meso Scale Discovery Blocker A was added to each well. The plates were incubated at room temperature for one hour with shaking. During the block incubation, a standard curve of M22 anti-TSHR antibodies and Kl-70 anti-TSHR antibodies was prepared in diluent-100 starting at 2000 ng / mL with 4-fold dilutions for eleven total concentrations. Patient samples were diluted fifty-fold in diluent-100 by adding 4 pL of serum to 196 pL diluent-100. After the blocking incubation, the plates were washed three times with phosphate-buffered saline with Tween 20 at 150 pL / well using a platewasher. The antibody standard curves and each patient sample were added 25 pL / well to the appropriate wells in duplicate and incubated for two hours at room temperature with shaking. After incubation, the plates were washed with phosphate-buffered saline with Tween 20 three times and then incubated with a sulfo-tagged anti-human IgG antibody that detects the Fab region of the IgG at 0.8pg / mL. The plates were incubated for 1.5 hours at room temperature with shaking then finally washed three times with phosphate-buffered saline with Tween 20. Read Buffer B was added to each well and the plates were immediately read on the Meso Scale Discovery instrument.

[0592] Degraders effectively captured anti-TSHR autoantibodies from Graves' disease patient samples. See FIG. 7.EXAMPLE 15Patient sample screening (competition)

[0593] Multi array 96-well streptavidin plates were coated with 25 pL / well biotinylated ABT301 for one hour at room temperature with shaking at 700 rpm. The solution was dumped and 150 pL Meso Scale Discovery Blocker A was added to each well. The plates were incubated at room temperature for one hour with shaking. During the block incubation, patient samples were diluted fifty-fold by mixing 16 pL serum with 784 pL diluent-100 to create a 2x working solution. A no competition control was prepared in diluent-100. A lx sample solution was prepared by adding 60 pL of each 2x patient sample to 60 pL of diluent-100 no compound control. After the blocking incubation, the plates were washed three times with phosphate-buffered saline with Tween 20 at 150 pL / well using a plate-washer. The samples were added 25 pL / well to the appropriate wells in duplicate and incubated for two hours at room temperature with shaking. After incubation, the plates were washed with phosphate-buffered saline with Tween 20 three times and then incubated with a sulfo-tagged anti-human IgG antibody that detects the Fab region of the IgG at 0.8 pg / mL. The plates were incubated for 1.5 hours at room temperature with shaking then finally washed three times with phosphate-buffered saline with Tween 20. Read Buffer B was added to each well and the plates were immediately read on the Meso Scale Discovery instrument.

[0594] Degraders effectively captured anti-TSH receptor autoantibodies from Graves' disease patient samples. See FIG. 8.EXAMPLE 16

[0595] AGN302 -001 is a sortase-ligation based antibody-drug-conjugate (ADC). The molecule consists of one single chain (ABT301, peptide), which is conjugated to a pentaglycine modified TBT302 via sortase-catalyzed ligation. The BAR is 0.9-1.1.

[0596] Purification method by Ni Excel. The antibody-drug-conjugate solution was purified by Ni Excel to remove SortaseA and unconjugated protein. The instrument used was AKTA pure. The columnwas HisTrap excel, 5 mL. Equilibration buffer was 20 mM sodium phosphate, 500 mM NaCI, 10 mM imidazole pH 7.4. Elution buffer = 0.2M NaOH. Flow rate was 4 mL / min. Wavelength was 280 nm.

[0597] Purification method for antibody-drug-conjugate by Amicon’ Ultra Centrifugal Filter Unit. An ultrafiltration membrane having a molecular weight of 30 kDa is preferable to concentrate the sample. The Amicon Ultra Centrifugal Filter Unit was used according to the following procedure (1) dH2O wash the centrifugal filter unit. (2) The centrifugal filter unit was equilibrated with phosphate-buffered saline, pH 7.4. (3) Add up sample to the Amicon’ Ultra filter device and centrifuged to concentrate. (4)Collected and filtered sample with 0.22 pm membrane.

[0598] Ultraviolet-visible platform by Nanodrop to determine protein concentration of in-process sample and final antibody-drug-conjugate. (1) 750 nm was set up as baseline. (2) The ultraviolet absorption at 280 nm and 220 nm were measured respectively. (3) The calculation method was based on Beer-Lambert Law A=E*C*I.

[0599] A28o-EmAb28o*C[mAb]*l, where E - molar extinction coefficient, C - molar concentration, and I - light path (Nanodrop = 0.1 cm) BAR determination by LC-MS

[0600] LC-MS was performed using a combination of Agilent 1260 series HPLC system and TOF mass spectrometry. BAR was calculated based on the peak abundance of the deconvoluted mass.Preparation of sample used in LC-MS analysis (deqlycosylation)

[0601] Denaturation. Transfer sample into 1.5 mL tube and incubate at 75°C for 5 min.

[0602] Deqlycosylation. Transfer 15 pg of denatured samples into a 1.5 mL tube, add 3 pL RapidPNGase F (non-reducing format, 5X) enzyme buffer and 0.5 pL Rapid PNGase F (non-reducing format) enzyme for de-glycosylation and some H2O to ensuring the total volume is 15 pL, then incubate this solution at 50°C for 15 min. The obtained sample was used in an LC-MS analysis.

[0603] LC-MS / MS method. A high-pressure liquid chromatography analysis was carried out under the following measurement conditions.

[0604] Aggregation determination by SEC-HPLC. Size-exclusion chromatography was performed using an Agilent 1260 series HPLC system with the TSK gel G3000SWXL Size-exclusion chromatography column (7.8x300 mm, 5 pm) at 25°C. The mobile phase was consisted of 78 mM KH2PO4, 122 mM K2HPO4, 250 mM KCI, 15% IPA at pH 7.0±0.1. The flow rate was set at 0.75 mL / min. Sample loading was 40~50 pg per injection. Samples were detected at 280 nm and 370 nm with an ultraviolet light detector.The retention time of the aggregation peak was recorded based on its relative molecular weight. And the aggregation level was determined by the relative area of the peak at 280 nm.

[0605] Residual free drug determination by LC-MS. The residual free drug level was determined by LC-MS. After protein precipitation, supernatant was loaded to InfinityLab Poroshell 120 SB-C18, 4.6 x 100 mm, 2.7 pm column, and eluted by a gradient of increasing the organic mobile phase. The percentage of residual free drug was quantified via peak area by comparing it to external standard curve.

[0606] Solvent I preparation. Weighed 10 g NaCI was added to the pre-mixed organic solvent of 30 mL MeOH and 50 mL CAN. The components were mixed and stirred for at least one hour, then allowed the solution to stand for at least one hour before use. The supernatant was the saturated sodium chloride solution.

[0607] Preparation of standard curve. The stock standard linker-drug solution was diluted to 40 pM with DMSO for standard curve preparation. Then, the standard curve and sample were prepared.MW"* 40782.6 Da, mAb concentration 7.47 mg / mL and antibody-drug-conjugate BAR value 1.00 were used for the calculation.

[0608] Endotoxin determination. The endotoxin level was determined by Endosafe® N exgen -PTS™.

[0609] Diluted antibody-drug-conjugate sample with endo-free water, pipetted 25 pL of diluted sample into each of the four reservoirs of the PTS Cartridge. The reader drew and mixed the sample with the LAL reagent in the sample channels in addition to the LAL reagent plus positive product control in the spike channels. The sample was combined with the chromogenic substrate then incubated. After mixing, the optical density of the wells was measured and analyzed against an internally archived standard curve.Conjugation methods

[0610] Procedure for bulk conjugation. (1) To 50 mL tube (Greiner, GN227270) containing 74.44 mg of ABT301 in original buffer (phosphate-buffered saline, pH7.4, 8.18 mg / mL) were added with 5.08 mg / mL SortaseA (0.01 equivalents) solution, 5X conjugation buffer (50mM HEPES, 150mM NaCI, 10% glycerol(V / V), pH 7.5), TBT302 (40 mM in DMSO) (sixteen equivalents) into the reactions, mixed well and incubated at 4°C for overnight. The protein concentration in the reaction was 3 mg / mL.

[0611] (2) The crude conjugation was purified with AKTA pure chromatography. The conjugate and free linker-payload were flowed through. The SortaseA and unconjugated protein with His-tag were bounded to the column and eluted by 0.2M NaOH. Pooled flow through liquid together and performed buffer exchange to phosphate-buffered saline, pH7.4, 5% DMSO for 50 DV follow by phosphate-buffered saline, 2 mM EDTA, pH7.4 for 10 DV with Amicon® Ultra Centrifugal Filter Unit (30 kDa, 4*15mL).

[0612] (3) The purified sample was filtered with 0.22 pm filter.

[0613] (4) The quality control tests (including concentration, SEC-HPLC, HIC-HPLC, LC-MS, free drug and endotoxin level) were performed.

[0614] Results and discussion of bulk conjugation. After conjugation, the reaction mixture is purified by Ni Excel. The eluate was exchange buffered to phosphate-buffered saline, 2 mM EDTA, pH7.4 use with Amicon® Ultra Centrifugal Filter Unit (30 kDa, 4*15mL). The solution was then filtered on a0.22 pm membrane.

[0615] Results and discussion of stability tests. The material is stored the sample under -70°C for one hour. The material is then allowed to thaw by letting the vial to sit at room temperature for sufficient amount of time until completed thawed. Collect the analytical characterization under different freeze-thaw cycles (1, 2, 3).

[0616] The antibody-drug-conjugate AGN302-001 was successfully generated. All the results meet targeting quality. Sample was stable under three freeze-thaw cycles.EXAMPLE 17

[0617] The monoclonal, biotinylated, Graves' patient-derived human anti-THS receptor antibody M22 was administered to all mice at a dose level of 0.4 pg per animal by intravenous injection. This dose level was chosen to match the approximate average concentration of anti-TSH receptor in Graves' patients (~200 ng / ml). Forty-five minutes later, either phosphate-buffered saline vehicle or test articles AGN301 or AGN302 were administered via intravenous or subcutaneous injection. The control groups received the vehicle in the same manner as the testing groups. Individual doses were calculated based on the most recent body weight measurement.

[0618] The anti-TSH receptor antibody (M22) concentrations in each sample were quantified by Meso Scale Discovery. M22 antibody levels were referenced to the M22 antibody concentration before test article administration.EXAMPLE 18Graves' disease assay

[0619] The inventors tested an IMMULITE® 2000 / 2000 XPi TSI automated and quantitative TSI assay (Siemens) (U.S. Food & Drug Administration-approved) to assay the presence of thyroid stimulating immunoglobulins (TSI) that bind to the TSH receptor on the thyroid cells and stimulate the uncontrolled production of thyroid hormones. The assay is commercially available from Siemens Medical Solutions USA, Inc. (Malvern, PA, USA)

[0620] This Chemiluminescent Immunoassay (CLIA) is also available from Cleveland Clinic (Cleveland, OH, USA), CPT Code 84445.

[0621] The assay description by the Cleveland Clinic implies that the test selectively measures thyroid stimulating immunoglobulin (TSI), rather than non-stimulating immunoglobulin. The assay uses a chimeric human TSHR (Mc4) where "the TSH binding site is intact, but its membrane-proximal part has been replaced by rat luteinizing hormone-choriogonadotropin receptor [...] to preclude "other" TSHR- binding (but non-stimulating) Abs of binding".

[0622] The Cleveland Clinic brochure also claims that "blocking antibodies, such as Kl-70, bind to the receptor more N-terminally and 155° away from where stimulatory antibodies bind. See Sanders et al., Crystal structure of the TSH receptor (TSHR) bound to a blocking-type TSHR autoantibody. J. Mol. Endocrinol. 46(2), 81-99 (February 15, 2011).

[0623] The TSHR-Mc4 construct was first described by Tahara et al., Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor / LH-CG receptor chimeras than either TSHor immunoglobulins from idiopathic myxedema patients. Biochem. Biophys. Res. Commun, 179, 70-77 (1991), a human TSHR protein with residues 261-370 substituted with a sequence from the rat luteinizing hormone / choriogonadotropin (LH / CG) receptor - another GPCR involved in hormone signaling.

[0624] Available structures with patient-derived stimulating (M22) and blocking (Kl-70) antibodies show that both immunoglobulin Fabs bind largely overlapping, non-contiguous epitopes, contained within residues ~ 20-260 which are included in the Mc4 construct. If anything, M22 binds more C- terminally (membrane-proximal), which seems opposite to the design depicted in the manufacturer's brochure above. See Sanders et al., J. Mol. Endocrinol. 46(2), 81-99 (February 15, 2011).

[0625] The "blocking antibodies Kl-70 and M22 bind a largely overlapping epitope. The binding mode of Kl-70 and M22 Fabs shows a rotation about the TSHR vertical axis, so that the orientations of the Kl-70 and M22 Fab HCs and LCs are opposite.EXAMPLE 19Pharmacokinetics and in vivo activity ofM22 antibody in elevating thyroxine levels following single-dose odministrotion in BALB / c mice

[0626] M22 elevotes T4 levels in the mouse model. M22 antibody administered intravenously increases T4 levels in vivo in a dose-dependent manner. The maximum T4 elevation was observed at twenty-four hours post-treatment. At eight hours post-dose, only 2 pg and 5 pg doses significantly elevated T4 levels. The 0.4 pg dose showed only a trend toward increased T4 at the 24-hour time point.

[0627] M22 ontibody increoses T4 levels in o dose-dependent monner. The peak effect was observed at twenty-four hours post-treatment, where all doses led to elevated T4 levels. At the eight- hour mark, only the 2 pg and 5 pg doses produced a significant increase. The 0.4 pg dose, which was used for PK / PD studies, showed just a trend toward increased T4 at twenty-four hours.

[0628] Estimoted depletion of M22 ontibody. Approximately sixty-two hours post-dose, calculated based on the slope derived from the last two time points' values.

[0629] In vivo efficocy on thyroxin (T4) level. AGN301 and AGN302 were dosed subcutaneously after the administration of randomly biotinylated M22 (bt-M22). M22 depletion was observed twenty- five minutes after drug dosing. AGN302 showed M22 rebound at the 24-hour time point. AGN301 sustained M22 suppression without rebound. Both agents effectively prevented M22-induced T4 elevation.

[0630] Histopathological examination. Thyroids were collected at the end of this study for histopathological examination. Thyroid tissues from animals treated with M22 showed weak eosin staining intensity and a large numbers of follicles with colloid depletion. M22 stimulates thyroid follicular cells to release T4 from the follicular lumen cause colloid depletion, reflected as decreased eosin staining. Thyroid follicles from animals treated with AGN301 or AGN302 showed strong eosin staining and preserved colloid in the lumens. Both AGN301 and AGN302 block the stimulatory effect of M22 and prevent T4 release from thyroid follicles.EXAMPLE 20Peptides

[0631] The inventors investigated the use of peptides as anti-TSH receptor autoantibody-binding moieties (bait). The inventors identified five TSH receptor peptides (contiguous and non-contiguous residues) for development based on the literature. See FaRbender et al., Thyroid, 29(2), 258-267 (2019), Diana et al., J. Autoimmun., 122, 102666 (August 2021), Pearce et al., Thyroid, 29(7), 1003-1011 (July 2019), and Jansson et al., Endocrinology, 159(9), 3446-3457 (September 1, 2018).

[0632] AGN400 is a P19-0GN3 anti-TSHR degrader. The P19 peptide is a circular peptide ABT400 CHQEEDFRVTC (SEQ ID NO: 18) (cysteine-cysteine linkage). P19 retains three residues of TSH receptor (EED 34-36), previously described as necessary for TSHR-M22 binding. GN3 was added to the C-terminus to avoid interference. The llmer P19 peptide has previously been shown to be therapeutic in mouse GD

[0633] ABT401 (P836) is a head-to-tail peptide. The P836 peptide is TKLDAVYLNKNKG (SEQ ID NO: 19), derived from the TSH receptor sequence amino acids 200-213. The P836 peptide previously demonstrated therapeutic effects in mouse model of Graves' disease.

[0634] ABT402 (ATX-GD-59) (1st) has the sequence KKKKYVSIDVTLQQLESHKKK (SEQ ID NO: 20). ATX-GD-59 is a mixture of two TSH receptor peptides. The prophylactic treatment of ATX-GD-59 had previously been shown to tolerize HLA-DR transgenic mice to development of anti-TSH receptor antibodies following immunization with LRD.

[0635] ABT403 (ATX-GD-59) (2nd) has the sequence GLKMFPDLTKVYSTD (SEQ ID NO: 21).

[0636] ABT404 (P12). The P12 linear peptide sequence is KFLGIFNPGDTIELIFFIDPGTLKLYN, (SEQ ID NO: 21), derived from the TSH receptor sequence. The P12 peptide previously demonstrated therapeutic effects in mouse model of Graves' disease.

[0637] Control peptide ABT404 (P14) is a linear peptide with the sequence ofFRVTSKDPGTEILKLTQPG (SEQ ID NO: 22).

[0638] These peptides were conjugated to cellular binding moieties for testing. These peptides were also biotinylated for Meso Scale Discovery assays.

[0639] The compositions of matter (agents, TRAPs™) containing peptides as anti-TSH receptor autoantibody-binding moieties have not developed as far as the compositions of matter (agents, TRAP™s) containing thermostable TSHR-LRD as anti-TSH receptor autoantibody-binding moieties. Five TSH receptor peptides informed by the literature (contiguous and non-contiguous residues) failed to bind selectively to tool monoclonal antibodies (by Meso Scale Discovery, surface plasmon resonance) nor enriched IgG from patient samples.

[0640] For ABT400, ABT401, ABT402, and ABT403 surface plasmon resonance assays, no binding was detected by the tested peptides to M22 antibody up to 10 pM. For ABT404 surface plasmon resonance assays, minimal binding of ABT404 was detected starting at 10 pM.EXAMPLE 21TSH binding

[0641] The bifunctional degraders bind human thyroid stimulating hormone (TSH). This binding was demonstrated by surface plasmon resonance binding assay. The Fc-fusion degrader binds with greater affinity to M22 antibody than TSH.

[0642] Surface plasmon resonance binding assays were performed to test binding of TSH to bifunctional degraders. For surface plasmon resonance, the bifunctional degrader AGN301 binds with greater affinity to autoantibodies than TSH.

[0643] Mouse TSH, rat TSH, cynomolgus monkey TSH, and human TSH have a high sequence consensus among the species, at least 71.9% sequence identity. Residues in the binding interface of TSH and TSH receptor are highly conserved among species. This sequence identity suggests that the bifunctional degraders could interact with TSH from these other species.EXAMPLE 22Additional bifunctional degrader testing

[0644] LRD-bait binds patient-derived activating antibody (M22) and blocking antibody (Kl-70) MAbs as measured by Meso Scale Discovery, surface plasmon resonance, and Biolayer Interferometry.

[0645] LRD-bait only samples were detected in clinical grade anti-TSHR ELISA assays.

[0646] LRD-bait only and LRD-bait degrader acted as decoys in in a cAMP functional assays, as measured by M22 antibody stimulation in CHO-K1 TSHR+ cells. cAMP measured using Promega cAMP- GloMax® assay. The assay used M22 antibody at fixed concentration 6 nM. The data were combined from two independent experiments with two technical replicates each. The inventors observed that dimeric LRD is similar to Fc fusion in cAMP stimulation assays. ABT301 and AGN301 perform better in vitro and in vivo than ABT302 and AGN302

[0647] The inventors are testing dimeric version to recapitulate these properties without the Fc portion.

[0648] LRD-bait degraders bound IgG from patient samples, as measured in Meso Scale Discovery assays, the methods of which are described above.

[0649] The inventors successfully obtained ternary complex data for M22 antibody, ASGPR, AGN301 and AGN302.

[0650] In cellular internalization assays, the methods of which are described above, the inventors observed successful cellular internalization of M22 antibody by AGN302 and Kl-70 antibody by AGN302. The inventors observed successful cellular internalization of M22 antibody by AGN301.

[0651] In pharmacodynamic assays, the methods of which are described above, the inventors observed successful in vivo depletion (>90%) of added M22 antibody in mouse circulation after addition of AGN301 intravenously. The inventors observed successful in vivo depletion of added M22 antibody in mouse circulation after addition of AGN301 subcutaneously.

[0652] As further measured by surface plasmon resonance assay, the inventors observed that AGN302 retains binding to M22 antibody compared to LRD-bait. The inventors measured AGN302 at KD= 5.3 ± 3.5. The inventors measured ABT302 at KD= 30.2 nM.

[0653] As further measured by surface plasmon resonance assay, the inventors observed that thermostable LRD versus wild-type LRD binding to M22 antibody. The inventors measured ABT303 at KD= 24.4 nM. The inventors measured ABT302 at KD= 30.2 nM.EXAMPLE 23Additional bifunctional degrader testing

[0654] Additional bifunctional degraders were synthesized for testing.

[0655] The inventors tested bifunctional degraders with extended LRD in cAMP stimulation assays.Previous data showed that the extended LRD modestly improves decoy affect in blocking M22 antibody from binding to TSH receptor. In this EXAMPLE, the inventors measured cAMP using Promega cAMP-GloMax assay. The assay used M22 antibody at a fixed concentration of 6 nM. The results were:

[0656] The inventors tested bifunctional degraders with dimeric LRD in cAMP stimulation assays.Previous data showed that the dimeric LRD baits could recapitulate the lower EC50 of the Fc fusion bait in blocking M22 antibody stimulation of TSH receptor. In this EXAMPLE, the inventors measured cAMP using Promega cAMP-GloMax assay. The assay used M22 antibody at a fixed concentration of 6 nM.Data were combined from two independent experiments with two technical replicates each. The results were:

[0657] In an endocytosis degradation assay, the conditions included the use of a HEK293-ASGPR1 cell line. M22 antibody was directly conjugated to LysoLight-Deep Red (LLDR, Invitrogen). The data points were mean + SEM from two technical replicates.

[0658] The inventors observed that dimeric LRD baits with single GN3 did not endocytose well (AGN330 and AGN331), possibly due to steric hindrance with ASGPR. The extended LRD with the rigid GSPG linker (AGN328) was more potent than the more flexible GGGS linker AGN329.

[0659] The rest of the dual LRD baits had similar EC5o values but with variable peak heights(AGN301, AGN501, AGN332, AGN333, AGN502). See FIG. 18.EXAMPLE 24T4 level and thyroid histopathology

[0660] The inventors tested the in vivo efficacy of AGN301 or AGN302 dosed subcutaneously (SC) following intravenous administration of randomly biotinylated M22 antibody (bt-M22). The inventors tested reduced doses of AGN301 or AGN302 to determine the minimal dose of these bifunctional degraders in in vivo efficacy.

[0661] The inventors tested the in vivo efficacy on thyroxin (T4) level, AGN301 or AGN302 dosed subcutaneously (SC) following intravenous administration of randomly biotinylated M22 antibody (bt- M22). The 8 mpk dose of AGN301 and 0.3 mpk dose of AGN302 effectively prevented M22-induced a T4 elevation. The 8 mpk dose of AGN301 showed only minimal M22 rebound. The 0.8 mpk dose of AGN301 showed a significant M22 antibody rebound at the eight-hour time point. AGN302 sustained M22 suppression without rebound even at this low dose. Thyroids were collected at the end of assays for histopathological examination.

[0662] The inventors tested the in vivo efficacy, on thyroids, of AGN301 or AGN302 dosed subcutaneously (SC) following intravenous administration of randomly biotinylated M22 antibody (bt- M22). See FIG. 19.

[0663] For M22 antibody-treated animals, thyroid tissues exhibited weak eosin staining and a high number of follicles with depleted colloid, indicative of increased follicular activity.

[0664] For AGN301-treated animals, thyroid follicles showed strong eosin staining and preserved colloid within the lumen, but this effect was observed only at the high dose.

[0665] For AGN302-treated animals, thyroid follicles displayed strong eosin staining and well- preserved colloid in the lumen, evident even at the low dose.

[0666] To test M22 antibody deposition in these thyroids, each animal received 200 pg of M22 antibody via intravenous injection. Thirty minutes later, the treatment group received 100 pg of AGN301 via intravenous administration. Tissues were collected one hour after M22 dosing.

[0667] For M22-treated animals, M22 antibody signal was predominantly observed in the inter- follicular spaces and outside the thyroid follicles, marked by relatively weak F-actin staining compared to the luminal side.

[0668] For AGN302-treated animals: M22 signal was minimal within thyroid tissues, suggesting effective clearance of M22 by AGN302.

[0669] Following AGN302 dosing in the mouse, M22 antibody was internalized in hepatocytes and largely colocalized with a lysosomal marker. Each mouse received 200 mg of M22 antibody via intravenous injection. Thirty minutes later, the treatment group received 100 mg of AGN302 via intravenous administration. Tissues were collected one hour after M22 dosing.

[0670] For vehicle-treated animals, M22 antibody signal was minimally detected in the liver, with weak staining localized to the peripheral regions of hepatocytes. No cytoplasmic IgG signal was observed.

[0671] For AGN302-treated animals, the M22 signal was predominantly localized within the cytoplasm of hepatocytes. The cytoplasmic IgG signal co-localized with LAMP-1, suggesting AGN302 facilitates endocytosis and lysosomal degradation of M22 antibody in the mouse liver.EQUIVALENTS

[0672] Persons having ordinary skill in the biomedical art will recognize or be able to determine using no more than routine experimentation many equivalents to the specific procedures described in this specification. These equivalents are within the scope of this invention and are covered by the following claims. For example, pharmaceutically acceptable salts other than those specifically disclosed in the description and Examples in this specification can be used. Furthermore, it is intended that specific items within lists of items, or subset groups of items within larger groups of items, can be combined with other specific items, subset groups of items, or larger groups of items whether there is a specific disclosure in this specification identifying such a combination.Further listed embodiments.

[0673] 1. A composition of matter comprising a binding moiety that binds to anti-TSH receptor antibody, a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cellsthrough asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on surface degrading cells, and a linker moiety (optionally a single peptide linkage or a single chemical linkage) connecting the anti-TSH receptor antibody-binding moiety and the cellular receptor-binding moiety.

[0674] 2. The composition of matter of embodiment 1, having a structure of AGN101, AGN102, or a pharmaceutically acceptable salt thereof, wherein each of a and b is independently an integer of 1 or greater, each AT or ABT is an anti-TSH receptor antibody-binding moiety or a fragment thereof, L is a linker moiety, and each TBT is independently a cellular receptor-binding moiety that 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.

[0675] 3. The composition of matter of embodiment 2, wherein a is 1, b is 3, and each TBT comprises an N-acetyl-D-galactosamine (GalNAc) moiety.

[0676] 4. The composition of matter of embodiment 1, wherein the anti-TSH receptor antibodybinding moiety is selected from the Markush group consisting of TSH receptors, mutant TSH receptors, fragments comprising one or more epitopes of a TSH receptor, and polypeptide comprising one or more epitopes of a TSH receptor.

[0677] 5. The composition of matter of embodiment 4, further comprising a VHH moiety conjugated to the linker moiety, wherein one or two TSH receptors, mutant TSH receptors, or fragments thereof, or one or two polypeptides comprising one or more epitopes of a TSH receptor are conjugated to one or each of the protein chains of the VHH moiety.

[0678] 6. The composition of matter of embodiment 1, wherein the anti-TSH receptor antibodybinding moiety comprises IgGl or an antigen-binding fragment connected to the linker L at an amino acid residue selected from K246 and K248 of an IgGl heavy chain and amino acid residues corresponding thereto, or the anti-TSH receptor antibody-binding moiety comprises lgG2 or a fragment thereof connected to the linker at an amino acid residue selected from K251 and K253 of an lgG2 heavy chain and amino acid residues corresponding thereto, or the anti-TSH receptor antibody-binding moiety comprises lgG4 or an antigen-binding fragment thereof, connected to the linker at an amino acid residue selected from K239 and K241 of an lgG4 heavy chain and amino acid residues corresponding thereto.

[0679] 7. The composition of matter of embodiment 6, wherein the anti-TSH receptor antibodybinding moiety comprises IgGl and is over 90% connected to the linker L at a K248 as compared with any other linkage with a lysine of the antibody.

[0680] 8. The composition of matter of embodiment 6, wherein the anti-TSH receptor antibodybinding moiety comprises IgGl and has two linkers at an amino acid connected to residue selected from K246 and K248 of each of the IgGl heavy chains and amino acid residues corresponding thereto.

[0681] 9. The composition of matter of embodiment 1, wherein the cellular receptor-binding moiety comprises an ASGPR binding group connected through an amine group.

[0682] 10. The composition of matter of embodiment 1, wherein the cellular receptor-binding moiety comprises an ASGPR binding group according to the chemical structurepharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof. Groups Ri, R2, R3, and X may be the same as described in International Patent Publication WO 2019 / 199634 and International Patent Publication WO 2019 / 199621.

[0683] 11. The composition of matter of embodiment 1, wherein the cellular receptor-binding moiety has the structuregroup optionally substituted with 1-5 halo groups (preferably RA is a methyl or ethyl group optionally substituted with from 1-3 fluoro groups), ZA is -(CH2)IM, -0-(CH2)IM, S-(CH2)IM, NRM-(CH2)IM, C(0)-(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) where IM and RM are the same as above, and ZB is absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)IM-NRM, where IM and RM are the same as above.

[0684] 12.The composition of matter of embodiment 1, wherein the ASGPR binding group is N- acetyl-D-galactosamine.

[0685] 13. A pharmaceutical composition comprising a composition of matter any of the preceding embodiments 1-12 and a pharmaceutically acceptable excipient.

[0686] 14. A composition comprising a first composition of matter comprising an anti-TSH receptor antibody-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 on surface degrading cells, and a linker moiety connecting the anti-TSH receptor antibody-binding moiety and the cellular receptor-binding moiety, and at least one additional composition of matter 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 on surface degrading cells, and a linker moiety connecting the cellular receptor-binding moiety and capable of binding to an anti- TSH receptor antibody-binding moiety.

[0687] 15. A method of removing anti-TSH receptor autoantibody in a patient or subject in need comprising administering to the patient or subject an agent of any of embodiments 1-12.

[0688] 16. A method of treating a disease state or condition associated with the upregulation of anti-TSH receptor autoantibody in a patient by administering to the patient an effective amount of an agent (TRAP™) of any of embodiments 1-12.

[0689] 17. A method of treating Graves' disease by administering to a Graves' disease patient an effective amount of an agent (TRAP™) of any of embodiments 1-12.REFERENCES

[0690] Persons having ordinary skill in the biomedical art can use these patents, patent applications, and scientific references as guidance to predictable results when making and using the invention.Patent literature

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[0695] Inti. Pat. Publ. WO 2006 / 016121 Al (RSR Ltd.) discloses TSH receptor mutants carrying, e.g., a point mutation at position 255 which are less stimulated by TSH receptor autoantibodies. WO 2006 / 016121A1 discloses a mutated TSH receptor preparation including at least one point mutation which can be used in the differential screening and identification of patient serum stimulating TSH receptor autoantibodies, patient serum blocking TSH receptor autoantibodies and TSH in a sample of body fluid from a patient being screened.

[0696] Inti. Pat. Publ. WO 2008 / 099185A1 discloses the isolation and characterization of a human monoclonal antibody 5C9 to the TSH receptor that is an effective antagonist of TSH and of stimulating TSH receptor autoantibodies in patient sera.

[0697] Inti. Pat. Publ. WO 2010 / 073012 A2 (RSR Ltd.) discloses TSH receptor mutants, which are more thermostable than the wild-type receptor, e.g., TSH receptor LRD C-CAP, which contains amino acids 1-409 of the human TSH receptor having amino acids 306-384 removed. TSH receptor mutants,such as TSHR260, are provided. Labeling with alkaline phosphatase is proposed. WO 2010 / 073012 also describes an assay to measure TSH receptor autoantibodies based on the bridging principle whereby divalent antibodies bind to the TSH receptor coated onto an ELISA plate well with one arm and to liquid phase TSHR260-alkaline phosphatase with the other arm to form a bridge. WO 2010 / 073012 further discloses the isolation and characterization of a further human monoclonal autoantibody (Kl-18) with powerful stimulating activity and a human monoclonal autoantibody (Kl-70) that is a potent TSH receptor antagonist from the peripheral blood lymphocytes of a patient.

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[0776] Throughout this application, several publications are referenced by author name and date or by patent or patent publication number. The disclosures of these publications are incorporated in their entireties by reference into this application to describe the state of the art more fully as known to persons having ordinary skill in the biomedical art as of the date of the invention described and claimed in this specification. However, the citation of a reference in this specification should not be construed as an acknowledgment that this reference is prior art to the present invention.

[0777] All patents and publications cited throughout this specification are incorporated in their entireties by reference to disclose and describe the materials and methods that might be used with the technologies described in this specification. The publications discussed are provided only for their disclosure before the filing date. They should not be construed as an admission that the inventors maynot antedate this disclosure under prior invention or for any other reason. If there is an apparent discrepancy between a prior patent or publication and the description provided in this specification, the specification (including any definitions) and claims shall control. All statements about the date or contents of these documents are based on the information available to the applicants. These statements are no admission to the correctness of the dates or contents of these documents. The publication dates in this specification may differ from the actual publication dates. If there is an apparent discrepancy between a publication date in this specification and the actual publication date supplied by the publisher, the actual publication date shall control.SEQUENCE LISTINGSequence Number (ID) : 1 Length: 330Molecule Type: AAFeatures Location / Quali tiers : source, 1. .330 mol type, protein organism, Homo sapiens Residues :ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 120PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 180STYRWSVLT VLHQDWINGK EYKCRVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 240LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300QQGNVFSCSV MHEATHNHYT QKSLSLSPGK 330Sequence Number (ID) : 2 Length: 326Molecule Type: AAFeatures Location / Quali tiers : source, 1. .326 mol type, protein organism, Homo sapiens Residues :ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP APPVAGPSVF 120LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVQFNWYVDG VEVHNAKTKP REBQFNSTFR 180WSVLTVLHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG OPREPQVYTL PPSREEMTKN 240QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN 300VFSCSVMHEA THNHYTQKSL SLSPGK 326Sequence Number (ID) : 3 Length: 327Molecule Type: AA Features Location / Quali tiers : source, 1. .327 mol type, protein organism, Homo sapiens Residues :ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KTGPPCPSCP APEFLGGPSV 120FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY 180RWSVLTVLH QDWINGKEYK CKVSNKGLPS SIEKTISKAK GOPREPQVYT LPPSQEEMTK 240NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 300NVFSCSVMHE ATHNHYTQKS LSLSLGK 327Sequence Number (ID) : 4Length: 764Molecule Type: AAFeatures Location / Quali tiers : source, 1. .764 mol type, protein organism, ] imo sapiensResidues :MRPADLLQLV LLLDLPRDLG GMGCSSPPCE CHQEEDFRVT CKDIQRIPSL PPSTQTLKLI 60ETHLRTIPSH AFSNLPNISR IYVSIDLTLQ QLESHSFYNL SKVTHIEIRN TRNLTYIDPD 120ALKELPLLKF LGIFNTGLKM FPDLTKVYST DIFFILEITD NPYMTSIPVN AFQGLCNETL 180TLKLYNNGFT SVQGYAFNGT KLDAVYLNKN KYLTVIDKDA FGGVYSGPSL LDVSQTSVTA 240LPSKGLEHLK ELIARNTWTL KKLPLSLSFL HLTRADLSYP SHCCAFKNQK KIRGILESLM 300CNESSMQSLR QRKSVNALNS PLHQEYEENL GDSIVGYKEK SKFQDTHNNA HYYVFFEEQE 360DEIIGFGQEL KNPQEETLQA FDSHYDYTIC GDSEDMVCTP KSDEFNPCED IMGYKFLRIV 420VWFVSLLALL GNVFVLLILL TSHYKLNVPR FLMCNLAFAD FCMGMYLLLI ASVDLYTHSE 480YYNHAIDWQT GPGCNTAGFF TVFASELSVY TLTVITLERW YAITFAMRLD RKIRLRHACA 540IMVGGWVCCF LLALLPLVGI SSYAKVSICL PMDTETPLAL AYIVFVLTLN IVAFVIVCCC 600YVKIYITVRN PQYNPGDKDT KIAKRMAVLI FTDFICMAPI SFYALSAILN KPLITVSNSK 660ILLVLFYPLN SCANPFLYAI FTKAFQRDVF ILLSKFGICK RQAQAYRGQR VPPKNSTDIQ 720VQKVTHDMRQ GLHNMEDVYE LIENSHLTPK KQGQISEEYM QTVL 764Sequence Number (ID) : 5 Length: 260Molecule Type: AA Features Location / Quali tiers : source, 1. .260 mol type, protein organism, Homo sapiens Residues :MRPADLLQLV LLLDLPRDLG GMGCSSPPCE CHQEEDFRVT CKDIQRIPSL PPSTQTLKLI 60ETHLRTIPSH AFSNLPNISR IYVSIDLTLQ QLESHSFYNL SKVTHIEIRN TRNLTYIDPD 120ALKELPLLKF LGIFNTGLKM FPDLTKVYST DIFFILEITD NPYMTSIPVN AFQGLCNETL 180TLKLYNNGFT SVQGYAFNGT KLDAVYLNKN KYLTVIDKDA FGGVYSGPSL LDVSQTSVTA 240LPSKGLEHLK ELIARNTWTL 260Sequence Number (ID) : 6 Length: 238Molecule Type: AAFeatures Location / Quali tiers : source, 1. .238 mol type, protein organism, Homo sapiens Residues :GCSSPPCECH QEEDFRVTCK DIQRIPSLPP STQTLKLIET HLRTIPSHAF SNLPNISRIY 60VSIDLTLQQL ESHSFYNLSK VTHIEIRNTR NLTYIDPDAL KELPLLKFLG IFNTGLKMFP 120DLTKVYSTDI FFILEITDNP YMTSIPVNAF QGLCNETLTL KLYNNGFTSV QGYAFNGTKL 180DAVYLNKNKY LTVIDKDAFG GVYSGPSLLD VSQTSVTALP SKGLEHLKEL IARNTWTL 238Sequence Number (ID) :Length: 239 Molecule Type: AA Features Location / Quali tiers : source, 1. .239 mol type, protein organism, synthetic constructResidues :MGCSSPPCEC HQEEDFRVTC KDIQRIPSLP PSTQTLKLIE TCLRTIPSHA FSNLPNISRI 60 YVSIDVTLQQ LESHSFYNLS KVTHIEIRNT PNLTYIDPDA LKELPLLKFL GIFNTGLKMF 120PPLTKVYSTE IFFILEITDN PYMTSIPRNA FQGLCNETLT LKLYNNGFTS VQGYAFNGTK 180LDAVYLNKNK YLTVIDKDAF GGVYSGPSLL DVSQTSVTAL PSKGLEHLKE LRARNTWTL 239Sequence Number (ID) : 8 Length: 232Molecule Type: AA Features Location / Quali tiers : source, 1. .232 mol type, protein organism, synthetic constructResidues :EPKSADKTHT CPPCPAPEAA GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF 60NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALAAPIEKT 120ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 180PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 232Sequence Number (ID) : 9 Length: 5Molecule Type: AA Features Location / Quali tiers : source, 1. .5 mol type, protein organism, synthetic constructResidues :GGGGSSequence Number (ID) : 10 Length: 13 Molecule Type: AA Features Location / Quali tiers : source, 1. .13 mol type, protein organism, synthetic construct- DISULFID, 2> note, Cys2 and Cysl2 form a disulfide linkage that make the peptide have a cyclic structure.- DISULFID, 12> note, Cys2 and Cysl2 form a disulfide linkage that make the peptide have a cyclic structure.- MOD_RES, 1> note, GN3 moietyResidues :DCAWHLGELV WCT 13Sequence Number (ID) : 11Length: 764Molecule Type: AAFeatures Location / Quali tiers : source, 1. .764 mol type, protein organism, synthetic construct Residues : MRPADLLQLV LLLDLPRDLG GMGCSSPPCE CHQE...

Claims

1. CLAIMSWhat is claimed:

1. A composition of matter comprising: a binding moiety that can bind to anti-TSH receptor antibody, wherein the binding moiety comprises at least one leucine rich domain (LRD) of human TSH receptor or a variant thereof, a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface of degrading cells, and optionally, a linker moiety (optionally a single chemical linkage) connecting the anti-TSH receptor autoantibody-binding moiety and the cellular receptor-binding moiety.

2. The composition of matter of Claim 1, wherein the leucine rich domain comprises amino acids 22-260 of the human TSH receptor.

3. The composition of matter of Claim 1, wherein the leucine rich domain variant comprises amino acids 22-260 of the human TSH receptor, as modified to make the leucine rich domain thermostable.

4. The composition of matter of Claim 1, wherein the binding moiety that can bind to anti-TSH receptor antibody comprises two leucine rich domains of human TSH receptor or variants thereof.

5. The composition of matter of Claim 1, wherein the leucine rich domain variant has 95% sequence identity to the human TSH receptor.

6. The composition of matter of Claim 1, wherein the anti-TSH receptor autoantibody-binding moiety is selected from the Markush group consisting of TSH receptors, mutant TSH receptors, fragments comprising one or more epitopes of a TSH receptor, and polypeptide comprising one or more epitopes of a TSH receptor.

7. The composition of matter of Claim 6, further comprising an IgG Fc moiety or a VHH moiety conjugated to the linker moiety, wherein one or two TSH receptors, mutant TSH receptors, or fragments thereof, or one or two polypeptides comprising one or more epitopes of a TSH receptor are conjugated to one or each of the protein chains of the VHH moiety.

8. The composition of matter of Claim 1, having a structure of:[AGN102], or a pharmaceutically acceptable salt thereof, wherein: each of a and b is independently an integer of 1 or greater; each AT or ABT is an anti-TSH receptor autoantibody-binding moiety or a fragment thereof;L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety that binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface of degrading cells in a patient or subject.

9. The composition of matter of Claim 8, wherein a is 1, b is 3, and each TBT comprises an N-acetyl-D-galactosamine (GalNAc) moiety.

10. The composition of matter of Claim 1, having a structure of:[AGN103], or a pharmaceutically acceptable salt thereof, wherein: is a moiety comprising at least one leucine rich domain (LRD) of human TSH receptor or a variant thereof;polypeptide moiety, which is a means for connecting andoptional polypeptide moiety connectinganconnected tvia a side chain amino group of a lysine residue of to formeachis an asialoglycoprotein receptor ("ASGPR") binding moiety, which is either present or absent, provided that at least oneis present.HN -11 The composition of matter of Claim 10, wherein ' — ' is present.HN -12. The composition of matter of Claim 10, wherein ' — ' is absent.

13. The composition of matter of Claim 10, having a structure of:

14. The composition of matter of Claim 10, having a structure of:or a pharmaceutically acceptable salt thereof, wherein eachis the same or different and is a moiety comprising one leucine rich domain (LRD) of human TSH receptor or a variant thereof.

15. The composition of matter of Claim 10, wherein the composition of matter comprises at least six16. A composition of matter having the structure:Formula (I), wherein,(A) each is a moiety comprising at least one leucine rich domain (LRD) of human TSH receptor or a variant thereof;each is an optional polypeptide moiety, which is a means for connecting and( C ).( C ) each is a peptide moiety having SEQ ID NO: 26 or a sequence having( C )95% sequence identity therewith, wherein the moieties are linked together via-?-s — s-f— two disulfide bridges ’ as shown in Formula (II), wherein one disulfide bridge links cysteine residues located in a position corresponding to position 11 of SEQ ID NO: 26 of each moiety( c ), and wherein the other disulfide bridge links cysteine residues located in a( C ) position corresponding to position 14 of SEQ ID NO: 2 6of each moiety ;eachis a means for connecting, whereineach is connected to via a side chain amino group of a lysine residueformeachis an asialoglycoprotein receptor ("ASGPR") binding moiety, which is either present or absent, provided that at least oneis present.(T)17. The composition of matter of any one of Claims 10 to 15, wherein each has the sequence of SEQ ID NO: AGN301 or a sequence having 95% sequence identity therewith.(T)18. The composition of matter of any one of Claims 10 to 15, wherein each has the sequence of SEQ ID NO: AGN302 or a sequence having 95% sequence identity therewith.

19. The composition of matter of any one of Claims 10 to 18, whereinis present.

20. The composition of matter of any one of Claims 10 to 19, whereinis absent.

21. The composition of matter of any one of Claims 10 to 19, wherein eachis a single chemical bond or a peptide connecting moiety comprising an amino acid selected from the group consisting of G, E, L, P, Q, S, and T.

22. The composition of matter of any one of Claims 10 to20, wherein each comprises the sequence of SEQ ID NO: 9.

23. The composition of matter of any one of Claims 10 to 21, wherein eachcomprises the sequence of SEQ ID NO: 49.The composition of matter of any one of Claims 10 to 22, wherein eachcomprises a moiety selected from the group consisting of:wherein,X2are independently CH2, O, S, NR4, C(O), S(O), S(O)2, S(O)2O, OS(O)2, or OS(O)2O;X3are independently O, S, NR4, wherein R4is H or a C1-C3 alkyl; and k and n are independently 1 to 25.

25. The composition of matter of any one of Claims 10 to 23, wherein eachfurther comprises a moiety selected from the group consisting of:wherein,X2are independently CH2, O, S, NR4, C(O), S(O), S(O)2, S(O)2O, OS(O)2, or OS(O)2O;X3are independently O, S, NR4, wherein R4is H or a C1-C3 alkyl; k and n are independently 1 to 25.

26. The composition of matter of any one of Claims 10 to 24, wherein eachcomprises one or more -(O)C-[(CH2)nO]m(CH2)nNH-, - [(CH2)nO]m(CH2)nNHC(O)[(CH2)nO]m-, and-[(CH2)nO]m(CH2)n{NHC(O)[(CH2)nO]m}P(CH2)nC(O)NH-, wherein each m and n are independently 1 to 10.

27. The composition of matter of any one of Claims 10 to 25, wherein eachcomprises one or more -[(CH2)n-O]m-, wherein each m and n are independently 1 to 10.

28. The composition of matter of any one of Claims 10 to 26, wherein eachcomprises -(CH2)n-O-(CH2CH2O)n-(CH2)n-, wherein each n is independently 1 to 10.

29. The composition of matter of any one of Claims 10 to 27, wherein all three moietiespresent, wherein each30. The composition of matter of any one of Claims 10 to 27, wherein two moietiespresent, wherein eachis the same as or different from the otherThe composition of matter of any one of Claims 10 to 27, wherein one moietyis present.

32. The composition of matter of any one of Claims 10 to 30, wherein at least onecomprises an N-acetyl-D-galactosamine ("GalNAc") group having the structure:Formula (II)33. The composition of matter of any one of Claim 10 to 31, wherein eachacetyl-D-galactosamine ("GalNAc") group having the structure:

34. The composition of matter of any one of Claim 10 to 32, wherein at least one ofcomprises a group having the structure:Formula (III), wherein,ZBis absent, -(CH2)IM-, -C(=O)-(CH2)IM-, or -C(=O)-(CH2)IM-NRM-;RM is H or C1-C3 alkyl; and each occurrence of IM is independently 1, 2, or 3.

35. The composition of matter of any one of Claims 10 to 33, wherein at least oneconsisting of:Formula (V)36. The composition of matter of any one of Claims 10 to 34, wherein the lysine residue ofis located in a position corresponding to position 31 or position 33 of SEQ IDNO: 26.

37. The composition of matter of any one of Claims 10 to 35, wherein the cysteine residues in positions 46 and 106 of SEQ ID NO: TBD are connected to form a disulfide bridge, and wherein the cysteine residues in positions 152 and 210 of SEQ ID NO: 26 are connected to form a disulfide bridge.

38. The composition of matter of Claim 10, wherein the composition of matter is AGN301 .( C )39. The composition of matter of Claim 10, wherein the comprises a portion of an Fc.( . C . )40. The composition of matter of Claim 10, wherein is absent.

41. The composition of matter of Claim 10, wherein at least one moiety ( c J ismutated so that the moieties do not dimerize.

42. The composition of matter of Claim 10, wherein the composition of matter is AGN302.

43. The composition of matter of Claim 1, wherein the binding moiety is selected from the group consisting of ABT301 (SEQ ID NO: 24), ABT302 (SEQ ID NO: 23), and ABT303.

44. The composition of matter of Claim 10, wherein the composition of matter is selected from the group consisting of AGN304, AGN305, AGN306, AGN307, AGN308, AGN309, AGN310, AGN311, AGN312, AGN313, AGN314, AGN316, AGN317, AGN318, AGN319, AGN320, AGN322, AGN323, AGN324, AGN325, AGN326, AGN327, AGN328, AGN329, AGN330, AGN331, AGN332, and AGN333.

45. The composition of matter of Claim 1, wherein the cellular receptor-binding moiety comprises an ASGPR binding group according to the chemical structure:

46. The composition of matter of Claim 1, wherein the cellular receptor-binding moiety is selected from the group consisting of TBT301 and TBT302.

47. The composition of matter of Claim 1, for use in treating a medical condition.

48. The composition of matter of Claim 47, wherein the medical condition is selected from the group consisting of Graves' disease, Graves orbitopathy, and thyroid eye disease.

49. A pharmaceutical composition, comprising the composition of matter of Claim 1 and a pharmaceutically-acceptable excipient.

50. A composition comprising: a first composition of matter comprising an anti-TSH receptor antibody-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 on the surface of degrading cells, and a linker moiety connecting the anti-TSH receptor antibody-binding moiety and the cellular receptor-binding moiety, and at least one additional composition of matter 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 on surface degrading cells, and a linker moiety connecting the cellular receptor-binding moiety and capable of binding to an anti-TSH receptor antibody-binding moiety.

51. A method of removing anti-TSH receptor autoantibody in a subject comprising administering to the subject a composition of matter of any of Claims 1-46.

52. A method of treating a disease state or condition associated with the upregulation of anti-TSH receptor autoantibodies in a patient by administering to the patient an effective amount of a composition of matter of any of Claims 1-46.

53. A method of treating Graves' disease, Graves orbitopathy, and thyroid eye disease in a patient by administering to a patient an effective amount of a composition of matter of any of Claims 1-46.

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