Bifunctional degraders

Abstract

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

Description

Bifunctional decomposition agents The technical field of invention

[0001] The invention generally relates to medicinal preparations characterized by the inactive ingredients used, for example, carriers or inert additives, which target or modify agents chemically related to the active ingredient. The inactive ingredient is chemically related to the active ingredient. For example, a drug-polymer associated with the inactive ingredient is considered a modifying agent. The modifying agent is an antibody, immunoglobulin, or a fragment thereof, for example, an Fc fragment. The modifying agent is an antibody or immunoglobulin that carries a binding site to at least one antigen. The antibody targets a receptor, a cell surface antigen, or a cell surface determinant, specifically bifunctional molecules containing a circulating protein binding fragment linked by a ligand group to a cell receptor binding fragment for the treatment of diabetes and other insulin disorders. Technical background of the invention

[0002] Diabetes often has an autoimmune component. Neutralizing antibodies produced against insulin reduce the effectiveness of insulin in diabetic patients. Several β-cell proteins are targets of autoimmune responses in type 1 diabetes (T1D). The contribution comes from protein products of the insulin gene: proinsulin precursor (PPI), proinsulin (PI), or insulin. Children and the majority of adults with type 1 diabetes show early generation of C-chain antibodies to proinsulin and native insulin. Autoantibodies generated against insulin reduce the effectiveness of insulin in diabetic patients.

[0003] There remains a need in the field of biomedicine for new drugs capable of treating or slowing the progression of diabetes and other insulin disorders. There is also a need for treatments to remove immunoneutralizing antibodies to insulin and insulin replacement therapies. Invention Disclosure

[0004] The invention provides formulations of a substance (factors, TRAPs) that bind to neutralizing antibodies for proinsulin, insulin, and insulin replacement therapies. These selective agents, in turn, substantially reduce or eliminate only the pathogenic types of neutralizing autoantibodies generated by an unwanted immune response, while leaving the majority of other antibodies unbound in the patient receiving the agents.

[0005] In a single embodiment, the invention provides a composition of a material (agent, TRAP) comprising: A binding molecule with a target that can bind to antibodies that are anti-insulin. A cleavage binding molecule that can bind to liver cells or other lysed cells via hepatocyte-specific alactic glycoprotein receptors (ASGPRs) or other cell receptors on the surface of lysed cells in a patient or someone undergoing treatment, and A linker fragment that connects the binding fragment and the cell-receptor binding fragment, where the linker fragment can be a single peptide bond or a larger linker fragment. The formulation consists of an autoantibody that is either anti-proinsulin or bifunctional antiinsulin. Unlike pan-IgG or poly-IgG lysis agents, this formulation avoids the possibility of immunosuppression.

[0006] In another embodiment, the composition of the substance (the agent, TRAP) has the structural formula: Where [CPBM] is a target binding fragment that can bind to an antibody that is anti-proinsulin or anti-insulin; [CRBM] is a cell receptor binding cleavage that binds to liver cells or other cells through non-associated glycoprotein receptors or other receptors on the surface of liver cells and other lysed cells; Each [CON] is a selectively conductive chemical molecule which, when present, connects directly to [CPBM] or [CRBM] or acts to connect [LINKER] to [CPBM] or [CRBM] and [LINKER] is a chemical molecule that is covalently attached to one or more [CRBM] and / or [CPBM] groups, or A pharmaceutically acceptable salt, a stereoisomer, a solute, or a polymorphic form thereof.

[0007] In some incarnations, the composition of the material (the agent, TRAP) has the structural formula: R CN −(No)y−R CC , [ AGN101 ] ، [ AGN102 ] ، [ AGN103 ] or [ AGN104 ] ، Or a salt of that.

[0008] In some embodiments, the composition of the material (the agent, TRAP) has the structural formula of the formula AGN105 : [ AGN105 ] Or a salt of that, as the composition of the material may contain additional elements described in this specification.

[0009] In another embodiment, the composition of the material is chosen from among a set consisting of the structures established in the following table. Table 1 AGN302 Mutant insulin with GN3 AGN304 PT0004- TBT 6171 Native insulin pro-progenitor structure constructed with a conjugate of αGN3 at the C-terminus. Sortase, U2073499G0-8, BAR = 1. AGN305 PT0004- TBT 6171 The structure of a mutant insulin precursor construct with a conjugate of αGN3 at the C-terminus. Sortase, U2073499G0-8, BAR = 1. AGN310 TRAP insulin. Target binding fragment (lns-004), prepared by AGN3-conjugated chemisorption of the ASGPR (triGalNAc) ligand. The synthetic insulin mode has an affinity similar to mAb49. Molecular weight: 7424.236. Sequence: GIVEQ[CC*TSIC]SLYQLENYC**N α-GN-triazol-(pent-4-inoic acid)-FVNQHLC*GSHLVEALYLVC**GERGAFYTPKT (sequence number: 43). Phenylalanine-to-alanine mutation (F to A). AGN4002 α(GalNAc ASGPR binding material)3 AGN4003 α(GalNAc ASGPR amide propionic ligand)3 AGN4004 β (GalNAc ASGPR amide propionic ligand)3 AGN4006 α(GalNAc ASGPR amide propionic ligand)3 AGN4007 β(GalNAc ASGPR amide propionic bonding agent)3 AGN401 He constructed a synthetic C-peptide facility (D amino acids) with the αGN3 protease, without binding to any of the C-peptide-specific antibodies. AGN402 He constructed a synthetic C-peptide facility (D amino acids) with αGN3 protease, without binding to any of the C-peptide-specific antibodies. AGN405 PT0004- TBT 6171 Native insulin progenitor prepared by C-terminal αGN3 conjugated gene recombinant product (triGalNAc), conjugated by sortase U2073499G0-8, BAR = 1. AGN406 Peptide-C with βGN3 (L amino acids). Peptide-C with a protease site, synthetic peptide-C degradation factor. AGN407 Peptide-C with the βGN3 protease (L amino acids). Peptide-C without the protease site, synthetic peptide-C degradation factor. AGN408 PT0009- TBT6170 The constructed structure of the original PT-0009-01 antibody with αGN3 at the N-terminus ( TBT6170 Molecular weight = 13319.04 Daltons, Sortase = U2073499G0-8. BAR = 0.82. IC 50 = 1.2 nanomolar. AGN409 PT0009- TBT6170 The antibody specific to the original proinsulin PT-0009-02, conjugated to αGN3 ( TBT6170 (Terminal N), molecular weight = 13323.34 Daltons, sortase = U2073499G0-8, BAR = 1. AGN410 PT0011- TBT6170 The conjugate of the original antibody PT-0011-01-αGN3 (N-terminus), molecular weight = 13242.87 Daltons, sortase = U2073499G0-8, BAR = 1. AGN411 PT0011- TBT6170 The structure of a mutant insulin precursor construct with the αGN3 conjugate at the N-terminus (N-terminus), molecular weight = 13247.69 Daltons, sortase = U2073499G0-8, IC 50 = 3.1 nanomolar. AGN412 Synthetic mutagenic insulin using β-GN3. Molecular weight = 7424.24 Daltons. AGN413 Fc-insulin-βGN3 conjugate AGN414 The link from proinsulin-Fc-G4S fusion ( ABT307 (Sequence No. 17) + βGN3 ( TBT4438 ) Molecular weight = 77525.74 Daltons. AGN415 The link from proinsulin-Fc-5 x G4S fusion ( ABT308 ) (Sequence number: 18) + βGN3 (TBT4438) Molecular weight = 80047.84 Daltons. AGN416 Synthetic mutant insulin using αGN3 on the A chain, molecular weight = 7424.24 Daltons, AGN417 ABT 410 -TBT 403 TRAP (Traditional Insulin AGN417) is a conjugate of the ASGPR (N-terminus) proinsulin. Synonym: GN3-proinsulin conjugate. Molecular weight: 11921.84. AGN417 demonstrated a 30% reduction in endogenous insulin-specific autoantibodies in 14-week-old NOD mice. AGN417 exhibited good subcutaneous bioavailability. The intravenous group showed rapid but robust detection of the hemolysis factor, in contrast to the subcutaneous group, where sustained detection was observed between 2 and 8 hours. The subcutaneous group demonstrated 76% bioavailability, indicating efficient absorption over time. Both subcutaneous (10 mg / kg) and intravenous (3 mg / kg) administration resulted in a significant reduction in blood glucose (BGL) levels at 30 minutes. AGN418 ABT410-TBT405 Short (G4S) linkage with βGN3. Molecular weight = 11871.59 Daltons. AGN419 ABT411-TBT404 Insulin precursor with βGN3 at the C-terminus. Molecular weight = 12509.77 Daltons. AGN420 ABT412-TBT403 The long βGN3 spacer at the N-terminus of insulin precursor. Molecular weight = 12267.07 Daltons. AGN421 ABT7714-TBT403 The TRAP mutant insulin precursor ΒGN3 at the N-terminus, with a double mutant insulin precursor (V3L for the A chain and F25A for the B chain) and a triGalNAc conjugation with the peptide. Molecular weight = 11859.59 Daltons. In vivo tolerance and blood glucose levels were assayed in NOD mice, 0.23 years (12 weeks) old. No toxic signals were observed. AGN422 ABT7714-TBT405) TRAP mutant proinsulin. ΒGN3 at the N-terminus - a non-nociceptive double mutant proinsulin (V3L for chain A and F25A for chain B). The amide is located between triGalNAc and the peptide. Molecular weight = 11809.86 Daltons AGN423 Insulin-Fc conjugate (efsitora alfa)- TBT4438 (βGN3-MATE) AGN6567 AGN417 with punctuation marks 13 and N15 AGN6601 The mutant insulin precursor prepared by the efflux gene recombination product technique suppressed αGN3 (triGalNAc) at the C-terminus, conjugated by sortase U2073499G0-8, BAR = 1. AGN7525 Short link (G4S) with αGN3. AGN7526 Long link (G4S x 3) with αGN3. AGN7849 Long spacer at the N end in βGN3, the nigra proinsulin. AGN7850 Long spacer at the N end in βGN3 proinsulin. AGN7865 The spacer at the N end in βGN3 proinsulin. AGN8123 This factor has the structural formula of AGN421 With a terminal G→A mutation, Refold scores may not reach the same level as other insulin analogs due to the B24 F→A mutation (sequence number: 38). AGN8138 ABT410-TBT7988 AGN8139 ABT410-TBT7989 AGN8140 ABT410-TBT7990 AGN8141 ABT410-TBT7991 AGN2640 β(GalNAc ASGPR binding material)3

[0010] In another embodiment, the target-binding component is insulin, an alternative form of insulin, or an insulin analog.

[0011] In another embodiment, the target-binding component is an insulin progenitor, a variant of an insulin progenitor, or an insulin progenitor analog.

[001] In another embodiment, the target binding fragment is an established structure for insulin-IgG1 Fc fusion.

[002] In another embodiment, the target-binding fragment is an insulin-IgG1 Fc fusion protein. This Fc fusion protein can be genetically expressed and produced from a mammalian cell system due to the Fc domain, which also allows for the purification of the genetically expressed protein. The Fc fusion protein can then be conjugated with the ASGPR binding molecule via the MATE reagent, resulting in a bifunctional lysis factor molecule. Individuals with typical biomedical skills can introduce LALA mutations in the Fc region to attenuate the immune effector function by inactivating FcgR. Similarly, individuals with typical biomedical skills can introduce PA mutations to attenuate the immune effector function by inactivating c1q binding. It can use a DNA sequence encoding insulin-IgG1 Fc profusion protein to produce IgG1 Fc profusion protein return product technology in mammalian systems.

[003] In another embodiment, the target binding fragment is a precursor structure created for insulin-IgG1 Fc fusion.

[004] In another embodiment, the target-link segment is chosen from a set consisting of constructs established in the following table: Table 2 ABT 306 Alk-Original Insulin ABT307 G4S linker for proinsulin-Fc fusion, 71006.170 Dalton, proinsulin-IgG1 Fc fusion, G4S linker, hIgG1 hinge region (C > A), LALA-PA (sequence no.: 48). ABT308 5x G4S linker for proinsulin-Fc fusion, 73528.457 Dalton, proinsulin-IgG1 Fc fusion, 5x G4S linker, hIgG1 hinge region (C > A), LALA-PA (sequence no.: 49). ABT309 The LALA mutation (in the Fc domain) does not prevent discrimination by AGN301 (IgG hemolysis factor) ABT311 Homo sapiens sequence 1.18 ABT 311 The coding marker for the native insulin precursor is the sortase at the N-terminus (sequence number: 10). Molecular weight = 11127. ABT 312 The coding marker for the mutant proinsulin sortase at the N-terminus (sequence number: 6). Modification F25A. Molecular weight = 11051. Contains the human spp. 1.18 sequence (sequence number: 45). ABT313 The coding marker for the mutant proinsulin is sortase at the N-terminus. Molecular weight = 11141. Homo sapiens sequence 1..18. ABT314 The His coding sign for the native insulin protozoan is at the C-terminus. (Sequence number: 7). Molecular weight = 10842. ABT315 The coding mark for the native insulin precursor is the C-terminus. (Sequence No.: 8). ABT401 The LALA mutation (in the Fc domain) does not prevent discrimination by AGN301 (IgG hemolysis factor) ABT403 mAb49 (Original), Creative Biolabs, FAMAB-0225WJ, with mouse-human Fab-IgG3 Fc. ABT404 gamma 3 chimeric heavy chain mAb49, VH / VL mouse (sequence no.: 34) [495 AA], with human IgG1 CH / CL with kappa chimeric light chain mAb49 (sequence no.: 33) [215 AA]. ABT405 MAb49 VH / VL mouse-IgG1 CH / CL human. The Fc regions contain “LALA / PA” mutations to activate the immune effector function (binding to FcgR, c1q). ABT406 An antibody that is anti-mouse C-peptide (native) ABT407 An antibody that is anti-peptide-C with mouse VH / VL and human IgG1 CH / CL ABT408 The antibody is anti-peptide-C, mouse VH / VL-human IgG1 CH / CL. The Fc regions contain "LALA / PA" mutations to activate the immune effector function (binding to FcgR, c1q). ABT409 An antibody that is anti-peptide-C, a monoclonal antibody for mouse VH / VL-human IgG3 CH / CL. ABT410 Native insulin pro-binding sortase at the N end (clean / native) (sequence number: 44). ABT 411 Pre-insulin-binding sortase at the C-terminus (clean / original). ABT412 Achieving optimal insulin precursor molecule levels to improve conjugation efficiency ABT 413 Human insulin. ABT 414 Insulin efsitora alfa (insulin-Fc). Synonym: LY3209590 (Sequence No.: 16). MedChemExpress, catalogue # HY-P99665. See Moyers et al., J. Pharmacol. Exp. Ther., 382, ​​346-355 (2022) (Eli Lilly). ABT 415 Lispro insulin. ABT 416 The coding mark for the mutant proinsulin at the N-terminus (sequence number: 11) is sortase. ABT417 Mutant insulin precursor with the sortase marker at the N-terminus (sequence number: 22). Mutant B-chain insulin F25A. ABT 418 Fc-proinsulin fusion protein + hIgG1 Fc-LALA / PA, mouse Ig kappa signal, proinsulin, G4S ligand, hIgG1 (C>A)-Fc-LALA / PA hinge region. (Sequence No.: 30) ABT 419 Insulin proprotein (sequence number: 4). ABT 420 Proinsulin proprotein (sequence number: 5). Creative Biomart, Shirley, NY, USA, Cat. NO: Proinsulin-01H. R&D Systems, Minneapolis, MN, USA. ABT 421 PT0003, the original proinsulin with the coding mark His at the C end (sequence number: 7). (GenScript U608R952G0-15). ABT 422 PT0004. Native insulin progenitor, coding mark sortase at the C-terminus (sequence number: 8). (GenScript U608R952G0-21). ABT 423 PT0009, the original proinsulin, coding tag His at the N end (sequence number: 9) (GenScript) ABT 424 PT0011, the sortase coding mark for the native proinsulin at the N-terminus (sequence number: 10) (GenScript) ABT6396 Sequence number: 49 ABT7714 Mutant insulin progenitor (V3L for the A series and F24A for the B series) ABT17384 Mutant proinsulin + lysine mutation ABT17385 Mutant insulin pro-methane + methionine ABT17386 Mutant insulin pro-cysteine

[005] In another embodiment, the target binding fragment additionally includes a C-terminal rigid linker for the unconjugated cysteine ​​compounds in the target fragment to conjugate: His6-SPSTPPTPSPSTPPC (sequence number: 37).

[006] In another embodiment, the cell receptor binding molecule includes an ASGPR binding group according to the chemical formula: [ TBT101 ] or [ TBT102 ] ، The cell receptor binding segment contains additional elements described in this specification.

[007] In another embodiment, the cell receptor binding fragment is selected from a set consisting of constructed constructs listed in the following table. Table 3 TBT103 Maleimide βGN3 cell-binding cleavage for genetically modified cysteine-based conjugation. TBT104 Cleavage of αGN3 with polyglycine for sortase-based conjugation. TBT402 TBT403 βGN3-GLPETGG-NH2 (Sequence No.: 50). TBT404 GGGGG-Pra(β-GN3)-NH2. TBT405 PEG3 links TBT7714 To obtain AGN422 (Ins-030), glycine to alanine. TBT6170 To modify the insulin precursor at the N-terminus. TBT6171 To modify the insulin precursor at the C-terminus. TBT4438 TBT501 The sortase coding sign at the C end with the His6 coding sign. GGGGSGGGGSLPETGGHHHHHH (Sequence No.: 20). TBT502 A solid C-term link for unpaired cysteine ​​conjugation. HHHHHHSPSTPPTPSPSTPPC (Sequence No.: 21). TBT7990 TBT7991 TBT7988 TBT7989

[008] In another embodiment, the invention provides a method for preparing the composition from the material.

[009] In another application, the invention provides a method for removing antibodies that are anti-proinsulin and antiinsulin in a patient or subject by administering the agent to the patient or subject. The administered bifunctional hemolytic agent removes anti-proinsulin and antiinsulin autoantibodies, whether IgG1, IgG2, or IgG4, from the bloodstream by binding to the autoantibody and to the high-potency ASGPR receptor on hepatocytes.

[0010] In another embodiment, the invention provides a method for treating a pathological condition or condition associated with the upregulation of antibodies that are anti-proinsulin and antiinsulin in a treated subject or patient by giving the treated subject or patient an effective amount of the agent.

[0011] In another embodiment, the invention provides a method for treating diabetes in a patient by administering a therapeutically effective dose of the agent. The methods for following a treatment course for insulin-treated diabetes are known to persons with ordinary skill in the field of biomedicine. In a specific embodiment, the diabetes being treated is type 1 diabetes. The methods for following a treatment course for insulin-treated type 1 diabetes are also known to persons with ordinary skill in the field of biomedicine.

[0012] In another specific manifestation, anti-drug neutralizing antibodies obtained against insulin reduce the effectiveness of insulin in diabetic patients. Removing these anti-drug antibodies corrects the antibody-related insulin resistance in these patients. Removing these anti-drug antibodies is beneficial for treating life-threatening insulin resistance, brain damage, or cellular starvation.

[0013] In another specific manifestation, the majority of children and adults with type 1 diabetes exhibit early generation of C-chain antibodies to proinsulin in addition to native insulin. Removal of autoantibodies directed against proinsulin alleviates insulin-related inflammation, particularly inflammation directed at the pancreatic islets of Langerhans, which is partly due to cytotoxicity resulting from an antibody-dependent cell-cell reaction (ADCC) directed against the patient's pancreas. Removal of autoantibodies directed against proinsulin can correct antibody-related insulin resistance in patients. Insulin resistance impairs glucose uptake and IgG-induced β-cell death. See Liu et al., Diabetes, 71(4), 722–732 (April 1, 2022). Active intervention with these agents can reduce β-cell loss and decrease pancreatic inflammation.

[0014] In another specific manifestation, the agent is beneficial for treating animals with diabetes. Many companion animals, such as dogs and cats, in the United States suffer from diabetes. Over 10% of diabetic dogs in the United States are treated therapeutically with human insulin. These dogs often exhibit neutralizing antibodies that reduce insulin concentrations and antibody bivalents.

[0015] In another embodiment, the invention provides a pharmaceutical formulation including the agent and at least one pharmacologically acceptable excipient.

[0016] In another embodiment, the invention provides a formulation that includes the factor and at least one additional factor that includes a moiety capable of binding to that which constitutes the antibody moiety of the first compound.

[0017] On one hand, targeting anti-insulin or anti-proinsulin autoantibodies with TRAPs offers several advantages. In human type 1 diabetes, autoantibodies are often present at low concentrations, e.g., ~1000 ng / mL (0.01% of total IgG). The targeted autoantibodies are well-defined and detectable using available clinical diagnostic methods to track the effectiveness of parameter reduction. A single-drug therapy approach can target several type 1 diabetes-related factors that induce β-cell damage, including anti-insulin and anti-proinsulin autoantibodies. The targeted autoantibodies are primarily of the IgG1 subclass, with a half-life of approximately 29 days. Therefore, infrequent dosing may be effective.

[0018] Many of the elements, properties, aspects, and benefits of the invention become clearer from the following detailed description of particular embodiments of the invention, with drawings. Brief description of the drawings

[001] For the sake of clarity, some representations of the invention are shown in the drawings described below. Similar numbers in the drawings indicate similar elements. The invention is not limited to the specific formats, dimensions, and tools shown.

[002] Figure 1 shows two schematic diagrams of the processing of proinsulin and insulin.

[003] Figure 2 shows the structural formula for a single embodiment of bifunctional molecules (factors, TRAPs).

[004] Figure 3 shows a process for preparing a single embodiment of bifunctional molecules (factors, TRAPS) from a complete IgG antibody and a reagent with an affinity by conjugating the IgG antibody to the reagent.

[005] Figure 4 shows a table (Table 4) of the R groups of the MATE reagents and the dual-functional MoDE end compounds.

[006] Figure 5 represents a comparison of the stable heavy chain sequences of human IgG1 (sequence 1), IgG2 (sequence 1), and IgG4 (sequence 3), which support human therapeutic and human-compatible antibodies. Sequence differences in IgG2 and IgG4 from IgG1 are noted. The hinge region and lower hinge regions are noted, along with the N-glycosylated treatment site, N297. Numbering follows the EU numbering scheme (IMGT). Sequences significant for binding to FcgR, C1q, and FcRn are noted. Some of the increased ADCC mutations are shown above or below the sequences, as noted: Xencor's S239D, A330L, I332E [red, above the sequences]; Genentech's S298A, E333A, K334A; And Eli Lilly / AME's P247I,A339D / Q. Examples of FcRn binding mutations for extended half-life are also shown, as noted: MedImmune's YTE mutation (M252Y,S254T,T256E), PDL's T250Q, M428L mutation, Sally Ward's (University of Southampton) H433K mutation, N434Y mutation, and Genentech's N434A mutation are shown.

[007] Figure 6 represents a diagram of the chemical reaction that conjugates insulin with GN3 to produce an unprocessed insulin peptide αGN3.

[008] Figure 7 is a diagram showing several examples of GN3-conjugated C-peptide. Figure 7a shows the first example of GN3-conjugated C-peptide. Figure 7b shows the second example of GN3-conjugated C-peptide.

[009] Figure 8 shows a diagram of several examples of insulin peptides conjugated to GN3. Figure 8a shows an example of native insulin with its B-chain conjugated to GN3. Figure 8b shows an example of heterozygous insulin with its B-chain conjugated to GN3. Detailed description of the invention

[0010] The following detailed description is provided to assist persons with ordinary skill in the field of biomedicine. Typical representations are described. However, these representations are only representative. This disclosure is not limited to this, but defines the scope of the attached protective elements. Persons with ordinary skill in the field of biomedicine may make modifications and changes to the representations described in this specification without exceeding the scope and content of this disclosure. Applicability in the industrial field .

[0011] The invention provides a formulation of a medically useful substance (agent) for the treatment or slowing of the progression of diabetes and other insulin disorders. The removal and degradation of circulating antibodies that are anti-insulin and anti-proinsulin should improve the health of patients with diabetes by increasing normal insulin levels and making it available to the patient.

[0012] Previously, the removal or suppression of pathogenic antibodies was used with general immunosuppressive therapies, including TNF inhibitors, JAK inhibitors, FcRn recycling inhibitors, MTOR inhibitors, anti-diabetic corticosteroids, bis-selective hemolytic agents, cyclophosphamide, and other agents. These therapies resulted in marked susceptibility to microbial, viral, and other infections, and an increased incidence of cancer, particularly lymphomas, through reduced immune surveillance. Selective removal of pathogenic antibodies, while sparingly preserving non-pathogenic antibodies, allows for normal hormonal immune responses, including the normal response to vaccination, resistance to infection, and other important effector functions of the retained non-autoreactive antibodies. This approach provides an anti-inflammatory drug without the other undesirable complications of immunosuppression.Determining the extent of immunoneutralizing antibody reduction to insulin is performed easily and specifically by commercially available diagnostic tests for detecting these antibodies.

[0013] There are approximately 300,000 children under the age of 20 in the United States with type 1 diabetes. Insulin is an autoantigen in the autoimmune pathogenesis underlying type 1 diabetes.

[0014] Many adults with diabetes have insulin resistance linked to autoantibodies. In some of them, this is a life-threatening condition.

[0015] Many domestic animals have insulin resistance linked to autoantibodies. Approximately 165,000 dogs and 300,000 cats in the United States suffer from diabetes. More than 10% are treated with human insulin.

[0016] Following endocytosis of bifunctional glycolytic factor and insulin-binding protein (IBP), they are released from ASGPR via calcium depletion of the endosomal granule and changes in the protonation of the amino acid at the binding site due to a decrease in pH. ASGPR is then recycled back to the hepatocyte surface. The endocytotic proteins are then translocated to mature endosomals, which integrate with lysosomes. Lysosomal proteases then degrade the endocytotic proteins, permanently removing them from the bloodstream. Definitions

[0017] For the sake of consistency, the meanings of certain terms and expressions used in the specification, examples, and attached elements of protection are listed below. Unless otherwise stated or implied from the context, these terms and expressions shall have the following meanings. These definitions, in turn, help to describe the embodiments but are not intended to limit the invention protected by the elements of protection.

[0018] As used in this application, unless otherwise provided in this specification, each term shall have the meaning given below. Additional definitions are provided during the application. Where a term is not specifically defined in this specification, that term shall be given a meaning commonly understood in the field of biomedicine, applied in a context consistent with its use in describing the invention.

[0019] Indefinite and definite articles have the apparent meaning of one or more of, that is, at least one of, the grammatical forms of the article unless the context dictates otherwise. For example, "element" means one element or more than one element.

[0034] The term "ABT" has the meaning described in this specification for a binding fragment that is itself an antibody, a variant of an antibody, or a binding fragment to an antigen. In some specific formulations of this specification, ABT binds to insulin.

[0020] The term “active ingredient” has the meaning provided by the United States Food & Drug Administration for any ingredient that provides pharmacological activity or other direct effect in the diagnosis, treatment, relief, cure, or prevention of disease, or to activate the structural formula or any function of a human or animal body.

[0021] The term “ADCC” has a recognized meaning in the field of biological medicine for cell-mediated antibody-dependent cytotoxicity, which represents a cell-mediated immune defense mechanism according to which an effector cell of the immune system kills a target cell whose membrane surface antigens are bound to specific antibodies.

[0055] The term “ADCP” has the commonly accepted meaning in the field of biological medicine of antibody-dependent cell phagocytosis, an immune clearance mechanism according to which tumor cells are targeted with antibodies to promote their clearance from the body by phagocytic immune cells.

[0022] The term "agent" has the meaning commonly understood in the field of biomedicine of a substance useful for performing a function. Several useful functions in the field of biomedicine are described in this specification.

[0056] The term “alleviates” has the meaning commonly understood in the field of biomedicine for a process by which the severity of a sign or symptom of a disorder is reduced. A sign or symptom may be alleviated without being eliminated or eradicated. Administering pharmaceutical preparations or formulations specific to the invention may or may not eliminate a sign or symptom, although elimination is not required. Effective doses should be expected to reduce the severity of a sign or symptom.

[0057] The terms "effective dose" and "therapeutally effective dose" have the commonly accepted meaning in the field of biomedicine of a dose effective in achieving the intended purpose. The effect can be detected by any assay method known in the field. The precise effective dose for a patient depends on the patient's body weight, size, and health; the nature and severity of the condition; and the therapeutic agent or combination of therapeutic agents chosen for administration. Therapeutally effective doses for a condition can be determined through routine trial and error, which falls within the skill and judgment of the treating physician. In this example, the disease or condition being treated is tendinitis.

[0058] The term "analogue" has the established meaning in biomedicine of a molecule that is not identical but has a corresponding function or structural properties. Biochemical modifications can increase an analog's protease resistance, membrane permeability, or half-life without warning, for example, by binding to a ligand. Analogues of insulin are well-known in biomedicine, such as fast-acting insulins, also known as rapid-acting insulins.

[0059] The term "antibody that is anti-drug (ADA)" has the commonly accepted meaning in the field of biomedicine. Many ADAs are IgG4 antibodies, such as those found in factor VIII hemophilia.

[0060] The term "anti-insulin antibody" has the meaning commonly used in the field of biomedicine and includes insulin autoantibodies (IAAs). Anti-C-peptide antibodies are commercially available. See recombinant mouse anti-C-peptide antibody (CB2931), available from Creative Biolabs, Shirley, NY, USA, product number CBMAB-MD1611-LY. See also Mouse Anti-C-Peptide Monoclonal Antibody (CBFYR0644), available from Creative Biolabs, product number CBMAB-R0644-FY. See also human (chimeric) Anti-Insulin Recombinant Antibody (clone mAb49), available from Creative Biolabs, product number FAMAB-0225WJ. The inventors found that synthetic insulin mode has similar affinity values ​​to mAb49.

[0061] The term "antigen-binding fragment" has the meaning commonly used in the field of biomedicine for (1) a fragment of an intact antibody that binds to the same antigen recognized by the full-length antibody, such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, sFv, or other fragments composed of variant regions; or (2) any synthetic or genetically modified protein that functions as an antibody by binding to a specific antigen to form a complex. The term antigen-binding fragment of an antibody includes single-chain antibodies.

[0062] The term "ASGPR-specific binding group" has the established meaning in the field of biomedicine for a binding group that binds to the hepatocyte's ASGPR receptor. The ASGPR binding group selectively binds to the hepatocyte's ASGPR receptor on the cell surface. In many specification models of this particular, the ASGPR binding group represents a component of a bifunctional factor as a cell receptor-binding fragment that is covalently linked to the antibody-binding fragment via a linker group or directly. Through this ASGPR fragment, bifunctional factors complex with a circulating protein, for example, and bind to hepatocytes. After the bifunctional factor complex with the circulating protein binds to a hepatocyte or other cell, the circulating protein is engulfed by phagocytosis, where it is lysed by lysosomes.

[0063] The term "asylamide glycoprotein receptor" (ASGPR) has the established meaning in the field of biomedicine for lectins that bind to alylamide glycoproteins and glycoproteins from which sialic acid has been removed to expose the galactose subunits. These cellular receptors are found on mammalian liver cells and other cells, such as glandular cells of the gallbladder and stomach. ASGPR removes the targeted glycoproteins from the bloodstream.

[0064] The term "at least one of", when it precedes a list of items, modifies the entire list of items and does not modify the individual items in the list.

[0065] The term "AT" has the meaning described in this specification for an antibody cleavage. In some specific formulations of this specification, AT is bound to insulin.

[0066] The term “autoantibody” has the meaning commonly understood in the field of biological medicine.

[0067] The term “binding fragment” has the commonly accepted meaning in the field of biomedicine of a fragment on a binding protein, for example, an antibody, a variant of an antibody, or a binding fragment of an antigen, that binds to insulin.

[0068] The term “cell receptor-binding fragment” has the meaning commonly understood in the field of biomedicine. In many specification of this specification, the cell receptor-binding fragment is a specific binding group to an asymmetric glycoprotein receptor (ASGPR).

[0069] The term "cellular receptor" has the commonly accepted meaning in the field of biomedicine of a protein on the surface of a cell that binds to a compound, for example, a binding molecule, for example, a protein, in solution or on another cell. In general, the binding of a binding molecule to a receptor induces one or more biological responses. In this specification, the asymmetric glycoprotein receptor (ASGPR) is a cellular receptor on the surface of liver cells or other cells that binds to an asymmetric glycoprotein or its derivative.

[0070] The term "chimeric-compatible" has the meaning commonly used in the field of biomedicine. Chimeric antibodies are prepared by combining variable domains from one species, such as mouse, with fixed domains from another species, such as human. Using this biotechnological process, chimeric antibodies retain the specificity and affinity for the foreign antigen.

[0071] The terms “combination therapy” and “consecutive therapy” have the meaning commonly understood in the field of biomedicine of administering a combination described in this specification and at least a second agent as part of a specific treatment regimen designed to provide the beneficial effect of the combined action of these therapeutic agents. The beneficial effect of the combination may include, but is not limited to, the combined effect of the pharmacokinetic or pharmacodynamic effects resulting from the combination of therapeutic agents. Typically, the administration of these therapeutic agents in combination is carried out over a defined period of time, usually minutes, hours, days, or weeks, depending on the combination chosen. The term “combination therapy” includes the administration of the therapeutic agents, as described above, in additional combination with other biologically active ingredients and drug-free therapies, for example, surgery or radiation therapy. Where combination therapy additionally includes drug-free therapy, the drug-free therapy may be carried out at any convenient time if a beneficial effect of the combined action of the combination of therapeutic agents is achieved.For example, in favorable cases, a beneficial effect is achieved when non-drug therapy is temporarily removed from the administration of therapeutic agents, perhaps by days or even weeks.

[0072] The term "compatibility limiting region (CDR)" has the conventional meaning in biomedicine of a polypeptide region specific to the heavy chain or light chain of an antibody that acts as a limiter for the binding of the antibody to the antigen. Each antibody heavy chain has three CDRs. Each antibody light chain has three CDRs, usually different from the three CDRs on the antibody heavy chain. People with basic biomedical skills can calculate the structure of a CDR using a standard numbering method known as the Kabat numbering scheme. Other numbering schemes, such as Chothia, IMGT, Martin (an improved version of Chothia), and other standard numbering schemes, are also used by people with basic biomedical skills.

[0073] The term “includes”, the term “includes”, the term “entails”, and the term “included” define the properties, regions, integers, steps, operations, elements and / or components mentioned, but does not preclude the existence or addition of one or more other properties, regions, integers, steps, operations, elements, components, and / or combinations thereof.

[0074] The term "diabetes" has the meaning commonly used in the field of biological medicine. See Wikipedia, the free encyclopedia, Dipeptidyl peptidase-4 inhibitor (online, accessed July 3, 2024). In type 1 diabetes, damage to the insulin-producing beta cells of the pancreas results in an inability to control blood glucose levels, as insulin signals the uptake of glucose by cells. Type 1 diabetes is a systemic disease that can lead to serious complications. Hyperglycemia (high blood sugar) is a symptom of diabetes. A person with diabetes is someone whose blood sugar is elevated or expected to be elevated, and therefore someone who wants to prevent high or low blood sugar levels.

[0075] The term "diabetic macular edema" (DME) has the established meaning in the field of biomedicine for diabetic retinopathy. See Landry et al., Assay Drug Dev. Technol. 11(5), 326-32 (June 2013). Small bulges, called microaneurysms, form in the blood vessels, causing fluid to leak into the retina.

[0076] The term "EC" 50 "The commonly accepted meaning in the field of biological medicine of the concentration of the binding compound required to achieve 50% of maximum receptor activation.

[0077] The term “Fc-III-4c” has the commonly accepted meaning in the field of biomedicine for a polypeptide region in the crystallizable region of a fragment (Fc region), the tail region, of an antibody.

[0078] The term “Fc-M” has the commonly accepted meaning in the field of biomedicine for a polypeptide region in the crystallizable region of the fragment (Fc region), the tail region, of an antibody.

[0079] The term “Fc” shall have the meaning commonly understood in the field of biological medicine.

[0080] The term “FcB-1” has the commonly accepted meaning in the field of biomedicine for a polypeptide region in the crystallizable region of the fragment (Fc region), the tail region, of an antibody.

[0081] The term “FcB-2” has the commonly accepted meaning in the field of biomedicine for a polypeptide region in the crystallizable region of the fragment (Fc region), the tail region, of an antibody.

[0082] The terms “first,” “second,” “third,” etc., have the simple meaning of describing many elements, components, regions, layers, and / or sectors, and these elements, components, regions, layers, and / or sectors should not be defined by these terms. These terms are used only to distinguish one element, component, region, layer, or sector from another. A first element, component, region, layer, or sector may be called a second element, component, region, layer, or sector without deviating from the teachings of the present incarnations.

[0083] The term "hepatocyte" has the accepted meaning in the field of biological medicine for a cell of the main visceral tissue of the liver. Hepatocytes constitute 55-65% of the liver's mass.

[0084] The term "human-compatible" has the established meaning in the field of biomedicine of a protein, for example, an antibody, that is genetically modified to closely resemble the polypeptide structure of a human counterpart. A rodent-derived variant domain of an antibody can be combined with a stable domain of human origin, thus preserving the rodent antibody's characteristics. The human domain does not need to originate directly from a human, as it is first synthesized in a human. Instead, human domains can be obtained from rodents whose genomes contain human immunoglobulin genes. An antibody can be partially or fully human-compatible. In one approach, there are four general steps used to make a monoclonal antibody human-compatible.These steps consist of (1) identifying the predicted nucleotide and amino acid sequence of the light and heavy variable domains of the initiating antibody; (2) designing the humanized antibody, i.e., determining the frame region of the antibody to be used during the humanized antibody matching process; (3) actual humanized matching methods / techniques; and (4) inoculation into and genetic expression of the humanized antibody.

[0085] The term antibody "IgG" has the commonly accepted meaning in the field of biomedicine. Each IgG molecule consists of the basic tetra-chain immunoglobulin structure—two heavy γ (gamma) chains and two identical light chains (either kappa or lambda)—and bears two identical antigen-binding sites. There are four subclasses of IgG, each with slight variations in its H chains but with distinct biological properties.

[0086] The term “IgG1” has the commonly accepted meaning in the field of biomedicine of an IgG antibody where the C region of the gamma-1 chain of Ig is a protein that is encoded in humans by the IGHG1 gene.

[0087] The term “IgG2” has the commonly accepted meaning in the field of biomedicine of an IgG antibody where the C region of the gamma-2 chain of Ig is a protein that is encoded in humans by the IGHG2 gene.

[0088] The term "IgG4" in the field of biomedicine refers to an IgG antibody where the C region of the gamma-4 chain of Ig is a protein encoded in humans by the IGHG4 gene. IgG4 has a weak effector function; it cannot bind complement.

[0089] The term "insulin efsitora alfa" (LY-3209590) refers to a selective insulin receptor (IR) fusion protein consisting of human IR fusion factors combined with the crystallizable (Fc) domain of the G2 (lgG2) fragment of human immunoglobulin, with a molecular weight of 64.1 kDa. Insulin efsitora alfa is being developed as a potential treatment for diabetes mellitus. The protein may be used as a selective or comparator compound for the insulin-Fc fusion protein and the insulin-Fc fusion protein progenitor described in this specification.

[0090] The term "insulin growth factor" (IGF) has the meaning commonly used in the field of biomedicine. Human IGF-1 means containing the amino acid sequence corresponding to native human IGF for human tissue. Human IGF-1 may include fusion proteins.

[0091] The term "insulin lispro" has the established meaning in the field of biomedicine for a rapid-acting insulin analog, a synthetic version of insulin, used to lower blood glucose levels in people with diabetes. It acts more quickly and for a shorter duration than regular human insulin. See Islam, Khanna, Patel, & Zito, Insulin Lispro, In: StatPearls [Internet] (Treasure Island (FL), StatPearls Publishing). In insulin lispro (Eli Lilly), the proline positions at position B28 and the lysine positions at position B29 in the B-chain are reversed. In insulin lispro, these changes reduce the strength of the interactions that hold the insulin dimers together, allowing for faster dissociation of the hexavalent compounds and thus absorption of the monomers. These analogs and formulations result in a faster onset of action, a quicker time to peak activity, and a shorter duration of action compared to regular human insulin, making them optimal for administration at mealtimes.

[0092] The term "insulin receptor" has the commonly accepted meaning in the field of biomedicine. The insulin receptor contains two major subunits: α and β. A single disulfide bridge exists between the α and β subunits, between Cys647 in the insertion domain and Cys872. The α subunit has five major domains: L1 (AA 28–174), CR (AA 182–339), and L2 (AA 340–497), and two fibronectin subunits: FnIII-1 (building block 624–726) and FnIII-2 (757–842). The two α subunits are linked by four disulfide bonds. The insulin receptor β (IRA) has two different isoforms (IRA and IRB), based on the gene variant for exon 11. IRA differs from IRA by including exon 11. The 12-amino acid sequence (building blocks 745–756) derived from exon 11 is present in IRA but not in IRA. Functionally, the isoforms have different uptake and recycling rates. IRA exhibits a higher uptake and recycling rate than IRA.While insulin receptor A exhibits greater affinity for IGFs than IRB, both isoforms have similar affinities for insulin, and the insulin they bind to is very different from proinsulin. Because of these differences, insulin receptor B binds preferentially to metabolic and differential signaling. Primarily, insulin receptor A functions to promote cell growth, proliferation, and survival. See Beneit et al., Cardiovasc. Diabetol., 15, 161 (2016).

[0093] The term "insulin-like growth factor 2" (IGF-2) has the established meaning in the field of biomedicine for a well-characterized, naturally occurring peptide secreted by the liver and circulating in the blood. IGF-2 is an insulin-like growth regulator and mitotic promoter.

[0094] The term "insulin" has the commonly accepted meaning in the field of biological medicine. Insulin can be produced by chemical synthesis.

[0095] The term “IVIG” has the commonly accepted meaning in the field of biological medicine for intravenous immunoglobulin administration.

[0096] The term “binding fragment” has the commonly accepted meaning in the field of biomedicine of a fragment of a chemical compound that links one fragment of the chemical compound to another fragment of the same compound.

[0097] The term "MoDE" has the specific meaning relating to molecular decomposition agents. See International Patent Publications WO 2019 / 199634 (Yale University) and WO 2019 / 199621 (Yale University).

[0098] The term “fraction” has the medical-biological meaning of a defined chemical group or unit with a specific structural formula or activity. A fragment generally refers to a part of a molecule. In some incarnations, a binding fragment retains one or more of the required structural features, properties, functions, or characteristics—for example, three-dimensional structure, antigen quality, ability to bind to an antigen, or immunological functions, etc.—comparable to its corresponding fusion protein, for example, an antibody. In some incarnations, the fragment is monovalent. In some incarnations, it is bivalent. In others, it is polyvalent.

[0099] The term "monotherapy" has the commonly accepted meaning in the field of biomedicine of administering a single active or therapeutic compound to a patient. Monotherapy typically involves administering a therapeutically effective dose of an active compound.

[0100] The term “Multimodal Antibody Enhancer (MATE or MATES)” has the special meaning attributed to it. See International Patent Bulletin WO 2021 / 102052 (Kleo Pharmaceuticals).

[35] The term “nanoparticle” has the commonly accepted meaning in the field of biomedicine. The term “antibody” refers to immunoglobulin molecules and the immunoactive parts of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that binds specifically to an antigen. The term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, and a variety of forms including full-length antibodies and their antigen-binding parts; Including immunoglobulin molecule, monoclonal antibody, chimeric antibody, CDR-grafted antibody, humanized antibody, Fab, Fab′, F(ab′)2, Fv, disulfide-bound Fv, scFv, single-band antibody, bivalent antibody, nano-sized antibody, multispecific antibody, e.g., trispecific antibody, bispecific antibody, bispecific antibody, anti-self antibody, binding fragment with a functionally active adhesive top, and / or bifunctional hybrid antibodies.

[0101] The term "above" has a clear and simple meaning. When an element is referred to as being above another element, it can be in direct contact with the other element, or there may be overlapping elements between them. When an element is referred to as being "directly above" another element, there are no overlapping elements.

[0102] The term “or” as used in this specification means “and / or”. The term “and / or” as used in this specification includes all combinations consisting of one or more of the relevant listed elements.

[0103] The term "other degenerative cells" has the meaning commonly used in the field of biomedicine. ALS (associated glycoprotein receptors) are found on glandular cells of the gallbladder and stomach, as well as on liver cells.

[0104] The term “partially human-compatible” has the established meaning in the field of biomedicine for a protein, for example, an antibody, that is genetically modified to closely resemble the polypeptide structure of its human counterpart. A rodent-specific variant domain of an antibody can be combined with a fixed, human-origin domain, thus preserving the rodent antibody's specificity. The human-origin domain does not need to come directly from a human, as it is first synthesized in a human. Instead, human domains can be obtained from rodents whose genomes contain human immunoglobulin genes. An antibody can be partially or fully human-compatible.

[0105] The term "peripheral blood mononuclear cell (PBMC)" has the commonly accepted meaning in the field of biological medicine for any peripheral blood cell with a round nucleus, including lymphocytes (T cells, B cells, NK cells) and mononuclear cells.

[0106] The term “pharmaceuticalally acceptable excipient” has the meaning commonly understood in the field of biomedicine of an excipient useful in the preparation of a pharmaceutical formulation that is generally considered safe, non-toxic, and not biologically or otherwise undesirable, and includes an excipient acceptable for veterinary and human pharmaceutical use. “Pharmaceuticalally acceptable excipient,” as used in specifications and safeguards, includes both a single excipient and more than one. A full discussion of pharmaceutically acceptable excipients is available in Remington’s, Pharmaceutical Sciences, 23rd edition (Elsevier, 2020).

[0107] The term “pharmaceuticalally acceptable” shall have the meaning commonly understood in the field of biomedicine of those compounds, anions, cations, substances, formulations, carriers, and / or dosage forms which, within the scope of sound medical decision, are appropriate for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, consistent with an acceptable benefit / risk ratio.

[0108] The term "proinsulin" has the established meaning in the field of biomedicine, referring to a precursor to mature insulin. Proinsulin is commercially available from R+D systems. It can also be produced chemically. Chemically synthesized proinsulin is comparable to commercial proinsulin for surface plasmon resonance (SPR) assays. Proinsulin does not bind to the insulin receptor.

[0109] The term “protein-binding fragment” has the commonly accepted meaning in the field of biomedicine of a region of a chemical structure, for example, a polypeptide region of a chemical structure, that is specifically bound to a protein, for example, a specific protein.

[0110] The term "ROC" has the commonly accepted meaning in the field of biomedicine of a distinctive approach to the work of a receiver.

[0111] The terms "under treatment" and "patient" shall have the meaning commonly understood in the field of biological medicine. The term "patient" includes human patients and other mammalian patients receiving either preventative or pharmacological treatment.

[0112] The term "TBT" has the meaning described in this specification for a target binding molecule, a binding molecule to a cellular receptor. In some specific implementations of this specification, TBT is associated with ASGPR.

[0113] The term "TRAP" has the meaning described in this specification for the targeted removal of an abnormal protein. TRAP stands for bifunctional lysis factor.

[0114] The term “treat” and “treat” shall have the meaning commonly understood in the field of biological medicine of dealing with and caring for a patient to combat a disease, condition, or disorder and shall include administering a formulation described in this specification to relieve the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder.

[0115] The term "type 1 diabetes" has the accepted meaning in the field of biological medicine for a chronic autoimmune disease that occurs when the immune system attacks the insulin-producing cells in the pancreas, leading to autoimmune destruction of the beta cells in the pancreatic islets. Insulin cannot be synthesized or secreted into the bloodstream. Other autoantibodies targeting the islet cells may also be present in patients with type 1 diabetes.

[0116] The term "type 2 diabetes" has the established meaning in the field of biological medicine of progressive loss of insulin receptors leading to hyperglycemia. This hyperglycemia, in turn, prompts increased insulin production by beta cells to compensate. The accumulation of amyloid in the pancreatic islets likely impairs their anatomy and physiology. Eventually, the beta cells degenerate, leading to a decline in insulin over time.

[0117] The term “pan-antibody binding fragment” has the commonly accepted meaning in the field of biomedicine of a polypeptide region of an antibody binding protein that binds to a class of antibodies, rather than a specific group of antibodies.

[0118] Some of the embodiments are described below with reference to structural formulas and diagrams, to explain aspects of the description.

[0119] Unless otherwise defined, all technical and scientific terms used in this specification shall have the same meaning as normally understood by persons with ordinary skill in the field of biomedicine.

[0120] This specification does not relate to the process of human cloning, methods of modifying the genetic identity of the human sex line, the use of human embryos for industrial or commercial purposes, or procedures for modifying the genetic identity of animals to cause them to suffer without any essential medical benefit to humans or animals resulting from such processes. Methods of administration .

[0121] The ideal delivery pattern depends on where the treatment takes place, whether in a hospital or outside (outpatient). Methods for measuring improvement in a patient undergoing treatment after the removal of antibodies that are anti-insulin .

[0122] There are several commercially available assays for measuring insulin levels in patients undergoing treatment. As those with a basic understanding of biomedicine know, a stable and normal insulin level indicates continued health and well-being for a patient with diabetes or other insulin disorders. The chemical structural formula of the compound (the agent, TRAP ) .

[0123] In embodiment, the invention provides a composition of a substance (agent) that includes: A binding molecule that can bind to an antibody that is anti-insulin, A cleavage binding to a cell receptor that binds to liver cells or other lysed cells via hepatocyte-specific alactic glycoprotein receptor (ASGPR) or other cell receptors on the surface of lysed cells in a patient or a patient undergoing treatment, and A link that connects the antibody fragment and the cell receptor binding fragment.

[0124] In some embodiments, the invention provides a composition of a material (agent) whose structural formula is selected from the Markush set of structural formulas consisting of: R CN −(No)y−R CC , [ AGN101 ] ، [ AGN102 ] ، [ AGN103 ] or [ AGN104 ] Or a pharmaceutically acceptable salt of it. In these structural formulas, a, b, and c individually may be an integer of 1 or greater. In some incarnations, each cell-receptor binding molecule individually has the structural formula −(R CN −(Xaa)y−R CC Or a picture of salt from it.

[0125] In some incarnations, the agent has the structural formula specific to the formula. AGN101 or AGN103 : [ AGN101 ] [ AGN103 ] or Or a salt that is pharmaceutically acceptable, such as: Each of a, b and c individually is 1-200; Each AT individually is a link segment; L is a connecting element; and Each TBT individually is a binding molecule to a cellular receptor, The binding fragment is an antigen-binding protein, a variant of an antigen-binding protein, or an antigen-binding fragment of it.

[0126] In some incarnations, an agent includes one and no more than one binding strand. In some incarnations, one and no more than one binding strand is attached to a linker strand. In some incarnations, a is 1. In some incarnations, a is 2 or more. In some incarnations, one and no more than one cell-receptor binding strand is attached to a linker strand. In some incarnations, b is 1. In some incarnations, two or more cell-receptor binding strands are attached to a single linker strand. In some incarnations, b is 2 or more. In some incarnations, an agent includes one and no more than one cell-receptor binding strand. In some incarnations, c is 1. In some incarnations, b is 1 and c is 1. In some incarnations, a is 1, b is 1, and c is 1. In some incarnations, a factor includes two or more parts of the target association. In some incarnations, b is 2 or more and c is 1. In some incarnations, b is 2 or more and c is 2 or more.In some incarnations, b is 1 and c is 2 or more.

[0127] In some translocations, each cell receptor-binding molecule in a factor is homologous. In some translocations, each ligand connecting a cell receptor-binding molecule to an antibody molecule is homologous. In some translocations, the TBT in the factors is homologous. In some translocations, −L−(TBT) is present. b Identical.

[0128] Persons with ordinary skill in the field of biomedicine know that many techniques can be used to conjugate antibody fragments with target-binding fragments, for example, certain techniques are used to prepare antibody-drug conjugates according to this specification. In some inventions, the target-binding fragments are joined to antibody fragments by certain types of groups and / or structural amino acid units. In some inventions, the target-binding fragments are joined to selectively lysine structural units by linker fragments. In some inventions, the target-binding fragments are joined to selectively cysteine ​​structural units by linker fragments. In some inventions, the target-binding fragments are joined to selectively unnatural structural amino acid units by linker fragments. In some inventions, the invention provides techniques for selectively joining target-binding fragments to selectively certain structural amino acid units by linker fragments.In some incarnations, available technologies selectively connect the target-binding fragments to specific types of structural amino acid units, for example, lysine structural units, via linker fragments. In some incarnations, available technologies selectively connect the target-binding fragments to antibody fragment sites via linker fragments. In some incarnations, available technologies selectively connect the target-binding fragments to specific types of structural amino acid units at selectively sites via linker fragments. In some incarnations, the target-binding fragments are selectively connected to K246 and K248 of the IgG1 heavy chain and their corresponding structural amino acid units via linker fragments. In some incarnations, the target-binding fragments are selectively connected to K251 and K253 of the IgG2 heavy chain and their corresponding structural amino acid units via linker fragments. In some incarnations, the target-binding fragments are connected to K239 and K241 of the heavy chain and their corresponding structural amino acid units optionally via the linker fragments.In some incarnations, a cell receptor-binding molecule is attached to a specific amino acid subunit or a selectively selected site via a ligand. In other incarnations, each cell receptor-binding molecule is attached individually to a specific amino acid subunit or a selectively selected site via a ligand. As people with a basic understanding of biomedicine know, an antibody may have more than one site—for example, one on each of more than one chain, such as one heavy chain or all of the heavy chains. In some incarnations, an antibody molecule has two heavy chains, and one or both of the corresponding amino acid subunits are attached individually to a cell receptor-binding molecule via a ligand. In some incarnations, one or more chains are attached. In some incarnations, the ligand is a 1. In other incarnations, both are attached. In some incarnations, c is 2. In some incarnations, both the target binding strands and / or both the link strands (if any) are identical.

[0129] In some embodiments, the invention provides a composition of a substance (agent) in the formula AGN105 : [ AGN105 ] Or a salt that is pharmaceutically acceptable, such as: Each Xaa individually is a building block of an amino acid or an amino acid analog; t is a range of 0-50; z is 1-50; L is a connecting part; TBT is a cellular receptor binding fragment; Each R c Separately, it is −L a −R'; Each of a and b individually is 1-200; Each L a Separately, it is a covalent bond, or a divalent group with a selectively chosen substitution from the C1-C group. 20 Aliphatic or C1-C group 20 Heteroliphatic aliphatic with 1-5 heteroatoms, where one or more of the methylene units of the group are optionally and individually replaced by −C(R')2−, −Cy−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, or −C(O)O−; Each −Cy− ring individually is a monocyclic, bicyclic, or bivalent polycyclic group with selective substitution, where each monocyclic ring is individually selected from the C ring. 3-20 aliphatic ring, C ring 6-20 Aryl, a heterocyclic aryl ring consisting of 5-20 atoms with 1-10 separately selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon, and a heterocyclic cyclic ring consisting of 3-20 atoms with 1-10 separately selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R' individually is −R, −C(O)R, −CO2R, or −SO2R; Each R individually is −H, or a set with an optional substitution selected from C 1-30 Alifaty, C 1-30 A heterocyclic aliphatic compound containing 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon. 6-30 Ariel, C 6-30 Ariel Alivati, C 6-30 A heteroaliphatic aryl with 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; a heteroaliphatic aryl with 5-30 atoms with 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; and a heterocyclic cycloate with 3-30 atoms with 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon, or The two R groups are optionally taken together and separately to form a covalent bond, or: Two or more R groups are taken on the same atom, optionally and separately from the atom, to form a ring with optional substitution, consisting of 3–30 atoms, monocyclic, bicyclic, or polycyclic, containing, in addition to the atom, 0–10 separately selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; or Two or more R groups are taken on two or more atoms selectively and separately with their respective overlapping atoms to form a ring with selectively substitution, consisting of 3-30 atoms, monocyclic, bicyclic, or polycyclic, which, in addition to the overlapping atoms, contains 0-10 separately selected heteroatoms of oxygen, nitrogen, silver, phosphorus and silicon.

[0130] Further guidance is provided in International Patent Application WO 2025 / 035052 (Biohaven Therapeutics Ltd.), the contents of which are included for reference. Table 5 AGN301 IgG (reference molecular) lysis factor [F] is associated with c -BP2-PR3] with an antibody from a rabbit. AGN302 Mutant insulin with GN3 AGN 401 He constructed a synthetic C-peptide facility (D amino acids) with the αGN3 protease, without binding to any of the C-peptide-specific antibodies. AGN 402 He constructed a synthetic C-peptide facility (D amino acids) with αGN3 protease, without binding to any of the C-peptide-specific antibodies. AGN403 βGN3 constructs a synthetic C-peptide (D amino acids) without binding to any of the C-peptide-specific antibodies. βGN3 constructs a synthetic C-peptide (D amino acids) without binding to any of the C-peptide-specific antibodies. C-peptide with βGN3 (D amino acids). AGN404 βGN3 constructs a synthetic C-peptide (D amino acid) without binding to any of the C-peptide-specific antibodies. The C-peptide is then synthesized with the βGN3 protease (D amino acid). AGN303 A native insulin precursor, lns-003, was prepared via chemical synthesis conjugated to GN3 (triGalNAc) (as a reference) and bound to a murine antibody. This synthetic insulin mode has an affinity similar to mAb49. AGN3 10 TRAP insulin. Target binding fragment (lns-004), prepared by chemical synthesis conjugated to the αGN3 of the ASGPR (triGalNAc) binding complex. The synthetic insulin mode has an affinity similar to mAb49. Molecular weight: 7424.236. Sequence: GIVEQ[CC*TSIC]SLYQLENYC**N α-GN-triazol-(pent-4-inoic acid)-FVNQHLC*GSHLVEALYLVC**GERGAFYTPKT (sequence number: 43). Phenylalanine-to-alanine mutation (F to A). AGN 405 PT0004- TBT 6171 Native insulin progenitor prepared by C-terminal αGN3 conjugated gene recombinant product (triGalNAc), conjugated by sortase U2073499G0-8, BAR = 1. AGN 6601 The mutant insulin precursor prepared by the efflux gene recombination product technique suppressed αGN3 (triGalNAc) at the C-terminus, conjugated by sortase U2073499G0-8, BAR = 1. AGN3 04 PT0004- TBT 6171 Native insulin pro-structural structure established with a conjugate of αGN3 at the C-terminus. Sortase, U2073499G0-8, BAR = 1. AGN3 05 PT0004- TBT 6171 The structure of a mutant insulin precursor construct with a conjugate of αGN3 at the C-terminus. Sortase, U2073499G0-8, BAR = 1. AGN 406 Peptide-C with βGN3 (Amino acids L. Peptide-C with protease site, synthetic peptide-C degradation factor. AGN 407 Peptide-C with βGN3 protease (L amino acids). Peptide- C Without the protease site, the peptide-degrading factor C Synthetic. AGN 408 PT0009- TBT6170 The constructed structure of the original PT-0009-01 antibody with αGN3 at the N-terminus ( TBT6170 Molecular weight = 13319.04 Daltons, Sortase = U2073499G0-8. BAR = 0.82. IC 50 = 2.1 nanomolar. AGN 409 PT0009- TBT6170 The antibody specific to the original proinsulin PT-0009-02, αGN3 ( TBT6170 ) conjugate (N end), molecular weight = 13323.34 Daltons, sortase = U2073499G0-8, BAR = 1. AGN 410 PT0011- TBT6170 The conjugate of the original antibody PT-0011-01-αGN3 (N-terminus), molecular weight = 13242.87 Daltons, MW = 13242.87 Daltons, sortase = U2073499G0-8, BAR = 1. AGN 411 PT0011- TBT6170 The structure of a mutant insulin precursor construct with the αGN3 conjugate at the N-terminus (N-terminus), molecular weight = 13247.69 Daltons, sortase = U2073499G0-8, IC 50 = 3.1 nanomolar. AGN412 Synthetic mutagenic insulin using β-GN3. Molecular weight = 7424.24 Daltons. AGN 413 Fc-insulin-βGN3 conjugate AGN414 The linker from proinsulin-Fc-G4S fusion (ABT307) (sequence number: 17) + βGN3 ( TBT4438 ) Molecular weight = 77525.74 Daltons. AGN415 The link from proinsulin-Fc-5 x G4S fusion ( ABT308 ) (Sequence number: 18) + βGN3 (TBT4438) Molecular weight = 80047.84 Daltons. AGN416 Synthetic mutant insulin using αGN3 on the A chain, molecular weight = 7424.24 Daltons, AGN 417 ABT 410 -TBT 403 TRAP (Traditional Insulin AGN417) is a conjugate of the ASGPR (N-terminus) proinsulin. Synonym: GN3-proinsulin conjugate. Molecular weight: 11921.84. AGN417 demonstrated a 30% reduction in endogenous insulin-specific autoantibodies in 14-week-old NOD mice. AGN417 exhibited good subcutaneous bioavailability. The intravenous group showed rapid but robust detection of the hemolysis factor, in contrast to the subcutaneous group, where sustained detection was observed between 2 and 8 hours. The subcutaneous group demonstrated 76% bioavailability, indicating efficient absorption over time. Both subcutaneous (10 mg / kg) and intravenous (3 mg / kg) administration resulted in a significant reduction in blood glucose (BGL) levels at 30 minutes. AGN7525 Short link (G4S) with αGN3. AGN7526 Long link (G4S x 3) with αGN3. AGN418 ABT410-TBT405 Short (G4S) linkage with βGN3. Molecular weight = 11871.59 Daltons. AGN419 ABT411-TBT404 Insulin precursor with βGN3 at the C-terminus. Molecular weight = 12509.77 Daltons. AGN420 ABT412-TBT403 The long βGN3 spacer at the N-terminus of insulin precursor. Molecular weight = 12267.07 Daltons. AGN421 TBT7714-TBT403 The TRAP mutant insulin precursor ΒGN3 at the N-terminus, with a double mutant insulin precursor (V3L for the A chain and F25A for the B chain) and a triGalNAc conjugation with the peptide. Molecular weight = 11859.59 Daltons. In vivo tolerance and blood glucose levels were assayed in NOD mice, 0.23 years (12 weeks) old. No toxic signals were observed. AGN422 TBT7714-TBT405 TRAP mutant proinsulin. ΒGN3 at the N-terminus - a non-nociceptive double mutant proinsulin (V3L for chain A and F25A for chain B). The amide is located between triGalNAc and the peptide. Molecular weight = 11809.86 Daltons AGN7849 Long spacer at the N end in βGN3 proinsulin click. AGN7850 Long spacer at the N end in βGN3 proinsulin. AGN7865 The spacer at the N end in βGN3 proinsulin. AGN423 Insulin conjugate - Fc (efsitora alfa)- TBT4438 (βGN3-MATE) AGN8123 It has the specific structural formula of AGN421 With a terminal G→A mutation, Refold scores may not reach the same level as other insulin analogs due to the B24 F→A mutation (sequence number: 38). AGN2640 β (GalNAc ASGPR binding material)3 AGN4002 α(GalNAc ASGPR binding material)3 AGN4003 α(GalNAc ASGPR amide propionic ligand)3 AGN4004 β (GalNAc ASGPR amide propionic ligand)3 AGN4006 α(GalNAc ASGPR amide propionic ligand)3 AGN4007 β (GalNAc ASGPR amide propionic ligand)3 AGN4586 AGN3764 AGN6567 AGN6567 AGN417 With punctuation marks 13 and N15 AGN8140 ABT410-TBT7990 AGN8141 ABT410-TBT7991 AGN8138 ABT410-TBT7988 AGN8139 ABT410-TBT7989

[0131] A GN401 αGN3, constructs a synthetic C-peptide facility (D amino acids), without binding to any of the C-peptide-specific antibodies.

[0132] AGN404 β-GN3, constructs a synthetic C-peptide facility (D amino acids), without binding to any of the C-peptide-specific antibodies.

[0133] AGN406 β-GN3, a synthetic C-peptide (L amino acid) construct.

[0134] الجسم المضاد أحادي النسيلة من الببتيد C المضاد لفأر. K D = 9.7 نانو مولار. الببتيد C المضاد لفأر الخاص بناتج عودة الاتحاد الجيني. K D = 15 ميكرو مولار. امتصاص AGN406 الببتيد-C IC 50 = 4.2 نانو مولار.

[0135] AGN407 β-GN3 (protease), a structural component of synthetic C-peptide (L amino acids).

[0136] الجسم المضاد أحادي النسيلة من الببتيد C المضاد لفأر. K D = 7.5 نانو مولار. الببتيد C المضاد لفأر الخاص بناتج عودة الاتحاد الجيني. K D = 7.9 ميكرو مولار. امتصاص AGN407 الببتيد-C IC 50 = 7.9 نانو مولار.

[0137] AGN412 Sequence: GIVEQ[CC*TSIC]SLYQLENYC**N β-GN-triazole-(pent-4-inoic acid)-FVNQHLC*GSHLVEALYLVC**GERGAFYTPKT (Sequence No.: 43).

[0138] AGN416 Sequence: αGN-triazole-(pent-4-inoic acid)-GIVEQ[CC*TSIC]SLYQLENYC**FVNQHLC*GSHLVEALYLVC**GERGAFYTPKT (Sequence No.: 43). Part of the goal connection .

[0139] In some incarnations, the target-binding fragment includes a selected fragment of the Markush group consisting of one or more structural amino acid units, a peptide fragment, a cyclic peptide fragment, a peptide containing one or more natural structural amino acid units, and a peptide containing one or more unnatural structural amino acid units.

[0140] In some incarnations, each linkage fragment in an agent may be of the same linkage fragment or a pharmaceutically acceptable salt of it.

[0141] In some incarnations, the binding molecule includes a selected Markush group consisting of one or more structural amino acid units, a peptide molecule, a cyclic peptide molecule, a peptide containing one or more normal structural amino acid units, and a peptide containing one or more unnatural structural amino acid units.

[0142] Many antibody-specific binding fragments, including pan-antibody binding fragments, may be used in accordance with the instructions of this specification. Specific antibody-specific binding fragments and techniques for identifying or evaluating antibody-specific binding fragments are described in International Order Booklets WO 2019 / 023501 (Kleo Pharmaceuticals, Inc.) and WO 2019 / 136442 (Kleo Pharmaceuticals, Inc.), both of which are included in this specification in their entirety for reference. It is known to persons with ordinary skill in biomedicine that additional biomedical techniques may be appropriate for identifying or evaluating antibody-specific binding fragments in accordance with this specification. In some embodiments, an antibody-specific binding fragment includes one or more structural amino acid units, each of which is either normal or abnormal.

[0143] In some incarnations, a binding molecule, for example, a protein binding molecule, for example, an antibody binding molecule, for example, a pan-antibody binding molecule, the structural formula of ABT101 Or a salt-like image of it, where: Each of R 1 R 3 and R 5 A separate hydrogen atom or a group with a selectively chosen substitution from the C ring 1-6 Aliphatic, a cyclic carbon ring consisting of 3-8 saturated or partially unsaturated monocyclic atoms; phenyl, a cyclic carbon ring consisting of 8-10 aromatic bicyclic atoms; a monocyclic heterocyclic ring consisting of 4-8 saturated or partially unsaturated atoms with 1-2 separately selected heteroatoms of nitrogen, oxygen, or sulfur; a monocyclic heterocyclic aromatic ring consisting of 5-6 atoms with 1-4 separately selected heteroatoms of nitrogen, oxygen, or sulfur; or a bicyclic heterocyclic aromatic ring consisting of 8-10 atoms with 1-5 separately selected heteroatoms of nitrogen, oxygen, or sulfur; or: R is taken 1 and R 1’ Optionally with the carbon atom interlaced with them to form a saturated or partially unsaturated cyclic spirocarbon ring consisting of 3-8 atoms with optional substitution or a saturated or partially unsaturated heterocyclic spirocarbon ring consisting of 3-8 atoms with 1-2 separately selected heteroatoms of nitrogen, oxygen, or sulfur; R is taken 3 and R 3’ Optionally with the carbon atom interlaced with them to form a saturated or partially unsaturated cyclic spirocarbon ring consisting of 3-8 atoms with optional substitution or a saturated or partially unsaturated heterocyclic spirocarbon ring consisting of 3-8 atoms with 1-2 separately selected heteroatoms of nitrogen, oxygen, or sulfur; Group R is taken 5 and group R 5’ The atom attached to the same carbon atom selectively with the overlapping carbon atom to form a saturated or partially unsaturated cyclic spirocarbon ring of 3-8 atoms with selective substitution, or a saturated or partially unsaturated heterocyclic spirocarbon ring of 3-8 atoms with 1-2 separately selected heteroatoms of nitrogen, oxygen, or sulfur; or My two R groups are taken 5 Optionally, with its own interlacing atoms, it forms a straight or branched divalent hydrocarbon chain, saturated or unsaturated, with optional substitution by C. 1-10 One to three of the chain's methylene units are individually and optionally replaced by –S–, –SS–, –N(R)–, –O–, –C(O)–, –OC(O)–, –C(O)O–, –C(O)N(R)–, –N(R)C(O)–, –S(O)–, –S(O)2–, or –Cy 1 – where each –Cy 1 - Separately, it is a heterocyclic arynyl composed of 5-6 atoms, with the heterocyclic atoms chosen separately from nitrogen, oxygen, or sulfur; Each of R 1’ R 3’ and R 5’ Separately, it is hydrogen or optionally C 1-3 My aliphatic has a replacement; Each of R 2 R 4 and R 6 Separately, it is hydrogen, or optionally C 1-4 My aliphatic language has a replacement, or: R is taken 2 and R 1 Optionally with its own overlapping atoms to form a saturated or partially unsaturated single-ring heterocyclic ring with optional substitution consisting of 4-8 atoms with 1-2 separately selected heteroatoms of nitrogen, oxygen, or silver; R is taken 4 and R 3 Optionally, with its own interlacing atoms, to form a saturated or partially unsaturated monocyclic heterocyclic ring with optional substitutions of 4-8 atoms, containing 1-2 separately selected heteroatoms of nitrogen, oxygen, or sulfur; or Group R is taken 6 and R group 5 Adjacent to it optionally with its own overlapping atoms to form a saturated or partially unsaturated single-ring heterocyclic ring with optional substitution consisting of 4-8 atoms with 1-2 separately selected heterocyclic atoms of nitrogen, oxygen, or silver; L 1 It is a trivalent bond fragment; and Each of m and n individually is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0144] In some incarnations, L 1 It is a trivalent group with a selective substitution of C1-C 20 Aliphati or C1-C 20 A heterocyclic aliphatic with 1–5 heteroatoms, where one or more of the methylene group units are optionally and individually replaced by −C(R')2−, −Cy−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, or −C(O)O−. In some incarnations, L 1 It is –(CH2CH2O) 2-4 – or –(CH2CH2O)2–.

[0145] Many antibody-binding fragments, including pan-antibody-binding fragments, may be used in accordance with the instructions of this specification. Some antibody-binding fragments and techniques for identifying or evaluating antibody-binding fragments are described in International Application Bulletins WO 2019 / 023501 (Kleo Pharmaceuticals, Inc.) and WO 2019 / 136442 (Kleo Pharmaceuticals, Inc.), both of which are included in this specification in their entirety for reference. Persons with ordinary skill in biomedicine know that additional biomedical techniques may be appropriate for identifying or evaluating antibody-binding fragments in accordance with this specification. In some embodiments, an antibody-binding fragment includes one or more structural amino acid units, each of which is either normal or abnormal.

[0146] In some incarnations, selected sites for the antibody fragments are used for conjugation. In some incarnations, K246 or K248 of an antibody factor (EU numbering, or corresponding structural units) are considered conjugation sites. In some incarnations, the conjugate site is K246 for a heavy chain (unless otherwise specified, sites in this specification include the corresponding structural units in, for example, a modified sequence, longer sequences, shorter sequences, rearranged sequences, etc.). In some incarnations, the site is K248 for a heavy chain. In some incarnations, the site is K288 or K290 for a heavy chain. In some incarnations, the site is K288 for a heavy chain. In some incarnations, the site is K290 for a heavy chain. In some incarnations, the site is K317. In some incarnations, the antibody fragment is a fragment of an IgG1 antibody. In some incarnations, the antibody fragment is a fragment of an IgG2 antibody. In some incarnations, the antibody fragment is a fragment of an antibody.In some incarnations, the formulation includes a multi-group MATE, where the antibody fragments of the MATE multi-group are individually an antibody fragment of an IgG1, IgG2, or IgG4 antibody, or a fragment thereof.

[0147] In some incarnations, the antibody's heavy chains are selectively conjugated / numbered over the light chains.

[0148] In some incarnations, approximately 10%–100% of all, or nearly all, of the fragments of interest are conjugated, for example, target-binding fragments, to fragments of a specific antibody type, e.g., IgG1, or fragments thereof, at one or more particularly specific sites, typically one or two particularly specific sites, e.g., K246 and K248 of the IgG1 heavy chain and their corresponding structural amino acid subunits. In some incarnations, approximately 10%–100% of all, or nearly all, of the fragments of interest are, for example, target-binding fragments, to fragments of IgG2 antibodies or fragments thereof at K251 and K253 of the IgG2 heavy chain and their corresponding structural amino acid subunits. In some incarnations, approximately 10%-100% of all, or largely all, of the fragments of interest are, for example, target-binding fragments, conjugated with IgG2 antibody fragments or fragments thereof at K239 and K241 of the heavy chain and their corresponding structural amino acid units.In some formulations, approximately 10%-100% of all, or largely all, of the fragments of interest are conjugated, for example, for a multi-factor combination, for a combination, etc., with the antibody fragments of IgG1, IgG2, and / or IgG4 antibodies, or fragments thereof, for example, to conjugate products of IgG1 antibodies or fragments thereof (the antibody fragments are specific to IgG1 antibodies or fragments thereof), IgG2 antibodies or fragments thereof (the antibody fragments are specific to IgG2 antibodies or fragments thereof), IgG4 antibodies or fragments thereof (the antibody fragments are specific to IgG4 antibodies or fragments thereof), or to conjugate products of IVIG (where certain available techniques described in this specification are used, selective conjugation with IgG1, IgG2 and IgG4). In some incarnations, the percentage is approximately 10% or more. In some incarnations, the percentage is approximately 20% or more. In some incarnations, the percentage is approximately 25% or more. In some incarnations, the percentage is approximately 30% or more. In some incarnations, the percentage is approximately 40% or more.In some incarnations, the percentage is approximately 50% or more. In some incarnations, the percentage is approximately 60% or more. In some incarnations, the percentage is approximately 65% ​​or more. In some incarnations, the percentage is approximately 70% or more. In some incarnations, the percentage is approximately 75% or more. In some incarnations, the percentage is approximately 80% or more. In some incarnations, the percentage is approximately 85% or more. In some incarnations, the percentage is approximately 90% or more. In some incarnations, the percentage is approximately 95% or more. In some incarnations, the percentage is approximately 100%.

[0149] Further guidance is provided in International Patent Application WO 2025 / 035052 (Biohaven Therapeutics Ltd.), the contents of which are included for reference. Table 6 ABT401 The LALA mutation (in the Fc domain) does not prevent discrimination by AGN301 (IgG hemolysis factor) ABT309 The LALA mutation (in the Fc domain) does not prevent discrimination by AGN301 (IgG hemolysis factor) ABT311 The rational human being YNITDHGSLIYSFSECAF 1.. 18 ABT 312 The coding marker for the mutant proinsulin precursor is sortase at the N-terminus (sequence number: 6). Modification F25A. Molecular weight = 11051. Homo sapiens GSLIYSFSECAFTGPLRP 1..18. ABT313 The coding marker for the mutant proinsulin is sortase at the N-terminus. Molecular weight = 11141. Homo sapiens FSECAFTGPLRPFFSPGF 1..18. ABT314 The His coding sign for the native insulin protozoan is at the C-terminus. (Sequence number: 7). Molecular weight = 10842. ABT315 The coding mark for the native insulin precursor is the C-terminus. (Sequence No.: 8). ABT 306 Alk-Original Insulin ABT307 G4S linker for proinsulin-Fc fusion, 71006.170 Dalton, proinsulin-IgG1 Fc fusion, G4S linker, hIgG1 hinge region (C > A), LALA-PA (sequence no.: 48). ABT308 5 x G4S linker for proinsulin-Fc fusion, 73528.457 Dalton, proinsulin-IgG1 Fc fusion, 5 x G4S linker, hIgG1 hinge region (C > A), LALA-PA (sequence no.: 49). ABT 402 Monoclonal antibody. ABT403 mAb49 (original), Creative Biolabs, FAMAB-0225WJ, with mouse-human Fab-IgG3 Fc ABT404 gamma 3 chimeric heavy chain mAb49, VH / VL mouse (sequence no.: 34) [495 AA], with human IgG1 CH / CL with kappa chimeric light chain mAb49 (sequence no.: 33) [215 AA]. ABT405 MAb49 VH / VL mouse-IgG1 CH / CL human. The Fc regions contain "LALA / PA" mutations to activate the immune effector function (binding to FcgR, C1q). ABT406 An antibody that is anti-mouse C-peptide (native) ABT407 An antibody that is anti-peptide-C with mouse VH / VL and human IgG1 CH / CL ABT408 The antibody is anti-peptide-C, mouse VH / VL-human IgG1 CH / CL. The Fc regions contain "LALA / PA" mutations to activate the immune effector function (binding to FcgR, C1q). ABT409 An antibody that is anti-peptide-C, a monoclonal antibody for mouse VH / VL-human IgG3 CH / CL. ABT410 Native insulin pro-binding sortase at the N end (clean / native) (sequence number: 44). ABT 411 Pre-insulin-binding sortase at the C-terminus (clean / original). ABT412 Achieving optimal insulin precursor molecule levels to improve conjugation efficiency ABT 413 Human insulin. ABT 414 Insulin efsitora alfa (insulin-Fc). Synonym: LY3209590 (Sequence No.: 16). MedChemExpress, catalogue # HY-P99665. See Moyers et al., J. Pharmacol. Exp. Ther., 382, ​​346-355 (2022) (Eli Lilly). ABT 415 Lispro insulin. ABT 416 The coding mark for the mutant proinsulin at the N-terminus (sequence number: 11) is sortase. ABT417 Mutant insulin precursor with the sortase marker at the N-terminus (sequence number: 22). Mutant B-chain insulin F25A. ABT 311 The coding marker for the native insulin precursor is the sortase at the N-terminus (sequence number: 10). Molecular weight = 11127. ABT 418 Fc-proinsulin fusion protein + hIgG1 Fc-LALA / PA, mouse Ig kappa signal, proinsulin, G4S ligand, hIgG1 (C>A)-Fc-LALA / PA hinge region. (Sequence No.: 30) ABT 419 Insulin proprotein (sequence number: 4). ABT 420 Proinsulin proprotein (sequence number: 5) Creative Biomart, Shirley, NY, USA, Cat. NO: Proinsulin-01H. R&D Systems, Minneapolis, MN, USA ABT 421 PT0003, the original proinsulin with the coding mark His at the C end (sequence number: 7). (GenScript U608R952G0-15). ABT 422 PT0004, the original proinsulin, coding mark sortase at the C-terminus (sequence number: 8). (GenScript U608R952G0-21). ABT 423 PT0009, the original proinsulin, coding tag His at the N end (sequence number: 9) (GenScript) ABT 424 PT0011, the sortase coding mark for the native proinsulin at the N-terminus (sequence number: 10) (GenScript) ABT6396 Sequence number: 49 ABT7714 Mutant proinsulin (V3L for chain A and F24A for chain B) ABT17384 Mutant proinsulin + lysine mutation ABT17385 Mutant insulin pro-methane + methionine ABT17386 Mutant insulin pro-cysteine Link parts

[0150] In some incarnations, the fragments are optionally connected to each other via linker fragments. In some incarnations, an interactive array, e.g., RG, is connected to a cell receptor-binding fragment, e.g., TBT, via a linker, e.g., L RM In some incarnations, a line may include, for example, L G Also, on one or more links, for example, L LG1 L LG2 L LG3 L LG4 And so on, to connect many parts. In some incarnations, L LG This is a linking segment described in this specification. In some incarnations, L LG1 This is a linking segment described in this specification. In some incarnations, L LG2 This is a linking segment described in this specification. In some incarnations, L LG3 This is a linking segment described in this specification. In some incarnations, L LG4 This is a linking segment described in this specification. In some incarnations, L RM This is a linking segment described in this specification. In some incarnations, L PM It is an L. In some incarnations, L is PM This is a linking segment described in this specification. In some incarnations, L PM It is an L.

[0151] Link fragments of multiple types and / or for multiple purposes may be used, for example, those used in antibody-drug conjugates, etc., in accordance with this specification.

[0152] Bond strands can be bivalent or polyvalent depending on how they are used. In some incarnations, a bond strand is bivalent. In other incarnations, it is polyvalent and consists of more than two strands.

[0153] In some incarnations, the linking hemistich is, for example, L z For example, L PM L RM L LG L LG1 Furthermore, it is a term or includes L.

[0154] In some incarnations, the linking part has the structural form specific to the formula LNK101 : NH(R a1 )−L a1 −C(R a2 )(R a3 )−L a2 −COOH. [ LNK101 ] Or a salt that is pharmaceutically acceptable, such as: Each of R a1 R a2 R a3 Separately, it is −L a −R'; L is a a1 and L a2 Separately, it is an L a ; Each L a Separately, it is a covalent bond, or a divalent group with a selected C1-C selective substitution. 20 Aliphati or C1-C 20 A heterocyclic aliphatic with 1-5 heteroatoms, where one or more of the group's methylene units are optionally and individually replaced by −C(R')2−, −Cy−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, or −C(O)O−; Each −Cy− ring individually is a monocyclic, bicyclic, or bivalent polycyclic group with selective substitution, where each monocyclic ring is individually selected from the C ring. 3-20 Aliphatic, C-ring 6-20 Aryl, a heterocyclic aryl ring consisting of 5-20 atoms with 1-10 separately selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon, and a heterocyclic cyclic ring consisting of 3-20 atoms with 1-10 separately selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R' individually is −R, −C(O)R, −CO2R, or −SO2R; Each R individually is −H, or a set with an optional substitution selected from C 1-30 Alifaty, C 1-30 A heterocyclic aliphatic compound containing 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon. 6-30 Ariel, C 6-30 Ariel Alivati, C 6-30 A heteroaliphatic aryl with 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; a heteroaliphatic aryl with 5-30 atoms with 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; and a heterocyclic cycloate with 3-30 atoms with 1-10 individually selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon, or The two R groups are optionally taken together and separately to form a covalent bond, or: Two or more R groups are taken on the same atom, optionally and separately from the atom, to form a ring with optional substitution, consisting of 3–30 atoms, monocyclic, bicyclic, or polycyclic, containing, in addition to the atom, 0–10 separately selected heteroatoms of oxygen, nitrogen, sulfur, phosphorus, and silicon; or Two or more R groups are taken on two or more atoms, chosen individually and separately, with their respective overlapping atoms, to form a ring with a chosen substitution, consisting of 3-30 atoms, monocyclic, bicyclic, or polycyclic, containing, in addition to the overlapping atoms, 0-10 separately chosen heteroatoms of oxygen, nitrogen, silver, phosphorus, and silicon.

[0155] In some incarnations, a structural amino acid unit has the structural formula −N(R) a1 -L a1 −C(R a2 (R) a3 -L a2 −COO− or a salt form of it.

[0156] Further guidance is provided in International Patent Application WO 2025 / 035052 (Biohaven Therapeutics Ltd.), the contents of which are included for reference. MATES

[0157] Persons with ordinary skill in the field of biomedicine may use MATES materials and methods as a guide to expected results when preparing and using the invention.

[0158] This agent may be called a MATE agent or simply MATE. MATE agents are described, for example, in international patent application WO 2021 / 102052 (Kleo Pharmaceuticals, Inc.), the contents of which are included for reference. In some formulations, an agent comprises an antibody fragment, a cell receptor-binding fragment, and a ligand that connects the antibody fragment and the cell receptor-binding fragment. Cellular receptor-binding junction.

[0159] Several of the binding fragments to a receptor, according to the incarnations for the present invention, are described in WO 2019 / 19962 (Bostal Drug Delivery Co., Ltd), WO 2019 / 199634 (Yale University), WO 2021 / 072246 (Yale University), and WO 2021 / 072246 (Yale University), each of which is included in this whole specification for reference.

[0160] In embodiment, the cell receptor binding cleft may include a non-associated glycoprotein receptor (ASGPR) binding group connected via an amine group to the binding cleft.

[0161] The amine group may be a major alkylamine group or a minor alkylamine group, each of which has a selective substitution on the amine group with a C1-C3 alkyl group.

[0162] The cell receptor binding cleft may include an ASGPR binding group according to the chemical formula: ، [ TBT101 ] or ، [ TBT102 ] Where X is 1-4 atoms long and includes groups O, S, N(R) N1 ) or C(R N1 (R) N1 So that: When X is 1 atom long, X is O, S, N(R) N1 ) or C(R N1 (R) N1 ) When X is 2 atoms long, no more than 1 atom of X is O, S or N(R) N1 ) When X is 3 or 4 atoms long, no more than two of the X atoms are O, S or N(R) N1 ) where R N1 It is an H or C1-C3 alkyl group with optional substitution of 1-3 halo groups; R1 and R3 separately consist of: H, -(CH2) K OH, -(CH2) K OC1-C4 Alkyl, with 1-3 halo groups optionally substituted, C1-C4 Alkyl, with 1-3 halo groups optionally substituted, -(CH2) K -Vinyl, O-(CH2) K -Vinyl, -(CH2) K -Alkenyl, -(CH2) K -COOH, -(CH2) K C(O)O-C1-C4 is an alkyl with optional substitution of 1-3 halo groups, OC(O)-C1-C4 Alkyl, with 1-3 halo substitutions, -C(O)-C1-C4, or R1 and R3 each constitute a set These alkyl groups have selective substitutions of up to three halo groups, C1-C4 alkyl groups, each of which has selective substitution of one to three halo groups or one or two hydroxyl groups, or O-C1-C4 groups. In an alkyl group, each alkyl group has a selective substitution of one to three halo groups or one or two hydroxyl groups; and K alone is an integer ranging from 0 to 4, or R1 and R3 each form a group according to the chemical formula: where R 7 It is an O-C1-C4 alkyl group, with a selective substitution of 1 to 3 halo groups, 1 or 2 hydroxyl groups, or R 7 It is -NR Group N3 R N4 or a; or Each of R1 and R3 individually constitutes a set according to the structural formula: , , , , or Grouped according to chemical formula: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or or R1 and R3 each constitute a set where it is It is a saturated cyclic carbon group C3-C8; R C Not present, H, C1-C4 alkyl with optional substitution of 1-3 halo groups or 1-2 hydroxyl groups, or a group according to the structural formula: Where R4, R5 and R6 are individually H, halo(F, Cl, Br, I), CN, NR N1 R N2 -(CH2) K OH, -(CH2) K OC1-C4 alkyl, has a selective substitution of 1-3 halo groups, C1-C3 alkyl, has a selective substitution of 1-3 halo groups, -O-C1-C3- alkyl, has a selective substitution of 1-3 halo groups, -(CH2) K COOH, -(CH2) K C(O)O-C1-C4 is an alkyl with 1-3 halo substitutions; OC(O)-C1-C4 is an alkyl with 1-3 halo substitutions; -C(O)-C1-C4 is an alkyl with 1-3 halo substitutions; or R C It is a group , group , group or group , Where each of R N R N1 and R N2 A separate H or C1-C3 alkyl group with optional substitution of one to three halo groups or one or two hydroxyl groups; K alone is an integer ranging from zero to 4; K' is an integer from 1 to 4; R N3 It is an H, or C1-C3 alkyl group with a selective substitution of 1-3 halo groups or 1 or 2 hydroxyl groups; and R N4 It is an H, C1-C3 alkyl group with a selective substitution of 1-3 halo groups or 1 or 2 hydroxyl groups, or it is R N4 It is a set, where K is preferably 1; It represents a linker group that includes at least one binding strand and links at least one binding strand to the binding strand at the cellular receptor through a selective binding strand, or It represents a linker set containing at least one or more functional sets that can be used to covalently link the linker set to at least one linker fragment or an optional linker fragment; R2 is a set where R N1 And K are similar to the above; R AM It is an H, C1-C4 alkyl group with optional substitution by up to three halo groups and one or two hydroxyl groups, -(CH2) group K COOH, -(CH2) group K C(O)O-C1-C4 alkyl group has 1-3 halo substitutions, OC(O)-C1-C4 alkyl group has 1-3 halo substitutions, F -C(O)-C1-C4 alkyl group has 1-3 halo substitutions, -(CH2) group K -NR N3 R N4 where R N3 It is an H, or C1-C3 alkyl group with a selective substitution of 1-3 halo groups or 1 or 2 hydroxyl groups; and R N4 It is an H, C1-C3 alkyl group with an optional substitution of 1-3 halo groups or 1 or 2 hydroxyl groups, or a group, or R2 is a set , where R TA These are H, CN, NR N1 R N2 -(CH2) K OH, -(CH2) K OC1-C4 alkyl, has an optional substitution of 1-3 halo groups, C1-C4 alkyl, has an optional substitution of 1-3 halo groups, -(CH2) K COOH, -(CH2) K C(O)O-C1-C4 is an alkyl with 1-3 halo substitutions; OC(O)-C1-C4 is an alkyl with 1-3 halo substitutions; -C(O)-C1-C4 is an alkyl with 1-3 halo substitutions; or R TA It is a C3-C group 10 An aryl or heteroaryl group consisting of three to ten atoms, containing up to five heteroaryl atoms, each of the aforementioned aryl or heteroaryl groups has an optional substitution of up to three (preferably 1) CN, NR N1 R N2 -(CH2) K OH, -(CH2) K OC1-C4 alkyl, has a selective substitution of 1-3 halo groups, C1-C3 alkyl, has a selective substitution of 1-3 halo groups or 1 or 2 hydroxyl groups, -O-C1-C3 alkyl, has a selective substitution of 1-3 halo groups, -(CH2) K COOH, -(CH2) K C(O)O-C1-C4 is an alkyl with 1-3 halo groups optionally substituted, OC(O)-C1-C4 is an alkyl with 1-3 halo groups optionally substituted, or -(CH2) K C(O)-C1-C4 is an alkyl with optional substitution of 1-3 halo groups, or R TA It is a group , group , group , group , group It has optional replacement with up to three C1-C3 alkyl groups, or optional replacement with up to three halo groups. R TA A lesson about a group, Where each of R N R N1 and R N2 A separate H or C1-C3 alkyl group has an optional substitution of one to three halo groups or one or two hydroxyl groups, and each group is -(CH2). K It has an optional substitution of 1-4, preferably 1 or 2, of C1-C3 alkyl groups. It has an optional substitution of 1-3 fluoro groups or 1-2 hydroxyl groups. K is a separate unit of 0-4.

[0163] Further guidance is provided in International Patent Application WO 2025 / 035052 (Biohaven Therapeutics Ltd.), the contents of which are included for reference. Table 7 TBT103 βGN3 maleimide cell-binding cleavage for genetically modified cysteine-based conjugation. TBT104 αGN3 is cleaved with polyglycine for a sortase-based conjugation. TBT402 TBT403 βGN3-GLPETGG-NH2. TBT404 GGGGG-Pra(β-GN3)-NH2. TBT405 Ins-030) PEG3 links TBT7714 To obtain AGN422 Glycine to alanine. TBT6170 Modification at the N-terminus of insulin protist. TBT6171 Modification at the C-terminus of insulin protist. TBT4438 TBT501 The sortase coding sign at the C end with the His6 coding sign. GGGGSGGGGSLPETGGHHHHHH (Sequence No.: 20). TBT502 A solid C-term link for unpaired cysteine ​​conjugation. HHHHHHSPSTPPTPSPSTPPC (Sequence No.: 21). TBT7990 TBT7991 TBT7988 TBT7989 The second factor .

[0164] In another embodiment, the invention provides a formulation that includes the agent and at least one additional agent that includes a fragment capable of binding to the antibody that forms the antibody fragment of the first compound.

[0165] In some formulations, the first formulation is a formulation containing a first factor as described in this specification. In some formulations, the second factors individually contain a second reactive group. In some formulations, the second formulation is a formulation containing a combination of factors as described in this specification, where each cell-receptor binding fragment is an individual reactive group as described in this specification. In some formulations, the second formulation is an antibody formulation, where the antibodies in the formulation are not chemically modified. In some formulations, the second formulation is an IVIG preparation. In some formulations, the product formulation is a formulation containing a combination of factors as described in this specification, where each cell-receptor binding fragment is an individual cell-receptor binding fragment as described in this specification.

[0166] In some incarnations, the cell receptor-binding molecule of a product factor is the cell receptor-binding molecule of a first factor. In some incarnations, the antibody molecule of a product factor is the antibody molecule of a second factor. In some incarnations, a second factor is an antibody factor, for example, a monoclonal antibody, an antibody in a polyclonal antibody, an antibody in an IVIG preparation, etc. In some incarnations, a second reactive group is a functional group specific to a structural amino acid unit, for example, −NH2 of lysine, −SH of cysteine, etc. In some embodiments, a second reactant is −NH2 of a lysine subunit, for example, of a selected subunit of K246 and K248 specific to the corresponding IgG1 heavy-chain amino acid subunits, K251 and K253 of the IgG2 heavy-chain and corresponding amino acid subunits, and K239 and K241 of the heavy-chain and corresponding amino acid subunits. In some embodiments, the invention provides selective reactivity at specific amino acid subunits of the antibody fragments.

[0167] In some inventions, a second reactive group is selectively attached to an antibody fragment via a ligand. In some inventions, a second reactive group is selectively attached to an antibody fragment via a ligand. In some inventions, a second reactive group is selectively attached to a specific site(s) of an antibody fragment, for example, a specific site(s) selected from K246 and K248 of the IgG1 heavy chain structural amino acid units, K251 and K253 of the IgG2 heavy chain and their corresponding structural amino acid units, and K239 and K241 of the heavy chain and their corresponding structural amino acid units. In some inventions, the invention provides selective reactions at specific structural amino acid units of antibody fragments.

[0168] In some embodiments, the invention provides agents, each of which individually includes an antibody binding fragment that binds to an antibody factor, a reactive group, a cell receptor binding fragment, and optionally one or more linker fragments that connect those groups / fragments. In some embodiments, these agents are useful as reaction partners (e.g., first agents) to accompany the fragments of interest (e.g., target binding fragments), reactive groups (e.g., second reactive groups), and agents containing antibody fragments (e.g., second agents). In some embodiments, the invention provides agents to accompany the fragments of interest with antibody fragments in several agents or antibody factors (e.g., monoclonal antibody factors, polyclonal antibody factors, IVIG-specific antibody factors, etc.). In some embodiments, each of the available agents includes a cell receptor binding fragment, an interaction group, an antibody binding fragment, and optionally one or more linker fragments(s) that connect those fragments.In some embodiments, the binding molecule of an antibody forms part of a leaving group that is released after contact of this agent, e.g., factor I, with an antibody molecule, e.g., from factor II, and the interaction of an reactant group of this agent, e.g., a first reactant group from factor I, with an reactant group from an antibody molecule, e.g., a second reactant group from factor II, such as −NH2 of a Lys subunit of an antibody protein. In some embodiments, available technologies can provide improved conjugation efficiency, high selectivity, and / or fewer (sometimes, a single) steps for conjugating product agents. In some embodiments, an available agent, e.g., factor I, is a compound with the formula. AGN110 Or a salt of it: LG−RG−LRM−TBT, [ AGN110 ] Or salt from it, where: LG represents a group that includes a binding fragment of an antibody; RG represents an interactive group; LRM represents a link; and TBT is a cellular receptor binding molecule.

[0169] In some incarnations, L G It consists of, or includes, an antibody binding fragment as described in this specification, and a ligand that connects an antibody binding fragment to an RG.

[0170] In some incarnations, L G It is or includes R LG −L LG -, where R LG It is or includes a binding molecule to an antibody, and is L LG It is a linking segment as described in this specification. In some incarnations, L G It is ABT-L LG In some incarnations, L LG It is −L LG1 −L LG2 - where each of L LG1 and L LG2 Separately, it is a linking segment as described in this specification. In some incarnations, L LG It is −L LG1 −L LG2 −L LG3 - where each of L LG1 L LG2 and L LG3 The term "separately" refers to a linking segment described in this specification. In some incarnations, L is... LG It is −L LG1 −L LG2 −L LG3 −L LG4 - where each of L LG1 L LG2 L LG3 and L LG4 Separately, it is a linking segment as described in this specification. In some incarnations, L is linked LG1 By R LG In some incarnations, L is linked LG1 By cleaving the cell-receptor junction. In some incarnations, L LG It is −L LG1 - An interactive set includes L LG2 L LG3 and L LG4 In some incarnations, L LG It is −L LG1 −L LG2 - An interactive set includes L LG3 and L LG4 In some incarnations, L LG It is −L LG1 −L LG2 −L LG3 - An interactive set includes L LG4 In some incarnations, both L LG1 L LG2 L LG3 and L LG4 Separately, it is an L.

[0171] In some incarnations, L LG4 It is a covalent bond. In some incarnations, L is not LG4 It is a covalent bond. In some incarnations, L LG4 It is −O−. In some incarnations, it is L LG4 It is −N(R')−. In some incarnations, it is L LG4 It is −NH−. In some incarnations, it is L LG4 It is −N(CH3)−. In some incarnations, it is L LG4 It is −N(R')−, and L is LG3 It is −O−. In some incarnations, R' is C 1-6 Alkyl has optional substitution. In some incarnations, L is LG4 It is −S−.

[0172] In some incarnations, R LG It is or includes an antibody binding molecule. In some incarnations, R is LG It is a protein that contains a binding cleavage protein. In some incarnations, R is LG It is or includes an antibody binding molecule. In some incarnations, R is LG It is a binding molecule to an antibody. In some incarnations, R is the binding molecule. LG It is a binding cleavage protein. In some incarnations, R LG It is a binding fragment of an antibody.

[0173] In some incarnations, R LG It is ABT101 R c −(Xaa)z−, a nucleic acid molecule fragment, or a small molecule fragment. In some incarnations, R is LG It is or includes ABT101 In some incarnations, R LG It is or includes Rc−(Xaa)z−. In some incarnations, R is LG It is a small molecule fragment or includes a subunit. In some incarnations, R is LG It is a peptide factor or contains a peptide component. In some incarnations, R is the active ingredient. LG It is a component of, or includes, a nucleic acid component. In some incarnations, R is the nucleic acid component. LG It is a component of, or includes an aptemer. In some incarnations, the binding molecule of an antibody is a component of, or includes an aptemer. ABT101 In some incarnations, the binding cleavage protein is a protein that consists of or includes a protein. ABT101 In some embodiments, the binding molecule of an antibody is a component of, or includes, an antibody. ABT101 In some incarnations, the binding molecule of an antibody is Rc−(Xaa)z−. In some incarnations, the binding molecule of a protein is Rc−(Xaa)z−. In some incarnations, the binding molecule of an antibody is Rc−(Xaa)z−.

[0174] In some embodiments, the target binding fragments may be conjugated with the antibody fragments optionally via the linker fragments using techniques described in US Patent Publication No. 2020 / 0190165.

[0175] In some embodiments, where a specific protective (PG), leaving (LG), or conversion condition is identified, individuals with a basic understanding of biomedicine know that other protective groups, leaving groups, and conversion conditions are relevant and should be investigated. These groups and conversions are described in Greene's Protecting Groups in Organic Synthesis (John Wiley & Sons, 2014), which is included in full in this specification for reference.

[0176] In some incarnations, the leaving groups include, but are not limited to, halogen groups, for example, fluoride, chloride, bromide, iodide, sulfonate groups, for example, mesylate, tosylate, benzenesulfonate, brusylate, nosylate, triflulate), diazunium, and the like.

[0177] In some formulations, the oxygen-protecting group includes carbonyl groups, hydroxyl groups, and so on. Hydroxyl groups are well-known in biomedicine and include those described in Greene's Protecting Groups in Organic Synthesis (John Wiley & Sons, 2014), which is included in full in this specification for reference. Examples of suitable hydroxyl groups include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, aryl alkyl ethers, and alkyl oxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfons.Examples of specific compounds include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenyl methoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylene dithio)pentanoate, bivaloate (trimethylacetyl), crotenes, 4-methoxy-crootenes, benzoates, p-benylbenzoate, 2,4,6-trimethylbenzoates, carbonates such as methyl, 9-fluorinyl methyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, phenyl, allyl, and p-nitrobenzyl. Examples of syl ethers include trimethylsilyl, triethylsilyl, t-butyl dimethylsilyl, t-butyl diphenylsilyl, triisopropylsilyl, and other trialkyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyl oxycarbonyl ethers or their derivatives. Alkyl oxyalkyl ethers include acetal compounds such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyl oxymethyl, β-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers.Examples of aryl alkyl ethers include: benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2-picolyl and 4-picolyl.

[0178] Amino acid protection groups are well-established in the field of biomedicine and include those described in Greene's Protecting Groups in Organic Synthesis (John Wiley & Sons, 2014), which is included in its entirety in this specification for reference. Suitable amino acid protection groups include, but are not limited to, alkylamines, carbamates, cyclic imides, allylamines, amides, and similar compounds. Examples of such groups include t-butyl oxycarbonyl (BOC), ethyl oxycarbonyl, methyl oxycarbonyl, trichloroethyl oxycarbonyl, allyl oxycarbonyl (Alloc), benzyl oxocarbonyl (CBZ), allyl phthalimide, benzyl (Bn), fluorinyl methylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, and the like.

[0179] Individuals with a basic understanding of biomedicine know that available agents may contain one or more stereocenters and may exist as racemic or stereoisomer mixtures. They also know that there are many well-established biomedical methods for separating isomers to obtain stereoenriched or stereopure isomers of these compounds, including, but not limited to, HPLC, polarized HPLC, fractional crystallization of stereoisomer salts, kinetic enzymatic hydrolysis (e.g., by lipases or esterases) of fungi, bacteria, or animals, and the formation of covalent stereoisomer derivatives using a reagent that has been stereoenriched.

[0180] Persons with a basic understanding of biomedicine know that many of the functional groups present in the compounds of this specification, such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles, can be interconverted by well-established biomedicine techniques, including, but not limited to, reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, hyogenation, drying, partial hydrolysis, and hydrolysis. See Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th The edition (John Wiley & Sons, 2001), which is fully included in this specification as a reference, may be used for these interconversion processes. These processes may require one or more of the techniques mentioned previously, and specific methods for synthesizing the compounds for this specification are described below in the Examples section.

[0181] As people with basic knowledge in the field of biomedicine know, contact between reaction partners is generally carried out under conditions and for a sufficient duration to obtain the desired results, for example, the formation of product factors and their formulations to the required extent. Various reaction conditions / times may be evaluated and used if suitable for the purposes required according to this specification; specific conditions, reaction times, evaluation, etc., are described in the examples.

[0182] In some incarnations, a generated factor, for example, the MATE factor of a product, has the structural formula of the formula. AGN101 or AGN102 In some incarnations, a cell receptor-binding molecule in a product factor (e.g., MATE) is analogous to a cell receptor-binding molecule in a reaction partner (e.g., Factor I containing a cell receptor-binding molecule) used to prepare a product factor. In some incarnations, an antibody molecule in a product factor (e.g., MATE) is analogous to an antibody molecule in a reaction partner (e.g., Factor II containing an antibody molecule) used to prepare a product factor.

[0183] In some embodiments, the ligand fragments or a portion thereof may be transferred to the target-binding fragments and / or antibody fragments of the interaction partners, e.g., L RM In the form AGN110 Or a salt of it. In some incarnations, a linking strand is in a producer factor (which may be called L). PM For example, L in the form AGN101 or AGN102 , is or includes a binding molecule in an interaction partner, for example, one between an interaction group and a binding molecule to a cell receptor, for example, L RM In some incarnations, L PM It is or includes L RM In some incarnations, L PM It is −L RM −L RG2 In some incarnations, L RG2 It is −C(O)−. In some incarnations, it is L RG2 It is −C(O)−, and it is linked to −NH− of a target factor cleavage, for example, −NH− in a side chain of a lysine structural unit of a protein cleavage, which in some incarnations is an antibody cleavage.

[0184] In some embodiments, the invention provides products specific to available processes, with lower levels of damage to antibody fragments compared to processes involving steps performed to remove the antibody-binding fragment but not the primary conjugation of the fragments of interest, e.g., target-binding fragments. In some embodiments, the formulations of the available product agent have high homogeneity, e.g., with regard to the number of cell receptor-binding fragments of the antibody fragment shape, and / or the positions of the structural amino acid units in the antibody fragments conjugated to the fragments of interest, compared to the reference product formulations, e.g., those from techniques without the use of antibody-binding fragments, or the use of an additional step(s) to remove the antibody-binding fragment, e.g., without the use of the reaction partners described in this specification, which include a reaction group positioned between an antibody-binding fragment and a cell receptor-binding fragment.

[0185] In some embodiments, the invention provides a product factor consisting of an antibody fragment, a cell receptor-binding fragment, and optionally a ligand that links an antibody-binding fragment and a cell receptor-binding fragment. In some embodiments, the invention provides combinations of these factors. Pharmacologically acceptable excipients .

[0186] Suitable formulations for non-intravenous administration, such as intra-articular (into joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes of administration, include sterile isotonic aqueous and non-aqueous injection solutions, which may contain antioxidants, buffer solutions, bacteriostatic agents, and solutes that make the formulation isotonic with the target recipient's blood, and sterile aqueous and non-aqueous suspensions, which may include suspending agents, dissolving agents, thickening agents, stabilizing agents, and preservatives. When applying the invention, formulations may be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, intravesically, or intrasacrally. Non-intravenous, oral, and intravenous administration are the preferred routes of administration. Formulations of the compounds may be presented in sealed containers as single doses or multiple doses, such as ampoules and vials.

[0187] Solutions or suspensions for intradermal, subcutaneous, or non-injectable administration may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol compounds, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben compounds; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering solutions such as acetate compounds, citrate compounds, or phosphate compounds; and adjusting agents such as sodium chloride or dextrose. The pH may be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide. Preparations for intradermal administration may be packaged in ampoules, single-use syringes, or multi-dose vials made of glass or plastic. Multiple factors .

[0188] In some embodiments, the invention provides a formula that includes a multi-factorial composition, where each factor individually includes: Splitting an antibody, It binds to a cellular receptor, and Optionally, a linker segment connects an antibody binding segment and a cell receptor binding segment.

[0189] In some incarnations, the product factors are MATE factors. In some incarnations, an antibody cleavage factor includes the IgG Fc region. In some incarnations, an antibody cleavage is linked to a cell receptor-binding cleavage via a selectively amino group through a ligand. In some incarnations, this is achieved through a lysine structural unit where the amino group of the side chain is linked to a selectively cell receptor-binding cleavage via a ligand, for example, forming −NH−C(O)− as part of an amide group, a carbamate group, etc.

[0190] In some embodiments, the invention provides a formulation that includes a multi-factorial set, each of which individually comprises: Splitting an antibody, It binds to a cellular receptor, and Optionally, a linker segment connects the antibody segment and the segment that binds to the cell receptor; The antibody fragments of polyclonal factors share a common amino acid sequence, and polyclonal factors share a common cell-receptor binding fragment with at least one common structural amino acid unit from the common amino acid sequence; Approximately 1%-100% of all factors that include an antibody fragment containing a common amino acid sequence and a cell receptor-binding fragment are polyclonal factors.

[0191] In some embodiments, the invention provides a formulation that includes a multi-factorial set, each of which individually includes: Splitting an antibody, It binds to a cellular receptor, and Optionally, a linker segment that connects an antibody segment and a cell receptor binding segment; Where the factors of the polyclonal group share the same or largely the same antibody cleft, and at least one common cellular receptor-binding cleft site; and Approximately 1%-100% of all factors that include both an antibody fragment and a cell receptor binding fragment are multigroup-specific factors.

[0192] Further guidance is provided in International Patent Application WO 2025 / 035052 (Biohaven Therapeutics Ltd.), the contents of which are included for reference. Methods for evaluating the chemical structural formula and the specific function of the agent .

[0193] HDX-MS The detection of hydrogen molecules on the surface of a protein or protein complex is measured. The first step involves the exchange of exposed hydrogen molecules with deuterium. Hydrogen molecules embedded in the structural formula, for example at the binding site of an antibody-antigen complex, are not exchanged as readily.

[0194] Comparing deuterium uptake between free states and protein-specific complex states via mass spectrometry provides valuable information about binding regions.

[0195] HDX-MS is continuing to be implemented. The inventors are modeling the interaction of the Fc antibody and the bifunctional lysis factor to predict the predicted regions in parallel with preventing HDX-MS.

[0196] Preparation of protein sequences In one method for protein sequence preparation, eight digestion solutions were prepared using five enzymes (pepsin, Lys C, trypsin, chymotrypsin, and Asp N). The digestion solutions of the sample were treated with disulfide reduction, cysteine ​​inhibition, and then enzyme digestion. The digestion solutions were analyzed by LC-MS / MS using a Thermo-Fisher Orbitrap Fusion™ mass spectrometer. Peptides were identified from the LC-MS / MS data using denovatase peptide sequence preparation and then assembled into antibody sequences. Assays used to determine proof of concept .

[0197] The inventors used these assays to determine the proof of concept of the bifunctional factors of an autoantibody that is anti-insulin / proinsulin specific to the invention.

[0198] Biophysical assay . Surface plasmon resonance assay (insulin receptor) The inventors produced a GN3-conjugated mutant insulin, which is being tested for binding to the insulin receptor using surface plasmon resonance assays. Additional GN3-conjugated mutant insulin compounds are being produced synthetically.

[0199] Insulin receptors are also produced synthetically. IR-forward of qPCR primer sequence: GCAACATCACCCACTACCTGGT (sequence no.: 39). IR-reverse of qPCR primer sequence: GAATGGTGGAGACCAGGTCCTC (sequence no.: 40).

[0200] Cellular assay . Insulin receptor assay iLite ( DT-40 ) Principle of the assay: Superexpression of the insulin receptor and transduction of a non-cone STAT5 recognition promoter and a STAT5 expression vector. Subsequently, insulin signaling via the insulin receptor leads to STAT5 phosphorylation, dimerization, and transcriptional activation of a booster factor, which in turn leads to luciferase production.

[0201] Insulin receptor associated with human insulin via surface plasmon resonance The biophysical evaluation of insulin / IGF1 binding to its respective receptors has been demonstrated. Weak binding of insulin precursor constructs to the insulin receptor has been observed.

[0202] A diabetic mouse model that is not obese ( NOD ) The NOD mouse model is described by Jorns et al., Diabetologia, Volume 57, pages 512–521 (December 6, 2014). NOD mice represent one of the best-studied mouse models of spontaneous type 1 diabetes. The immunological progression of diabetes in NOD mice is similar to that in humans. Disease progression is driven by immune cells (macrophages, dendritic cells, NK cells, B cells, and T cells). Insulin-mediated inflammation is confirmed at 12–14 weeks of age. T cells are the predominant islet-filtering cells, which in turn destroy B cells, leading to the development of diabetes / hyperglycemia.

[0203] In humans, autoantibodies against multiple β-cell proteins are identical in more than 90% of newly diagnosed type 1 diabetes patients. The prevalence of autoantibodies to the islet is associated with age at diagnosis.

[0204] mouse pattern NOD Human-compatible immunodeficiency vaccine for type 1 diabetes Several immunodeficient models have been described previously in the field of biomedicine. SCID mice that have reconstituted PBMCs from type 1 diabetic patients can produce autoantibodies against diabetes-associated antigens. See Petersen et al., Diabetes (1994). Genetic modification of the core NOD / SCID gene map allows hybridization with human lymphocytes. Table 8 Commercially available NOD / SCID mice Source Interest NOD SCID. NOD.CB17-Prkdcscid / NCrCrl. Immunodeficient common donor mice. The "gold standard" in humanized mice. The core genetic map of NOD results in defective natural killer (NK) cell function. For the SCID mutation, homozygous animals had impaired T-cell and B-cell lymphoid development. NOD.Cg- Prkdc scid Il2rg tm1Wjl / SzJ. Strain #:005557. The common name is NSG. It is also known as NOD / SCID IL2Rgammanull, NOD / SCID IL2Rgnull, NOD / SCID gamma Severe immunodeficiency allows for the cross-fertilization of mice with humans through hybridization with human CD34+ HSCs or PBMCs. Mutations in the gamma chain of the IL2 receptor (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) lead to severe adaptive and innate defects (most pronounced immunodeficiency). NOD.CB17- Prkdc scid Il2rg tm1 / Bcgen. Catalog number 112469. The common name is huPBMC-B-NDG mice

[0205] Pediatric patients are a good pool of autoantibodies. PBMCs from type 1 diabetic patients and control groups are commercially available. In vitro, B cells generate a polyclonal array of autoantibodies. A humanized mouse model can demonstrate the lysis of patient-specific autoantibodies.

[0206] Diabetic canines The inventors administered a bifunctional lysate factor to diabetic dogs to treat their diabetes. Diabetic dogs are typically treated with human insulin and usually exhibit neutralizing antibodies that reduce insulin concentrations and the effectiveness of the bivalent antibodies. The inventors were able to perform an assay to confirm that the canine polyclonal antibody response to human insulin likely mimics the human autoantibody response.

[0207] The inventors can also perform an assay to confirm the measurement of hypoglycemia in dogs.

[0208] A comparison based on MesoScale Discovery (MSD) To detect low levels of human insulin autoantibodies, detailed protocols are described in the scientific literature in the field of biomedicine. See Yu, Methods in Molecular Biology (2016) and Gu et al., J. Clin. Cellular Immunology (2017). An advantage of using an MSD-based assay is its increased sensitivity and the low serum sample size required. With ELISA-based assays, antigens are bound to the solid-phase, i.e., the ELISA plate. This binding limits the identification of the sticky peak of the conjugate antigen by the tested antibodies. Only one antigen can be identified per eye. MesoScale Discovery-based assays can be developed for up to ten different autoantibodies. These assays also allow for higher throughput. Table 9 Reference standard antibodies and other biological agents Abcam, Catalogue # ab254262. Anti-ASGPR non-glycoprotein receptor 1 / HL-1 (Anti-ASGPR Ab). Cell Signaling Technology, catalogue # 14364S. Rabbit IGF-2 receptor / monoclonal antibody CI-M6PR (D3V8C) (Anti-IGF2 Ab), Cell Signaling Technology, catalogue # 17174S. The antibody that is anti-IGF1 to the rabbit α (D3A2W) monoclonal antibody to the IGF-1 receptor. Cell Signaling Technology, catalogue # 23413S. Rabbit XP monoclonal antibody to insulin receptor B (E9L5V). Cell Signaling Technology, catalogue # 23413S. Rabbit XP® monoclonal antibody to insulin receptor B (E9L5V) (Anti-insulin B Ab), Cell Signaling Technology, Catalogue # 74118S. Rabbit monoclonal antibody to the insulin receptor α (D3U7I). This antibody is anti-insulin. Cell Signaling Technology, catalogue # 88175S. The antibody that is anti-monoclonal to rabbit FcRn (E6D1S). Cell Signaling Technology, catalogue # 9750S. Rabbit monoclonal antibody to IGF-1 β receptor (D23H3) XP® (Anti-IGF1 B Ab), Genscript mouse anti-human insulin monoclonal antibody IgG2a (clone: ​​6E9F1), GenScript, catalogue # A01715. This antibody detects human insulin and somewhat distinguishes insulin pro-insulin, without cross-reactivity with C-peptide. This antibody had a better cytotoxicity signal compared to human mAb49. LS Bio, Catalogue # LSC331895-200. A rabbit monoclonal antibody that is anti-human insulin (IHC, Western blot). M6PR antibodies rabbit. CST, catalog #14364S mAb13. Creative Biolabs, catalogue # FAMAB-0223WJ (poor binding vis SPR). mAb13 is a monoclonal antibody that is anti-human insulin derived from an IDDM patient being treated with insulin produced by human gene recombination via EBV mutation and somatic cell hybridization techniques. mAb13 can be used in applications such as ELISA and Western blot. mAb48. Creative Biolabs, catalogue # FAMAB-0224WJ-S(P); scFv fragment (IgG3 subtype) (poor binding in surface plasmon resonance assays). mAb48 is a monoclonal antibody that is anti-human insulin derived from an IDDM patient being treated with insulin produced by human gene recombination via EBV transformation and somatic cell hybridization techniques. mAb48 can be used in applications such as ELISA and Western blot. mAb49 Fab fragment. Creative Biolabs, Catalogue # FAMAB-0225WJ-F(E). This product is a Fab fragment of a monoclonal antibody that is anti-human insulin, derived from three IDDM patients treated with human gene recombination insulin, via EBV transformation and somatic cell hybridization techniques. This antibody fragment can be used in applications such as ELISA and Western blot. mAb49. Creative Biolabs, catalogue # FAMAB-0225WJ-F(E)). Surface plasmon resonance results show binding to insulin / proinsulin. The anti-human insulin gene return product antibody (clonal mAb49); Fab fragment (described as an IgG1 subtype) – but internally confirmed as human IgG3 / chimeric. mAb49 is a monoclonal antibody that is anti-human insulin derived from an IDDM patient being treated with human gene return product insulin via EBV transformation and somatic cell hybridization techniques. mAb49 can be used in applications such as ELISA and Western blot. Sino, catalogue # 11038-CR007. The anti-insulin antibody to the product of gene recombination, monoclonal rabbit. Sino, catalogue # 11038-T08. An anti-insulin, polyclonal rabbit antibody. Sino, catalogue # 11038-T24. An antibody that is anti-insulin, polyclonal from rabbit. ThermoFisher, Catalogue # A20173. Alexa Fluor 647 Protein. The main sequence of the insulin receptor. The long homologous image P06213-1|INSR_HUMAN of the insulin receptor OS = Homo sapiens OX=9606 GN=INSR PE=1 SV=4 (Sequence No.: 31). Short homologous image P06213-2, INSR_HUMAN of insulin receptor OS = Homo sapiens OX=9606 GN=INSR PE=1 SV=4 (Sequence No.: 32). Kappa light chain of mouse anti-peptide C antibody (sequence number: 35) [219 AA] Gamma 1 heavy chain of the mouse anti-peptide C antibody (sequence number: 36) [437 AA] The anti-C peptide monoclonal antibody (CBFYR0644) (CAT#: CBMAB-R0644-FY) is a mouse antibody that recognizes C peptide. The CBFYR0644 antibody can be used for immunoassay techniques such as radioimmunoassay, EIA, and IHC. Anti-C peptide monoclonal mouse antibody (CBFYR0645) (CBMAB-R0645-FY). The antibody to the mouse gene recombinant product antipeptide C (CBCNC-012). Mouse anti-peptide C antibody to gene recombination product (CB2931) (CBMAB-MD1611-LY) Invitrogen. Anti-proinsulin C-peptide monoclonal antibody (C-PEP-01). Anti-proinsulin C-peptide monoclonal antibody (Product # MA1-19159). See also 05-1109 Sigma-Aldrich. Anti-proinsulin C-peptide monoclonal antibody C-PEP-01. See Hilgert et al., Hybridoma 10(3) 379-386 (1991). Table 10 Detection kits ThermoFisher A20173, AF647 Numbering Kit ThermoFisher A1023,5AF-488 Numbering Kit MedChemExpress HY-14710,2Tri-GalNAc biotin (comparative reagent) Abbexa Kit: Human insulin autoantibody (IAA) CLIA Abbexa Kit: Mouse insulin autoantibody (IAA) CLIA

[0209] Commercial kits are available to measure insulin, proinsulin, or autoantibodies that are anti-insulin. Table 11 Insulin / Proinsulin Detection seller Quality comparison detection range MesoScale Discovery, catalog # K151S5D Human, a primate, descended from a dog Electrochemical fluorescence (ECL) 0.006 - 19.5 micro-international units / mL MesoScale Discovery, catalog # K1516MK Human insulin progenitor Electrochemical fluorescence (ECL) 0.054 - 130 picomolar Novus Bio, catalog # NBP2-60078-1 Kit human insulin ELISA Table 12 Insulin / Proinsulin Detection Quality Seller ELISA detection range Human anti-insulin IgG Eagle Biosciences, catalog #3806 0.1 – 20 units / mL human insulin antagonist Aviva Systems Biology, catalog # OKEH00908 3.12-200 nanograms / milliliter mouse antiinsulin LS Bio, catalog #LSF74131-1 3.12 - 200 nanograms / milliliter mouse antiinsulin Abbexa, catalogue # Abx053161 0.156 - 10 nanograms / milliliter Antiinsulin from a dog American Research Products, catalog # E08I0019 5.0-100 nanograms / milliliter Statistical analysis . Quantitative variables for a normal distribution can be expressed as means and standard deviations (SDs) and compared using independent samples t-tests, as demonstrated by Zhang et al. (October 2019). For non-normally distributed variables, individuals with typical biomedical skills can use a median and interquartile range and analyze them using the Mann-Whitney U test. Categorical data are summarized as percentages. A two-sided p-value < 0.05 is considered statistically significant. All statistical tests can be performed using SPSS version 16.0. Identifying the binding sites of the antibody .

[0210] Several techniques are available for identifying, evaluating, or characterizing antibody binding fragments, including protein-binding fragments (e.g., pan-antibody binding fragments), and / or for use in existing techniques, such as those described in International Patent Application WO 2019 / 023501 (Kleo Pharmaceuticals, Inc.). The techniques for these techniques are included in this specification for reference. In some embodiments, an antibody binding fragment is a fragment (e.g., a small molecule fragment, a peptide fragment, a nucleic acid fragment, etc.) that can selectively bind to IgG and, when used in existing techniques, can provide and / or induce ADCC and / or ADCP. In some embodiments, peptide display techniques (e.g., phase display, acellular display, etc.) can identify antibody binding fragments.In some embodiments, the binding molecule of an antibody is a molecule, for example, a small molecule molecule, a peptide molecule, a nucleic acid molecule, etc., that can bind to IgG and selectively can compete with known antibody binding molecule ...

[0211] Individuals with a basic understanding of biomedicine know that antibodies with multiple properties and activities—for example, antibodies specific to different antigens, those with selective modifications, and so on—may be targeted by the antibody-binding moieties described in this specification. In some formulations, these antibodies include those administered to a patient for therapeutic purposes. In some formulations, the antibody-binding moieties described in this specification may bind antibodies to different antigens and be useful for conjugating the moieties of interest with multiple antibodies.

[0212] In some incarnations, the binding molecule of an antibody, for example, is a Meditope factor. See US Application Bulletin 2019 / 0111149 (Gardiner et al.).

[0213] In some incarnations, an antibody binding molecule, for example, an antibody binding molecule, can bind to human IgG. In some incarnations, an antibody binding molecule, for example, an antibody binding molecule, can bind to a selected antibody from the Markush group of antibodies consisting of IgG, IgG1, IgG2, IgG3, and IgG4 from rabbit. In some incarnations, an antibody binding molecule, for example, an antibody binding molecule, can bind to IgG1, IgG2, and IgG4.

[0214] In some incarnations, it is used In a reference technique as a non-antibody binding molecule. In some incarnations, the chemical group used in the reference technique as a non-antibody binding molecule is chosen from the Markush group of chemical groups consisting of CH3−, CH3C(O)−, CH3C(O)NH−, CH3C(O)NHCH2−, CH3CH2−, CH3CH2NH−, and CH3CH2NHC(O)−.

[0215] In some embodiments, the antibody's binding strands (e.g., antibody-specific binding strands) are bound to targets (e.g., antibody-specific binding strands) with a K value. D It is approximately 1 millimolar - 1 picomolar or less. In some incarnations, the value of K is D About 1mM, 0.5mM, 0.2mM, 0.1mM, 0.05mM, 0.02mM, 0.01mM, 0.005mM, 0.002mM, 0.001mM, 500nM, 200nM, 100nM, 50nM M, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or less. In some embodiments, K represents D Selected affinity values ​​from the Markush range consist of approximately 1 mM or less, approximately 0.5 mM or less, approximately 0.1 mM or less, approximately 0.05 mM or less, approximately 0.01 mM or less, approximately 0.005 mM or less, approximately 0.001 mM or less, approximately 500 nM or less, approximately 200 nM or less, approximately 100 nM or less, approximately 50 nM or less, approximately 20 nM or less, approximately 10 nM or less, approximately 5 nM or less, approximately 2 nM or less, and approximately 1 nM or less. In some incarnations, the binding moieties of the antibody to the IgG antibody are valued at Ki. D Described in this specification.

[0216] Individuals with a basic understanding of biomedicine know that antibodies with multiple properties and activities—for example, antibodies specific to different antigens, those with selective modifications, and so on—may be used with the specific antibody binding moieties described in this specification. In some formulations, these antibodies include those administered to a patient for therapeutic purposes. In some formulations, the antibodies used with the specific antibody binding moieties include antibodies against different antigens. In some formulations, the antibodies used with the specific antibody binding moieties include antibodies whose specific antigens are not present on the surface or membrane of the target cells. In some formulations, the antibodies used with the specific antibody binding moieties include antibodies that do not target antigens present on the surface or membrane of the target cells.In some formulations, the antigens present on the surface of the target cells may interfere with the structural formula, morphology, and / or one or more properties and / or activities of the antibodies used that bind to those antigens. In some formulations, the antibodies used are those in IVIG. In some formulations, IVIG may be administered before, concurrently with, or subsequent to an agent or combination. Antibodies from multiple immunoglobulin structural formulas may be used. In some formulations, one or more IgG subclasses are used. In some formulations, the antibodies used are selected from the Markush group of antibody classes consisting of IgG1, IgG2, IgG3, and IgG4. In some formulations, the antibodies used are IgG1 and IgG2. In some formulations, the antibodies used are IgG1, IgG2, and IgG4. In some formulations, the antibodies used are or include IgG1, IgG2, IgG3 and IgG4.The antibodies used may interact with multiple types of receptors, for example, those genetically expressed by multiple types of immune cells. In some incarnations, the antibodies used can interact efficiently with multiple types of Fc receptors and provide the required immune activity. In some incarnations, the antibodies used can utilize immune cells. In some incarnations, the antibodies used interact efficiently with the human Fcγ receptor IIIA. In some incarnations, the antibodies used interact efficiently with the human Fcγ receptor IIIA on macrophages. In some incarnations, macrophages are used to provide ADCC or ADCP activity against a virus, for example, SARS-CoV-2, and / or cells infected with it. In some incarnations, NK cells are used to provide immune activity. In some incarnations, the antibodies used interact efficiently with the human Fcγ receptor IIIA. In some incarnations, the antibodies used interact efficiently with the human Fcγ receptor IIIA on dendritic cells.In some embodiments, the antibody components in the agents of this specification include one or more of the properties, structural formulas and / or activities of the antibodies used that are described in this specification. Specific mutation strategies to reduce the Fcγ receptor and bind to c1q / effector function, and to improve binding to FcRn / antibody-specific detection. Boom strategies FC To reduce the future Fcγ And the connection with c1q .

[0364] The inventors mutate an antibody, a variant of an antibody, or a binding fragment to an antigen in the Fc region to insert the LALA mutation using methods commonly used in biomedicine, first described by the Winter group in the 1990s. In this example, LALA = L234A / L235A.

[0365] The inventors mutate an antibody, a variant of an antibody, or a binding fragment to an antigen in the Fc region to insert a LALA mutation using established biomedical methods first described by the Winter group in the 1990s, and then P-injected mutations, such as those introduced by the Roche team in 2016, like the technique for adding P329G and P329G fused to 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 even LALA alone invalidates binding to C1q. P329A was tested alone and showed that it abolishes binding to C1q and reduces binding to FcgR. This demonstrates that P329G / LALA further reduces binding to the Fcγ receptor beyond what LALA alone does.

[0366] The inventors also mutated an antibody, a variant of an antibody, or a binding fragment to an antigen in other regions. N297A / Q removes the normal N-linked glycosyltransferase site in the articular region. Mutation strategies FC For improved connection with FcRn To prolong the investigation .

[0367] There are many techniques and mutation sets publicly available to increase the binding of Fc to FcRn. Table 13 Examples of genetically modifying the Fc sequence to modify half-life. Job company or source Mutations or changes Increased half-life Medlmmune lgG1·M252Y, S254T, T256E Increased half-life Protein Design Labs lgG1 T250Q, M428L Increased half-life Genentech lgG1 N434A Increased half-life Sally Ward lgG1·H4J3K, N434Y Increased half-life Deny Roopenian lgG1 T307A.E380A,N434A Increased half-life Xeocor Xtend ™ technol o gy IgG endogenous reducer Sally Ward lgG1 M252Y, S254T, T256E, H433K, N434F, 436H Reduced half life Deny Roopenian lgG1·I253A Reduced half life Ell Lilly lgG1 P2571, N434H or Q376V, N434H

[0368] An earlier summary is provided by Strohl, Optimization of Fc-mediated effector functions of monoclonal antibodies. Current Opinion in Biotechnology, 20(6), 685-691 (2009).

[0369] For an example of the insertion of the YTE region into a protein (M252Y / S254T / T256E), see Acqua et al., Journal of Immunology 169(9), 5171–5180 (2002). For a recent example combining LALA and YTE, see Cobb et al., bioRxiv, 2021–09 (2021). Further references with detailed information on genetic modification are also available.

[0370] For an example of the insertion of the LS region into a protein (M428L / N434S), see Zalevsky et al., Nature Biotechnology, 28(2), 157-159 (2010). The structural formula of the constructs of the linkage complex resulting from gene recombination .

[0371] A revealing substance for GN3 sortase A basic protocol for the conjugation of sortases involving the ASPGR binding agent for sortases. The sortase coding tag at the C-terminus (LPETGG) of the conjugation (GN3 / binding).

[0372] The reagent material from GN3 Maleimide (Maleimide-GN3 / linker), with cysteine ​​for conjugation at the C-terminus.

[0373] Biophysical / biochemical activity assay to determine the amount of binding to the target A biological molecule is coupled to the surface of a sensor chip as a bonding compound. When the hydrolysis product in solution flows onto the bonding compound, the bonding to the sensor chip surface induces a change in the refractive index proportional to the bonded mass.

[0374] An assay was performed using a Biacore S200 assay instrument. This instrument has high sensitivity and low-to-medium throughput. A dose of a heterogeneous fragment of a crosslinking compound ranging from 1 nanomolar to 1 micromolar was administered, exhibiting single-loop kinetics.

[0375] Absorption assay based on ASGPR A bioluminescent HEK293 cell-based assay was used to evaluate the uptake of the tertiary complex by cells in vitro. This endocytosis assay measures the accumulation of bifunctional lysis factor (BNF) in HEK293 cells via the BNF mechanism described in this specification. A component of this assay is the determination of receptor gene expression on the surface of the HEK293 cells used in the assay.

[0376] Decomposition assay The decay assay using this mechanism is considered a direct, low-throughput Western blot assay or a high-throughput assay using an activated fluorescence quenching agent probe.

[0377] Other off-target assays include measuring cytotoxicity by in vitro toxicity using HepG2 cells, for example, in the CellTiter-Glo assay; measuring hemagglutination by red blood cell reactions; measuring PBMCs; and measuring off-target binding states in a specially formulated C-type pectin plate. Signaling in HEK293 cells is enhanced compared to HepG2 cells when measuring signal buildup, not lysis. Western blot assays can measure lysis in HEK cells and other cell lines.

[0378] The following steps may include a competitive assay with FC3 (Kifi). Manufacturing agent . Table 14 purification steps number Steps buffer solution 1 Dissolution Dissolution regulator solution 50 mM Tris-HCl, 150 mM NaCl, 1 mM TCEP, pH 8.0 2 washing Washing solution, A Contains 0.5% Triton X-100 and 10 mM EDTA Washing regulator solution, B ddH2O Washing regulator solution, C Contains 2% Triton X-114 3 Change in physical properties The buffer solution for changing the physical properties 50 mM Tris-HCl, 7 mM Gu-HCl, 50 mM DTT, pH 8.0 4 Refolding المحلول المنظم لإعادة الطي، A 25 مللي مولار Tris-HCl، 6 مولار يوريا، 500 مللي مولار NaCl، 50 مللي مولار جليسين، 5 مللي مولار GSH، 0.5 مللي مولار GSSH، الرقم الهيدروجيني 9.5 المحلول المنظم لإعادة الطي، B 25 مللي مولار Tris-HCl، 4 مولار يوريا، 500 مللي مولار NaCl، 50 مللي مولار جليسين، 5 مللي مولار GSH، 0.5 مللي مولار GSSH، الرقم الهيدروجيني 9.5 المحلول المنظم لإعادة الطي، C 25 مللي مولار Tris-HCl، 2 مولار يوريا، 500 مللي مولار NaCl، 50 مللي مولار جليسين، 5 مللي مولار GSH، 0.5 مللي مولار GSSH، الرقم الهيدروجيني 9.5 المحلول المنظم لإعادة الطي، D 25 مللي مولار Tris-HCl، 500 مللي مولار NaCl، 50 مللي مولار جليسين، 5 مللي مولار GSH، 0.5 مللي مولار GSSH، الرقم الهيدروجيني 9.5 5 العمود Q المحلول المنظم للتوازن 25 مللي مولار Tris-HCl، 166 مللي مولار NaCl، 10 مللي مولار جليسين، الرقم الهيدروجيني 9.5 المحلول المنظم للتصفية التتابعية المحلول المنظم للتوازن باستخدام 166~500 مللي مولار NaCl

[0379] تعرض عملية التصنيع الدايمر / التكتل إلى الخطر أثناء التنقية. طرق إعداد العديد من العوامل .

[0217] The agents for this specification may be prepared or isolated by synthetic and / or semi-synthetic methods or gene recombination methods in accordance with this specification. In some incarnations, polypeptide agents, for example, peptide agents for cell-receptor cleavage, may be prepared using biological gene expression systems. In some incarnations, the available agents are prepared synthetically. In some incarnations, the available agents are prepared using specific techniques described in International Patent Application WO 2019 / 023501 (Kleo Pharmaceuticals, Inc.), which is included in its entirety in this specification for reference.

[0218] Several techniques, for example, those for preparing antibody-drug conjugates, may be used in the preparation of MATE agents. In many of these techniques, conjugation is not selective with respect to the sites of the structural amino acid unit, and the product formulations typically contain multiple types of agents that may differ from one another with respect to the number of conjugated target-binding fragments and / or conjugation sites. In some embodiments, the invention provides techniques that can be used for the selective conjugation of target-binding fragments at the sites of the structural amino acid unit.

[0219] In some incarnations, the invention provides a method of synthesis, which includes the steps for: Factor I, containing a cellular receptor-binding fragment, selectively binds to a first reactive group via a first link, interacts with factor II, containing an antibody fragment selectively binds to a second reactive group via a second link. The first reactive group then interacts with the second reactive group, and The formation of a product factor that includes a cell receptor binding fragment and a selective antibody binding fragment via a linker.

[0220] In some incarnations, the invention provides a method of synthesis, which includes the steps for: A first formulation consisting of a multi-group of first factors, each of which individually includes a cleavage fragment that binds to a cell receptor selectively linked to a first reactive group via a first linker fragment, comes into contact with a second formulation consisting of a multi-group of second factors, each of which individually includes a cleavage fragment that binds selectively to a second reactive group via a second linker fragment. The product formulation is made up of a multi-product set, each of which includes a cell receptor binding fragment and a selective antibody binding fragment via a linker. Examples

[0221] The invention is further illustrated by non-exclusive examples. Example 1 Proinsulin and proinsulin fusion protein sequences IgG1 Fc .

[0380] The inventors produced proteins numbered with His and Sortase. See the list of sequences.

[0381] Genetic expression of the Fc fusion construct in E. coli is enhanced by the use of a fusion protein. Genetic expression of the Fc fusion construct with a high-purity material yields good results. ASPGR is conjugated via the reagent MATE to obtain a lysis factor molecule. LALA and PA mutations, which produce distinct chemical subunits, can attenuate the immunomodulatory effector's function. Quality control may be relevant to any issues related to disulfide reactions, as insulin may misfold with the Fc receptor and cysteine ​​compounds in Fc, causing aggregation. This misfolding can be detected by native protein gel assays and size-based exclusion assays.

[0382] Individuals with basic skills in biomedicine are familiar with the preparation of insulin-Fc profusion proteins. These insulin-Fc profusion proteins can be either selective or reference compounds. Individuals with basic skills in biomedicine are also capable of preparing insulin-Fc fusion proteins. Table 15 protein the focus buffer solution PT0003, Original Proinsulin, coding tag His at the C-terminus 0.25 مجم / ملليلتر 25 مللي مولار Tris-HCL، 500 مللي مولار NaCl، 50 مللي مولار جليسين، الرقم الهيدروجيني 9.5 PT0003، طليعة الأنسولين الأصلي علامة الترميز His عند الطرف C 0.71 مجم / ملليلتر 50 مللي مولار Tris-HCl، 150 مللي مولار NaCl، 10% من جليسرول، الرقم الهيدروجيني 8.0 PT0004، طليعة الأنسولين الأصلي علامة الترميز sortase عند الطرف C 0.75 مجم / ملليلتر 50 مللي مولار Tris-HCl، 150 مللي مولار NaCl، 10% من جليسرول، الرقم الهيدروجيني 8.0 PT0004، طليعة الأنسولين الأصلي علامة الترميز sortase عند الطرف C 0.36 مجم / ملليلتر 25 مللي مولار Tris-HCL، 500 مللي مولار NaCl، 50 مللي مولار جليسين، الرقم الهيدروجيني 9.5

[0383] التحقق من Abs الانتقائية المرتبطة ببروتين طليعة الأنسولين Fc Both rabbit and mouse anti-insulin antibodies bind to the Fc fusion protein or recognize it via the proinsulin cleavage. The LALA mutation (in the Fc domain) does not prevent discrimination by anti-insulin antibodies.

[0384] يرتبط كل من الجسم المضاد من الأرانب والفئران المضاد للأنسولين ببروتين الاندماج Fc أو يتعرف عليه عبر شطر طليعة الأنسولين) المثال 2 مقايسة رنين البلازمون السطحي – النتائج الأولى .

[0385] يمكن قياس ألفة الارتباط الخاصة بشطر الارتباط بواسطة رنين البلازمون السطحي (نظامي Biacore™ وIBIS-MX96) وقياس التداخل للطبقة البيولوجية (نظامي ForteBio™ وOctet™).

[0386] The assay sequence plans include target engagement via surface plasmon resonance, trivalent complex formation, cell-dependent assays, and endocytosis assays. Additional bridging assays are used to confirm lysis. SPR / TR-FRET (ASGPR)  TR-FRET (trivalent complex, biophysical)  uptake  TR-FRET in cells (lysis).

[0387] Confirm the connection by Surface plasmon resonance Fc was immobilized on the CM5 chip via amine coupling immediately prior to the assays. A 1 nM to 1 μM binding molecule dose was administered and characterized by single-loop kinetics. (First run, single-loop kinetics, steady-state K) D (Nano-molar): 22. Second run, single-loop kinematics, K-state stability D (nanomolar): 24. The inventors verify a commercial antibody that binds to human Fc. At the discriminant n = 2, K was obtained. D It is approximately nanomolar. No binding response was observed for the full-length IgG1-3 and IgG1-3 Fc regions at concentrations up to 1 μM. R indicates max Theoretical to an active surface (a measure of data quality and an indicator of a valid mounting method). Table 16 The reagent used in the assay molecular weight note Insulin (human) 5807.57 Daltons MCE Insulin Lispro 5807.63 Daltons MCE Human IGF-1 receptor treated with biotinyl / CD221 protein, His, Avitag™ 106600 Daltons IGR-H82E3 Human IGF-1 receptor / protein CD221, Fc Tag IGR-H5253 Human IGF-1 protein, His coding tag, excellent grade IG1-H5245 Human insulin receptor treated with butinyl / protein CD220 (28-944), His, Avitag™ (MALS and SPR verified) 108200 Dalton INR-H82E6 Human insulin receptor / protein CD220 (28-944), coding tag His (MALS and SPR verified) 106400 Daltons INR-H52Ha The α antibody that is anti-insulin receptor [83-7] Abcam ab36550 Table 17 Surface plasmon resonance assays Step stabilizing Level (RU) Capture Level (RU) 1 The α antibody that is anti-insulin receptor [83-7] in acetate buffer solution at pH 5.0 5041.8 Human insulin R / protein CD220 (28-944), coding marker His 170 2 The α antibody that is anti-insulin receptor [83-7] in acetate buffer solution at pH 5.0 5132.1 Human insulin R / protein CD220 (28-944), coding marker His 374.8 3 The α antibody that is anti-insulin receptor [83-7] in acetate buffer solution at pH 5.0 5203.8 Human insulin R / protein CD220 (28-944), coding marker His 574.1 4 Neutravidin, in an acetate buffer solution at pH 5.0 9965.8 Human insulin R processed with butinyl / protein CD220 (28-944), His, Avitag 737.1 5 Neutravidin, in an acetate buffer solution at pH 5.0 9951.1 Human insulin R processed with butinyl / protein CD220 (28-944), His, Avitag 1106.3 6 Neutravidin, in an acetate buffer solution at pH 5.0 9616.3 Human insulin R processed with butinyl / protein CD220 (28-944), His, Avitag 1472.1 7 Neutravidin, in an acetate buffer solution at pH 5.0 9592.7 Human IGF-1 R treated with biotinyl / protein CD221, His, Avitag 699.6 8 Neutravidin, in an acetate buffer solution at pH 5.0 9563.5 Human IGF-1 R treated with biotinyl / protein CD221, His, Avitag 1389.2 Table 18 Summary table Type level of installation ka (molar-1 second-1) K D (Second-1) K D (Kinematics) (Molar) K D Stability (Molar) R max 1:1 R max Theory Insulin (human) Human insulin receptor / CD220, His coding tag 170.0 4.88 E+05 1.07 E-01 2.20 E-07 3.34 E-07 9.2 9 374.8 4.97 E+05 9.17 E-02 1.84 E-07 3.77 E-07 19.5 20 574.1 5.30 E+05 6.90 E-02 1.30 E-07 3.87 E-07 29.7 31 Human insulin receptor treated with butinyl / CD22 737.1 3.36 E+05 1.04 E-01 3.11 E-07 3.41 E-07 62.1 40 1106.3 3.12 E+05 9.44 E-02 3.03 E-07 3.66 E-07 98.7 59 1472.4 1.78 E+06 3.93 E-01 2.21 E-07 3.85 E-07 133.5 79 Human IGF-1 receptor treated with butinyl / CD221 699.6 1.15 E+05 6.35 E-02 5.54 E-07 2.39 E-06 21.0 38 1389.2 6.43 E+04 6.65 E-02 1.03 E-06 2.78 E-06 50.8 76 Insulin Lispro Human insulin receptor / CD220, His coding tag 170.0 5.14 E+05 1.13 E-01 2.21 E-07 3.38 E-07 8.2 9 374.8 4.37 E+05 9.51 E-02 2.18 E-07 4.07 E-07 17.1 20 574.1 3.80 E+05 7.97 E-02 2.10 E-07 4.19 E-07 26.2 31 Human insulin receptor treated with butinyl / CD22 737.1 2.94 E+05 1.09 E-01 3.71 E-07 3.60E-07 56.7 40 1106.3 1.40 E+06 3.66 E-01 2.62 E-07 3.93E-07 88.9 59 1472.4 1.33 E+06 3.59 E-01 2.70 E-07 3.99E-07 120.9 79 Human IGF-1 receptor treated with butinyl / CD221 699.6 1.33 E+05 5.80 E-02 4.37 E-07 2.84E-06 19.5 38 1389.2 8.27 E+04 6.30 E-02 7.63 E-07 3.36E-06 46.9 76 Surface plasmon resonance assays To bind to insulin

[0388] The inventors tested the dendrocal autoantibodies that are anti-insulin mAb13, mAb48, and mAb49. Assessment 1: (Completed)

[0389] sensor chip : CM5.

[0390] Proteins: 1 The α antibody, which is anti-insulin receptor [83-7], was fixed on the CM5 chip using amine coupling with 5000 RU.

[0391] Proteins: 2 Human insulin receptor (replica A) / CD220 protein (28-944), His coding tag from Acro Biosystems, up to ~150, 300, and 600 RU. Replica B from R&D Systems, human insulin receptor R for gene recombination product / CD220 protein (aa 28-956), CF.

[0392] Analysis results Human insulin and insulin lispro, 5 micromolar maximum, diluted twice, MCK 1+10+1, 90 seconds on, 180 seconds off.

[0393] Operating regulating solution : 10 mM HEPES at pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assessment 2: (Completed)

[0394] sensor chip : CM5+Neutravidin.

[0395] proteins : Captured a human insulin receptor treated with butinyl / protein CD220 (28-944), His, Avitag from Acro Biosystems on a chip SA at ~750, 1000, and 1500 RU.

[0396] Analysis results Human insulin and insulin lispro, 5 micromolar maximum, diluted twice, MCK 1+10+1, 90 seconds on, 180 seconds off.

[0397] Operating regulating solution : 10 mM HEPES at pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assessment 3: (Completed)

[0398] sensor chip : CM5+Neutravidin.

[0399] proteins The human IGF-1 receptor, treated with butinyl / CD221 protein, His, Avitag from Acro Biosystems, was captured on an SA chip at ~750 and 1250 RU. IGF-1 receptors can be synthesized. The forward IGF1R sequence is qPCR-specific: CCTGCACAACTCCATCTTCGTG (sequence number: 41). The reverse IGF1R sequence is qPCR-specific: CGGTGATGTTGTAGGTGTCTGC (sequence number: 42).

[0400] Analysis results Human insulin and insulin lispro, 10 micromolar maximum, diluted twice, MCK 1+10+1, 90 seconds on, 180 seconds off.

[0401] Operating regulating solution : 10 mM HEPES at pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assay 4 :

[0402] sensor chip : CM5.

[0403] proteins Three autoantibodies that are anti-insulin were fixed on the CM5 chip using amine coupling at ~750 and 1250 RU.

[0404] Analysis results Human insulin and insulin lispro, 5 micromolar maximum, diluted twice, MCK 1+10+1, 120 seconds on, 600 seconds off.

[0405] Operating regulating solution : 10 mM HEPES at pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assay 5 :

[0406] sensor chip Protein A.

[0407] proteins : Capture three autoantibodies that are anti-insulin on a protein A chip down to ~750 and 1250 RU.

[0408] Analysis results Human insulin and insulin lispro, 5 micromolar maximum, diluted twice, MCK 1+10+1, 120 seconds on, 600 seconds off.

[0409] Operating regulating solution : 10 mM HEPES, pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Example 3 Pharmacodynamics .

[0410] study AGN310 In diabetic mice that are not obese ( NOD ) Four mice were in the group. Two additional mice were excluded from the group because they had too low antibody levels to begin the study. The researchers observed a 42% depletion rate. AGN310 Based on the area under the curve, there was a 50% depletion if the background signal was taken into account.

[0411] In another analysis, he removed AGN310 With an efficiency of 39% of the antibodies that are anti-insulin (2 mg / kg) in hairless mice.

[0412] It was possible AGN310 It performed well when administered at a dose of 3 mg / kg IV in hairless mice, with no adverse events or changes in glucose levels observed when measured twelve hours after the dose. Example 4 Absorption assays and trivalent complex formation .

[0413] Measurement conditions: Cell lineage: HEK293-ASGPR (clonal 3F7) Culture media: DMEM + 10% FBS + 1% P / S Antibodies: Stable at 100 nanomolar (all numbered using AF647) Anti-insulin monoclonal rabbit antibody, Sino, Cat#11038-CR007 Anti-insulin monoclonal mouse antibody, Genscript, Cat#A01715 (clonal 6E9F1) Factors: AGN301 Only (fixed at 50 nM) Reading: Triple complex formation  Flow cytometry measurement (IntelliCyt iQue Screener Plus) Absorption  High-content imaging device (Operetta)

[0414] tested molecules :

[0415] AGN301 IgG lysis factor (reference molecule)

[0416] ABT307 : Insulin-IgG1 Fc profusion, G4S ligand, hIgG1 hinge zone (C > A), LALA-PA.

[0417] ABT308 : Insulin-IgG1 Fc profusion, 5 x G4S ligand, hIgG1 (C > A) hinge region, LALA-PA.

[0418] AGN303 Original insulin with GN3

[0419] AGN302 Mutant insulin with GN3

[0420] ABT306 Original Alk-insulin without GN3 (peptide only)

[0421] ABT304 Original insulin precursor

[0422] ABT305 Mutant insulin precursor

[0423] Test that antibodies that are anti-insulin (from rabbits versus from mice) recognize proinsulin fusion proteins Fc.

[0424] Conclusions Anti-insulin agents are effective. A monoclonal antibody from a rabbit, but not from a mouse, can be used as an alternative with an IgG hemolytic factor. AGN301 No peptide other than GN3 was shown. ABT306 ) absorption or formation of a tertiary complex. Anti-proinsulin agents do not ( ABT304 / ABT305 ) by absorption or it becomes complicated with antibodies that are anti-insulin. Example 5 Factors associated with insulin-specific autoantibodies and proinsulin, leading to uptake, hepatic breakdown, and correction of glucose homeostasis .

[0425] Autoantibodies and autoantiproinsulin antibody MoDEs form triple complexes, exhibiting uptake in vitro and resulting in clearance in the organism without binding to the insulin receptor or the IGF1 receptor.

[0426] A strong and selective reduction of these autoantibodies was observed in mouse pharmacokinetic / pharmacodynamic assays.

[0427] Primary efficacy and toxicity studies The agents deplete naturally occurring insulin-specific autoantibodies in non-obese diabetic (NOD) mice.

[0428] Conclusion The reduction of neutralizing antibodies in NOD mice suggests the potential for restoring glucose homeostasis in type 1 diabetic patients. Example 6 Technical report on the production of insulin prototypes

[0429] Summary . It is considered ABT410 An insulin precursor molecule with a leading sequence H27R at the N-terminus and a TEV recognition site. It was expressed ABT410 As inclusion bodies in the BL21(DE3) strain, after refolding and appropriate buffer solution replacement, the target protein was cleaved by TEV protease and then purified by SP HP resins. The purified protein was prepared in 50 mM Tris / HCl, 150 mM sodium chloride, 10% glycerol, pH 7.5 by UF / DF. The assay panel included protein concentration by Bradford, reducing and non-reducing SDS-PAGE, SEC-HPLC, endotoxin level, and LC-MS.

[0430] family cell E. coli BL21(DE3) competitor cell

[0431] Cryptogram carrier name : pET28a(+).

[0432] protein sequence :

[0433] ABT410 (قبل اهتضام TEV) MGMTMITNSPEISHHHHHHHHHHQLISEARENLYFQGGGGGFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSL QKRGIVEQCCTSICSLYQLENYCNG (المتوالية رقم: 19).

[0434] ABT410 (After TEV) GGGGGFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGASLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCNG.

[0435] Depreciation factor : 0.651 after digestion.

[0436] molecular weight : 9736.98 Daltons (-SH, after Al-Ahtizam).

[0437] pI 5.20 after digestion.

[0438] Conversion steps :

[0439] (1) Add ~100 ng of plasmids to 100 µL of BL21(DE3) competing cells.

[0440] (2) Perform a thirty-minute incubation on ice.

[0441] (3) Thermal shock for forty-five seconds at 42 degrees Celsius in a water bath.

[0442] (4) Perform a two-minute incubation on ice. (5) Add 900 microliters of LB medium (BD-Cat.244620) and shake for forty-five minutes at 37°C, 220 rpm.

[0443] (6) Post the shift in a panel from LB- Kanamycin + agar and overnight incubation at 37°C, in an upside-down position.

[0444] Gene expression :

[0445] (1) Select lysates in 100 ml of LB medium with 50 micrograms / ml of kanamycin + antibiotic.

[0446] (2) Carry out an incubation at 30°C, 200 rpm for an overnight period as a seed blister.

[0447] (2) Inoculate using 10 ml of crystallization seeds in 1 liter of TB medium (BD-Cat.243820) containing 4 ml of glycerol, incubate at 37°C, 220 rpm.

[0448] (3) When OD arrives 600 To 1.0~1.2 (~2.5 hours), add IPTG to reach a final concentration of 1 mM, and incubate for an additional sixteen hours at 18°C.

[0449] (4) In one example, the cells were clustered when the OD reached 600 The concentration of soluble protein was 8.69. The volume of soluble protein was 26.8 mg / mL. The volume of soluble protein was 90.0 mL. The amount of soluble protein was 2412.0 mg. The titer was 482.4 mg / L.

[0450] (5) Collect the cells by centrifugation at 8000 Gs, fifteen minutes.

[0451] (6) Cell pellets can be collected at -80 degrees Celsius.

[0452] Preparing the included bodies :

[0453] (1) Melt the gene expression pellets at room temperature for ten-twenty minutes.

[0454] (2) Resuspend the cells in a freshly prepared lysis buffer solution at a ratio of 1:5 (WITH V).

[0455] (3) In a magnetic stirring medium, stir for thirty minutes at room temperature until the cell pellet solution is completely dissolved.

[0456] (4) Homogenization method at 1000 bar for two to three minutes.

[0457] (5) Perform a centrifuge at 12,000 GJ for one hour at 4°C.

[0458] (6) Carefully remove the floating portion and resuspend the included objects using a wash buffer solution to a volume equal to their original size. Stir for thirty minutes at room temperature until all cell remnants have dissolved.

[0459] (7) Perform a centrifuge at 12,000 Gs for one hour at 4°C.

[0460] (8) Repeat step 6 and step 7 one more time.

[0461] (9) Discard the floating portion and resuspend the included objects with the dissolution buffer at a ratio of 1:10 (WITH V). Stir overnight at 4°C.

[0462] (10) Perform a centrifuge of the dissolved included objects at 40,000 acceleration due to gravity for one hour at 4 degrees Celsius.

[0463] (11) Collect the floating part using a 0.2 micrometer filter.

[0464] (12) Determine the protein concentration by Pierce Coomassie assay in addition to a protein-determining reagent following the manufacturer’s instructions.

[0465] Dissolution regulator solution : 50 مللي مولار Tris / HCl، الرقم الهيدروجيني 8.0، 5 مللي مولار EDTA، 1% من Triton X-100.

[0466] Washing regulator solution : 50 مللي مولار Tris / HCl، الرقم الهيدروجيني 8.0، 5 مللي مولار EDTA.

[0467] Dissolution buffer solution 50 mM Tris / HCl, pH 8.0, 8 M urea

[0468] In one example, from 1 liter of pellets, 482.4 mg of protein was obtained.

[0469] Refolding . For a quantity of 1 liter of refolding :

[0470] (1) Dilute the sample with altered physical properties at 10 mg / mL using the solubility buffer solution.

[0471] Regulatory solution for refolding : 20 مللي مولار Tris / HCl،10 مللي مولار جليسين، 1 مللي مولار EDTA، 1 مللي مولار GSH، 5 مللي مولار GSSG، الرقم الهيدروجيني 10.0.

[0472] (2) Add DTT to the dissolved inclusion bodies up to a final concentration of 10 mM and mix thoroughly.

[0473] (3) Perform a thirty-minute incubation at 37°C to break up the incorrectly paired disulfide bonds before refolding.

[0474] (4) Slowly drip 50 mL of the reduced included bodies (~500 mg of protein) into 1 liter of freshly prepared recombination buffer solution on a magnetic stirrer (medium power).

[0475] (5) Carry out an incubation at 4 degrees Celsius for twenty-four hours, stirring slowly.

[0476] (6) Run SDS-PAGE and LC-MS to characterize the refolded protein.

[0477] Based on the LC-MS specification, three pairs of disulfide bonds (-6 Dalton / -6H) were formed after overnight refolding. The results also showed that the first 'Met' was successfully removed.

[0478] Replacing the buffer solution ( UF / DF ) and digestion TEV :

[0479] (1) The refolded sample was first filtered through a 0.45 µm filter to remove the visible precipitate before ultrafiltration.

[0480] (2) The sample concentration was reached to ~3 mg / mL using a UF device.

[0481] (3) The concentrated sample in the buffer solution for TEV digestion was changed to a sixfold volume by a DF device.

[0482] Digestive regulator solution TEV : 25 mM Tris / HCl, 100 mM sodium chloride, pH 7.5, 10% glycerol.

[0483] Replacing the buffer solution with UF / DF A sample of SP HP can be replaced in a buffer solution of the final formulation at a volume six times that of a UF / DF device. For small-scale batch production, an SEC and a desalting column were used to replace the buffer solution.

[0484] (4) Add an endogenously produced TEV protease to the protein at a ratio of 1:20 (WITH W), and incubate the mixtures at room temperature overnight.

[0485] (5) Analyze digestion efficiency by SDS-PAGE.

[0486] In one example, SDS-PAGE gel showed that the majority of the target protein was cleaved by TEV protease.

[0487] purification SP HP :

[0488] (1) Add 10% acetic acid to adjust the pH of the sample to 4.5.

[0489] (2) Dilute the sample using ultrapure water to prepare a load conductivity of approximately 7-8 mS / cm.

[0490] (3) Filter the floating part using a 0.2 micrometer filter.

[0491] (4) Load the protein onto an SP Sepharose HP column (Cytiva) at a dynamic binding density of approximately 20 mg / mL (residence time ~2 mins - 5 mins) using an AKTA Pure device.

[0492] (5) Wash the SP HP column using buffer solution A at a volume five times the column volume (CV).

[0493] (6) Wash the SP HP column using buffer solution C at a volume five times that of the column, ensuring a contact time of 300 minutes.

[0494] (7) Wash the SP HP column using buffer solution A to a volume ten times that of the column.

[0495] (8) Perform sequential filtration of the protein in a graduated salt concentration. 0-100% of buffer solution B to a volume twenty times that of the column.

[0496] (9) Determine the protein concentration using Pierce Coomassie in addition to the protein determination reagent following the manufacturer’s instructions.

[0497] (10) Run the fractions at the peak in an unreducible SDS-PAGE gel and collect the fractions containing the purified target protein.

[0498] buffer solution A for SP HP : 50 mM NaAc-HAc, pH 5.0, 10 mM sodium chloride, 10% glycerol.

[0499] buffer solution B for SP HP 50 mM NaAc-HAc, pH 5.0, 1 M sodium chloride, 10% glycerol.

[0500] buffer solution C for SP HP 50 mM NaAc, pH 5.0, 10 mM Sodium Chloride, 10% Glycerol, 0.1% Triton-X114.

[0501] The parts in peak 1 that have good purity were collected for SP HP purification and exchanged in the buffer solution for the final formula.

[0502] In one example, the purity of SEC-HPLC reached 98.03%.

[0503] Formula buffer solution : 50 mM Tris / HCl, 150 mM sodium chloride, 10% glycerol, pH 7.5.

[0504] Product quality analysis The purified protein is subjected to a quality control assay, including reduced and non-reduced SDS-PAGE, SEC-HPLC, Bradford assay, LC-MS, and endotoxin level.

[0505] comparison Bradford The final protein concentration was determined using Pierce Coomassie plus the protein-determining reagent, following the manufacturer's instructions. The final concentration was controlled to be less than 5 mg / mL. If higher concentrations are required in the future, stability tests can be performed on the high-concentration proteins and buffer solutions.

[0506] SEC-HPLC SEC-HPLC analysis was performed on a Vanquish Flex Duo liquid chromatography instrument using a TSKgel G3000SWxl stainless steel column (7.8 x 300 mm). The mobile phase consisted of 50 mM sodium phosphate and 300 mM sodium chloride at pH 6.8. Each phase was filtered by sequential isometric filtration for 15 minutes at a flow rate of 1.0 mL / min. Sequential protein filtration was monitored by UV absorbance at 280 nm. The corresponding peaks for agglomerates, monomers, and low molecular weight (LMW) species were included to calculate the percentage of each species.

[0507] SDS-PAGE Non-reducing and reduced SDS-PAGE were performed using pre-cast 4-12% ThermoFisher Bis-Tris NuPAGE™ gel. The sample loading buffer (4X LDS) was from Invitrogen, and the gel activation buffer (20X MES) was from GenScript. Non-reducing samples were treated with 30 mM iodoacetamide and heated at 95°C for five minutes prior to analysis. Reduced samples were treated with 50 mM DTT and heated at 95°C for five minutes prior to analysis. Electrophoresis was then performed at a constant voltage of 180 V for forty minutes. The gel was stained with Coomassie blue for thirty minutes and decolorized with water for one hour.

[0508] Internal poison level The purified proteins were subjected to endotoxin testing using a Limulus Amoebocyte Lysate (LAL) kinetic turbidity assay. The purified proteins were diluted to the maximum approved dilution (MVD) in water containing LAL reagent (Charles River / W110), and the sample was then loaded into Charles River Laboratories EndoSafe LAL Cartridges (Charles River / PTS55F) and detected using a Charles River Laboratories Endosafe NexGen-MCS™ instrument.

[0509] LC-MS LC mass spectrometry assays were performed using an Agilent 6230 AdvanceBio LC / Transition Time system with a PL1912-1502, PLRP-S 1000 angstrom, 2.1 x 50 mm, 5 μm column (Agilent / PL1912-1502). The mass spectrometry data were analyzed using Bioconfirm 10.0 software, and molecules were identified based on their molecular masses. For unreducible mass spectrometry, the sample was directly injected. For reduced mass spectrometry, the sample was pre-treated with 50 mM DTT for 30 minutes at room temperature prior to injection.

[0510] The average yield of the final sample was approximately 20-30 mg / L. Example 7 Technical report on AGN417

[0511] be AGN417 It is a bifunctional lysis agent based on a sortase-linked complex (TRAP). The molecule consists of a single single-strand chain ( ABT410 The peptide is conjugated to the LPETG pentpeptide of TBT403 via sortase-catalyzed binding. The antibody-target ratio (BAR) is 1.

[0512] Successfully obtained AGN417 All results met the targeted quality standards. The sample remained stable under three freeze-thaw cycles.

[0513] purification method TRAP by AEX :

[0514] TRAP solution was purified by AEX to remove sortase A and the binder load.

[0515] (1) The measuring instrument was the AKTA pure 150 (Pure water generator Milli-Q Reference).

[0516] (2) The column was Capto Q Impres, 500 ml.

[0517] (3) Mobile phase A was 20 mM Tris, pH 8.3. Mobile phase B was 20 mM Tris, 1 M sodium chloride, pH 8.3.

[0518] (4) The gradient was B from 0% to 45% in four of the column size.

[0519] (5) The flow rate was 40 milliliters / minute.

[0520] (6) The wavelength was 280 nanometers and 220 nanometers.

[0521] Purification method for TRAP by UF / DF The UF / DF was pre-processed according to the following procedure:

[0522] (1) Assemble the UF / DF system and install the kit (Pellicon3 0.11m) 2 Cassette, Ultracel 10 kDa).

[0523] (2) Rinse the system with water, clean it using 0.1 molar sodium hydroxide for thirty minutes, and rinse it with water again.

[0524] (3) Add phosphate-buffered saline solution, pH 7.4, to the feedstock tank. Start the feedstock pump. Verify that the pH and conductivity of the system are balanced with the level of phosphate-buffered saline solution.

[0525] (4) Add the reaction mixture solution to the feedstock tank. Start the feedstock pump by partially closing the product retention valve and adjusting the pump speed. Dilute and filter the product using phosphate-buffered saline, pH 7.4 for 15 DVs.

[0526] (5) Fully open the product retention valve and collect all the product. Filter the product through a 0.22 µm membrane.

[0527] (6) Test the quality and concentration of the product.

[0528] Visible and ultraviolet spectroscopy platform by Nanodrop 2000 ( ThermoFisher, 1294881 To determine the protein concentration in the sample under investigation and TRAP Final :

[0529] (1) 750 nanometers was set as the base value.

[0530] (2) Ultraviolet absorption was measured at 280 nm and 220 nm respectively

[0531] (3) Calculation method based on Beer-Lambert Law A=E*C*l

[0532] A 280 =E mAb 280 *C [mAb] *l, where E represents the molar decay coefficient; c represents the molar concentration; and l represents the light path (Nanodrop: 0.1 cm)

[0533] to set BAR by LC-MS LC-MS was performed using a combination of an Agilent 1260 series high-performance liquid chromatography system and time-transition mass spectrometry (Agilent Technologies, HPLC 1260 / 6224 TOF). BAR was calculated based on the mass abundance of the unwrapped mass at the peak.

[0534] Preparing the sample used in the analysis LC-MS The antibody-drug conjugate solution was diluted to approximately 1 mg / mL in a total of 30 µL of water. The resulting sample was used for LC-MS analysis.

[0535] road LC-MS / MS High-performance liquid chromatography (HPLC) analysis was performed under the following measurement conditions. Equipment: Agilent 1260 HPLC (Agilent Technologies, HPLC 1260). Column: Agilent PLRP-S, 50 x 2.1 mm, 8 µm, 1000 angstroms. Detection wavelength: 280 nm, 214 nm. Column furnace temperature: 80 °C. Sampling medium temperature: 2–8 °C. Flow rate: 0.05 mL / min. Injection volume: 2 µg. Mobile phase A: 0.025% trifluoroacetic acid + 0.1% formic acid in water. Mobile phase B: 0.025% trifluoroacetic acid + 0.1% formic acid in acetonitrile. Gradient program for 10 minutes. Time 0.0 minutes at B = 5%; Time 0.7 minutes at B = 5%; Time five minutes at B = 45%; Time six minutes at B = 90%; Time seven minutes at B = 90%; Time 7.1 minutes at B = 5%; and Time 10.0 minutes at B = 5%.

[0536] Determination of agglomeration by size-based exclusion chromatography - High-performance liquid chromatography Volumetric exclusion chromatography (VOC) was performed using an Agilent 1260 series high-performance liquid chromatography system with a TSK gel G3000SWXL (7.8 × 300 mm, 5 μm) VOC column at 25 °C. The mobile phase was prepared from 78 mM KH₂PO₄, 122 mM K₂HPO₄, 250 mM potassium chloride, and 15% isopropanol at pH 7.0 ± 0.1. The flow rate was set at 0.75 mL / min. The sample load was 40–50 μg per injection. Samples were detected at 280 nm and 370 nm using a UV detector. The retention time of the agglomeration peak was recorded based on its relative molecular weight. The level of agglomeration was determined by the relative area of ​​the peak at 280 nm.

[0537] Identifying the property free of residual substances by LC-MS The level of the residual free drug was determined by LC-MS. After protein precipitation, the supernatant was loaded onto an InfinityLab Poroshell 120 SB-C18 column, 4.6 x 100 mm, 2.7 μm, and filtered sequentially by gradient to increase the organic mobile phase. The percentage of residual free drug across the peak area was determined by comparison to an external standard curve.

[0538] (1) Solvent preparation I Ten grams of sodium chloride were weighed into a premixed organic solvent consisting of 30 mL of methanol and 50 mL of acetonitrile, mixed, and stirred for at least one hour. The solution was then allowed to stand for at least one hour before use. The supernatant was the saturated sodium chloride solution.

[0539] (2) Preparing the standard curve First, the crude standard drug-binding solution was diluted to 40 µM using dimethyl sulfoxide to prepare the standard curve. The standard curve and sample were then prepared. MW was used. mAb = 9731 Daltons. TRAP BAR value = 0.91 for the calculation.

[0540] (3) Sample preparation The samples were prepared as follows. (The TRAP product concentration of 22.45 mg / mL was used for the calculation.) TRAP concentration = 0.50 mg / mL. V TRAP 3.30 µL. Dimethyl sulfoxide volume fraction = 0.1 V DMSO 15.00 microliters. The V value was 15.00. H2O 31.70 microliters. Solvent I amounted to 100.00 microliters.

[0541] Liquid chromatography variables for determining the free drug :

[0542] Column Agilent PLRP-S, 50 x 2.1 mm, 8 micrometers, 1000 angstroms.

[0543] detection wavelength : 280 nm, 220 nM.

[0544] Column furnace temperature 80 degrees Celsius.

[0545] Temperature of the sampling medium 2~8 degrees Celsius.

[0546] The flow rate was 0.4 mL / min. The pause time was thirteen minutes. The maximum pressure was 350 bar. The injection volume was 20 microliters.

[0547] mobile phases Mobile phase A represented 0.025% trifluoroacetic acid + 0.1% formic acid in water. Mobile phase B represented 0.025% trifluoroacetic acid + 0.1% formic acid in acetonitrile. The gradient was as follows: At 2.00 minutes, A represented 95.0% and B represented 5.0%. At 5.00 minutes, A represented 70.0% and B represented 30.0%. At 8.00 minutes, A represented 20.0% and B represented 80.0%. At 8.50 minutes, A represented 10.0% and B represented 90.0%. At 10:00, A represented 10.0% and B represented 90.0%. At 10:01, A represented 95.0% and B represented 5.0%. At 13:00, A represented 95.0% and B represented 5.0%.

[0548] Four-pole transition time variables: for determining free property The equipment was an Agilent 6530 QTOF. The polarization was positive. The gas temperature was 250°C. The drying gas flow rate was 13 L / min. The spray pressure was 45 psi. V Cap 3500 volts. Fragmentor reached 125 volts. Mass range was 150–8000 mass / charge. Acquisition rate was 1 spectrum / second.

[0549] Identifying internal toxins Endotoxin levels were determined using the Endosafe® NexGen-PTS™. The TRAP sample was diluted with endotoxin-free water, and 25 µL of the diluted sample was drawn into each of the four reservoirs of the Portable Test System (PTS) Cartridge using a pipette. The reader drew and mixed the sample with LAL reagent in the sample channels and with LAL reagent plus a positive control product in the transient pulse channels. The sample was then combined with the color-generating substrate and incubated. After mixing, the optical density of the eyes was measured and analyzed against an internally stored standard curve.

[0550] Conjugation buffer solution and formula buffer solution .

[0551] The buffer solution is conjugated AGN417 In 50 mM Tris, 150 mM sodium chloride, 10% glycerol, pH 7.5.

[0552] The buffer solution of the formula AGN417 In a phosphate-buffered saline solution, pH 7.4.

[0553] Procedure for mass conjugation .

[0554] (1) Two 2000 ml bottles (Corning, 430281) containing 5971.2 mg of were added ABT410 In the original buffer solution (50 mM Tris, 150 mM sodium chloride, 10% glycerol, pH 7.5) with a 5.08 mg / mL solution of sortase A (PT0008, 0.01 equator), TBT404 (40 mM in dimethyl sulfoxide) (eighteen equivalents) in the reactions, mixed thoroughly and incubated at 4°C overnight (seventeen hours). The protein concentration in the reaction was 3.21 mg / mL.

[0555] (2) The crude conjugate product was purified using AKTA pure 150 chromatography. The conjugate was ligated to the column in the load sampling process. Sortase A, labeled His, and the binder load flowed through it. The flow was collected through the liquid, and a buffer solution was exchanged with phosphate-buffered saline, pH 7.4, using UF / DF (3 kDa, 2 x 0.11 m²) for 15 DV.

[0556] (3) The purified sample was filtered using a 0.22 micrometer filter.

[0557] (4) Quality control testing is carried out (including concentration, volume-based exclusion chromatography-high-performance liquid chromatography, HIC-high-performance liquid chromatography, LC-MS, free drug, endotoxin level and osmosmosis).

[0558] Mass conjugation results The reaction conditions for mass conjugation are summarized in the following table. After conjugation, the reaction mixture is purified by AEX and the buffer solution is replaced with a phosphate-buffered saline solution, pH 7.4, using UF / DF (3 kDa, 2 x 0.11 M). 2 (10 DV), it was filtered using a 0.22 μm membrane. The molecular information about AGN417 The results of AKTA, LC-MS, free drug, endotoxin level, and osmolarity are shown in the following table.

[0559] Reaction conditions mass conjugation for AGN417 ( ABT410-TBT403 ) The Sortase A / monoclonal antibody ratio was 0.01. The monoclonal antibody / conjugate ratio was 18. The conjugate concentration was 3.21 mg / mL. The conjugate temperature and time were 4°C, overnight. The conjugate pH was 7–7.5.

[0560] Molecular information about AGN417 In one example, for AGN417 ( ABT410-TBT403 The molecular weight of BAR1 (amino acid) was 11921.21 Daltons. The adjustment was BAR = 1. The measured mass was 11921.92 Daltons.

[0561] Results for mass conjugation In one example, for AGN417 ( ABT410-TBT403 The concentration measured by UV spectroscopy was 22.45 mg / mL, the amount was 5948.45 mg, MS-BAR was 0.91. Purity by volume-exclusion chromatography was 97.40%, the free drug was < 1.0%; the endotoxin was < 0.089 EU / mg; and the osmolarity was 303 mOsmol / kg.

[0562] Results and discussion of reliability tests The sample was stored at -70°C for one hour to ensure freezing, then thawed by leaving the vial at room temperature for a sufficient period until complete thawing. The analytical chromatogram was then collected under different freeze-thaw cycles (1, 2, 3). The resulting ratios were BAR = 0.89–0.91. Table 19 Name of the material Information about the material LC-MS column Agilent Technologies, PLRP-S, 1000 angstroms, 8 micrometers, 50*2.1 mm, PL1912-1802 Size-based exclusion chromatography column TOSOH, TSKgel G3000SWXL, 7.8 × 300 mm, 5 µm, 08541 Column C18 Agilent Technologies, InfinityLab Poroshell 120 SB-C18, 4.6 × 100 mm, 2.7 µm, 685975-902 Millex-GP Syringe Filter Unit, 0.22 µm millipore-SLGPR33RB Acetonitrile Merck, Supelco-1.00030.4008 Phosphate-regulated saline solution, pH 7.4 Gibco, 10010031 Trifluoroacetic acid jkchemical-134753 Na2HPO4-7H2O Sigma-S2429-500G NaH2PO4 Sigma-S5011-500G (NH4)2SO4 Sigma-A4418-1KG 2-Propanol Sigma-34863 Sodium chloride Domestic Example 8 Preparation procedure TBT403 (Ins-011)

[0563] to prepare Intermediate compound 2 :

[0564] to a solution of Intermediate compound 1a (60.0 g, 400 mmol, 2.0 equivalents) in 2-methyltetrahydrofuran (450 mL) was added to (34.2 g, 200 mmol, 1.0 equivalents) in 2-methyltetrahydrofuran (160 mL) at 0°C. The mixture was stirred at 25°C for 2 hours. Thin-layer chromatography (DCM:methanol = 20:1, R) showed f The reaction was completed, and a new principal spot with lower polarization was detected (p < 0.70). HCl / EA (1 nM, 27.0 mL) was added to the reaction mixture and stirred for 30 minutes. The white precipitate was then removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the desired concentration. Intermediate compound 2 (Crude, 105.0 g, 370.6 mmol) in the form of a yellow oil. LC-MS: Retention time = 0.797 mins, Calculated MS: 283.14, Observed mass: [M + Na]+ = 306.1. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.23 - 7.41 (m, 5 H), 5.01 (s, 2 H), 4.60 (br s, 1 H), 3.45 - 3.52 (m, 6 H), 3.38 - 3.43 (m, 5 H), 3.14 (q, J = 5.94 Hz, 2 H), 2.53 - 2.55 (m, 1 H).

[0565] to prepare Intermediate compound 3 :

[0566] to a solution of Intermediate compound 2A (100.0 g, 257 mmol, 1.0 equation) in DCE (500 mL) TMSOTf (85.6 g, 385 mmol, 1.5 equations) was added and stirred at 60 °C for 2 hours. The reaction mixture was then cooled to room temperature (25 °C) and stirred for another 1 hour. Intermediate compound 2 (80.0 g, 282 mmol, 1.1 equivalents) and molecular sieves in 4 angstrom powder (50.0 g) in DCE (500 mL) were added to the reaction. The reaction mixture was stirred for thirty minutes in a nitrogen atmosphere. A solution of Intermediate compound 2A (100.0 g, 257 mmol, 1.0 equation) in DCE was distilled into the mixture at 0°C. The mixture was stirred for sixteen hours at 25°C in a nitrogen atmosphere. Thin-layer chromatography (DCM:methanol = 10:1, R) was performed. f = 0.42) That it was consumed Intermediate compound 2A The entire reaction was then fully exposed, revealing one new major spot with greater polarization. The reaction mixture was then filtered and washed with saturated NaHCO3 (500 mL), water (500 mL), and saline (500 mL). The organic layer was dried on anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, PE:EA = 3:1 to 1:6, then DCM:Methanol = 20:1) to obtain Intermediate compound 3 (90.0 g, 146.9 mmol, 91.6% purity, 57.2% yield) in the form of a yellow oil. LC-MS: Retention time = 0.860 min, Calculated MS: 612.25, Observed mass: [M + H]+ = 613.2. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.80 (d, J = 9.03 Hz, 1 H), 7.24 - 7.39 (m, 6 H), 5.22 (d, J = 3.51 Hz, 1 H), 4.95 - 5.05 (m, 3 H), 4.53 - 4.59 (m, 1 H), 3.99 - 4.06 (m, 3 H), 3.84 - 3.92 (m, 1 H), 3.73 - 3.82 (m, 1 H), 3.55 - 3.61 (m, 1 H), 3.45 - 3.53 (m, 7 H), 3.41 (t, J = 5.90 Hz, 2 H), 3.11 - 3.18 (m, 3 H), 2.10 (s, 3 H), 1.99 (s, 3 H), 1.89 (s, 3 H), 1.77 (s, 3 H).

[0567] to prepare Intermediate compound 4 :

[0568] To Pd / C (9.00 g, 10% purity) in a reaction flask (sterilized with argon gas three times), THF (180 mL) was slowly added, followed by a solution of trifluoroacetic acid (16.7 g, 147 mmol, 1.0 Eq). Intermediate compound 3 (90.0 g, 147.0 mmol, 1.0 equation) in THF (720 mL) was slowly reacted in a nitrogen atmosphere. The reaction mixture was then degassing and purged three times with nitrogen and hydrogen gas, and stirred at 25 °C for three hours in a hydrogen atmosphere (40 psi). Thin-layer chromatography (DCM:methanol = 10:1, Rc) clearly demonstrated the results. f = 0.20) That it was consumed Intermediate compound 3 The entire mixture was then identified, revealing a new major spot with greater polarization. The reaction mixture was dissolved in THF (100 mL), carefully filtered through siliceous soil in a nitrogen atmosphere, washed with THF (100 mL x 2), and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was then diluted with water (1000 mL), washed with DCM (300 mL x 3), and the aqueous layer was dehydrated to obtain the final product. Intermediate compound 4 (80.0 g, 139.0 mmol, 95.1% purity, 91.8% yield, salt of trifluoroacetic acid) as a white solid. LC-MS: Retention time = 0.484 min, Calculated MS: 478.22, Observed mass: [M + H]+ = 478.9. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.91 (br t, J = 9.03 Hz, 4 H), 5.21 (d, J = 3.26 Hz, 1 H), 4.96 (dd, J = 11.17, 3.39 Hz, 1 H), 4.54 (d, J = 8.53 Hz, 1 H), 3.98 - 4.08 (m, 3 H), 3.85 - 3.93 (m, 1 H), 3.75 - 3.84 (m, 1 H), 3.59 (br t, J = 5.14 Hz, 3 H), 3.50 - 3.56 (m, 6 H), 2.98 (br s, 2 H), 2.10 (s, 3 H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.78 (s, 3 H).

[0569] to prepare Intermediate compound 6 :

[0570] to a mixture of Intermediate compound 5 (60.0 g, 495.0 mmol, 1.0 equation) in DMSO (166 mL). Aqueous NaOH (5.0 M, 9.91 mL, 0.1 equation) was added by distillation at 0–15 °C for more than five minutes. After the addition, the mixture was stirred at 0–15 °C for five minutes, then further additions were made. Intermediate compound 5a (254.0 g, 1.98 mol, 287 mL, 4.0 equivalents) were added to the reaction mixture by distillation at 20 °C. The reaction mixture was stirred at 25 °C for sixteen hours. Thin-layer chromatography (DCM:methanol = 10:1, R) was performed. f = 0.7) That it was consumed Intermediate compound 5 The entire reaction was completed, revealing one new major spot with lower polarization. The resulting reaction mixture was then concentrated under reduced pressure to obtain a residue. This residue was dissolved in ethyl acetate (400 mL), quenched by adding water (400 mL), and extracted using ethyl acetate (400 mL x 3). The collected organic layers were washed with saline solution (300 mL x 2), dried on Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the Intermediate compound 6 (100.0 g, 197.8 mmol, 96.0% purity, 40.0% yield) in the form of a colorless oil. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 3.51 - 3.61 (m, 7 H), 3.17 (s, 5 H), 2.39 (t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H).

[0571] to prepare Intermediate compound 7 :

[0572] to a solution of Intermediate compound 6 Hydroxybenzotriazole (10.7 g, 79.1 mmol, 1.0 equator) was added to acetonitrile (400 mL). Then, 6a (16.5 g, 79.1 mmol, 1.0 equator) and DCC (16.3 g, 79.1 mmol, 1.0 equator) were added. The reaction mixture was then stirred at 25 °C for sixteen hours. The results were shown by thin-layer chromatography (PE:EA = 1:1, R f = 0.80) That it was consumed Intermediate compound 6 Completely. One new major spot was detected with lower polarization. Acetonitrile was evaporated to obtain the residual material. The residual material was purified by column chromatography (SiO2, PE:EA = from 10:1 to 1:1) to obtain Intermediate compound 7 (40.0 g, 57.4 mmol, 82.9% purity, 72.5% yield) as a white solid. LC-MS: Retention time = 1.151 min, Calculated MS: 696.38, Observed mass: [M + H] + = 697.3, [M + Na] + = 719.3. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.26 - 7.40 (m, 6 H), 7.06 (s, 1 H), 5.03 (s, 2 H), 3.49 - 3.61 (m, 14 H), 2.39 (br t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H).

[0573] to prepare Intermediate compound 8 :

[0574] A solution of Intermediate compound 7 (30.0 g, 43.0 mmol, 1.0 equation) in HCOOH (300 mL) at 25 °C for sixteen hours. Clear thin-layer chromatography (PE:EA = 1:1, R f (= 0.04) That it was consumed Intermediate compound 7 The entire solution was removed, revealing a single major new spot with greater polarization. The solvent was evaporated under reduced pressure, then co-evaporated with toluene (50 mL x 3) under reduced pressure, and dried under reduced pressure to obtain the residue. The residue was then purified by preparative HPLC (A: 0.1% trifluoroacetic acid / water, B: acetonitrile) to obtain Intermediate compound 8 (20.0 g, 37.8 mmol, 98.2% purity, 87.9% yield). 1 H NMR (400 MHz, DMSO- d 6) δ ppm 12.17 (br s, 3 H), 7.26 - 7.43 (m, 6 H), 7.06 (s, 1 H), 5.02 (s, 2 H), 3.49 - 3.65 (m, 14 H), 2.42 (br t, J = 6.27 Hz, 6 H). LC-MS: Detention time = 0.790 minutes, Calculated MS: 528.20, Observed mass: [M + H]+ = 529.2.

[0575] to prepare Intermediate compound 9 :

[0576] إلى محلول يتم تقليبه من Intermediate compound 8 (20.0 جم، 37.8 مللي مول، 1.0 مكافئ) و Intermediate compound 4 (78.5 جم، 132 مللي مول، 3.5 من المكافئات، ملح لحمض تراي فلورو أسيتيك) في DMF (400 ملليلتر) تمت إضافة هيدروكسي بنزوترايازول (20.4 جم، 151 مللي مول، 4.0 من المكافئات)، EDCI (29.0 جم، 151 مللي مول، 4.0 من المكافئات) وDIEA (22.0 جم، 170 مللي مول، 4.5 من المكافئات) تتابعيًا. وتم تقليب خليط التفاعل عند 25 درجة مئوية لمدة ساعتين. أوضح استشراب بطبقة رفيعة (DCM: ميثانول = 10: 1، R f = 0.4) أنه تم استهلاك Intermediate compound 8 The entire reaction was then revealed, revealing one major new spot with greater polarization. The reaction mixture was then slowly poured into a stirred solution of 0.5 mol / L cold hydrochloric acid (900 mL) and stirred for ten minutes. A white precipitate was formed and filtered. The aqueous phase was then extracted twice using DCM (600 mL x 2). The collected organic layers were washed with 5% NaHCO3 (450 mL), dried on Na2SO4, and concentrated under reduced pressure to obtain a residue. The residue was then purified by column chromatography (SiO2, DCM:methanol = 100:1 to 5:1) to obtain Intermediate compound 9 (58.0 g, 30.4 mmol, 82.7% purity, 80.3% yield) in the form of a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.92 (br t, J = 5.14 Hz, 3 H), 7.81 (d, J = 9.03 Hz, 3 H), 7.28 - 7.39 (m, 6 H), 7.13 (s, 1 H), 5.21 (d, J = 3.26 Hz, 3 H), 5.02 (s, 2 H), 4.97 (dd, J = 11.17, 3.39 Hz, 3 H), 4.54 (d, J = 8.53 Hz, 3 H), 4.03 (s, 9 H), 3.84 - 3.92 (m, 3 H), 3.75 - 3.81 (m, 3 H), 3.45 - 3.61 (m, 37 H), 3.39 (br s, 3 H), 3.18 - 3.23 (m, 6 H), 2.30 (br t, J =6.15 Hz, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H). LC-MS: Detention time = 3.455 minutes, Calculated MS: 1908.81, Observed mass: [M + 2H] 2+ = 955.7.

[0577] to prepare Intermediate compound 10 :

[0578] To Pd / C (4.6 g, 25.13 mmol, 10% purity, 1.0 equation) in a reaction flask (purified with nitrogen gas three times), methanol (230 mL) was slowly added, followed by a solution of trifluoroacetic acid (2.75 g, 24.1 mmol, 1.0 equation) and Intermediate compound 9 (46.0 g, 24.1 mmol, 1.0 equation) in methanol (230 mL) was slowly reacted in a nitrogen atmosphere. The reaction mixture was then degassing and purged three times using nitrogen and hydrogen gas, stirred at 25 °C for 2 hours in a hydrogen atmosphere (15 psi). Thin-layer chromatography (DCM:methanol = 10:1, Rc) was performed. f = 0.3) That a was consumed For the intermediate compound 9 The entire reaction was completed, revealing one new major spot with greater polarization. The reaction mixture was then dissolved in methanol (250 mL), carefully filtered through siliceous earth in a nitrogen atmosphere, the paste was washed with methanol (250 mL x 2), and the filtrate was concentrated under reduced pressure to obtain Intermediate compound 10 (Crude, 42.0 g, 22.4 mmol, 94.1% purity, 92.2% yield, salt of trifluoroacetic acid) in the form of a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.97 (br t, J = 5.44 Hz, 6 H), 7.81 - 7.88 (m, 3 H), 7.73 - 7.77 (m, 1 H), 5.19 - 5.26 (m, 3 H), 4.97 (dd, J = 11.13, 3.38 Hz, 3 H), 4.52 - 4.61 (m, 3 H), 3.99 - 4.06 (m, 10 H), 3.84 - 3.93 (m, 4 H), 3.74 - 3.82 (m, 4 H), 3.46 - 3.61 (m, 42 H), 3.38 - 3.42 (m, 8 H), 3.19 - 3.24 (m, 6 H), 2.28 - 2.34 (m, 6 H), 2.08 - 2.12 (m, 9 H), 1.98 - 2.02 (m, 9 H), 1.87 - 1.90 (m, 9 H), 1.75 - 1.80 (m, 9 H). LC-MS: Detention time = 1.585 minutes, Calculated MS: 1774.78, Observed mass: [M + H] + = 1775.7, [M + 2H] 2+ = 888.7.

[0579] to prepare Intermediate compound 12 :

[0580] DIEA (8.62 g, 66.6 mmol, 3.0 equivalents), EDCI (6.69 g, 33.3 mmol, 1.5 equivalents) and hydroxybenzotriazole (6.38 g, 33.3 mmol, 1.5 equivalents) were added to the solution Intermediate compound 10 (42.0 g, 22.2 mmol, 1.0 Eq, salt of trifluoroacetic acid) and 11a (7.77 g, 33.3 mmol, 1.5 Eq) in DMF (420 mL). The mixture was stirred at 25 °C for 2.0 h. LC-MS showed that the solution was consumed Intermediate compound 10 The entire reaction was visualized by LC-MS, revealing several new peaks and identifying the desired compound. The reaction mixture was then slowly poured into a stirred 0.5 M hydrochloric acid solution (cold, 500 mL). The aqueous phase was extracted using DCM (500 mL x 3). The collected organic layers were washed with 5% NaHCO3 (500 mL), dried on Na2SO4, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM:methanol = 100:1 to 5:1) to obtain the desired product. Intermediate compound 12 (34.0 g, 17.1 mmol, 93.3% purity, 77.0% yield) in the form of a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.88 - 7.94 (m, 3 H), 7.78 - 7.83 (m, 3 H), 7.71 - 7.76 (m, 1 H), 7.21 - 7.26 (m, 1 H), 5.19 - 5.23 (m, 3 H), 4.94 - 5.02 (m, 3 H), 4.51 - 4.59 (m, 3 H), 4.00 - 4.06 (m, 9 H), 3.91 - 3.93 (m, 2 H), 3.84 - 3.90 (m, 3 H), 3.72 - 3.81 (m, 5 H), 3.46 - 3.63 (m, 46 H), 3.37 - 3.43 (m, 9 H), 3.18 - 3.23 (m, 6 H), 2.27 - 2.34 (m, 6 H), 2.09 - 2.12 (m, 9 H), 1.98 - 2.01 (m, 9 H), 1.88 - 1.90 (m, 9 H), 1.75 - 1.79 (m, 9 H). LC-MS: Detention time = 0.501 minutes, Calculated MS: 1989.87, Observed mass: [M + 2H] 2+ = 996.3.

[0581] to prepare Target A043 :

[0582] to a solution of Intermediate compound 12 (34.0 g, 12.6 mmol, 1.0 equator) NaOMe (5.4 M in methanol, 9.91 mL, 4.2 equators) was added to 400 mL of methanol at 0°C. The solution was then stirred at 0°C for 0.5 hours. LC-MS showed that the solution was consumed. Intermediate compound 12 The entire reaction was performed, and several new peaks were visualized by LC-MS, revealing the desired compound. The reaction mixture was then adjusted to pH 6 using a 1.0 M hydrochloric acid solution at 0°C and washed three times with DCM (250 mL x 3). The aqueous layer was then dehydrated to obtain [the desired result]. Target A043 (Raw, containing sodium chloride, 20.0 g, 12.4 mmol, 96.3% purity) in the form of a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ ppm 7.92 - 7.98 (m, 3 H), 7.73 - 7.77 (m, 1 H), 7.61 - 7.65 (m, 3 H), 7.22 - 7.27 (m, 1 H), 4.59 - 4.64 (m, 3 H), 4.55 - 4.58 (m, 3 H), 4.49 - 4.52 (m, 3 H), 4.25 - 4.31 (m, 3 H), 3.90 - 3.94 (m, 2 H), 3.68 - 3.81 (m, 9 H), 3.63 - 3.67 (m, 4 H), 3.59 - 3.62 (m, 6 H), 3.58 - 3.62 (m, 7 H), 3.38 - 3.41 (m, 13 H), 3.17 - 3.24 (m, 7 H), 2.28 - 2.34 (m, 7 H), 1.78 - 1.82 (m, 9 H). LC-MS: Detention time = 0.501 minutes, Calculated MS: 1611.77, Observed mass: [M + H]+ = 1612.8, [M + 2H] 2+ = 807.2.

[0583] to prepare Intermediate compound 13 :

[0584] The peptide was synthesized using standard Fmoc chemistry.

[0585] (1) Resin preparation To the vessel containing Rink amide MBHA resin (sub: 0.27 mmol / g, 5.00 mmol, 18.5 g) and DMF (150 mL), bubbles were introduced using nitrogen gas for 2 hours at 25°C. Then, 20% piperidine was added to DMF (200 mL), and the mixture was bubbled again using nitrogen gas for 30 minutes at 25°C. The mixture was then filtered and washed using DMF (200 mL) * 5 before proceeding to the next step.

[0586] (2) Pairing A solution of Fmoc-GG-OH (3.54 g, 10.00 mmol, 2.00 eQU), HBTU (7.2 g, 9.5 mmol, 1.90 eQU), and DIEA (5.16 g, 40.00 mmol, 4.00 eQU) in DMF (100 mL) was added to the resin, and nitrogen gas bubbles were introduced for 30 minutes at 25 °C. The conjugation reaction was monitored using the ninhydrin assay. The resin was then washed with DMF (200 mL)*5.

[0587] (3) Removal of protection 20% piperidine / DMF (300.0 ml) was added and mixed at room temperature (25°C) for 30 minutes. The deprotection reaction was monitored using the ninhydrin test; if a blue or reddish-brown color appeared, the reaction was complete. The resin was then washed with DMF (300.0 ml)*5.

[0588] (4) Steps 2 through 3 were repeated for all other amino acids: (#7 in the following table). In the final step, the resin was washed with methanol (300.0 mL)*3 and dried by introducing nitrogen bubbles for three hours. Table 20 List of amino acids and corresponding reagents used on SPPS. # Materials Pairing detectors 1 Fmoc-Gly-Gly-OH (2.00 equivalents) HBTU (1.90 equivalent) and DIEA (4.00 equivalent) 2 Fmoc-Thr-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 3 Fmoc-Glu-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 4 Fmoc-Pro-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 5 Fmoc-Leu-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 6 Fmoc-Gly-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 7 4-pentenoic acid (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent)

[0589] Peptide cleavage and cyclization .

[0590] (1) The fission solution (TFA / TIS / Water, 95 / 2.5 / 2.5, v / v / v, 220 mL) was added to the vial containing the resin-bound side-chain protected peptide (RINK AMIDE MBHA resin, 22 g, 5.0 mmol) at 25°C and stirred for 2 hours.

[0591] (2) After filtration, the filtration output was collected.

[0592] (3) The filtrate was precipitated using cold isopropyl ether (1000 mL). After filtration, the solid was washed twice with isopropyl ether (500 mL), and the crude peptide was dried under reduced pressure for 2 hours to obtain intermediate compound 1 (5 g, crude) as a white solid. The crude peptide was then purified by preparative HPLC (a: 0.1% trifluoroacetic acid / water, b: acetonitrile), followed by lyophilization to obtain intermediate compound 13 (1.5 g, 90% purity, 42.3% yield) as a white solid.

[0593] to prepare TBT403 (Ins-011) :

[0594] to a solution of Target A043 (2.184 g, 1.354 mmol, 1.2 equivalents) and Intermediate compound 13 (0.8 g, 1.129 mmol, 1.0 equation) in DMF (1 mL) and water (10 mL). A solution of sodium ascorbate (670.827 mg, 3.386 mmol, 3.0 equations), TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, 490.430 mg, 1.129 mmol, 1 equation), and CuSO4-5H2O (0.4 M, 2.822 mL, 1.0 equation) was added in water (3 mL) at 0°C. The mixture was then stirred at 25°C for 1 hour. LC-MS showed that the solution was consumed. Intermediate compound 13 The entire mixture was analyzed by LC-MS, revealing several new peaks and identifying the desired compound. The mixture was then purified by preparative HPLC (A: 0.5% acetic acid / water, B: acetonitrile) followed by lyophilization to obtain the desired result. TBT403 (Ins-011) (1.42 g, 94% purity, 50.9% yield) as a white solid. LC-MS: Retention time = 0.15 min, Calculated MS: M av = 2321.4, observed mass: [M + H] + = 2321.9. Example 9 The procedure for compensation TBT405 ( Ins-030 )

[0595] To a solution of intermediate compound 15 (99.0 g, 52.4 mmol, 1.00 Eq, trifluoroacetic acid) in methanol (1.00 L), NaOMe (11.3 g, 210 mmol, 4.00 Eq). The mixture was stirred at 25 °C for half an hour. LC-MS showed that the molecule was consumed Intermediate compound 15 The reaction mixture was then concentrated under reduced pressure to remove methanol. The residue was diluted with water (500 mL) and washed with DCM (500 mL x 3), and the aqueous layer was dehydrated to obtain the crude product. The crude product was then purified by preparative HPLC (A: 0.5% acetic acid / water, B: acetonitrile) to obtain Target A001A (36.0 g, 25.7 mmol, 49.1% yield, 97.5% purity) in the form of a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.95 (br t, J = 5.38 Hz, 3 H), 7.63 (br d, J = 8.88 Hz, 3 H), 7.57 - 7.50 (m, 1 H), 4.28 (d, J = 8.51 Hz, 3 H), 3.84 - 3.70 (m, 6 H), 3.70 - 3.60 (m, 6 H), 3.59 - 3.46 (m, 43 H), 3.32 - 3.29 (m, 4 H), 3.25 - 3.19 (m, 6 H), 3.05 (s, 2 H), 2.31 (br t, J = 6.25 Hz, 6 H), 1.81 (s, 9 H). LC-MS: Detention time = 1.103 minutes, Calculated MS: 1397.5, It was found that: [M + H] + = 1397.5, [(M + 2H) / 2] 2+ = 699.6.

[0596] to prepare Intermediate compound 15 :

[0597] to a solution of Intermediate compound 14 (15 g, 44.333 mmol, 1.0 Eq) in DMF (50 mL). A solution of 2,3,5,6-tetrafluorophenol (18.406 g, 110.832 mmol, 2.5 Eq) in DMF (50 mL) was added at 0°C. Then, a solution of EDCI (21.247 g, 110.832 mmol, 2.5 Eq) in DMF (50 mL) was added to the mixture and stirred at 25°C for 2 hours. LC-MS showed that the mixture was consumed. Intermediate compound 14 The entire solution was analyzed by LC-MS, revealing several new peaks and identifying the desired compound. The crude solution was then purified by preparative HPLC (A: 0.1% TFA / water, B: acetonitrile) followed by lyophilization to obtain the desired result. Intermediate compound 15 (22 g, 97% purity, 75.8% yield) as a colorless oil. LC-MS: Retention time = 0.724 mins, Calculated MS: M av = 634.5, found: [M +H] + = 635.3. 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm 7.93 (tt, J =10.92, 7.40 Hz, 2 H), 3.77 (t, J =5.90 Hz, 4 H), 3.58 - 3.47 (m, 16 H), 3.02 (t, J =5.90 Hz, 4 H).

[0598] to prepare Intermediate compound 16 :

[0599] The peptide was synthesized using standard Fmoc chemistry (CTC resin).

[0600] (1) Resin preparation To the vessel containing CTC Resin (sub: 0.55 mmol / g, 15.00 mmol, 27.3 g) and Fmoc-Gly-Gly-OH (5.3 g, 15 mmol, 1.0 eQ) in DCM (200 mL), DIEA (4.0 eQ) was added by distillation and mixed for 2 hours with the introduction of nitrogen gas bubbles at 25 °C. Then, methanol (15.0 mL) was added, and nitrogen gas bubbles were introduced for another 30 minutes. The resin was then washed with DMF (100 mL) * 5, followed by the addition of 20% piperidine in DMF (200 mL) and nitrogen gas bubbles for 30 minutes at 25 °C to deprotect the Fmoc. The mixture was then filtered. The resin was washed again with DMF (100 mL) * 5 before proceeding to the next step.

[0601] (2) Pairing A solution of Fmoc-Thr-OH (17.9 g, 45 mmol, 3.0 Et.) and HBTU (16.3 g, 42.75 mmol, 2.85 Et.) in DMF (100 mL) was added to the resin with nitrogen gas bubbles. DIEA (6.0 Et.) was then added to the mixture by distillation, and nitrogen gas was introduced for 30 minutes at 25 °C. The conjugation reaction was monitored using the ninhydrin test; if no color was detected, conjugation was complete. The resin was then washed with DMF (200 mL) *5.

[0602] (3) Removal of protection 20% piperidine in DMF (200 ml) was added to the resin and the mixture was bubbled using nitrogen gas for thirty minutes at 25°C. The resin was then washed with DMF (200 ml) *5.

[0603] (4) Steps 2–3 were repeated for all other amino acids: (#3–5 in the following table). In the final step, the resin was washed with methanol (200.0 mL)*3 and dried by introducing nitrogen bubbles for three hours. Table 21 List of amino acids and corresponding reagents used on SPPS. # Materials Pairing detectors 1 Fmoc-Gly-Gly-OH (1.00 equivalent) DIEA (4.00 of the rewards) 2 Fmoc-Thr-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 3 Fmoc-Glu-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 4 Fmoc-Pro-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent) 5 Fmoc-Leu-OH (3.00 equivalents) HBTU (2.85 equivalent) and DIEA (6.00 equivalent)

[0604] Peptide cleavage and cyclization .

[0605] (1) The fission solution (TFA / TIS / Water, 95 / 2.5 / 2.5, v / v / v, 90 ml) was added to the vial containing the peptide bound to the side-chain-protected resin (CTC Resin, 30 g, 15.0 mmol) at 25°C and stirred for 2 hours.

[0606] (2) After filtration, the filtration output was collected.

[0607] (3) The filtrate was precipitated using cold isopropyl ether (1500 mL). After filtration, the solid was washed twice using isopropyl ether (500 mL), and the crude peptide was dried under reduced pressure for 2 hours to obtain Intermediate compound 1 (10 g, crude) in the form of a white solid. The crude peptide was then purified by preparative HPLC (A: 0.1% TFA / water, B: acetonitrile) followed by lyophilization to obtain Intermediate compound 16 (6.0 g, 93% purity, 69% yield) as a white solid. LC-MS: Retention time = 0.304 min, Calculated MS: M av = 572.6, found: [M +H]+ = 573.4.

[0608] to prepare Intermediate compound 17 :

[0609] to a solution of Intermediate compound 15 (13.296 g, 20.957 mmol, 2.0 equivalents) and DIEA (2.709 g, 20.957 mmol, 3.464 mL, 2.0 equivalents) in DMF (15 mL) were added Intermediate compound 16 (6 g, 10.478 mmol, 1.0 equation) in DMF (15 mL) was slowly infused at 25 °C in a nitrogen atmosphere. The mixture was stirred at 25 °C for 1 hour. LC-MS showed that the solution was consumed Intermediate compound 16 The entire mixture was analyzed by LC-MS, revealing several new peaks and identifying the desired compound. The mixture was adjusted to pH 5–6 using acetic acid. The crude solution was then purified by preparative HPLC (A: 0.5% acetic acid / water, B: acetonitrile) followed by lyophilization to obtain the desired result. Intermediate compound 17 (3.1 g, 95% purity, 28.4% yield) as a white solid. LC-MS: Retention time = 0.491 min, Calculated MS: M av = 1040.9, found: [M +H] + = 1041.7.

[0610] to prepare TBT405 ( Ins-030 ) :

[0611] to a solution of Target A001A (700 mg, 500.906 micromoles, 1.0 equation) and Intermediate compound 17 (548.89 mg, 500.906 µmol, 1.0 equations) DIEA (129.477 mg, 1.002 mmol, 2.0 equations) was added slowly at 0°C in a nitrogen atmosphere to 10 mL of DMF. The mixture was then stirred at 25°C for 1 hour. LC-MS showed that the DIEA was consumed. Intermediate compound 17 The entire mixture was analyzed by LC-MS, revealing several new peaks and identifying the desired compound. The mixture was adjusted to pH 5–6 using 1 M hydrochloric acid at 0°C. The crude solution was then purified by preparative HPLC (A: 0.5% acetic acid / water, B: acetonitrile) followed by lyophilization to obtain the desired result. TBT405 (Ins-030) (495.3 mg, 99.5% purity, 42.9% yield) as a white solid. LC-MS: Retention time = 0.328 min, Calculated MS: M av = 2272.4, observed mass: [M + H] + / 2 = 1136.7. Example 10 Biophysical assays . Surface plasmon resonance – results two .

[0612] Conditions for surface plasmon resonance assay of insulin growth factor receptor 1 :

[0613] buffer solution HSB-N with 0.005% of Tween-20, pH: 7.4, 90 seconds on, 180 seconds off.

[0614] Acro's human insulin growth factor receptor (His-Avitag) is held on an SA chip at ~750, 1000, and 1500 RU.

[0615] Insulin binding to the receptor is assessed (maximum 10 micromolar, dilution twice).

[0616] Surface plasmon resonance insulin growth factor receptor 1 An Avi-encoded receiver is held on an SA chip. The inventors performed an assay for insulin and insulin lispro.

[0617] The surface shows better activity at higher density (50% vs. 65% active)

[0618] The effects were sufficient for a selective assay.

[0619] Conclusion A robust surface plasmon resonance assay was developed for insulin-growth factor receptor 1 (IGF-1). Mutant insulin significantly reduced binding to IGF-1. Table 22 Surface plasmon resonance Future of IGF-1 AGN303 (Original insulin precursor) K D = 909 nanomolar AGN310 (Mutational insulin precursor) K D = 10.6 micromolar insulin K D = 1.2 micromolar Table 23 Surface plasmon resonance updates Sample Sample information IR-B K D IGF-1R K D (micromolar) mAb49 K D (micromolar) Peptide-C Ab K D (micromolar) ASGPR IC 50 (micromolar) insulin Comparison sample 0.34 micromolar 1.24 0.11 - - AGN417 -03 insulin prohelolytic factor 8.22 micromolar 18 0.80 0.20 9.8 AGN417 -06 insulin prohelolytic factor 6.82 micromolar 22 0.84 0.09 AGN421 -03 Mutating proinsulin hemolysis factor 22.5 micromolar NB 1.20 0.23 13.2 AGN8138 -01 AGN417 With punctuation marks 13 and N15 6.99 micromolar 17 0.91 0.16 4 AGN8139 -01 AGN417 With punctuation marks 13 and N15 6.48 micromolar 17 0.99 0.14 4.5 ABT17384 -01 Mutant proinsulin + lysine mutation 6.34 micromolar NB 0.24 0.07 - ABT17385 -01 Mutant insulin pro-methane + methionine 4.54 micromolar NB 0.22 0.08 - ABT17386-01 Mutant insulin pro-cysteine > 5.0 micromolar 22 3.13 0.07 - Example 11 Determining the total conjugate of AGN6567 In plasma K2EDTA rat by LC-MS / MS

[0620] Purpose. The LC-MS / MS method was developed to determine total antibody from AGN6567 In rat K2EDTA plasma, SIL TTPP was used as the internal standard (IS). This method is used for non-GLP studies. AGN6567 It is too large for direct LC-MS analysis. Its characteristic peptide, ELVAVITTDGSTNYADSVK (sequence number: 51), is used for quantitative determination. AGN6567 After the immunoprecipitation and digestion step in rat plasma. Table X Method summary Type of assay UPLC-MS / MS Vehicles AGN6567 Solid: No Internal Standards SIL ELVA Solid: Yes Type / Template rat plasma sequential filtering Graduation Sample processing Immune retention and trypsin digestion Sample size requirement 10 microliters Runtime 10.0 minutes Assay range (µg / mL) in rat K2EDTA plasma 0.1~20 Solution storage temperature (degrees Celsius) -70 degrees Celsius QC storage temperature (degrees Celsius) -70 degrees Celsius Injection volume 15 microliters Special conditions required Not available

[0621] Prepared at (As listed or equivalent). The following equipment, or equivalent, is used during the execution of the method: Table 23 Equipment Seller / Model LC-MS / MS Sciex API 6500+ with pumps and autosample device, Shimadzu HPLC valve From system centrifuge Beckman GS-6R A way to shake a nursery board VWR Method of custody Heated Microplate Vortexer KingFisher Purification System KingFisher Flex analytical balance Sartorius CP225D, capable of weighing 0.00001 g Micro scale Mettle Toledo, capable of weighing 0.000001 g Water system Sys# 56-2 PureProteome™ Magnetic Stand, 15 ml Millipore Plate 1 consists of 96 eyes KingFisher Deepwell 96 2ml Layer, V-shaped Bottom, Polypropylene A strip consisting of 96 ends KingFisher 96-pin magnetic strip DW A fine dish consisting of 96 holes KingFisher 96 KF Precision Dish (200 microliters)

[0622] Detectors and materials (As listed or equivalent). The following reagents and materials, or their equivalents, are used during the execution of the method: Table 24 Reagent Category methanol HPLC Acetonitrile HPLC Water purified formic acid GR K2EDTA plasma for rats nothing Dynabeads M-280 streptavidin from Invitrogen (catalog number: 60210) or equivalent nothing SBA, treated with biotinyl WOVUS27560 (Vector Laboratories) RapiGest nothing Phosphate-regulated saline solution with Tween 20X GR Phosphate-regulated saline solution 10X GR 1 molar Tris HCl pH 8.0 GR Trifluoroacetic acid GR Trypsin from Promega (V5113) Sequence preparation category Tween-20 GR

[0623] conditions HPLC for Sciex 6500+ The measurement instrument conditions may be adjusted to achieve the optimal response level. Table 25 Mobile phase A 0.1% formic acid in water Mobile phase B 0.1% formic acid in acetonitrile Automatic sampling device washing methanol Automatic sampling device temperature 4 degrees Celsius Chapter type Gradual flow rate 0.5 ml / min Gradual Column Waters Acquity, UPLC BEH Phenyl C18, 2.1 x 50 mm, 1.7 µm, Part No. 186002884 Column temperature 40 degrees Celsius Injection volume 15 microliters (may be adjusted based on sensitivity and platform) Detention time (in minutes) Switching valve Data acquisition time eight minutes

[0624] conditions MS / MS The measurement instrument conditions may be adjusted to achieve an optimal response. SIL ELVA peptide: EL*VAVITTDGSTNYADSVK (L*=Leu 13 C6, 15 N) Table 26 Mass spectrometer Sciex API 6500+ or ​​7500 interface ESI Ionization pattern positive Detection pattern MRM Decomposition product MS Transitions Data acquisition time four minutes Settings: General Voltia 5500 temperature 550 GS1 50 GS2 50 CUR 20 CAD 8 Settings: Compound-based Conj ELVA DP: 80 CE: 48 CXP: 10 EP: 10 SIL ELVA DP: 80 CE: 48 CXP: 10 EP: 10

[0625] preparations One dish containing 96 cells will require 4 mL of M-280 beads and four tubes of SBA, Biotinylated Antibody™. The preparations described below may be measured in ascending or descending order to provide larger or smaller amounts of reagents, mobile phases, etc.

[0626] 1. Phosphate-buffered saline buffer solution (washing buffer solution). Add 200 ml of 10x phosphate-buffered saline buffer solution to 1800 ml of purified water. Cool the buffer solution.

[0627] 2. 1 x Phosphate-buffered saline solution with Tween (disability-buffered saline solution). Mix 50 ml of 20 x Phosphate-buffered saline solution with Tween solution with 950 ml of purified water. Mix thoroughly and store at room temperature.

[0628] 3. Attach the reagent retention material to the Dynabeads M-280 Streptavidin beads.

[0629] 4. Coat the antibody onto Dynabeads M-280 Streptavidin beads.

[0630] 5. Perform the swirling rotation and fully reattach the magnetic beads.

[0631] 6. Transfer 5000 microliters (equal to 10 mg of Dynabeads M-280 Streptavidin beads) of Dynabeads M-280 Streptavidin beads into a 5 ml tube of LoBind® protein.

[0632] 7. Use a magnet to interlock the beads on the side of the tube and dispose of the buffer solution for storage.

[0633] 8. Wash the beads by adding 4000 microliters of phosphate-buffered saline solution with Tween and vortexing to re-suspend the beads.

[0634] 9. Use a magnet to interlock the beads on the side of the tube and remove the floating part.

[0635] 10. Repeat the washing again.

[0636] 11. Add 4000 microliters of phosphate-buffered saline solution with Tween and 100 microliters of SBA, Biotinylated Antibody (5 mg / mL) to the washed beads.

[0637] 12. Mix thoroughly and incubate at room temperature, stirring for 60 minutes. Use a magnet to remove any floating portion.

[0638] 13. Wash the beads two more times with phosphate-buffered saline with Tween and resuspend the beads using 5000 microliters of phosphate-buffered saline with Tween to a final concentration of 10 mg / mL in phosphate-buffered saline with Tween.

[0639] 14. The beads can be kept in a phosphate-buffered saline solution with Tween at 4°C.

[0640] 15. 10% acetonitrile in water (diluent). Add 10 ml of acetonitrile to 90 ml of purified water. Mix thoroughly. Store at room temperature.

[0641] 16. 300 mM Calcium Chloride (buffer solution for the assay). Dissolve 333 mg of Calcium Chloride in 10 mL of purified water. Mix thoroughly. Store at room temperature. Divide into equal portions as needed and store at -70°C.

[0642] 17. 50 mM Ammonium Bicarbonate (Basic Buffer Solution). Dissolve 1.98 g of ammonium bicarbonate in 500 mL of purified water. Store at room temperature for one month.

[0643] 18. RapiGest 1 mg / ml solution (natural properties alteration solution).

[0644] 19. Dissolve 50 mg (pre-weighted) of RapiGest powder in 40 mL of 50 mM ammonia bicarbonate and 10 mL of acetonitrile. Prepare small amounts in equal portions and store at -70°C for up to three months.

[0645] 20. 100 mM DTT (reducing solution). Dissolve 154 mg of DTT (MW 154) in 10 mL of purified water. Divide into equal portions as appropriate and store at -70°C.

[0646] 21. 100 mM iodoacetamide (alkylation buffer solution). Dissolve 185 mg of iodoacetamide in 10 mL of 50 mM ammonium bicarbonate solution. Divide into equal portions as appropriate and store at -70°C.

[0647] 22. 10% trifluoroacetic acid (acid buffer solution). Add 5 ml of trifluoroacetic acid to 45 ml of purified water. Whirl to mix thoroughly. Store at room temperature.

[0648] 23. Mobile Phase A: 0.1% formic acid in water (Mobile Phase A). Add 2 mL of formic acid to 2000 mL of purified water. Mix thoroughly. Store at room temperature.

[0649] 24. Mobile Phase B: 0.1% formic acid in acetonitrile (mobile phase B). Add 2 mL of formic acid to 2000 mL of acetonitrile. Mix thoroughly. Store at room temperature.

[0650] 25. Needle Wash: 0.1% formic acid in isopropanol / water 50 / 50 (needle wash). Add 2 ml of formic acid to 1000 ml of isopropanol and 1000 ml of purified water in a glass bottle. Store at room temperature for six months.

[0651] 26. Preparation of standard crude solutions

[0652] Standard Crude Solution (STD) 1: Use a pipette to withdraw 49 µL (8.16 mg / mL) of AGN6567 To 351 microliters of rat K2EDTA plasma to obtain a final concentration of 1 mg / mL for total antibody in rat K2EDTA plasma.

[0653] Standard Crude Solution (STD) 2: Use a pipette to draw 100 microliters of STD crude solution 1 into 900 microliters of rat K2EDTA plasma to obtain a final concentration of 100 micrograms / mL for total antibody in rat K2EDTA plasma.

[0654] Standard Crude Solution (STD) 3: Use a pipette to draw 100 microliters of STD crude solution 2 into 900 microliters of rat K2EDTA plasma to obtain a final concentration of 10 micrograms / mL for total antibody in rat K2EDTA plasma.

[0655] Standard Crude Solution (STD) 4: Use a pipette to draw 100 microliters of STD crude solution 3 into 900 microliters of rat K2EDTA plasma to obtain a final concentration of 1 microgram / mL for total antibody in rat K2EDTA plasma.

[0656] The exact volume of raw solution withdrawn by pipette should be calculated based on the actual antibody concentration of the reference standard solutions.

[0657] The crude solutions STD 1 and 2 are freshly prepared before use and disposed of after use.

[0658] Prepare standard solutions working with rat K2EDTA plasma as detailed in the following table: Table 27 Volume of source solution used (microliters) ID Standard Working Solution ID of the solution source Volume of source solution used (microliters) Add one volume (microliter) of K2EDTA plasma to a rat Total volume (microliters) Total antibody concentration in rat plasma K2EDTA (µg / mL) STD8 STD crude solution 2 80 320 400 20 STD7 STD crude solution 2 64 336 400 16 STD6 STD crude solution 2 48 352 400 12 STD5 STD crude solution 2 32 368 400 8 STD4 STD crude solution 3 80 320 400 2 STD3 STD crude solution 3 16 384 400 0.4 STD2 STD crude solution 4 80 320 400 0.2 STD1 STD crude solution 4 40 360 400 0.1 SST STD crude solution 4 40 360 400 0.1

[0659] Preparation of raw solutions for quality control The exact volume of raw solution withdrawn by pipette should be calculated based on the actual antibody concentration of the standard reference solutions.

[0660] Quality control solution QC Raw 1 Use a pipette to withdraw 49 microliters (8.16 mg / mL) of AGN6567 To 351 microliters of rat K2EDTA plasma to obtain a final concentration of 1 mg / mL for total antibody in rat K2EDTA plasma.

[0661] Quality control solution QC Raw 2 Use a pipette to draw 100 microliters of STD 1 crude solution into 900 microliters of rat K2EDTA plasma to obtain a final concentration of 100 micrograms / mL for total antibody in rat K2EDTA plasma.

[0662] Quality control solution QC ) Raw 3 Use a pipette to draw 100 microliters of STD 2 crude solution into 900 microliters of rat K2EDTA plasma to obtain a final concentration of 10 micrograms / mL for total antibody in rat K2EDTA plasma.

[0663] Quality control solution QC Raw 4 Use a pipette to draw 100 microliters of STD 3 crude solution into 900 microliters of rat K2EDTA plasma to obtain a final concentration of 1 microgram / mL for total antibody in rat K2EDTA plasma.

[0664] Preparation of Quality Control Samples. Quality control (QC) samples are prepared by fixing individual crude QC solutions in K2EDTA plasma from a rat-free environment, as described in the following table: Table 28 Volume of source solution used (microliters) ID Standard Working Solution ID of the solution source Volume of source solution used (microliters) Add one volume (microliter) of K2EDTA plasma to a rat Total volume (microliters) Total antibody concentration in rat plasma K2EDTA (µg / mL) AQL QC Raw QC Solution 1 300 100 100 750 HQC Raw QC Solution 2 60 340 400 15 MQC Raw QC Solution 2 24 376 400 6 LQC Raw QC Solution 3 12 388 400 0.3 LLOQ Raw QC Solution 4 40 360 400 0.1 Only LLOQ samples were used for verification. After preparation, each QC sample was divided into equal portions in individual tubes. QC samples were stored at -70°C. Always store QC samples under the same conditions as the study samples. Preparation of an internal standard solution

[0665] solution SIL ELVA raw Dissolve 1 mg of SIL ELVA in 1 ml of diluent to obtain a concentration of 1 mg / ml of SIL ELVA and store at -70°C.

[0666] solution IS Raw 1 Use a pipette to draw 20 microliters of crude SIL ELVA solution into 1980 microliters of diluent to obtain a final concentration of 10 micrograms / mL of SIL ELVA. Store at -70°C.

[0667] stabilizing IS Use a pipette to draw 50 microliters of crude IS 2 solution down to a total of 10 milliliters of diluent to obtain a final concentration of 50 ng / mL of SIL ELVA. Store at -20°C.

[0668] Extraction procedures Equilibrate the LC-MS / MS system with the mobile phases for at least 60 minutes. Then, dissolve the calibration standard samples, QC samples, unidentified samples, and blank samples in an ice-water bath. Thoroughly vortex the samples.

[0669] 1. Add 10 microliters of each sample (SST and standard solutions) to a 2 mL dish containing 96 cells. Add 10 microliters of each sample (double free, free, QC, and unknown samples).

[0670] 2. Add 35 microliters of 10 mg / mL anti-ID SBA-Biotin coated beads and 15 microliters of 300 mM CaCl2 solution to each eye.

[0671] 3. Add 150 microliters of phosphate-buffered saline with Tween to each eye. Then cover the dish, centrifuge for about 1 minute at 500 rpm, and vortex the dish for 30 seconds.

[0672] 4. Incubate all samples at room temperature for sixty minutes in a vortex stirring medium for an incubation dish at a speed of 1000 rpm.

[0673] 5. Process the plate using the KingFisher™ Flex Magnetic Particle Processor, washing the beads twice with 300 µL of phosphate-buffered saline. Then transfer the beads to a digestion plate containing 100 µL of Rapi-Gest solution.

[0674] 6. Add 10 microliters of 100 mM DTT to the digestion dish. Then centrifuge for 1 minute at 1000 rpm and vortex for 20 seconds.

[0675] 7. Cover the digestion plate with a leak-proof adhesive film and incubate it on a shaking medium at 60°C, 900 rpm for 1 hour. Then briefly centrifuge the plate at 800 rpm.

[0676] 8. Add 12 microliters of 100 mM iodoacetamide to each eye. Then cover the dish with a leak-proof adhesive film. Incubate the dish on a shaking medium in the dark at room temperature for thirty minutes at 1000 rpm.

[0677] 9. To prepare 0.1 mg / mL trypsin, dissolve the required number of modified trypsin tubes from the Sequence Preparation class. Add 150 µL of 50 mM ammonium to each tube (20 µg of trypsin) and mix thoroughly. Combine the contents of all tubes and mix thoroughly. This solution is prepared using a modern method. Approximately ten Sequence Preparation class trypsin tubes are required for a full 96-cell dish. Then, add 20 µL of 0.1 mg / mL trypsin to each cell and centrifuge for 1 minute at 1500 rpm.

[0678] 10. Centrifuge the dish at 4000 rpm for three minutes. Then stop the reaction by adding 10 µL of 10% trifluoroacetic acid. Centrifuge the dish at 1000 rpm for one minute, then shake at 1000 rpm for one minute.

[0679] 11. Add 20 µL of IS fixative solution to each vial except the blank double sample. Add 20 µL of diluent to the blank double sample. Shake the dish at 1000 rpm for 1 minute, then centrifuge at 1000 rpm for 1 minute.

[0680] 12. Inject 15 microliters of the total antibody digestion product into LC-MS / MS.

[0681] Data evaluation The retention time and peak area are determined by the Analyst Data Acquisition / Processing software. Decomposition product concentrations are obtained from a calibration curve generated by plotting the peak area versus concentration ratio. Concentrations are calculated using linear regression according to the following equation: y = ax + b.

[0682] where: y = Peak area ratio of analysis output / internal standard

[0683] a = slope of the corresponding standard curve

[0684] x = Concentration of the decomposition product (µg / mL)

[0685] b = intersection of the corresponding standard curve (with 1 / x) 2 (of weight). Example 12 The antibodies were numbered using a protein numbering kit. AF647

[0686] Numbering results CR007 The yield is 61.5% = (0.14 ml * 4.39 mg / ml) / 1 mg. CR007 concentration = 4.39 / 150,000 = 29.3 µM. AF647 = 79.8 µM. Nomenclature ratio = 79.8 µM / 29.3 µM = 2.72.

[0687] results Numbering 6E9F1 The yield is 60.6% = (0.13 ml * 4.66 mg / ml) / 1 mg. The concentration of 6E9F1 = 4.66 / 150,000 = 31.1 µM. AF647 = 78.0 µM. The numerization ratio = 78.0 µM / 31.1 µM = 2.50.

[0688] Numbering results ABT404 The yield is 60.6% = (0.13 ml * 4.66 mg / ml) / 1 mg. The concentration of 6E9F1 = 4.66 / 150,000 = 31.1 µM. AF647 = 78.0 µM. The numerization ratio = 78.0 µM / 31.1 µM = 2.50.

[0689] Numbering results ABT403 The yield was 58% = (0.25 ml * 2.32 mg / ml) / 1 mg. Concentration ABT403 = 2.32 / 155.031 = 15.0 microMolar. AF647 = 31.0 μM. Numbering ratio = 31.0 µM / 15.0 µM = 2.1.

[0690] Numbering results ABT404 The yield is 59.6% = (0.2 ml * 2.98 mg / ml) / 1 mg. Concentration ABT404 = 2.98 / 144.685 = 20.6 microMolar. AF647 = 40.5 μM. Numbering ratio = 40.5 µM / 20.6 µM = 1.97.

[0691] Numbering results ABT405 The yield is 62.2% = (0.2 ml * 3.11 mg / ml) / 1 mg. Concentration ABT405 = 3.11 / 144.464 = 21.5 microMolar. AF647 = 35.8 μM. Numbering ratio = 35.8 µM / 21.5 µM = 1.67

[0692] Numbering results ABT410 The yield is 55.2% = (0.12 ml * 4.60 mg / ml) / 1 mg. Concentration ABT410 = 4.60 / 144.680 = 31.8 microMolar. AF647 = 30.9 μM. Numbering ratio = 30.9 µM / 31.8 µM = 0.97.

[0693] Numbering results ABT407 The yield is 63.1% = (0.19 ml * 3.32 mg / ml) / 1 mg. Concentration ABT407 = 3.32 / 145.075 = 22.9 microMolar. AF647 = 27.4 microM. Numbering ratio = 27.4 µM / 22.9 µM = 1.2

[0694] Numbering results ABT408 The yield is 56.9% = (0.18 ml * 3.16 mg / ml) / 1 mg. Concentration ABT408 = 3.16 / 144.855 = 21.8 microMolar. AF647 = 27.3 microM. Numbering ratio = 88.1 µM / 23.9 µM = 1.25.

[0695] Numbering results ABT409 The yield is 58.5% = (0.25 ml * 2.34 mg / ml) / 1 mg. ABT409 concentration = 2.34 / 155.421 = 15.1 µM. AF647 = 16.5 µM. The coding ratio = 16.5 µM / 15.1 µM = 1.09.

[0696] results Numbering 6E9F1 The yield is 65.1% = (0.37 ml * 1.76 mg / ml) / 1 mg. The concentration of 6E9F1 in mice is 1.76 / 150,000 = 11.7 µM. AF647 = 48.6 µM. The coding ratio is 48.6 µM / 11.7 µM = 4.15.

[0697] Numbering results LS-C331895 The yield is 20% = (0.21 ml * 0.95 mg / ml) / 1 mg. Concentration ABT403 = 0.95 / 150,000 = 6.3 µM. AF647 = 58.3 µM. Numbering ratio = 58.3 µM / 6.3 µM = 9.25. Compared to other antibodies, the numbering ratio was only 20% (compared to ~56-65% for other antibodies). The numbering ratio is 9.25 (compared to ~0.97-4.15 for other antibodies). Example 13 Biophysical assays . Surface plasmon resonance – results three .

[0698] IGF1 receptor binding to IGF1 via surface plasmon resonance assay (Acro Biosystems)

[0699] The biophysical evaluation of insulin / IGF1 binding to its respective relevant receptors has been demonstrated. Example 14 Biophysical assays . Surface plasmon resonance – Results 4 .

[0700] Surface plasmon resonance assays can be used to measure anti-insulin and anti-proinsulin autoantibodies. An assay was performed to determine the binding affinity of two factors to the insulin receptor and to a human insulin antibody.

[0701] An assay for two TRAP factors was performed in the TR-FRET / SPR assay (ASGPR1). AGN303 (Original proinsulin) to obtain an IC result 50 = 1.2 millimolar. While it led AGN310 (Mutagenic proinsulin) to obtain an IC result 50 = 1.2 millimolar. Table 29 Surface plasmon resonance mAb49 AGN303 (Original insulin precursor) K D = 131 nanomolar AGN310 (Mutational insulin precursor) K D = 174 nanomolar insulin K D = 134 nanomolar AGN310 K D = 558 nanomolar AGN412 It was not disclosed AGN416 K D = 1.1 micromolar

[0702] Mutant insulin maintains a high affinity for mAb49.

[0703] comparison FRET Cell-based (numbered target) AF488 Or numbered with AF647 ) To evaluate a triplet complex on a cell surface. The inventors performed FRET-dependent uptake / lysis of POI (antibody labeled with DL650 and QC-1). Table 30 TR-FRET AGN303 (Original insulin precursor) IC 50 = 1.2 micromolar AGN310 (Mutational insulin precursor) IC 50 = 1.2 micromolar Example 15 Biophysical assays . Surface plasmon resonance – results five .

[0704] Insulin receptor selectivity assays The binding of the insulin receptor to human insulin can be measured via surface plasmon resonance assay.

[0705] It was found that the original proinsulin has a lower affinity for insulin receptors but maintains a similar affinity for insulin-specific antibodies.

[0706] The symmetrical image of the insulin receptor The insulin receptor B differs from the insulin receptor A by including exon 11. Both isoforms have similar affinity for insulin. However, the isoforms exhibit different functional properties. The insulin receptor A shows a higher affinity for IGFs and greater uptake and recycling rate than the insulin receptor B. Due to these differences, the insulin receptor is preferred for signaling metabolism and differentiation. The insulin receptor A primarily functions to promote cell growth, proliferation, and survival. Table 31 Surface plasmon resonance insulin receptor A Insulin receptor B AGN303 (Original insulin precursor) K D = 437 nanomolar K D = 226 nanomolar AGN310 (Mutational insulin precursor) K D = 9.4 micromolar K D = 2.8 micromolar insulin K D = 2.8 micromolar AGN310 K D = 3.7 micromolar AGN412 It was not disclosed AGN416 K D = 7.4 micromolar

[0707] Mutant insulin has a greatly reduced affinity for insulin A receptor and insulin B receptor. Example 16 Biophysical assays . Surface plasmon resonance .

[0708] An assay was also performed for two TRAP factors to determine the binding affinity of TRAP factors to C-peptide-specific antibodies (SPR). AGN406 (Peptide-C with a protease site, peptide-C synthesis degradation factor) to obtain an EC result 50 = 9.8 nanomolar. AGN407 (Peptide-C without the protease site, synthetic peptide-C degradation factor) to obtain an EC result 50 = 12.5 nanomolar. Example 17 In vitro cellular uptake assays – preliminary results

[0709] The inventors performed cellular uptake of insulin-specific antibodies in HEK293 cells.

[0710] mAb49 An antibody that is anti-human.

[0711] 6E9F1 An antibody that is anti-insulin from a mouse. Table 32 Biochemical selectivity (autoantibodies that are anti-insulin) AGN303 AGN310 mAb49 EC 50 = 39.68 nanomolar EC 50 = 67.21 nanomolar 6E9F1 EC 50 = 39.13 nanomolar EC 50 = 37.61 nanomolar mAb49: An antibody that is anti-human insulin 6E9F1: An anti-insulin antibody from a mouse

[0712] Insulin-specific antibody uptake is ASGPR-dependent and insulin receptor-independent.

[0713] This conclusion provides flexibility with antibodies that are anti-human insulin of different types in the design assays.

[0714] Factors ( TRAPs ) for which an assay was performed :

[0715] AGN301 IgG hemolysis factor (comparative sample of reference IgG hemolysis factor). Results: Continuously good positive control of uptake using HEK293-ASGPR cells (clone 3F7) + a rabbit antibody that is anti-human insulin.

[0716] AGN303 Original insulin with a comparison sample of a reference anti-insulin hemolysis factor GN3). A good continuous positive control of uptake using HEK293-ASGPR cells (clone 3F7) + an antibody from a mouse that is anti-human insulin.

[0717] AGN411 (Facility structure for mutation) > AGN408 (Original facility structure) for absorption, despite the presence of a lower signal with either. Neither induced AGN411 (Facility structure for mutation) or AGN408 (Original facility structure) based on absorption.

[0718] AGN304 Structure of an insulin precursor with GN3 (native) at the C-terminus

[0719] AGN305 Structure of an insulin precursor with GN3 (mutant) at the C-terminus

[0720] ABT306 Original Alk-insulin without GN3 (peptide only).

[0721] ABT307 : Insulin-IgG1 Fc profusion, G4S ligand, hIgG1 hinge zone (C > A), LALA-PA.

[0722] ABT308 : Insulin-IgG1 Fc profusion, 5 x G4S ligand, hIgG1 (C > A) hinge region, LALA-PA.

[0723] AGN411 Structure of an insulin progenitor with αGN3 (mutant) at the N-terminus

[0724] AGN412 Synthetic mutagenic insulin using βGN3. In comparison, it contains AGN310 On αGN3.

[0725] AGN413 : (Proinsulin Fc GN3) is also low without absorption.

[0726] AGN414 : Conjugated with proinsulin-Fc ( ABT307- TBT4438 )

[0727] AGN415 : Conjugated with proinsulin-Fc ( ABT308- TBT4438 )

[0728] AGN416 : Synthetic mutagenic insulin using αGN3 on the insulin A chain. In comparison, other synthetic insulin-GN3 compounds are synthesized with GN3 attached to the insulin B chain.

[0729] AGN417 Native insulin precursor with βGN3 on the N-terminus. Table 33 dose AGN414 1 micromolar as maximum concentration, 9 points, 3-fold dilution in 2x DMSO 50 nanomolar Ab from rabbit numbered AF647 dose AGN415 1 micromolar as maximum concentration, 9 points, 3-fold dilution in 2x 370 nanomolar AGN301 50 nanomolar Ab from rabbit numbered AF647 dose AGN414 1 micromolar as maximum concentration, 9 points, 3-fold dilution in 2x DMSO 50 nanomolar Ab from a mouse numbered AF647 dose AGN415 1 micromolar as maximum concentration, 9 points, 3-fold dilution in 2x 370 nanomolar AGN301 50 nanomolar Ab from a mouse numbered AF647

[0730] The rationale behind conducting further assays. Test whether it can be identified ABT307 and ABT308 (The insulin precursor part) is inhibited by either rabbit antibodies or mouse antibodies that are anti-insulin.

[0731] (1) It should be done AGN301 + Ab from a numbered rabbit (CR007) with uptake (and therefore a positive signal). If the proinsulin protein Fc is added, it will compete with AGN301 Therefore, it has a lower signal.

[0732] (2) He does AGN301 +6E9F1 Ab from an absorbance-labeled mouse (therefore, no signal). If the proinsulin fusion protein Fc is added, a signal should be obtained, since AGN301 It binds to the Fc portion of the Fc fusion protein, and Ab from a numbered mouse will bind to the insulin precursor portion.

[0733] ​​(3) The LALA mutation on the Fc proinsulin fusion protein should not interfere with binding to the IgG hemolysis factor, so the LALA mutation should not change the results.

[0734] Dosage range using ABT307 and ABT308 .

[0735] Both the mouse antibody (clonal 6E9F1) and the rabbit anti-insulin antibody (clonal CR007) are used at a constant concentration.

[0736] The test is performed on HEK293-ASGPR (3F7) cells.

[0737] Absorption assay conditions:

[0738] Cell lineages: HEPG2 and HEK293-ASGPR (clonal 3F7, 3C6, 1D4).

[0739] Culture media: DMEM + 10% bovine fetal serum + 1% P / S.

[0740] Cell density: constant at 7.5 kcal / eye.

[0741] Antibodies: Stable at 100 nM (all numbered using AF647). Various concentrations were also tested at 25 nM, 50 nM, and a final 100 nM. Anti-insulin monoclonal rabbit antibody, Sino, catalogue #11038-CR007. Anti-insulin monoclonal mouse antibody, Genscript, catalogue #A01715 (clone 6E9F1) at a numbering ratio of approximately 2.9.

[0742] Factors: AGN301 (Constant at 50 nM). Based on the results, the antibody concentration at 50 nM is sufficient for the absorbance assay. The assay was also tested. AGN301 (fixed at 370 nM).

[0743] Incubation: Incubation is carried out at 37 degrees Celsius for eighteen hours.

[0744] The inventors also carried out an incubation at 37 degrees Celsius for twenty-four hours.

[0745] Reading: Absorption on a high-content imaging device (Operetta)

[0746] When using a rabbit antibody that is anti-insulin (Sino, catalogue #11038-CR007): Rabbit monoclonal antibody + AGN301 (IgG lysis factor) for absorption. It competes AGN301 With the fusion protein IgG Fc in the presence of a rabbit monoclonal antibody. It competes ABT307 and ABT308 with AGN301 When a drop in the signal occurs.

[0747] When using a mouse antibody that is anti-insulin (Genscript, catalogue #A01715 (clonal 6E9F1)): Monoclonal mouse antibody + AGN301 (IgG lysis factor) for low absorption. Allows AGN301 By forming a complex of IgG Fc fusion proteins in the presence of a monoclonal mouse antibody. ABT307 and ABT308 With a mouse antibody that cleaves the insulin precursor protein. Then, it binds AGN301 By cleaving Fc from the Fc protein when included in the signal.

[0748] Conclusion: Both rabbit and mouse anti-insulin antibodies recognize the Fc fusion protein (via cleavage of the insulin precursor).

[0749] Absorption assay: HEK293-ASGPR cells (clonal 3F7). Four replicate compounds using a rabbit antibody that is anti-human insulin, labeled AF647, on HEK293-ASGPR 3F7.

[0750] shows AGN303 and AGN310 Using a rabbit antibody that is anti-human insulin and labeled AF647, it has a significant effect on HEK293-ASGPR 3F7. The results can be replicated compared to n = 1.

[0751] Ab numbered (100 nanomolar). AGN301 (IgG hemolytic agent at 50 nM). Table 34 Absorption assay 370 nanomolar AGN301 0.1% of DMSO 2.50 E+04 370 nanomolar AGN301 0.1% of DMSO 5.00 E+04 370 nanomolar AGN301 0.1% of DMSO 1.00 E+05 370 nanomolar AGN301 0.1% of DMSO 2.00 E+05

[0752] Cells used: HEK293-ASGPR 3F7 +100 nm mol CR007 HEK293 + 100 nm mol CR007, HEK293-ASGPR 3C6 + 100 nm mol CR007 HepG2 + 100 nm mol CR007, HEK293-ASGPR 1D4 + 100 nm mol CR007 HEK293, and HepG2. Table 35 Absorption assay 370 nanomolar AGN301 0.1% of DMSO 2,500 370 nanomolar AGN301 0.1% of DMSO 5,000 370 nanomolar AGN301 0.1% of DMSO 7,500 370 nanomolar AGN301 0.1% of DMSO 10,000

[0753] Cells used: HEK293-ASGPR 3F7 HEK293, HEK293-ASGPR 3C6, HepG2, and HEK293-ASGPR 1D4.

[0754] Conclusions: The results for the anti-insulin agents were as expected. A rabbit (not mouse) antibody can be used as an alternative to an IgG control agent. AGN301 No peptide other than GN3 is shown. ABT306 ) absorption. Anti-insulin agents do not ( AGN304 , AGN305 By absorption.

[0755] The following procedure: Perform an assay for a monoclonal antibody that is anti-C-peptide.

[0756] Results over time. A clear uptake signal was observed for HEK293-ASGPR 3F7, 3C6, and HepG2 S / B cells for HEK293-ASGPR 3F7 and 3C6, which peaks at six hours and lasts up to eighteen hours.

[0757] Based on the results, it shows AGN418 , AGN419 , AGN420 and AGN7865 All of these have good effects on HEK293-ASGPR 3F7 using CR007 and 6E9F1, but they show some effects when using ABT404 .

[0758] Absorption: AGN310 > AGN303 > AGN416 Weak signal with established proinsulin constructs: AGN414 , AGN415 , AGN408 and AGN411 Urge AGN310 For stronger absorption. And it did not encourage AGN301 On absorption.

[0759] Good signal using: AGN417 (Proinsulin βGN3) and insulin structures are associated with GN3 ( AGN303 , AGN310 and AGN416 Weak or no signal using alpha constructs created for proinsulin at the N-terminus: AGN408 and AGN411 Weak or no signal, or using constructs created for βGN3 mutant insulin: AGN412 . Table 36 Absorption assay results antibody Description AGN310 AGN417 ABT403 mAb49 (Original). Creative Biolabs (FAMAB-0225WJ) + + ABT404 mAb49 + + ABT405 mAb49 + + ABT406 Anti-peptide-C (original) - + ABT407 Anti-peptide-C - + ABT408 Anti-peptide-C - + ABT409 Anti-peptide-C - + Ab from a polyploid rabbit (LS-C331895) LSBio (LS- C331895) - + rabbit mAb (CR007) Sino (11038-CR007) +++ ++ mAb from mouse (6E9F1) Genescript (A01715) ++ ++ Example 18 In vitro cellular uptake assays – results two Table 37 Absorption assay The compound antibody AUC EC50-1 (Nano-Molar) EC50-2 (Nano-Molar) TOP-1 TOP-2 cell lineage AGN421 -002 CR007 2622665 26 758 1800 1797 HEK293T / ASGPR 3F7 AGN421 -003 CR007 3109184 20 706 2140 2149 HEK293T / ASGPR 3F7 AGN422 -002 CR007 1716017 35 555 1663 1659 HEK293T / ASGPR 3F7 AGN7849 -001 CR007 1393821 21 617 2148 2134 HEK293T / ASGPR 3F7 AGN7850 -001 CR007 1130832 26 453 2177 2157 HEK293T / ASGPR 3F7 AGN421 -002 6E9F1 780072 31 812 526 522 HEK293T / ASGPR 3F7 AGN421 -003 6E9F1 791142 27 758 571 566 HEK293T / ASGPR 3F7 AGN422 -002 6E9F1 915700 39 826 616 609 HEK293T / ASGPR 3F7 AGN7849 -001 6E9F1 553940 48 864 767 761 HEK293T / ASGPR 3F7 AGN7850 -001 6E9F1 444341 34 549 788 772 HEK293T / ASGPR 3F7 AGN421 -002 ABT404 395313 10 1470 147 148 HEK293T / ASGPR 3F7 AGN421 -003 ABT404 399679 14 551 240 243 HEK293T / ASGPR 3F7 AGN422 -002 ABT404 567260 27 920 368 367 HEK293T / ASGPR 3F7 AGN7849 -001 ABT404 108002 16 704 171 171 HEK293T / ASGPR 3F7 AGN7850 -001 ABT404 109995 14 367 240 240 HEK293T / ASGPR 3F7 AGN421 -002 CR007 2953484 33 992 1556 1542 HEK293T / ASGPR 3F7 AGN421 -003 CR007 2950178 32 943 1621 1605 HEK293T / ASGPR 3F7 AGN422 -002 CR007 1714877 31 625 1594 1583 HEK293T / ASGPR 3F7 AGN7849 -001 CR007 982078 27 789 1478 1451 HEK293T / ASGPR 3F7 AGN7850 -001 CR007 881298 17 584 1467 1456 HEK293T / ASGPR 3F7 AGN421 -002 6E9F1 1146907 31 869 790 783 HEK293T / ASGPR 3F7 AGN421 -003 6E9F1 1124227 30 855 798 790 HEK293T / ASGPR 3F7 AGN422 -002 6E9F1 953794 31 787 711 704 HEK293T / ASGPR 3F7 AGN7849 -001 6E9F1 849739 37 816 1241 1208 HEK293T / ASGPR 3F7 AGN7850 -001 6E9F1 695318 23 552 1197 1180 HEK293T / ASGPR 3F7 AGN421 -002 ABT404 316924 21 713 212 212 HEK293T / ASGPR 3F7 AGN421 -003 ABT404 314685 19 871 207 206 HEK293T / ASGPR 3F7 AGN422 -002 ABT404 335870 17 987 213 212 HEK293T / ASGPR 3F7 AGN7849 -001 ABT404 103418 20 474 194 193 HEK293T / ASGPR 3F7 AGN7850 -001 ABT404 102430 14 367 214 214 HEK293T / ASGPR 3F7 AGN418 CR007 1107853 35 665 1851 1820 HEK293T / ASGPR 3F7 AGN419 CR007 524322 38 375 1157 1142 HEK293T / ASGPR 3F7 AGN420 CR007 891180 36 669 1474 1446 HEK293T / ASGPR 3F7 AGN7865 CR007 984673 29 674 1565 1541 HEK293T / ASGPR 3F7 AGN418 6E9F1 645547 32 608 1083 1067 HEK293T / ASGPR 3F7 AGN419 6E9F1 740253 31 510 1359 1334 HEK293T / ASGPR 3F7 AGN420 6E9F1 662335 35 812 962 943 HEK293T / ASGPR 3F7 AGN7865 6E9F1 578833 30 806 843 824 HEK293T / ASGPR 3F7 AGN418 ABT404 45559 23 336 111 111 HEK293T / ASGPR 3F7 AGN419 ABT404 22181 20 299 65 65 HEK293T / ASGPR 3F7 AGN420 ABT404 43700 23 329 109 109 HEK293T / ASGPR 3F7 AGN7865 ABT404 45586 19 312 117 117 HEK293T / ASGPR 3F7 AGN418 CR007 1215721 24 709 1855 1836 HEK293T / ASGPR 3F7 AGN419 CR007 625856 34 336 1492 1470 HEK293T / ASGPR 3F7 AGN420 CR007 1157693 32 585 2012 1980 HEK293T / ASGPR 3F7 AGN7865 CR007 1131165 29 626 1905 1868 HEK293T / ASGPR 3F7 AGN418 6E9F1 655344 30 602 1109 1089 HEK293T / ASGPR 3F7 AGN419 6E9F1 712479 27 504 1296 1281 HEK293T / ASGPR 3F7 AGN420 6E9F1 630028 35 682 1009 990 HEK293T / ASGPR 3F7 AGN7865 6E9F1 692893 28 738 1041 1023 HEK293T / ASGPR 3F7 AGN418 ABT404 53266 20 305 129 129 HEK293T / ASGPR 3F7 AGN419 ABT404 26822 13 288 70 70 HEK293T / ASGPR 3F7 AGN420 ABT404 52443 17 321 120 121 HEK293T / ASGPR 3F7 AGN7865 ABT404 48139 13 289 119 119 HEK293T / ASGPR 3F7 AGN414 CR007 385583 13 316 917 896 HEK293T / ASGPR 3F7 AGN415 CR007 238771 14 412 486 473 HEK293T / ASGPR 3F7 AGN414 ABT404 44267 3.9 195 134 134 HEK293T / ASGPR 3F7 AGN415 ABT404 43848 4.8 228 119 118 HEK293T / ASGPR 3F7 AGN7525 CR007 501233 7.6 241 1463 1425 HEK293T / ASGPR 3F7 AGN7526 CR007 358780 5.9 295 870 857 HEK293T / ASGPR 3F7 AGN7525 ABT404 79582 4.0 98 354 353 HEK293T / ASGPR 3F7 AGN7526 ABT404 75581 3.8 110 327 325 HEK293T / ASGPR 3F7 AGN417 -003 ABT403 214317 21 321 270 270 HEK293T / ASGPR 3F7 AGN414 CR007 246693 13 276 729 707 HEK293T / ASGPR 3F7 AGN415 CR007 159414 11 315 375 368 HEK293T / ASGPR 3F7 AGN414 ABT404 38012 2.9 204 109 109 HEK293T / ASGPR 3F7 AGN415 ABT404 38428 3.4 277 95 94 HEK293T / ASGPR 3F7 AGN7525 CR007 335865 6 237 999 978 HEK293T / ASGPR 3F7 AGN7526 CR007 256983 5 264 681 671 HEK293T / ASGPR 3F7 AGN7525 ABT404 55657 2.6 113 233 231 HEK293T / ASGPR 3F7 AGN7526 ABT404 52579 2.6 94 251 251 HEK293T / ASGPR 3F7 AGN417 -003 ABT403 68865 30 343 93 94 HEK293T / ASGPR 3F7 AGN417 -003 6E9F1 1566311 33 987 959 950 HEK293T / ASGPR 3F7 AGN310 ABT403 361271 18 635 293 293 HEK293T / ASGPR 3F7 AGN417 ABT403 194965 25 378 288 289 HEK293T / ASGPR 3F7 AGN310 ABT404 820058 15 1039 362 363 HEK293T / ASGPR 3F7 AGN417 ABT404 338043 18 486 303 306 HEK293T / ASGPR 3F7 AGN310 CR007 10854609 20 1797 3300 3271 HEK293T / ASGPR 3F7 AGN417 CR007 2944204 29 825 1896 1888 HEK293T / ASGPR 3F7 AGN310 6E9F1 3129671 13 573 2482 2481 HEK293T / ASGPR 3F7 AGN417 6E9F1 1878996 26 878 1252 1242 HEK293T / ASGPR 3F7 AGN310 ABT405 186011 17 508 162 163 HEK293T / ASGPR 3F7 AGN417 ABT405 119172 21 377 149 151 HEK293T / ASGPR 3F7 AGN310 ABT406 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT406 188477 21 367 255 257 HEK293T / ASGPR 3F7 AGN310 ABT407 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT407 231673 31 347 410 409 HEK293T / ASGPR 3F7 AGN310 ABT408 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT408 247710 27 342 437 436 HEK293T / ASGPR 3F7 AGN310 ABT409 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT409 113737 29 324 232 232 HEK293T / ASGPR 3F7 AGN310 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN417 LSC331895 339797 17 749 253 250 HEK293T / ASGPR 3F7 AGN310 ABT405 200141 19 604 155 156 HEK293T / ASGPR 3F7 AGN417 ABT405 119762 21 394 153 155 HEK293T / ASGPR 3F7 AGN310 ABT406 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT406 186130 21 393 248 249 HEK293T / ASGPR 3F7 AGN310 ABT407 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT407 193310 30 361 330 330 HEK293T / ASGPR 3F7 AGN310 ABT408 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT408 226991 32 331 431 428 HEK293T / ASGPR 3F7 AGN310 ABT409 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT409 132062 28 343 245 245 HEK293T / ASGPR 3F7 AGN310 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN417 LSC331895 278697 16 593 250 248 HEK293T / ASGPR 3F7 AGN310 ABT403 632102 24 615 528 525 HEK293T / ASGPR 3F7 AGN417 ABT403 296613 22 365 452 454 HEK293T / ASGPR 3F7 AGN310 ABT404 10314457 19 741 366 368 HEK293T / ASGPR 3F7 AGN417 ABT404 3263784 16 560 334 336 HEK293T / ASGPR 3F7 AGN310 CR007 10314457 24 1747 3226 3190 HEK293T / ASGPR 3F7 AGN417 CR007 3263784 27 790 2188 2178 HEK293T / ASGPR 3F7 AGN310 6E9F1 3194185 15 651 2378 2374 HEK293T / ASGPR 3F7 AGN417 6E9F1 2247463 26 837 1491 1482 HEK293T / ASGPR 3F7 AGN412 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN417 FAMAB-0225WJ 358633 24 410 513 516 HEK293T / ASGPR 3F7 AGN408 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN411 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN412 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN417 FAMAB-0225WJ 212698 21 339 392 393 HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN303 FAMAB-0225WJ 262910 11 340 482 481 HEK293T / ASGPR 3F7 AGN310 FAMAB-0225WJ 571517 21 581 554 553 HEK293T / ASGPR 3F7 AGN416 FAMAB-0225WJ 364773 22 546 413 411 HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN303 FAMAB-0225WJ 213580 15 350 362 350 HEK293T / ASGPR 3F7 AGN310 FAMAB-0225WJ 447435 24 632 407 405 HEK293T / ASGPR 3F7 AGN416 FAMAB-0225WJ 281288 26 534 327 325 HEK293T / ASGPR 3F7 AGN414 FAMAB-0225WJ 52049 3.4 190 177 175 HEK293T / ASGPR 3F7 AGN415 FAMAB-0225WJ 66152 4.6 177 236 232 HEK293T / ASGPR 3F7 AGN408 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN411 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN412 CR007 1273315 142 4434 239 238 HEK293T / ASGPR 3F7 AGN416 CR007 5257312 28 673 3323 3308 HEK293T / ASGPR 3F7 AGN412 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN416 6E9F1 3634412 16 491 3177 3196 HEK293T / ASGPR 3F7 AGN412 CR007 2536472 145 4085 505 500 HEK293T / ASGPR 3F7 AGN416 CR007 9316249 18 1058 4455 4409 HEK293T / ASGPR 3F7 AGN412 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN416 6E9F1 6625680 15 564 5535 5543 HEK293T / ASGPR 3F7 AGN414 CR007 422068 6.0 283 1069 1050 HEK293T / ASGPR 3F7 AGN415 CR007 457281 9.2 289 1164 1136 HEK293T / ASGPR 3F7 AGN414 6E9F1 535352 4.0 269 1538 1517 HEK293T / ASGPR 3F7 AGN415 6E9F1 667341 6.7 277 1819 1784 HEK293T / ASGPR 3F7 AGN414 LSC331895 72589 1.3 59 518 520 HEK293T / ASGPR 3F7 AGN415 LSC331895 76247 1.4 70 505 506 HEK293T / ASGPR 3F7 AGN303 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN301 LSC331895 4398965 8.1 759 2565 2552 HEK293T / ASGPR 3F7 AGN310 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN304 LSC331895 1990188 34 939 2754 2690 HEK293T / ASGPR 3F7 AGN305 LSC331895 1083287 18 494 1980 1957 HEK293T / ASGPR 3F7 AGN301 LSC331895 4210248 9.4 650 2742 2756 HEK293T / ASGPR 3F7 AGN408 LSC331895 957957 34 760 1430 1405 HEK293T / ASGPR 3F7 AGN411 LSC331895 - 130 - 905 - HEK293T / ASGPR 3F7 AGN413 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN301 LSC331895 3574449 8.3 640 2247 2251 HEK293T / ASGPR 3F7 AGN414 FAMAB-0225WJ-F(E) 58283 4.3 433 109 108 HEK293T / ASGPR 3F7 AGN415 FAMAB-0225WJ-F(E) 65880 5.2 359 138 137 HEK293T / ASGPR 3F7 AGN303 FAMAB-0225WJ-F(E) 307229 10 655 200 200 HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN310 FAMAB-0225WJ-F(E) 214123 11 507 166 167 HEK293T / ASGPR 3F7 AGN304 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN305 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN408 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN411 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN413 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN414 CR007 219652 8.8 253 626 611 HEK293T / ASGPR 3F7 AGN415 CR007 254387 9.9 308 619 605 HEK293T / ASGPR 3F7 AGN414 6E9F1 434915 4.2 267 1139 1105 HEK293T / ASGPR 3F7 AGN415 6E9F1 478977 8.5 280 1250 1202 HEK293T / ASGPR 3F7 AGN301 CR007 7465550 17 690 4786 4785 HEK293T / ASGPR 3F7 AGN408 CR007 - 70 - 98 - HEK293T / ASGPR 3F7 AGN411 CR007 - 127 - 425 - HEK293T / ASGPR 3F7 AGN413 CR007 - 45 - 38 - HEK293T / ASGPR 3F7 AGN301 6E9F1 1687786 12 422 1828 1827 HEK293T / ASGPR 3F7 AGN408 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN411 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN413 6E9F1 - - - - - HEK293T / ASGPR 3F7 ABT307 CR007 - - - - - HEK293T / ASGPR 3F7 ABT308 CR007 - - 2.6 - 1210 HEK293T / ASGPR 3F7 ABT307 6E9F1 818081 0.34 213 1976 1972 HEK293T / ASGPR 3F7 ABT308 6E9F1 948577 0.61 137 2704 2704 HEK293T / ASGPR 3F7 AGN303 CR007 17025436 38 1451 6582 6547 HEK293T / ASGPR 3F7 AGN310 CR007 16219273 22 1478 5796 5717 HEK293T / ASGPR 3F7 AGN304 CR007 - - - - - HEK293T / ASGPR 3F7 AGN305 CR007 1383077 42 2030 541 526 HEK293T / ASGPR 3F7 AGN303 CR007 183786 17 167 64 64 HepG2 AGN310 CR007 228091 53 375 86 86 HepG2 AGN304 CR007 - - - - - HepG2 AGN305 CR007 263578 27 1178 55 57 HepG2 AGN301 6E9F1 - - - - - HepG2 AGN303 6E9F1 2208088 51 705 197 197 HepG2 AGN310 6E9F1 2442025 48 554 249 250 HepG2 ABT306 6E9F1 - - - - - HepG2 AGN304 6E9F1 - - - - - HepG2 AGN305 6E9F1 - - - - - HepG2 AGN301 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN303 6E9F1 5582416 22 784 5067 4993 HEK293T / ASGPR 3F7 AGN310 6E9F1 2900143 16 593 5825 5819 HEK293T / ASGPR 3F7 ABT306 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN304 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN305 6E9F1 1363009 8.2 1818 358 350 HEK293T / ASGPR 3F7 AGN402 CR007 - - - - - HepG2 AGN401 CR007 - - - - - HepG2 AGN403 CR007 - - - - - HepG2 AGN404 CR007 - - - - - HepG2 AGN303 CR007 - - - - - HepG2 AGN310 CR007 139533 53 364 56 56 HepG2 AGN304 CR007 - - - - - HepG2 AGN305 CR007 - - - - - HepG2 AGN402 CR007 - - - - - HEK293T / ASGPR 3F7 AGN401 CR007 - - - - - HEK293T / ASGPR 3F7 AGN403 CR007 - - - - - HEK293T / ASGPR 3F7 AGN404 CR007 - - - - - HEK293T / ASGPR 3F7 AGN303 CR007 22761749 32 1599 8595 8482 HEK293T / ASGPR 3F7 AGN310 CR007 25196241 16 1868 7869 7824 HEK293T / ASGPR 3F7 AGN304 CR007 - 71 - 137 - HEK293T / ASGPR 3F7 AGN305 CR007 1730240 28 2526 511 502 HEK293T / ASGPR 3F7 AGN4006 CR007 1488243 83 1341 796 798 HepG2 AGN301 CR007 4004648 74 1266 2613 2619 HepG2 AGN4006 T24 2179897 25 859 30 30 HepG2 AGN301 T24 2004469 39 818 138 138 HepG2 The density of cells inducing crystallization is 7500 cells / eye. Incubation period is eighteen hours. Example 19 Trigonal Complex Formation Assay - Initial results.

[0760] Trivalent Complex Formation Assay (Hep2)

[0761] 1. Two antibodies are numbered using AF647. Antibody numbering is fixed at 100 nanomolar.

[0762] An anti-insulin (IgG) antibody, a rabbit antibody to "T24". Numbering ratio = 0.49.

[0763] The antibody was an anti-insulin (IgG) monoclonal antibody from a rabbit, designated "CR007". Numbering ratio = 2.7.

[0764] 2. To assay the formation of a ternary complex in order to develop and validate the assay, two IgG factors were tested ( AGN4006 , AGN301 ) as reference compounds with two numbered antibodies from rabbits.

[0765] Tested Trace Agents (TRAPs):

[0766] AGN301 : IgG lysis factor (reference molecule). AGN301 + CR007 and AGN301 + T24.

[0767] AGN4006 : IgG lysis factor (reference molecule). AGN4006 + CR007 and AGN4006 + T24.

[0768] AGN302 : Mutant insulin with GN3.

[0769] AGN303 Original insulin with GN3

[0770] AGN304 Original insulin precursor

[0771] AGN305 Mutant insulin precursor

[0772] ABT306 Original Alk-insulin without GN3 (peptide only).

[0773] Logical reasoning. Testing whether it can be identified ABT307 and ABT308 (The proinsulin component) is inhibited by either rabbit antibodies or mouse antibodies that are anti-insulin.

[0774] (1) Constitutes AGN301 The +Ab from a numbered rabbit (CR007) forms a triplet complex, hence the positive signal. If the proinsulin Fc protein is added, it will compete with AGN301 Therefore, there is less signal.

[0775] (2) will not form AGN301 Adding the 6E9F1 Ab from a numbered mouse forms a triplet complex, therefore there will be no signal. If the proinsulin fusion protein Fc is added to the above, a signal should be obtained, since AGN301 It binds to the Fc portion of the Fc fusion protein, and the Ab from a numbered mouse binds to the insulin precursor.

[0776] (3) The LALA mutation on the insulin pro-fusion protein Fc should not interfere with the binding of IgG hemolytic factor, so the LALA mutation should not change the results.

[0777] Conditions for assaying the formation of the ternary complex :

[0778] cell lineages : HepG2 or HEK293-ASGPR (clonal 3F7).

[0779] growing media : DMEM + 10% of bovine fetal serum + 1% of P / S.

[0780] Antibodies (All are numbered using AF647).

[0781] Anti-insulin monoclonal rabbit antibody, Sino, Cat#11038-CR007. Numbering ratio = 2.8

[0782] Anti-insulin monoclonal mouse antibody, Genscript, Cat#A01715 (clonal 6E9F1). Numbering ratio = approximately 2.9.

[0783] Factors for which assays were performed: high concentration 10 µM, 3-fold dilution solutions, 9-point curve. In some assays, an assay was performed for AGN301 , fixed at 50 nM.

[0784] Reading: Flow cytometry (IntelliCyt iQue Screener Plus).

[0785] Results: Work AGN301 + CR007 is working well.

[0786] 100 nM will be used AGN301 With CR007 numbered as a comparison sample in the additional assays

[0787] Anti-insulin agents work as expected.

[0788] A rabbit monoclonal antibody can be used, but not a mouse monoclonal antibody for an IgG control factor. AGN301 .

[0789] Non-GN3 peptide ABT306 It did not show the formation of a triple complex or complex with antibodies that are anti-insulin.

[0790] A rabbit monoclonal antibody was shown + AGN301 (IgG lysis factor) forms a strong triple complex. It competes ABT307 and ABT308 with AGN301 When a drop in the signal occurs.

[0791] A monoclonal mouse antibody showed + AGN301 (IgG lysis factor) forms a weak triple complex. It binds ABT307 and ABT308 From mouse Ab, via a cleavage of the insulin precursor protein. Then it binds AGN301 By cleaving Fc from the protein Fc

[0792] Conclusion: Both a rabbit antibody and a mouse antibody that are anti-insulin recognize the Fc fusion protein via cleavage of the insulin precursor. Table 38 Triple complex formation: HepG2 cells AGN301 (10 micromolar maximum, 3 times, 9 points). 1% DMSO AGN303 (10 micromolar maximum, 3 times, 9 points). 370 nanomolar AGN301 AGN310 (10 micromolar maximum, 3 times, 9 points) ABT306 (10 micromolar maximum, 3 times, 9 points) AGN304 (10 micromolar maximum, 3 times, 9 points). 1% DMSO AGN305 (10 micromolar maximum, 3 times, 9 points). 370 nanomolar AGN301 Table 39 Triple complex structure: HEK293-ASGPR (clonal 3F7) AGN301 (10 micromolar maximum, 3 times, 9 points). 1% DMSO AGN303 (10 micromolar maximum, 3 times, 9 points). 370 nanomolar AGN301 AGN310 (10 micromolar maximum, 3 times, 9 points) ABT306 (10 micromolar maximum, 3 times, 9 points) AGN304 (10 micromolar maximum, 3 times, 9 points). 1% DMSO

[0793] shows AGN303 and AGN310 In addition, the mouse antibody that is anti-human insulin has little effect on HepG2.

[0794] It was for AGN301 The effect of using the antibody from a rabbit that is anti-human insulin, but no effect of using the mouse antibody that is anti-human insulin on HepG2.

[0795] It was for AGN301 In addition to the rabbit antibody that is anti-human insulin, a good triple complex is formed.

[0796] It was for AGN303 and AGN310 In addition to the rabbit antibody that is anti-human insulin, a weak triple complex is formed.

[0797] It was for AGN303 and AGN310 In addition to the mouse antibody that is anti-human insulin, a good triple complex is formed.

[0798] It was for AGN301 In addition to the mouse antibody that is anti-human insulin, a weak triple complex is formed, which is compatible with mouse Ab and IgG hemolysis factor.

[0799] The data were consistent with the results of the absorption assay.

[0800] was for AGN3764 , AGN303 and AGN310 In addition to an antibody from a rabbit that is anti-human insulin, a good triple complex is formed.

[0801] The data were consistent with the results of the absorption assay.

[0802] Of the tested compounds, none showed any effect when using either the human or rabbit antibody against HEK293-ASGPR 3F7. AGN301 Effect using an antibody from a rabbit.

[0803] Rabbit monoclonal antibody: AGN415 > AGN414 In a triple complex. Mouse monoclonal antibody: AGN415 > AGN414 In the formation of a triple complex. Both rabbit and mouse monoclonal antibodies work well with proinsulin-Fc conjugates.

[0804] Shows AGN414 and AGN415 Good effects were observed in HEK293-ASGPR 3F7 using an Arbet antibody (CR007) and a mouse antibody, which is consistent with the results of the absorption assay. Example 20 Tertiary Complex Formation Assays – Second Results .

[0805] Biophysical assays An assay for a tri-complex of TRAP factors was performed using a method previously described in this specification using fixed HepG2 or HEK293 cells. Table 40 Trigonal Complex Formation Assay The compound antibody AUC EC 50 -1 (nanomolar) EC 50 -2 (nanomolar) TOP-1 TOP-2 cell lineage AGN421 -002 CR007 293996623 11 481 250851 250929 HEK293T / ASGPR 3F7 AGN422 -002 CR007 149504588 11 343 218392 217739 HEK293T / ASGPR 3F7 AGN7849 -001 CR007 92134277 9.9 294 225017 222258 HEK293T / ASGPR 3F7 AGN7850 -001 CR007 63233426 6.7 185 215949 213209 HEK293T / ASGPR 3F7 AGN421 -002 6E9F1 62015311 9.5 283 113461 112331 HEK293T / ASGPR 3F7 AGN422 -002 6E9F1 51986270 7.7 233 114977 114728 HEK293T / ASGPR 3F7 AGN7849 -001 6E9F1 44497667 7.2 210 138543 137802 HEK293T / ASGPR 3F7 AGN7850 -001 6E9F1 266432227 4 137 104729 105083 HEK293T / ASGPR 3F7 AGN421 -002 ABT404 21775507 7.5 418 18396 18244 HEK293T / ASGPR 3F7 AGN422 -002 ABT404 11922865 5.7 196 13935 13969 HEK293T / ASGPR 3F7 AGN7849 -001 ABT404 2319845 5.9 93 12523 12560 HEK293T / ASGPR 3F7 AGN7850 -001 ABT404 1718671 3.4 125 8749 8721 HEK293T / ASGPR 3F7 AGN421 -002 CR007 414532586 16 584 338604 339211 HEK293T / ASGPR 3F7 AGN422 -002 CR007 250067753 13 377 301596 302996 HEK293T / ASGPR 3F7 AGN7849 -001 CR007 146091629 13 393 294267 292373 HEK293T / ASGPR 3F7 AGN7850 -001 CR007 101565767 8.5 246 276637 272258 HEK293T / ASGPR 3F7 AGN421 -002 6E9F1 124714692 11 332 195257 194754 HEK293T / ASGPR 3F7 AGN422 -002 6E9F1 87333279 9.7 238 185075 183965 HEK293T / ASGPR 3F7 AGN7849 -001 6E9F1 85719115 9.8 292 211700 208538 HEK293T / ASGPR 3F7 AGN7850 -001 6E9F1 52298223 6.9 162 185971 184974 HEK293T / ASGPR 3F7 AGN421 -002 ABT404 37288078 10 347 35562 35772 HEK293T / ASGPR 3F7 AGN422 -002 ABT404 21324694 5.3 184 25364 25846 HEK293T / ASGPR 3F7 AGN7849 -001 ABT404 7404549 5.5 178 22456 22673 HEK293T / ASGPR 3F7 AGN7850 -001 ABT404 5021407 2.8 115 18175 18427 HEK293T / ASGPR 3F7 AGN418 CR007 74026938 11 332 163550 161947 HEK293T / ASGPR 3F7 AGN419 CR007 36504926 13 235 112447 110712 HEK293T / ASGPR 3F7 AGN420 CR007 87104306 13 376 180377 178875 HEK293T / ASGPR 3F7 AGN7865 CR007 76049321 11 331 168367 166821 HEK293T / ASGPR 3F7 AGN418 6E9F1 31526124 10 218 99502 98099 HEK293T / ASGPR 3F7 AGN419 6E9F1 42435459 9.5 212 136707 134692 HEK293T / ASGPR 3F7 AGN420 6E9F1 43164894 9.8 252 116346 114812 HEK293T / ASGPR 3F7 AGN7865 6E9F1 35874436 9.4 223 107247 106164 HEK293T / ASGPR 3F7 AGN418 ABT404 2399627 5.7 139 8471 8512 HEK293T / ASGPR 3F7 AGN419 ABT404 1891383 6.1 140 8370 8391 HEK293T / ASGPR 3F7 AGN420 ABT404 3600963 6.7 188 10072 10202 HEK293T / ASGPR 3F7 AGN7865 ABT404 2622499 5.2 150 8911 8990 HEK293T / ASGPR 3F7 AGN418 CR007 65604385 14 345 142558 141972 HEK293T / ASGPR 3F7 AGN419 CR007 34318466 15 281 89466 88856 HEK293T / ASGPR 3F7 AGN420 CR007 69601410 15 366 146427 146045 HEK293T / ASGPR 3F7 AGN7865 CR007 62018359 13 337 135526 134793 HEK293T / ASGPR 3F7 AGN418 6E9F1 26236485 11 255 73464 72453 HEK293T / ASGPR 3F7 AGN419 6E9F1 33897945 11 224 104556 103109 HEK293T / ASGPR 3F7 AGN420 6E9F1 27844605 11 240 80480 79383 HEK293T / ASGPR 3F7 AGN7865 6E9F1 26883707 9.9 245 76034 74952 HEK293T / ASGPR 3F7 AGN418 ABT404 1903693 5.2 167 6112 5991 HEK293T / ASGPR 3F7 AGN419 ABT404 1476232 4.6 155 5595 5489 HEK293T / ASGPR 3F7 AGN420 ABT404 2365149 4.7 236 6094 6040 HEK293T / ASGPR 3F7 AGN7865 ABT404 2601915 5.2 228 6659 6625 HEK293T / ASGPR 3F7 AGN414 CR007 34940126 3.0 291 83778 82557 HEK293T / ASGPR 3F7 AGN415 CR007 24104416 2.2 336 51457 51301 HEK293T / ASGPR 3F7 AGN414 ABT404 5981406 0.9 183 16468 16449 HEK293T / ASGPR 3F7 AGN415 ABT404 4642357 1.1 209 12323 12245 HEK293T / ASGPR 3F7 AGN7525 CR007 31988735 1.9 229 90757 90120 HEK293T / ASGPR 3F7 AGN7526 CR007 27207269 1.3 297 62720 62751 HEK293T / ASGPR 3F7 AGN7525 ABT404 5845906 0.8 77 21629 21970 HEK293T / ASGPR 3F7 AGN7526 ABT404 6052835 0.8 121 18515 18648 HEK293T / ASGPR 3F7 AGN414 CR007 24873069 2.9 310 56451 56172 HEK293T / ASGPR 3F7 AGN415 CR007 17761448 2.7 382 35264 35328 HEK293T / ASGPR 3F7 AGN414 ABT404 3740636 1.0 159 11297 11332 HEK293T / ASGPR 3F7 AGN415 ABT404 2995554 1.0 222 7688 7681 HEK293T / ASGPR 3F7 AGN7525 CR007 27194361 2.1 288 64259 64051 HEK293T / ASGPR 3F7 AGN7526 CR007 20910871 1.6 320 46139 46187 HEK293T / ASGPR 3F7 AGN7525 ABT404 3519935 0.86 77 13917 14103 HEK293T / ASGPR 3F7 AGN7526 ABT404 3481239 0.80 108 11846 11923 HEK293T / ASGPR 3F7 AGN417 -003 ABT403 24533574 6.5 145 15511 15856 HEK293T / ASGPR 3F7 AGN417 -003 CR007 106446754 14 317 149981 149579 HEK293T / ASGPR 3F7 AGN310 CR007 180595640 3.8 536 129400 130336 HEK293T / ASGPR 3F7 AGN417 CR007 44541924 16 348 76014 76235 HEK293T / ASGPR 3F7 AGN310 6E9F1 34158932 6.7 161 109076 109330 HEK293T / ASGPR 3F7 AGN417 6E9F1 24504831 13 201 43090 42951 HEK293T / ASGPR 3F7 AGN310 ABT405 3572460 2.1 93 4665 4750 HEK293T / ASGPR 3F7 AGN417 ABT405 7233146 5.9 84 1652 1648 HEK293T / ASGPR 3F7 AGN417 ABT406 6329386 11 139 17209 17293 HEK293T / ASGPR 3F7 AGN417 ABT407 6523747 13 148 21660 21737 HEK293T / ASGPR 3F7 AGN417 ABT408 5787950 12 154 17345 17392 HEK293T / ASGPR 3F7 AGN417 ABT409 3009320 13 135 9896 9934 HEK293T / ASGPR 3F7 AGN417 LSC331895 15734490 3.9 131 14373 14374 HEK293T / ASGPR 3F7 AGN310 ABT403 18730287 8.1 262 30999 30978 HEK293T / ASGPR 3F7 AGN417 ABT403 5119973 12 160 11473 11396 HEK293T / ASGPR 3F7 AGN310 ABT404 17434080 5.9 278 28143 28278 HEK293T / ASGPR 3F7 AGN417 ABT404 6188478 7.6 160 9838 9926 HEK293T / ASGPR 3F7 AGN310 CR007 375880365 4.4 870 18436 181504 HEK293T / ASGPR 3F7 AGN417 CR007 63662337 14 339 101617 101846 HEK293T / ASGPR 3F7 AGN310 6E9F1 75696622 8.3 193 178103 178137 HEK293T / ASGPR 3F7 AGN417 6E9F1 33647655 12 213 65729 65562 HEK293T / ASGPR 3F7 AGN310 ABT405 4865364 3.1 204 6226 6262 HEK293T / ASGPR 3F7 AGN417 ABT405 5047547 5.1 125 2522 2567 HEK293T / ASGPR 3F7 AGN310 ABT406 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT406 7281303 9.9 124 17333 17446 HEK293T / ASGPR 3F7 AGN310 ABT407 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT407 13365856 15 172 36832 36918 HEK293T / ASGPR 3F7 AGN310 ABT408 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT408 11176078 15 169 35586 35681 HEK293T / ASGPR 3F7 AGN310 ABT409 - - - - - HEK293T / ASGPR 3F7 AGN417 ABT409 8134806 14 163 21271 21320 HEK293T / ASGPR 3F7 AGN310 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN417 LSC331895 39720375 6.7 134 24728 24844 HEK293T / ASGPR 3F7 AGN412 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN417 FAMAB-0225WJ 14178555 8.9 142 9221 9300 HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ 22511961 33 1475 9696 9509 HEK293T / ASGPR 3F7 AGN303 FAMAB-0225WJ 17307458 20 1013 10553 10347 HEK293T / ASGPR 3F7 AGN310 FAMAB-0225WJ 38255407 30 1018 19458 19560 HEK293T / ASGPR 3F7 AGN416 FAMAB-0225WJ 19267427 40 961 12692 12493 HEK293T / ASGPR 3F7 AGN408 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN411 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN412 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN417 FAMAB-0225WJ 9618328 14 317 7363 7331 HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ 39925358 5.8 954 19051 19023 HEK293T / ASGPR 3F7 AGN303 FAMAB-0225WJ 27369755 4.6 544 23801 23822 HEK293T / ASGPR 3F7 AGN310 FAMAB-0225WJ 60405203 10 682 47435 47110 HEK293T / ASGPR 3F7 AGN416 FAMAB-0225WJ 29836325 14 507 32582 32404 HEK293T / ASGPR 3F7 AGN414 FAMAB-0225WJ 5683938 0.22 513 9922 9868 HEK293T / ASGPR 3F7 AGN415 FAMAB-0225WJ 4942184 0.49 277 11567 11276 HEK293T / ASGPR 3F7 AGN408 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN411 FAMAB-0225WJ - - - - - HEK293T / ASGPR 3F7 AGN412 CR007 76458748 34 2819 18373 18387 HEK293T / ASGPR 3F7 AGN416 CR007 440252448 8.6 743 247344 248164 HEK293T / ASGPR 3F7 AGN412 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN416 6E9F1 260400828 11 365 310324 309723 HEK293T / ASGPR 3F7 AGN412 CR007 132707886 26 2116 38640 38786 HEK293T / ASGPR 3F7 AGN416 CR007 539139182 7.6 556 395230 397595 HEK293T / ASGPR 3F7 AGN412 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN416 6E9F1 358272147 9.9 309 467454 466336 HEK293T / ASGPR 3F7 AGN414 CR007 27222580 3.0 361 56188 55819 HEK293T / ASGPR 3F7 AGN415 CR007 33152013 3.4 394 65080 65165 HEK293T / ASGPR 3F7 AGN414 6E9F1 37571635 5.0 279 97248 95351 HEK293T / ASGPR 3F7 AGN415 6E9F1 41465544 5.7 278 103211 101093 HEK293T / ASGPR 3F7 AGN414 CR007 7865113 3.8 302 17937 18141 HEK293T / ASGPR 3F7 AGN415 CR007 7499782 4.1 265 20679 20480 HEK293T / ASGPR 3F7 AGN414 6E9F1 9225413 6.7 268 28064 27745 HEK293T / ASGPR 3F7 AGN415 6E9F1 13349047 4.4 274 35099 34485 HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN303 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN310 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN304 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN301 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN305 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN408 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN411 FAMAB-0225WJ-F(E) - - - - - HEK293T / ASGPR 3F7 AGN301 LSC331895 116665345 7.1 548 97872 97904 HEK293T / ASGPR 3F7 AGN303 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN310 LSC331895 - - - - - HEK293T / ASGPR 3F7 AGN304 LSC331895 8520959 17 712 13725 13308 HEK293T / ASGPR 3F7 AGN301 LSC331895 97854424 7.2 508 92885 93125 HEK293T / ASGPR 3F7 AGN305 LSC331895 3431920 9.1 331 9395 9050 HEK293T / ASGPR 3F7 AGN408 LSC331895 2198792 14 202 7794 7676 HEK293T / ASGPR 3F7 AGN411 LSC331895 2319326 10 473 4309 4142 HEK293T / ASGPR 3F7 AGN301 CR007 25207429 15 389 30858 31101 HEK293T / ASGPR 3F7 AGN408 CR007 - - - - - HEK293T / ASGPR 3F7 AGN411 CR007 - - - - - HEK293T / ASGPR 3F7 AGN413 CR007 - - - - - HEK293T / ASGPR 3F7 AGN301 6E9F1 25974715 21 294 17723 17742 HEK293T / ASGPR 3F7 AGN408 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN411 6E9F1 - - - - - HEK293T / ASGPR 3F7 AGN413 6E9F1 - - - - ...

Claims

1. A composition of matter (agent) comprising:a target-binding moiety that binds to an anti-insulin antibody,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 target-binding moiety and the cellular receptor-binding moiety.

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

3. The composition of matter of Claim 1, wherein the composition of matter is selected from the group consisting of AGN302, AGN304, AGN305, AGN310, AGN4002, AGN4003, AGN4004, AGN4006, AGN4007, AGN401, AGN402, AGN405, AGN406, AGN407, AGN408, AGN409, AGN410, AGN411, AGN412, AGN413, AGN414, AGN415, AGN416, AGN417, AGN418, AGN419, AGN420, AGN421, AGN422, AGN423, AGN6567, AGN6601, AGN7525, AGN7526, AGN7849, AGN7850, AGN7865, AGN8123, AGN8138, AGN8139, AGN8140, AGN8141, and AGN2640.

4. The composition of matter of Claim 1, wherein the target-binding moiety is an anti-insulin antibody-binding protein, an anti-insulin antibody-binding protein variant, or an anti-insulin antibody-binding fragment thereof.

5. The composition of matter of Claim 1, wherein the target-binding moiety is an insulin, an insulin variant, an insulin analogue, or an insulin fragment thereof.

6. The composition of matter of Claim 1, wherein the target-binding moiety is a proinsulin, a proinsulin variant, a proinsulin analogue, or a proinsulin fragment thereof.

7. The composition of matter of Claim 1, wherein the target-binding moiety is made by chemical synthesis.

8. The composition of matter of Claim 1, wherein the target-binding moiety is made by genetic engineering technology.

9. The composition of matter of Claim 1, wherein the target-binding moiety is an insulin-Fc fusion protein.

10. The composition of matter of Claim 1, wherein the target-binding moiety is selected from the group consisting of ABT306, ABT307, ABT308, ABT309, ABT311, ABT311, ABT312, ABT313, ABT314, ABT315, ABT401, ABT403, ABT404, ABT405, ABT406, ABT407, ABT408, ABT409, ABT410, ABT411, ABT412, ABT413, ABT414, ABT415, ABT416, ABT417, ABT418, ABT419, ABT420, ABT421, ABT422, ABT423, ABT424, ABT6396, ABT7714, ABT17384, ABT17385, and ABT17386.

11. The composition of matter of Claim 1, wherein the cellular receptor-binding moiety comprises a compound having a structure selected from the group consisting of:or ;where RA is a C1-C3 alkyl group 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, -O-(CH2)IM, S-(CH2)IM, NRM-(CH2)IM, C(O)-(CH2)IM-, a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH2)IMNRM group (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues); andZB is absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)IM-NRM.

12. The composition of matter of Claim 1, wherein the cellular receptor-binding moiety comprises N-acetyl-D-galactosamine.

13. The composition of matter of Claim 1, wherein the cellular receptor-binding moiety is selected from the group consisting of TBT103, TBT104, TBT402, TBT403, TBT404, TBT405 , TBT6170, TBT6171, TBT4438, TBT501, TBT502, TBT7990, TBT7991, TBT7988, and TBT7989.

14. The composition of matter of Claim 1 for use as a medicament.

15. The composition of matter of Claim 1 for use in removing an anti-insulin antibody from a subject.

16. The composition of matter of Claim 1 for use in treating a disease state or condition associated with the upregulation of an anti-insulin antibody in a patient.

17. The composition of matter of Claim 1 for use in treating Type 1 diabetes.

18. A pharmaceutical composition comprising a composition of matter of any of Claims 1-13 and a pharmaceutically acceptable excipient.

19. A method of making composition of matter of any of Claims 1-13.

20. A method of removing an anti-insulin antibody from a subject comprising the step of administering to the subject the agent of any of Claims 1-13.

21. A method of treating a disease state or condition associated with the upregulation of an anti-insulin antibody in a patient comprising the step of administering to the subject an effective amount of the agent of any of Claims 1-13.

22. A method of treating Type 1 diabetes in a patient comprising administering to the patient an effective amount of the agent of any of Claims 1-13.

23. A composition comprising:a first composition of matter comprising:a target-binding moiety that binds to an anti-insulin 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 degrading cells in a patient or subject, and a linker moiety linking the antibody moiety and the cellular receptor-binding moiety, andat least one additional composition of matter comprising a moiety capable of binding to the antibody that forms the antibody moiety of the first composition of matter.  

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