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WO2025210538A1 - Bifunctional degraders - Google Patents

Bifunctional degraders

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Publication number
WO2025210538A1
WO2025210538A1 PCT/IB2025/053467 IB2025053467W WO2025210538A1 WO 2025210538 A1 WO2025210538 A1 WO 2025210538A1 IB 2025053467 W IB2025053467 W IB 2025053467W WO 2025210538 A1 WO2025210538 A1 WO 2025210538A1
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WO
WIPO (PCT)
Prior art keywords
insulin
antibody
binding
moiety
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/053467
Other languages
French (fr)
Inventor
Sampat Ingale
Christian M. VIDAL
Brian M. LINHARES
Charlotte Bredo SPLIID
Ana M. ESTRELLA
Silvina DEL CARMEN
Brett RASILE
Bruce D. CAR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biohaven Therapeutics Ltd
Original Assignee
Biohaven Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biohaven Therapeutics Ltd filed Critical Biohaven Therapeutics Ltd
Publication of WO2025210538A1 publication Critical patent/WO2025210538A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant, and particularly to bifunctional molecules which contain a circulating protein binding moiety linked through a linker group to a cellular receptor- binding moiety to treat diabetes and other insulin disorders.
  • BACKGROUND OF THE INVENTION [002] Diabetes often has an autoimmune component. Neutralizing antibodies generated against insulin reduce the effectiveness of insulin in diabetic patients. Many ⁇ -cell proteins are the targets of autoimmune responses in Type 1 diabetes (T1D). A contribution comes from the protein products of the insulin gene: preproinsulin (PPI), proinsulin (PI), or insulin.
  • the invention provides a composition of matter (an agent, a TRAP) comprising: a target-binding moiety that can bind to anti-insulin antibodies, a cellular receptor-binding moiety that can bind to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, and a linker moiety connecting the binding moiety and the cellular receptor-binding moiety, wherein the linker moiety can be a single peptide bond or a larger linker moiety.
  • the composition of matter is a bifunctional anti-proinsulin and or anti-insulin autoantibody degrader.
  • the composition of matter (the agent, the TRAP) has the structure: R CN ⁇ (Xaa)y ⁇ R CC , , or a salt thereof.
  • the composition of matter (the agent, the TRAP) has the structure of formula AGN105: or a salt thereof, wherein the composition of matter may have additional elements described in this specification.
  • the composition of matter is selected from among group consisting of the constructs in the following TABLE. TABLE 1
  • the target-binding moiety is insulin, an insulin variant, or an insulin analogue.
  • the target-binding moiety is proinsulin, a proinsulin variant, or a proinsulin analogue.
  • the target-binding moiety is an insulin-IgG1 Fc fusion construct.
  • the target-binding moiety is a proinsulin-IgG1 Fc fusion protein.
  • the proinsulin-IgG1 Fc fusion protein can be expressed in and produced from a mammalian cell system because of the Fc domain, which also allows for purification of the expressed protein.
  • the proinsulin- IgG1 Fc fusion protein can then be conjugated to ASGPR-binder via MATE reagent, resulting in a bifunctional degrader molecule.
  • Persons having ordinary skill in the biomedical art can introduce LALA mutations into the Fc region to attenuate immune effector function via abrogation of FcgR.
  • Persons having ordinary skill in the biomedical art can also introduce PA mutations to attenuate immune effector function via abrogation of c1q binding. They can use a DNA sequence encoding a proinsulin-IgG1 Fc fusion protein for recombinant production of proinsulin-IgG1 Fc fusion protein in mammalian systems.
  • the target-binding moiety is a proinsulin-IgG1 Fc fusion construct.
  • the target-binding moiety is selected from among group consisting of the constructs in the following TABLE: TABLE 2 .
  • the cellular receptor-binding moiety comprises an ASGPR binding group according to the chemical structure: wherein the cellular receptor-binding moiety has additional elements described in this specification.
  • the cellular receptor-binding moiety is selected from among group consisting of the constructs listed in the following TABLE. TABLE 3
  • variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, thus keeping the specificity of the rodent antibody.
  • the human origin domain need not originate directly from a human because it is first synthesized in a human. Instead, human domains can
  • the antibody can be partially or completely humanized.
  • steps used to humanize a monoclonal antibody are (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process; (3) the actual humanizing methodologies/techniques; and (4) the transfection and expression of the humanized antibody.
  • the term “IgG” antibody has the biomedical art-recognized meaning.
  • insulin efsitora alfa means the selective agonist of insulin receptor (IR) that is a fusion protein composed of human IR agonists fused with the crystallizable (Fc) domain of human immunoglobulin G2 (lgG2) fragment, with a molecular weight of 64.1 kDa.
  • IR insulin receptor
  • Fc crystallizable domain of human immunoglobulin G2
  • IGF insulin growth factor
  • insulin receptor has the biomedical art-recognized meaning.
  • the insulin receptor has two main subunits: ⁇ and ⁇ .
  • the ⁇ subunit contains five main domains, L1 (AA 28–174) CR (AA 182–339), and L2 (AA 340–497), and two fibronectin subunits: FnIII-1 (residue 624–726) and FnIII-2 (757–842).
  • the two ⁇ -subunits are linked by four disulfide bonds.
  • the insulin receptor ⁇ has different isoforms (IRA and IRB), depending on the gene splicing of exon 11.
  • Insulin receptor B differs from insulin receptor A by including exon 11.
  • the 12-amino acid sequence (residues 745–756) derived from exon 11 is present in the insulin receptor B isoform but absent in the insulin receptor A isoform.
  • the isoforms have functionally different internalization and recycling. Insulin receptor A exhibits a greater internalization and recycling rate than insulin receptor B. Insulin receptor A exhibits a higher affinity for IGFs than IRB. Both isoforms have similar affinity for insulin Binding to these isoforms very different to proinsulin. Because of these differences, insulin receptor B is preferentially associated with metabolic and differentiating signals. Insulin receptor A mainly favors cell growth, proliferation, and survival. See Beneit et al., Cardiovasc. Diabetol., 15, 161 (2016).
  • IGF-2 insulin-like growth factor 2
  • IGF-2 has growth- regulating, insulin-like and mitogenic activities.
  • IGF-2 has growth- regulating, insulin-like and mitogenic activities.
  • IGF-2 has growth- regulating, insulin-like and mitogenic activities.
  • IGF-2 has growth- regulating, insulin-like and mitogenic activities.
  • IGF-2 has growth- regulating, insulin-like and mitogenic activities.
  • the term “insulin” has the biomedical art-recognized meaning. Insulin can be produced by chemical synthesis.
  • IVIG has the biomedical art-recognized meaning of the administration of intravenous immunoglobulin.
  • linker moiety has the biomedical art-recognized meaning of a moiety of a chemical compound that links one moiety of the chemical compound to another moiety of the same compound.
  • MoDE has the proprietary meaning of molecular degraders. See International Patent Publications WO 2019/199634 (Yale University) and WO 2019/199621 (Yale University).
  • moiety has the biomedical meaning of a defined chemical group or entity with a particular structure or activity.
  • a moiety generally refers to a part of a molecule.
  • a binding moiety maintains one or more desired structural features, properties, functions, or properties, e.g., 3-dimension structure, antigen specificity, antigen-binding capacity, or immunological functions, etc. comparable to its corresponding binding protein, e.g., an antibody.
  • a moiety is monovalent.
  • a moiety is bivalent.
  • a moiety is polyvalent.
  • the term “monotherapy” has the biomedical art-recognized meaning of the administration of a single active or therapeutic compound to a subject. Monotherapy usually is the administration of a therapeutically effective amount of an active composition.
  • MATE or MATES Multimodal Antibody Therapy Enhancers
  • the term “nanobody” has the biomedical art-recognized meaning.
  • the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
  • the term "on” has the plain meaning. When an element is referred to as being on another element, it can be directly in contact with the other element or intervening elements may be present therebetween. When an element is referred to as being “directly on” another element, there are no intervening elements present. [00102]
  • the term “or” as used in this specification means “and/or.” The term “and/or” as used in this specification includes all combinations of one or more of the associated listed items.
  • the term “other degrading cells” has the biomedical art-recognized meaning. Asialoglycoprotein receptors (ASGPRs) are on the glandular cells of the gallbladder and the stomach, as well as on hepatocytes.
  • ASGPRs Asialoglycoprotein receptors
  • the term “partially humanized” has the biomedical art-recognized meaning a protein, e.g., an antibody, is genetically engineered so it more closely resembles the polypeptide structure of the human homologue.
  • a variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, thus keeping the specificity of the rodent antibody.
  • the domain of human origin need not originate directly from a human because it is first synthesized in a human. Instead, human domains can be generated in rodents whose genome incorporates human immunoglobulin genes.
  • the antibody can be partially or completely humanized.
  • Proinsulin can also be produced by chemical synthesis.
  • the chemically synthesized proinsulin is comparable to commercial proinsulin for the surface plasmon resonance (SPR) assay.
  • Proinsulin does not bind to the insulin receptor.
  • protein binding moiety has the biomedical art-recognized meaning of a region of a chemical composition, e.g., a polypeptide region of a chemical composition, that specifically binds to a protein, e.g., a specific protein.
  • ROC has the biomedical art-recognized meaning of receiver operating characteristic curve.
  • the term “subject” and the term “patient” have the biomedical art-recognized meanings.
  • patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • TBT has the meaning described in this specification of target binding moiety, a cellular receptor-binding moiety. In some embodiments of this specification, the TBT binds to ASGPR.
  • TBT has the meaning described in this specification of a targeted removal of aberrant protein.
  • a TRAP is a bifunctional degrader.
  • the term “treating” and the “treat” has the biomedical art-recognized meaning of the management and care of a patient for combating a disease, condition, or disorder and includes the administration of a composition described in this specification to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder.
  • the term “Type 1 diabetes mellitus” has the biomedical art-recognized meaning of a chronic autoimmune disease that occurs when the immune system attacks the pancreas' insulin-producing cells, resulting in the autoimmune eradication of ⁇ -cells in pancreatic islets. Insulin can no longer be synthesized or be secreted into the blood.
  • the invention provides a composition of matter (agent) comprising: a binding moiety that can bind to 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.
  • the invention provides a composition of matter (an agent) having a structure selected from the Markush group of structures consisting of: R CN ⁇ (Xaa)y ⁇ R CC ,
  • each cellular receptor-binding moiety independently has the structure of ⁇ (R CN ⁇ (Xaa)y ⁇ R CC ) or salt form thereof.
  • one and no more than one cellular receptor-binding moiety is bonded to a linker moiety.
  • b is 1.
  • two or more cellular receptor-binding moiety is bonded to a single linker moiety.
  • b is 2 or more.
  • an agent comprises one and no more than one cellular receptor-binding moiety.
  • c is 1.
  • b is 1 and c is 1.
  • a is 1, b is 1 and c is 1.
  • an agent comprises two or more
  • each cellular receptor-binding moiety in an agent is the same.
  • each linker moiety connecting a cellular receptor-binding moiety to an antibody moiety is the same.
  • the TBT in agents is the same.
  • the ⁇ L ⁇ (TBT) b are the same.
  • the target binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues.
  • each binding moiety in an agent may be of the same binding moiety or a pharmaceutically acceptable salt thereof.
  • the binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues.
  • an antibody binding moiety comprises one or more amino acid residues, each independently natural or unnatural.
  • a binding moiety e.g., a protein binding moiety, e.g., an antibody binding moiety, e.g., a universal antibody binding moiety
  • a binding moiety has the structure of ABT101 or a salt form thereof, wherein: each of R 1 , R 3 and R 5 is independently hydrogen or an optionally substituted group selected from C 1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or: R 1 and R 1
  • R 5 groups are optionally taken with their intervening atoms to form a C 1-10 optionally substituted bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-3 methylene units of the chain are independently and optionally replaced with –S–, –SS–, –N(R)–, –O–, – C(O)–, –OC(O)–, –C(O)O–, –C(O)N(R)–, –N(R)C(O)–, –S(O)–, –S(O) 2 –, or –Cy 1 –, wherein each –Cy 1 – is independently a 5-6 membered heteroarylenyl with 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; each of R 1’ , R 3’ and R 5’ is independently hydrogen or optionally substituted C 1-3 aliphatic; each of R 2 , R 4 and R 6 is independently hydrogen, or optionally substituted C 1-4 aliphatic
  • L 1 is an optionally substituted trivalent group selected from C 1 -C 20 aliphatic or C 1 -C 20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
  • L 1 is –(CH 2 CH 2 O) 2-4 – or –(CH 2 CH 2 O) 2 –.
  • Some antibody binding moieties and technologies for identifying or assessing antibody binding moieties are described in International Patent Publications WO 2019/023501 (Kleo Pharmaceuticals, Inc.) and WO 2019/136442 (Kleo Pharmaceuticals, Inc.), each of which is incorporated in this specification in its entirety by reference. Persons having ordinary skill in the biomedical art know that additional technologies in the biomedical art may be suitable for identifying or assessing antibody binding moieties in accordance with this specification. In
  • an antibody binding moiety comprises one or more amino acid residues, each independently natural or unnatural.
  • selected locations of antibody moieties are used for conjugation.
  • K246 or K248 of an antibody agent are conjugation locations.
  • a conjugation location is K246 of heavy chain (unless otherwise specified, locations in this specification include corresponding residues in, e.g., modified sequence, e.g., longer, shorter, rearranged, etc., sequences.
  • a location is K248 of heavy chain.
  • a location is K288 or K290 of heavy chain.
  • a location is K288 of heavy chain.
  • a location is K290 of heavy chain. In some embodiments, a location is K317.
  • an antibody moiety is a moiety of an IgG1 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG2 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an antibody or a fragment thereof.
  • a composition comprises a plurality of MATE agents, wherein antibody moieties of the plurality of MATE agents are independently an antibody moiety of an IgG1, IgG2, or IgG4 antibody, or a fragment thereof. [00147] In some embodiments, antibody heavy chains are selectively conjugated/labeled over light chains.
  • moieties of interest e.g., target binding moieties
  • antibody moieties of a particular type of antibodies e.g., IgG1
  • particularly sites typically one or two particularly sites, e.g., K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto.
  • about 10%-100% of all, or substantially all, moieties of interest, e.g., target binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K239 and K241 of a heavy chain and amino acid residues corresponding thereto.
  • about 10%-100% of all, or substantially all, moieties of interest are conjugated to antibody moieties of IgG1, IgG2, and/or IgG4 antibodies, or fragments thereof, e.g., for conjugation products with IgG1 antibodies or fragments thereof (antibody moieties being of IgG1 antibodies or fragments thereof), IgG2 antibodies or fragments thereof (antibody moieties being of IgG2 antibodies or fragments thereof), IgG4 antibodies or fragments thereof (antibody moieties being of IgG4 antibodies or
  • a first composition is a composition comprising a first agent as described in this specification.
  • second agents independently comprise second reactive groups.
  • a second composition is a composition comprising a plurality of agents as described in this specification, wherein each cellular receptor-binding moiety is independently a reactive group as described in this specification.
  • a second composition is an antibody composition, wherein antibodies in the composition are not chemically modified.
  • the invention provides agents each independently comprising an antibody binding moiety that binds to an antibody agent, a reactive group, a cellular receptor-binding moiety, and optionally one or more linker moieties linking such groups/moieties.
  • agents are useful as reaction partners, e.g., first agents) for conjugating moieties of interest, e.g., target binding moieties, reactive groups, e.g., second reactive groups) to agents comprising antibody moieties, e.g., second agents).
  • LG is a group comprising an antibody binding moiety; RG is a reactive group; LRM is a linker; and TBT is a cellular receptor-binding moiety.
  • L G is or comprises an antibody binding moiety as described in this specification, and a linker which links an antibody binding moiety and RG.
  • L G is or comprises R LG ⁇ L LG ⁇ , wherein R LG is or comprises an antibody binding moiety, and L LG is a linker moiety as described in this specification.
  • L G is ABT-L LG ⁇ .
  • L LG is ⁇ L LG1 ⁇ L LG2 ⁇ , wherein each of L LG1 and L LG2 is independently a linker moiety as described in this specification.
  • L LG is ⁇ L LG1 ⁇ L LG2 ⁇ L LG3 ⁇ , wherein each of L LG1 , L LG2 and L LG3 is independently as linker moiety described in this specification.
  • L LG is ⁇ L LG1 ⁇ L LG2 ⁇ L LG3 ⁇ L LG4 ⁇ , wherein each of L LG1 , L LG2 , L LG3 and L LG4 is independently a linker moiety as described in this specification.
  • L LG1 is bonded to R LG .
  • L LG1 is bonded to cellular receptor-binding moiety.
  • L LG is ⁇ L LG1 ⁇
  • a reactive group comprises L LG2 , L LG3 and L LG4 .
  • L LG is ⁇ L LG1 ⁇ L LG2 ⁇
  • a reactive group comprises L LG3 and L LG4 .
  • L LG is ⁇ L LG1 ⁇ L LG2 ⁇ L LG3 ⁇
  • a reactive group comprises L LG4 .
  • each of L LG1 , L LG2 , L LG3 and L LG4 is independently L. [00171]
  • L LG4 is a covalent bond.
  • L LG4 is not a covalent bond. In some embodiments, L LG4 is ⁇ O ⁇ . In some embodiments, L LG4 is ⁇ N(R’) ⁇ . In some embodiments, L LG4 is ⁇ NH ⁇ . In some embodiments, L LG4 is ⁇ N(CH 3 ) ⁇ . In some embodiments, L LG4 is ⁇ N(R’) ⁇ , and L LG3 is ⁇ O ⁇ . In some embodiments, R’ is optionally substituted C 1-6 alkyl. In some embodiments, L LG4 is ⁇ S ⁇ . [00172] In some embodiments, R LG is or comprises an antibody binding moiety. In some embodiments, R LG is or comprises a protein binding moiety.
  • R LG is or comprises an antibody binding moiety. In some embodiments, R LG is an antibody binding moiety. In some embodiments, R LG is a protein binding moiety. In some embodiments, R LG is an antibody binding moiety. [00173] In some embodiments, R LG is ABT101, R c ⁇ (Xaa)z ⁇ , a nucleic acid moiety, or a small molecule moiety. In some embodiments, R LG is or comprises ABT101. In some embodiments, R LG is or comprises Rc ⁇ (Xaa)z ⁇ . In some embodiments, R LG is or comprises a small molecule moiety. In some embodiments, R LG is or comprises a peptide agent.
  • R LG is or comprises a nucleic acid agent. In some embodiments, R LG is or comprises an aptamer agent. In some embodiments, an antibody binding moiety is or comprises ABT101. In some embodiments, a protein binding moiety is or comprises ABT101. In some embodiments, an antibody binding moiety is or comprises ABT101. In some embodiments,
  • an antibody binding moiety is or comprises Rc ⁇ (Xaa)z ⁇ .
  • a protein binding moiety is or comprises Rc ⁇ (Xaa)z ⁇ .
  • an antibody binding moiety is or comprises Rc ⁇ (Xaa)z ⁇ .
  • target binding moieties may be conjugated to antibody moieties optionally through linker moieties using technologies described in published U.S. Patent Publication 2020/0190165.
  • PG protecting group
  • LG leaving group
  • transformation condition persons having ordinary skill in the biomedical art know that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated.
  • leaving groups include but are not limited to, halogens, e.g. fluoride, chloride, bromide, iodide, sulfonates, e.g. mesylate, tosylate, benzenesulfonate, brosylate, nosylate, triflate), diazonium, and the like.
  • an oxygen protecting group includes carbonyl protecting groups, hydroxyl protecting groups, etc.
  • Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4- oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy- crotonate, benzoate, p-benylbenzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl, 9- fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl.
  • silyl ethers examples include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers.
  • Alkyl ethers include methyl, benzyl, p- methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives.
  • ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, and 2-picolyl and 4-picolyl.
  • Amino protecting groups are well known in the biomedical art and include those described in Greene’s Protecting Groups in Organic Synthesis (John Wiley & Sons, 2014), the entirety of which is incorporated in this specification by reference.
  • Suitable amino protecting groups include, but are not limited to, aralkylamines, carbamates, cyclic imides, allyl amines, amides, and the like.
  • Examples of such groups include t-butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl (CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, and the like.
  • reaction partners are generally contacted with each other under conditions and for a time sufficient for production of the desired results, e.g., formation of product agents and compositions thereof to desired extents.
  • reaction conditions/reaction times may be assessed and used if they are suitable for desired purposes in accordance with this specification; certain such conditions, reaction times, assessment, etc. are described in the Examples.
  • an agent formed e.g., a product MATE agent
  • a cellular receptor-binding moiety in a product agent e.g., a MATE agent
  • a reaction partner e.g., a first agent comprising a cellular receptor-binding moiety
  • an antibody moiety in a product agent e.g., a MATE agent
  • a reaction partner e.g., a second agent comprising an antibody moiety
  • linker moieties or a part thereof connected to target binding moieties and/or antibody moieties may be transferred from reaction partners, e.g., L RM of formula AGN110 or a salt thereof.
  • L RG2 is ⁇ C(O) ⁇ , and is bonded to ⁇ NH ⁇ of a target agent moiety, e.g., ⁇ NH ⁇ in a side chain of a lysine residue of a protein moiety, which in some embodiments, is an antibody moiety.
  • a target agent moiety e.g., ⁇ NH ⁇ in a side chain of a lysine residue of a protein moiety, which in some embodiments, is an antibody moiety.
  • the invention provides products of provided processes, which have low levels of damage to antibody moieties compared to processes comprising steps performed for antibody binding moiety removal but not for substantial conjugation of moieties of interest, e.g. target binding moieties.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.
  • Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include these components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Plurality of agents the invention provides a composition comprising a plurality of agents, wherein each agent independently comprises: an antibody moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody binding moiety and a cellular receptor-binding moiety.
  • product agents are MATE agents.
  • an antibody agent moiety comprises IgG Fc region.
  • an antibody moiety is connected to a cellular receptor-binding moiety through an amino group optionally through a linker. In some embodiments, it is through a lysine residue wherein the amino group of the side chain is connected to a
  • the invention provides a composition comprising a plurality of agents each of which independently comprising: an antibody moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody moiety and a cellular receptor-binding moiety; wherein agents of the plurality share the same or substantially the same antibody moiety, and a cellular receptor-binding moiety at least one common location; and wherein about 1%-100% of all agents that comprise the antibody moiety and the cellular receptor-binding moiety are agents of the plurality.
  • agents of the plurality share the same or substantially the same antibody moiety, and a cellular receptor-binding moiety at least one common location; and wherein about 1%-100% of all agents that comprise the antibody moiety and the cellular receptor-binding moiety are agents of the plurality.
  • HDX-MS is continuing to be performed.
  • the inventors are modeling the antibody Fc-and bifunctional degrader interaction to predict paratope regions in parallel with HDX-MS.
  • Protein sequencing In one method of protein sequencing, eight digestions are prepared using five enzymes (pepsin, Lys C, trypsin, chymotrypsin, Asp N). The digestions for the sample are processed with disulfide reduction, cysteine blocking, and then enzyme digestion. Digestions were analyzed by LC-MS/MS using a Thermo-Fisher Orbitrap fusionTM mass spectrometer. Peptides are characterized from LC -MS/MS data using de nova peptide sequencing and then assembled into antibody sequences.
  • Assays used to determine proof of concept [00197] The inventors used these assays to determine proof-of-concept for the anti- insulin/proinsulin autoantibody bifunctional agents of the invention.
  • Biophysical assay Surface plasmon resonance assay (insulin receptor). The inventors produced mutant insulin conjugated with GN3 and is being tested for binding to the insulin receptor by surface plasmon resonance assay. Additional mutant insulins conjugated with GN3 are produced synthetically.
  • Insulin receptors are also produced synthetically. qPCR method primer sequence IR-forward: GCAACATCACCCACTACCTGGT (SEQ ID NO: 39). qPCR method primer sequence IR-reverse: GAATGGTGGAGACCAGGTCCTC (SEQ ID NO: 40).
  • FIG. 1 Cellular Assay. iLite Insulin reporter assay (DT-40). Assay principle: Overexpression of insulin receptor and transfection of a non-canonical STAT5 recognition motif and a STAT5 expression vector. Thus, insulin signaling via the insulin receptor results in STAT5 phosphorylation, dimerization, and transcriptional activation of a promoter leading to luciferase production.
  • Insulin receptor-binding to human insulin via surface plasmon resonance Biophysical assessment of insulin/IGF1 binding to their respective receptors was demonstrated. Weak binding of proinsulin constructs to insulin receptor
  • NOD Nonobese diabetic
  • MSD MesoScale Discovery
  • an antibody binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can selectively bind to IgG, and when used in provided technologies can provide and/or stimulate ADCC and/or ADCP.
  • peptide display technologies e.g., phase display, non-cellular display, etc., can identify antibody binding moieties.
  • an antibody binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can bind to IgG and optionally can compete with known antibody binders, e.g., protein A, protein G, protein L, etc.
  • an antibody binding moiety e.g., an antibody binding moiety
  • a Meditope agent moiety See United States Patent Publication 2019/0111149 (Gardiner et al.).
  • an antibody binding moiety e.g., an antibody binding moiety
  • an antibody binding moiety e.g., an antibody binding moiety
  • an antibody binding moiety e.g., an antibody binding moiety, binds to IgG1, IgG2, and IgG4.
  • antibody binding moieties bind to targets, e.g., antibody agents for antibody binding moieties) with a K D that is about 1 mM -1 pM or less.
  • a K D is about 1 mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, 0.01 mM, 0.005 mM, 0.002 mM, 0.001 mM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or less.
  • K D is an affinity selected from the Markush group of affinities consisting of about 1 mM or less, about 0.5 mM or less, about 0.1 mM or less, about 0.05 mM or less, about 0.01 mM or less, about 0.005 mM or less, about 0.001 mM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, and about 1 nM or less.
  • antibody binding moieties bind to IgG antibody agents with K D described in this specification.
  • antibodies of several properties and activities may be recruited by antibody binding moieties described in this specification.
  • such antibodies include antibodies administered to a subject, e.g., for therapeutic purposes.
  • antibodies recruited by antibody binding moieties comprise antibodies toward different antigens.
  • antibodies recruited by antibody binding moieties comprise antibodies whose antigens are not present on the surface or cell membrane of target cells.
  • antibodies recruited by antibody binding moieties comprise antibodies not targeting antigens present on surface or cell membrane of targets.
  • antigens on surface of target cells may interfere with the structure, conformation, and/or one or more properties and/or activities of recruited antibodies which bind such antigens.
  • recruited antibodies are those in IVIG.
  • IVIG may be administered before, concurrently with or subsequently to an agent or composition.
  • Antibodies of several types of immunoglobulin structures may be recruited.
  • one or more subclasses of IgG are recruited.
  • recruited antibodies are selected from the Markush group of antibody classes consisting
  • recruited antibodies can effectively interact with human Fc ⁇ receptor IIIA on macrophages.
  • macrophages are recruited to provide ADCC or ADCP activities toward a virus, e.g., a SARS-CoV-2 virus, and/or cells infected thereby.
  • NK cells are recruited to provide immune activities.
  • recruited antibodies can effectively interact with human Fc ⁇ receptor IIIA.
  • recruited antibodies can effectively interact with human Fc ⁇ receptor IIIA on dendritic cells.
  • antibody moieties in agents of this specification comprise one or more properties, structures and/or activities of recruited antibodies described in this specification.
  • Biophysical/biochemical potency assay to quantify target-engagement A biomolecule coupled to surface of sensor chip as ligand. As analyte is flowed in solution over immobilized ligand, binding to the sensor chip surface induces a change in refractive index proportional to bound mass.
  • An assay was done using a Biacore S200 instrument. This machine has a high sensitivity, low- medium throughput. A ligand variant fragment is dosed 1 nM to 1 ⁇ M and characterized by single cycle kinetics.
  • ASGPR-dependent uptake assay A HEK293 bioluminescent cell-based assay was used to assess ternary complex formation uptake by cells in vitro.
  • This on-mechanism endocytosis assay measures the accumulation of bifunctional degrader in HEK293 cells through the bifunctional degrader mechanism described in this specification.
  • a component of this assay is a determination of the receptor expression on the surfaces of the assay’s HEK293 cells.
  • Degradation assay is a direct, low-throughput Western blot measurement or a high-throughput assay using an activatable fluorescence-quencher probe.
  • Off-target assays include measurement of cytotoxicity through In vitro toxicity with HepG2 cells, e.g., in a CellTiter-Glo assay, measurement of hemagglutination by red blood cell interactions, measurement of PBMC, and measurement of off-target bindings in a house C-type lectin panel. Signal is boosted in the HEK293 versus HepG2 cells when measuring signal accumulation not degradation. Western assays can measure the degradation in HEK cells and other cell lines.
  • Next steps can include a competition assay with FC3 (qualitative). Manufacturing the agent. TABLE 14 P rifi ti n t H A 5 5
  • Agents of this specification may be prepared or isolated by synthetic and/or semi-synthetic methods or recombinant methods in accordance with this specification.
  • polypeptide agents e.g., cellular receptor-binding moiety peptide agents
  • provided agents are prepared synthetically.
  • provided agents are prepared using certain technologies described in International Patent Publication WO 2019/023501 (Kleo Pharmaceuticals, Inc.), which is incorporated in this specification in its entirety by reference.
  • the invention provides a method of synthesis, comprising the steps of: contacting a first agent comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker with a second agent comprising an antibody moiety linked to a second reactive group optionally through a second linker, wherein the first reactive group reacts with a second reactive group, and
  • AGN303 Native insulin with GN3
  • AGN302 Mutant insulin with GN3
  • ABT306 Alk-Native Insulin without GN3 (just peptide)
  • ABT304 Native Proinsulin
  • ABT305 Mutant Proinsulin
  • Non-GN3 peptide (ABT306) does not elicit internalization or ternary complex formation.
  • ABT410 is a proinsulin molecule with N-terminal H27R leading sequence and TEV recognition site. ABT410 was expressed as inclusion bodies in BL21(DE3) strain. After properly refolding and buffer exchange, target protein was cleaved by TEV protease, then purified by SP HP resins. The purified protein was formulated in 50 mM Tris/HCl, 150 mM sodium chloride, 10% glycerol, pH 7.5 by
  • UF/DF UF/DF.
  • the testing panel included protein concentration by Bradford, reducing and non-reducing SDS- PAGE, SEC-HPLC, endotoxin level and LC-MS.
  • Host cell E.coli BL21(DE3) competent cell
  • Coding vector name pET28a(+).
  • Protein Sequence [00433] ABT410 (before TEV digestion) MGMTMITNSPEISHHHHHHHHHHQLISEARENLYFQGGGGGFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAED LQVGQVELGGGPGAGSLQPLALEGSL QKRGIVEQCCTSICSLYQLENYCNG (SEQ ID NO: 19).
  • ABT410 (after TEV digestion) GGGGGFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCT SICSLYQLENYCNG.
  • Extinction coefficient 0.651 after digestion.
  • Molecular weight 9736.98 Da (-SH, after digestion).
  • pI 5.20 after digestion.
  • Refolding buffer 20 mM Tris/HCl,10 mM Glycine, 1 mM EDTA,1 mM GSH, 5 mM GSSG, pH 10.0.
  • Refolding buffer 20 mM Tris/HCl,10 mM Glycine, 1 mM EDTA,1 mM GSH, 5 mM GSSG, pH 10.0.
  • (2) Add DTT to solubilized inclusion bodies to a final concentration of 10 mM and mix completely.
  • Buffer exchange (UF/DF) and TEV digestion [00479] (1) Refolded sample was filtered by 0.45 ⁇ m filter firstly to remove visible precipitation before ultrafiltration. [00480] (2) Sample concentration was concentrated to ⁇ 3 mg/mL by UF device. [00481] (3) Concentrated sample was exchanged into TEV digestion buffer for six-fold volume by DF device. [00482] TEV digestion buffer: 25 mM Tris/HCl, 100 mM sodium chloride, pH 7.5, 10% glycerol. [00483] Buffer exchange by UF/DF: SP HP pool sample can be exchanged into final formulation buffer with six-fold volume by UF/DF device.
  • SP HP buffer C 50 mM NaAc, pH5.0, 10 mM sodium chloride, 10% glycerol, 0.1% Triton- X114.
  • Peak 1 fractions with good purity of SP HP purification were pooled and exchanged into final formulation buffer.
  • the purity of SEC-HPLC was 98.03%.
  • Formulation buffer 50 mM Tris/HCl, 150 mM sodium chloride, 10% glycerol, pH 7.5.
  • Product quality analysis The purified protein is subjected to quality control testing, including reducing and non-reducing SDS-PAGE, SEC-HPLC, Bradford assay, LC-MS, and endotoxin level.
  • Endotoxin level Purified proteins were subjected to endotoxin test in Limulus Amoebocyte Lysate (LAL) based kinetic turbidimetric test. Purified proteins were diluted based on maximum valid dilution (MVD) in LAL reagent water (Charles River/W110), then sample was loaded in Charles River Laboratories EndoSafe LAL Cartridges (Charles River/ PTS55F) and detected by Charles River Laboratories Endosafe NexGen-MCSTM instrument.
  • LAL Limulus Amoebocyte Lysate
  • Quality Control Stock Solutions The exact pipetting volume for stock solution should be calculated based on the actual antibody concentration of Reference Standard solutions.
  • Quality Control (QC) Stock 1 Freshly pipet 49 ⁇ L (8.16 mg/mL) of AGN6567 to 351 ⁇ L rat K 2 EDTA Plasma to give a final concentration of 1 mg/mL for total antibody in rat K 2 EDTA Plasma.
  • Quality Control (QC) Stock 2 Pipet 100 ⁇ L STD stock 1 to 900 ⁇ L rat K2EDTA Plasma to give a final concentration of 100 ⁇ g/mL for total antibody in rat K 2 EDTA Plasma.
  • Quality control (QC) samples are prepared by spiking individual QC stock solutions into blank rat K 2 EDTA Plasma described in the TABLE below: TABLE 28 Volume of source solution used ( ⁇ L) e
  • SIL ELVA Stock Dissolve 1 mg of SIL ELVA into 1 mL of diluent to give a concentration of 1 mg/mL for SIL ELVA and store at -70°C.
  • IS Stock 1 Pipet 20 ⁇ L of SIL ELVA stock to 1980 ⁇ L diluent to give a final concentration of 10 ⁇ g/mL SIL ELVA. Store at -70 o C.
  • AF647 78.0 ⁇ M.
  • AF647 31.0 ⁇ M.
  • AF647 30.9 ⁇ M.
  • AF647 27.4 ⁇ M.
  • AF647 48.6 ⁇ M.
  • Insulin receptor A exhibits a higher affinity for IGFs and a greater internalization and recycling rate than Insulin receptor B. Because of these differences, Insulin receptor B is preferentially associated with metabolic and differentiating signals. Insulin receptor A mainly favors cell growth, proliferation, and survival. TABLE 31 Surface plasmon resonance
  • mAb49 Human anti-Insulin antibody.
  • 6E9F1 Mouse anti-Insulin antibody. TABLE 32 Biochemistry selectivity (anti-insulin autoantibodies) ependent of insulin receptors. [00713] This finding provides flexibility with anti-human Insulin antibodies from different species in designing assays.
  • AGN303 Native insulin with GN3 Reference anti-insulin degrader control). Continued good positive control for internalization with HEK293-ASGPR (clone 3F7) cells + mouse anti-human insulin an. [00717] AGN411 (mutant construct) > AGN408 (native construct) for internalization, although minimal signal with either. Neither AGN411 (mutant construct) or AGN408 (native construct) induced internalization. [00718] AGN304: Proinsulin construct with C-terminal GN3 (Native) [00719] AGN305: Proinsulin construct with C-terminal GN3 (Mutant)
  • ABT306 Alk-native insulin without GN3 (peptide only).
  • ABT307 Proinsulin-IgG1 Fc fusion, G4S linker, hIgG1 hinge (C > A), LALA-PA.
  • ABT308 Proinsulin-IgG1 Fc fusion, 5x G4S linker, hIgG1 hinge (C > A), LALA-PA.
  • AGN411 Proinsulin construct with N-terminal ⁇ GN3 (mutant)
  • AGN412 Synthetic Mutant insulin with ⁇ GN3. By comparison, AGN310 has ⁇ GN3.
  • AGN414 Proinsulin-Fc conjugate (ABT307-TBT4438)
  • AGN415 Proinsulin-Fc conjugate (ABT308-TBT4438)
  • AGN416 Synthetic Mutant insulin with ⁇ GN3 on insulin A chain. By comparison, other synthetic insulin-GN3 compounds synthesized have GN3 attached to insulin B chain.
  • AGN417 Native proinsulin with ⁇ GN3 on N-terminal.
  • AGN301 + labeled rabbit Ab should internalize (therefore positive signal) If add Proinsulin Fc protein, will compete with AGN301, and therefore have less signal
  • AGN301 + labeled mouse 6E9F1 Ab should internalize (therefore no signal).
  • AGN303 native proinsulin
  • Synthetic insulin MoDE has similar affinity to mAb49.
  • AGN310 mutant proinsulin
  • Synthetic insulin MoDE has similar affinity to mAb49.
  • Insulin agents can be captured with GalNAc-binding lectin VVL and detected with an anti- insulin antibody.
  • Proinsulin agents can be captured with an anti-insulin antibody and detected with anti- C-peptide antibody (provides higher sensitivity than VVL).
  • CD1 female mouse CD1 Strain CRL#: Crl:CD1(ICR).
  • Age and weight range at start of study Animals aged six-eight weeks with body weight of approximately 25-30 g.
  • the mouse is a well characterized system for drug efficacy evaluation.
  • Administration of agents [00861] Dose: 1 mg/kg or 8.8 mg/mL.
  • mice On day 0, animals is weighed and randomly assigned to groups. All groups of animals receive their treatment of 1 mg/kg AGN417, as described in the TABLE below, either intravenously or subcutaneously. After a defined period, three animals from each group will undergo blood collection at two time points: the first time point involves collecting around 100 ⁇ l of blood from the submandibular vein, and the second will involve cardiac exsanguination following carbon dioxide asphyxiation. Blood samples from individual animals is collected in K3EDTA tubes, and plasma is separated. All samples are stored at -80°C until further analysis. [00870] Non-fasting blood glucose levels is monitored by collecting a small blood drop from a tail nick at the tip of the tail.
  • the blood is applied to a One-Touch test strip and measured using a One-Touch Ultra 2 Glucometer in three mice per group at the following time points: pre-dose, five minutes, ten minutes, fifteen minutes, thirty minutes, one hour, and two hours post-dose. TABLE 46 . After dosing, weights is recorded daily.
  • Plasma levels of AGN417 are determined by in-house developed MSD. Data are presented in tabular form. A standard pharmacokinetic analysis is to be performed for each dose group. EXAMPLE 25 Pharmacodynamics. [00875] Study design: 13.3 nmol/kg anti-insulin autoantibodies – 6E9F1 intravenously (2 mg/kg).133 nmol/kg AGN310 intravenously – 1:10 ratio. [00876] An assay in mice showed a 39% depletion by AGN310, based on area under the curve. [00877] The inventors performed a pharmacodynamics evaluation of AGN310 in nude mice. Nude mouse plasma causes no interference in antibody detection. [00878] Degrader interference could be prevented by diluting samples to below 10 ng/mL antibody. EXAMPLE 26 Pharmacodynamics. NOD mouse insulin inhibition. TABLE 47 l f i
  • TRAP agents were administered to NOD mice to assay NOD mouse insulin inhibition in vivo.
  • AGN310 and AGN417 were administered, each both intravenously and subcutaneously.
  • NOD mice were assayed for body weight and glucose levels (PharmaLegacy).
  • Body weight taken at each dosing, every five days.
  • Non-fasting blood glucose taken every two weeks via tail vein bleed (9-10 AM).
  • AGN303 (native proinsulin) and AGN310 (mutant proinsulin) Insulin agents were well- tolerated when administered at 3 mg/kg intravenously in pre-diabetic NOD mice, with no observed adverse events and no changes in glycemia when measured at twelve hours-post dose.
  • EXAMPLE 29 AGN310 extended tolerability in NOD female mice.
  • the objective of this EXAMPLE was to investigate the longer-term effects of multi-dosing IV AGN310 on NOD female mice.
  • mice given AGN417 intravenous and subcutaneous doses up to 3 mg/kg were well-tolerated, but exposure correlated with transient reduction in blood glucose levels of approximately 50 mg/dL. At no point during pharmacokinetic studies did blood glucose fall below reference range intervals. Blood glucose levels returned to vehicle control animal levels when food was provided. The current study will evaluate the dose-response relationship of AGN417 in rats. The low dose of 15 mg/kg is a low multiple of previous doses in mice, and the mid and high doses of 50 and 150 mg/kg study will explore tolerability and attempt to build a dose-response relationship.
  • the subcutaneous dose of 50 mg/kg is intended to understand the subcutaneous bioavailability and pharmacokinetic profile relative to intravenous administration.
  • the Wistar Han IGS rat was chosen as the animal model for this assay because it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies.
  • Assays in laboratory animals provide a basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
  • the housing set-up is as specified in the United States Department of Agriculture Animal Welfare Act (9 C.F.R., Parts 1, 2 and 3) and as described in the Guide for the Care and Use of Laboratory Animals (National Research Council, Current edition; Office of Laboratory Animal Welfare, Current edition). Animals are separated during designated procedures/activities or separated as required for monitoring and/or health purposes, as deemed appropriate by Study Director and/or staff veterinarian.
  • Bone marrow smear evaluation Bone marrow smears are collected and prepared. Evaluation of stained smears may be performed. TABLE 61 Bioanalytical Sample Collection, Target Time Post-dose on Days 1 and 7 r ical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations, accuracy, and precision are performed. [00909] Toxicokinetic evaluation. TK parameters are generated using the concentration units provided by the bioanalytical lab. Concentration values below the limit of quantitation are treated as zero for the purposes of TK data analysis. TABLE 62 P b i d
  • composition of matter of Embodiment 1, wherein the cellular receptor binding moiety is a low-density lipoprotein receptor-related protein 1 (LRP1), a low-density lipoprotein receptor (LDLR), a Fc ⁇ receptor I-binding group, a FcRn binding group, a transferrin receptor binding group, or a macrophage scavenger receptor binding group.
  • LRP1 low-density lipoprotein receptor-related protein 1
  • LDLR low-density lipoprotein receptor
  • Fc ⁇ receptor I-binding group a FcRn binding group
  • transferrin receptor binding group a transferrin receptor binding group
  • macrophage scavenger receptor binding group a macrophage scavenger receptor binding group.
  • composition of matter of Embodiment 25, wherein binding moiety comprises a peptide moiety that binds to a specific amino acid residue of the glycan-specific IgG antibody comprises a peptide moiety that binds to a specific amino acid residue of the glycan-specific IgG antibody.
  • a composition comprising: a first composition of matter comprising: an antibody moiety, a cellular receptor binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, and a linker moiety linking the antibody moiety and the cellular receptor binding moiety, and at 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.
  • ASGPR asialoglycoprotein receptors
  • Non-patent literature [00983] Abhinandan & Martin, Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Molecular Immunology, 45(14), 3832–3839 (2008)., providing the Martin (enhanced Chothia) CDR definitions and numbering used for biologics. [00984] Acqua et al., Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences. The Journal of Immunology, 169(9), 5171-5180 (2002). [00985] Aguirre, et.al., Insulin-like growth factor 1 in the cardiovascular system. Rev. Physiol. Biochem. Pharmacol., Vol.175, pages 1-45 (2016).
  • This publication shows the schematic structure of insulin, IGFs, and their receptors. Resemblances between insulin and IGFs allow them to cross-interact with each other’s receptors. IGF2 can also interact with IGF1R, hybrid receptors, and the insulin receptor, with a lower affinity. IGF1 can also bind to IGF2R, albeit with less affinity to that of its putative receptor. [00986] Akashi et al., Inter. Immu. (1997). In NOD mouse model, insulin autoreactive B cells induce diabetes, while B cell deficiency or depletion protects from Type 1 diabetes and allows for normoglycemia. [00987] Al-Lazikani, Lesk, & Chothia, C. (1997).
  • Burrack, Martinov, & Fife, T cell-mediated beta cell destruction Autoimmunity and alloimmunity in the context of Type 1 diabetes. Front Endocrinol (Lausanne), 8, 343 (December 5, 2017). Type 1 diabetes results from destruction of pancreatic ⁇ cells by T cells of the immune system. Despite improvements in insulin analogs and continuous blood glucose level monitoring, there is no cure for
  • INSR and IGF1R are composed of two ⁇ dimers which associate to form heterotetrameric complexes.
  • the ⁇ dimers are linked together by disulfide bonds and two dimers are also linked by disulfide bonds to form the tetramer.
  • the ⁇ subunit is the extracellular portion of the receptor while the ⁇ subunit spans the membrane and its cytoplasmic portion interacts with IRS proteins which are key intracellular mediators of insulin/IGF signaling.
  • Single ⁇ dimers are derived from separate genes and the INSR has two splice variants, INSR-B and INSR-A. Each variant shares the same membrane-spanning ⁇ subunit (dark blue) but differs in the extracellular ⁇ subunit (light pink or dark pink, respectively).
  • the IGF1R has different ⁇ and ⁇ subunits compared to the INSR (dark green). These combinations of ⁇ dimers allow for hybrid receptors, which bind insulin, IGF1, and IGF2 with differing affinities.
  • ⁇ dimers allow for hybrid receptors, which bind insulin, IGF1, and IGF2 with differing affinities.
  • ECL electrochemiluminescence
  • Immune cell infiltration into pancreatic islets involves mostly CD4+ & cytotoxic CD8+ T cells (leading to antigen spreading), but growing evidence of B cell infiltration & role in Type 1 diabetes pathogenesis.
  • Hu et al. Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor–related protein 1–mediated integrin signaling. The Journal of Clinical
  • Insulin and insulin precursors play role in pathogenesis of Type 1 diabetes, as mediated by CD4+ and CD8+ T cell responses to autoantigens.
  • PPI insulin precursors
  • PI insulin precursors
  • Michel, Boitard, & Bach Insulin autoantibodies in non-obese diabetic (NOD) mice.
  • NOD non-obese diabetic mice.
  • Clinical and Experimental Immunology 75(3), 457 (1989).
  • Moyers et al. Preclinical characterization of LY3209590, a novel weekly basal insulin Fc- fusion protein. J. Pharmacol. Exp. Ther., 382, 346-355 (September 2022) (Eli Lilly).
  • LY320959 is produced by Eli Lilly and Company, Indianapolis, Indiana and San Diego, California. [001039] Muguruma et al., Kinetics-based structural requirements of human immunoglobulin G binding peptides. ACS Omega, 4, 14390 ⁇ 14397 (2019). [001040] Mustafaoglu et al., Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule, Analyst, 141(24), 6571–6582 (November 28, 2016). [001041] Narendran, Mannering, & Harrison, Proinsulin—a pathogenic autoantigen in type 1 diabetes. Autoimmune Reviews, 2(4), 204-10 (June 1, 2003).
  • PA modification Residues METDTLLLWV LLLWVPGSTG DFVNQHLCGS HLVEALYLVC GERGFFYTPK TRREAEDLQV 60 GQVELGGGPG AGSLQPLALE GSLQKRGIVE QCCTSICSLY QLENYCNGGG GSEPKSADKT 120 HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE 180 VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALAAPIE KTISKAKGQP 240 REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 300 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 344 Sequence Number (ID): 31 Leng
  • Molecule Type AA Features Location/Qualifiers: - source, 1..19 > mol_type, protein > organism, synthetic construct Residues: ELVAVITTDG STNYADSVK 19
  • composition of matter (the agent, the TRAP) has one of the structures:
  • Extracellular Protein Targeting Ligand refers to a target-binding moiety that can bind to an anti-proinsulin or anti-insulin antibody.
  • X 1 is 1 to 5 groups independently selected from O, S. N(R 6 ), arid C(R 4 XR 4 ), wherein if X ! group then X 1 is O, S, N(R”), or C(R 4 )(R 4 ), if X 1 is 2 groups then no more than 1 group of X 1 , S. or N(R 6 ), if X 1 is 3, 4, or 5 groups then no more than 2 groups of X 1 are O, S, orN(R & );
  • R* is selected from
  • heteroatoms independently selected from N. O, and S, each of which aryl, heterocycle., and heteroaryl is optionally substituted with 1, 2, 3, or 4 substituents;
  • Civ hydrogen, R u *, alkyl-CXP)- ⁇ . -C(O)-R ⁇ alkyl, haioalkyl, “()C(O)R ⁇ and ⁇ NRS-CCOJR’ 0 ;
  • R 10 is selected from aryl, alkyl-NR s -C(O)-R alkyl-aryl, alkyl -heteroaryl with 1 , 2, or 4 heteroatoms, alkyl -cyano, alkyLOR 6 , alkyl-NRV, NlV-hiR 6 -C(O)R ⁇ NRM(O>R 3 , alkenyl, ally 1, alkynyl, -N'R ( '-a1kenyi, -O-alkenyl, -NR ⁇ -alkynyL -NRMieteroatyl, ⁇ NR/’-aryl, -O-heteroaryL -O-ary! , and -O-alkynyl. each of which R’" is optionally substituted with 1 , 2, 3, or 4 substituents;
  • R ' at each occurrence is independently selected from hydrogen, alkyl, heteroalkyl, haioalkyl (including - €Fg -CHF ; , -CH>F, -ClfaCF?, -CH2CH2F, and -CF?CF-)> arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, hetcroaryl. heterocycle, -PR* 8 , and -NR S R 9 ;
  • R'' is independently selected at each occurrence from hydrogen, heteroalkyi, alkyl, haioalkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycle, -OR 6 , -NIV’R', C(0)R 3 , S(O)R 3 , C(S)R 3 , and S(O)’R 3 ,
  • R* andR' are independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, haioalkyl, heteroatyl, heterocycle, -alkyl-OR 8 , - alkyl-MVR 9 , C(0)R 3 , S(O)R 3 , C(S)R 3 , and S(O) -RA
  • R* and K;‘ are independently selected at each occurrence from hydrogen, beteroalkyl. alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroatyl, and heterocycle;
  • each Linker' is a bond or a moiety that covalently links the ASGPR ligand to Linker’ 3 ;
  • Linker 0 is a bond or a moiety that covalentfy links Linker* to an Exlracellular Protein Targeting Ligand

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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 DEGRADERS FIELD OF THE INVENTION [001] The invention relates generally to medicinal preparations characterized by the non-active ingredients used, e.g. carriers or inert additives, targeting or modifying agents chemically bound to the active ingredient, the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant, and particularly to bifunctional molecules which contain a circulating protein binding moiety linked through a linker group to a cellular receptor- binding moiety to treat diabetes and other insulin disorders. BACKGROUND OF THE INVENTION [002] Diabetes often has an autoimmune component. Neutralizing antibodies generated against insulin reduce the effectiveness of insulin in diabetic patients. Many β-cell proteins are the targets of autoimmune responses in Type 1 diabetes (T1D). A contribution comes from the protein products of the insulin gene: preproinsulin (PPI), proinsulin (PI), or insulin. Children and most adults with type I diabetes show early generation of antibodies to the proinsulin C-chain and native insulin. Autoantibodies generated against insulin reduce the effectiveness of insulin in diabetic patients. [003] There remains a need in the biomedical art for new medicines capable of treating or slowing down the progression of diabetes and other insulin disorders. Therapies for removing immunoneutralizing antibodies to insulin and insulin replacement therapies also remain needed. SUMMARY OF THE INVENTION [004] The invention provides compositions of matter (agents, TRAPs) that bind to neutralizing antibodies to proinsulin, insulin and insulin replacement therapies. These selective agents significantly reduce or eliminate only the pathogenic species of neutralizing autoantibodies generated by an undesirable immune response while leaving most of the other antibodies unbound in the subject to whom the agents are administered.
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[005] In one embodiment, the invention provides a composition of matter (an agent, a TRAP) comprising: a target-binding moiety that can bind to anti-insulin antibodies, a cellular receptor-binding moiety that can bind to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, and a linker moiety connecting the binding moiety and the cellular receptor-binding moiety, wherein the linker moiety can be a single peptide bond or a larger linker moiety. The composition of matter is a bifunctional anti-proinsulin and or anti-insulin autoantibody degrader. This composition of matter, unlike pan-IgG or multi-IgG degraders, avoids the potential for immunosuppression. [006] In another embodiment, the composition of matter (the agent, the TRAP) has the structure: ulin or anti-insulin antibody; [CRBM] is a cellular receptor-binding moiety that binds to hepatocytes or other cells through asialoglycoprotein receptors or other receptors on the surface of hepatocytes and other degrading cells; each [CON] is an optional connector chemical moiety which, when present, connects directly to [CPBM] or to [CRBM] or connects the [LINKER] to [CPBM] or to [CRBM] and [LINKER] is a chemical moiety that covalently attaches to one or more [CRBM] and/or [CPBM] groups, or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof.
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[007] In some embodiments, the composition of matter (the agent, the TRAP) has the structure: RCN−(Xaa)y−RCC, , or a salt thereof. [008] In some embodiments, the composition of matter (the agent, the TRAP) has the structure of formula AGN105: or a salt thereof, wherein the composition of matter may have additional elements described in this specification. [009] In another embodiment, the composition of matter is selected from among group consisting of the constructs in the following TABLE. TABLE 1
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TABLE 1 AGN304 PT0004-TBT6171. Native proinsulin construct with C-terminal αGN3 conjugate. - 4 30125-WO-PCT
TABLE 1 AGN416 Synthetic mutant insulin with αGN3 on A chain, MW = 7424.24 Da, h d
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[0010] In another embodiment, the target-binding moiety is insulin, an insulin variant, or an insulin analogue. [0011] In another embodiment, the target-binding moiety is proinsulin, a proinsulin variant, or a proinsulin analogue. [001] In another embodiment, the target-binding moiety is an insulin-IgG1 Fc fusion construct. [002] In another embodiment, the target-binding moiety is a proinsulin-IgG1 Fc fusion protein. The proinsulin-IgG1 Fc fusion protein can be expressed in and produced from a mammalian cell system because of the Fc domain, which also allows for purification of the expressed protein. The proinsulin- IgG1 Fc fusion protein can then be conjugated to ASGPR-binder via MATE reagent, resulting in a bifunctional degrader molecule. Persons having ordinary skill in the biomedical art can introduce LALA mutations into the Fc region to attenuate immune effector function via abrogation of FcgR. Persons having ordinary skill in the biomedical art can also introduce PA mutations to attenuate immune effector function via abrogation of c1q binding. They can use a DNA sequence encoding a proinsulin-IgG1 Fc fusion protein for recombinant production of proinsulin-IgG1 Fc fusion protein in mammalian systems. [003] In another embodiment, the target-binding moiety is a proinsulin-IgG1 Fc fusion construct. [004] In another embodiment, the target-binding moiety is selected from among group consisting of the constructs in the following TABLE: TABLE 2 .
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TABLE 2 ABT404 mAb49 chimeric heavy chain gamma 3, mouse VH/VL (SEQ ID NO: 34) [495 AA], with
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TABLE 2 ABT17386 Mutant proinsulin + cysteine r or t e unpare cystenes n t e target moety or conjugaton: s - ( Q : 7). [006] In another embodiment, the cellular receptor-binding moiety comprises an ASGPR binding group according to the chemical structure: wherein the cellular receptor-binding moiety has additional elements described in this specification. [007] In another embodiment, the cellular receptor-binding moiety is selected from among group consisting of the constructs listed in the following TABLE. TABLE 3
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TABLE 3 TBT4438 . matter. [009] In another embodiment, the invention provides a method of removing anti-proinsulin and anti-insulin antibodies in a subject or patient by administering the agent to the patient or subject. The administered bifunctional degrader agent removes anti-proinsulin and anti-insulin autoantibodies, whether IgG1, IgG2, or IgG4, from circulation by binding to the autoantibody and to a high capacity ASGPR receptor on hepatocytes. [0010] In another embodiment, the invention provides a method of treating a disease state or condition associated with the upregulation of anti-proinsulin and anti-insulin antibodies in a subject or patient by administering to the subject or patient an effective amount of the agent. [0011] In another embodiment, the invention provides a method of treating diabetes in a patient by administering to the patient a therapeutically effective amount of the agent. Methods of following the course treatment for insulin-treated diabetes are known to persons having ordinary skill in the biomedical art. In a particular embodiment, diabetes being treated is Type 1 diabetes. Methods of following the course treatment for insulin-treated Type 1 diabetes are known to persons having ordinary skill in the biomedical art. [0012] In another particular embodiment, anti-drug, neutralizing antibodies generated against insulin reduce the effectiveness of insulin in diabetic patients. Removal of anti-drug antibodies corrects anti-drug antibody-related insulin resistance in patients. Removal of anti-drug antibodies is useful for treating life-threatening insulin resistance, encephalopathy, or cellular starvation. [0013] In another particular embodiment, almost all children and most adults with Type I diabetes, show early generation of antibodies to the proinsulin C-chain as well as native insulin. Removal of autoantibodies directed against proinsulin mitigates insulitis, and specifically inflammation directed to
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the pancreatic islets of Langerhans, which is in part caused by a secondary antibody-dependent cell- mediated cytotoxicity (ADCC) directed against β-cells of the patient’s pancreas. Removal of autoantibodies directed against proinsulin can correct anti-drug antibody-related insulin resistance in patients. Insulin resistance abrogates glucose uptake insulin resistance and IgG-mediated 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 reduce pancreatic inflammation. [0014] In another particular embodiment, the agent is useful for treating diabetic animals. Many companion dogs and companion cats in the United States have diabetes mellitus. More than 10% of diabetic dogs in the United States are therapeutically treated with human insulin. These dogs frequently develop immune-neutralizing antibodies which decrease insulin concentrations and antidiabetic efficacy. [0015] In another embodiment, the invention provides a pharmaceutical composition including the agent and at least one pharmaceutically acceptable excipient. [0016] In another embodiment, the invention provides a composition including the agent and at least one additional agent comprising a moiety capable of binding to that forms the antibody moiety of the first compound. [0017] In one aspect, the targeting of anti-insulin autoantibodies or anti-proinsulin autoantibodies with TRAPs has several advantages. In humans Type 1 diabetes, autoantibodies are often found in low concentrations, e.g., ~1000ng/mL (0.01% of total IgG). The target autoantibodies are well-defined and detectable with available clinical diagnostics to track efficacy of lowering titers. A single drug approach can target several Type 1 diabetes-associated factors that induce β cell destruction, including autoantibodies to anti-insulin autoantibodies and anti-proinsulin autoantibodies. The target autoantibodies are primarily of the IgG1 subclass with a half-life of twenty-nine days. Thus, infrequent dosing may be effective. [0018] Several objects, features, aspects, and advantages of the invention become more apparent from the following detailed description of embodiments of the invention, along with the drawings.
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BRIEF DESCRIPTION OF THE DRAWINGS [001] For illustration, some embodiments of the invention are shown in the drawings described below. Like numerals in the drawings indicate like elements throughout. The invention is not limited to the precise arrangements, dimensions, and instruments shown. [002] FIG.1 shows two schematics of proinsulin and insulin processing. [003] FIG.2 shows the structure of one embodiment of the bifunctional molecules (agents, TRAPs). [004] FIG.3 shows a process of preparation of one embodiment of the bifunctional molecules (agents, TRAPS) from a full IgG antibody and a reagent having affinity to the by conjugating the IgG antibody to the reagent. [005] FIG.4 is a table (TABLE 4) of R-Groups for MATE reagents and bifunctional MoDE final compounds. [006] FIG.5 is a comparison of sequences of the constant heavy chains of human IgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 1), and IgG4 (SEQ ID NO: 3), which support human and humanized therapeutic antibodies. Sequence differences in IgG2 and IgG4 from IgG1 are noted. The hinge and lower hinge regions, with the N-glycosylation site, N297, are noted. Numbering is according to EU numbering scheme (IMGT). Sequences important for FcgR binding, C1q, and FcRn are noted. Some increased ADCC mutants are shown above or below the sequences, as noted: Xencor’s S239D, A330L, I332E [red, above sequences]; Genentech’s S298A, E333A, K334A; and Eli Lilly/AME’s P247I,A339D/Q. Examples of FcRn- binding mutants for prolongation of half-life are also shown, as noted: MedImmune’s YTE mutant (M252Y,S254T,T256E), PDL’s T250Q, M428L mutant, Sally Ward (University of Southampton) H433K mutant, N434Y mutant, and Genentech’s N434A mutant are shown. [007] FIG.6 is a schematic of the chemical reaction conjugating insulin with GN3 to produce an αGN3 wild-type insulin peptide. [008] FIG.7 is a schematic of several examples of C-peptide conjugated to GN3. FIG.7A is a first example of C-peptide conjugated to GN3. FIG.7B is a second example of C-peptide conjugated to GN3. [009] FIG.8 is a schematic of several examples of insulin-peptide conjugated to GN3. FIG.8A is an example of native insulin with its B chain conjugated to GN3. FIG.8B is an example of a variant insulin with its B chain conjugated to GN3.
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DETAILED DESCRIPTION OF THE INVENTION [0010] The following detailed description is provided to aid persons having ordinary skill in the biomedical art. Exemplary embodiments are described. However, these embodiments are only exemplary. This disclosure is not limited thereto but is defined by the scope of the appended claims. Persons having ordinary skill in the biomedical art may make modifications and variations in the embodiments described in this specification without departing from the spirit or scope of this disclosure. Industrial Applicability. [0011] The invention provides a medically useful composition of matter (agent) for treating or slowing down the progression of diabetes and other insulin disorders. The removal and degradation of circulating anti-insulin and anti-proinsulin antibodies should improve the health of diabetic subjects by increasing the natural levels and availability of the subject’s insulin. [0012] The removal or suppression of pathogenic antibodies previously used general immune- suppressive therapies including TNF inhibitors, Jak inhibitors, FcRn recycling inhibitors, MTOR inhibitors, glucocorticoid receptor antagonists, pan-selective bispecific degraders, cyclophosphamide and other agents resulting in marked susceptibility to microbial, viral, and other infections and an increased incidence of cancer, especially lymphoma, through decreased immune-surveillance. Selective removal of pathogenic antibodies while sparing nonpathogenic antibodies, enables normal humoral immune responses including normal response to vaccination, resistance to infection and other important effector functions of the spared, non-autoreactive antibodies. This approach provides an anti-inflammatory medicine without other unwanted immune-mediated or immunosuppression-mediated consequences. The determination of lowering of immunoneutralizing antibodies to insulin is readily and specifically determined by commercially available diagnostic tests for the detection of these antibodies. [0013] There are about 300,000 childhood diabetes patients in the United States who are less than twenty years of age. Insulin as an autoantigen in the immune pathogenesis of Type I diabetes. [0014] Many diabetic adults have autoantibody-associated insulin resistance. In some, this is a life- threatening disease. [0015] Many domestic animals have autoantibody-associated insulin resistance. About 165,000 dogs and 300,000 cats in the United States have diabetes mellitus. Over 10% are treated with human insulin. [0016] After the bifunctional degrader and the bound insulin protein are endocytosed, they are released from the ASGPR through depletion of calcium from the endosome and changes in binding site
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amino acid protonation changes due to a decrease in pH. The ASGPR is recycled back to the hepatocyte surface. Endocytosed proteins are trafficked to late endosomes, which are fused with lysosomes. Lysosomal proteases then degrade endocytosed proteins, permanently removing them from circulation. Definitions [0017] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are listed below. Unless stated otherwise or implicit from context, these terms and phrases shall have the meanings below. These definitions aid in describing embodiments but are not intended to limit the claimed invention. [0018] As used in this application, except as otherwise provided in this specification, each term shall have the meaning set forth below. Additional definitions are set forth throughout the application. Where a term is not specifically defined in this specification, that term is given a biomedical art- recognized meaning applying that term in context to its use in describing the invention. [0019] The articles "a" and "an" have the plain meaning of one or to more than one, i.e., at least one, of the grammatical object of the article unless the context indicates otherwise. For example, "an element" means one element or more than one element. [34] The term “ABT” has the meaning described in this specification of a binding moiety that is itself an antibody, an antibody variant, or an antigen-binding fragment thereof. In some embodiments of this specification, the ABT binds to insulin. [0020] The term “active Ingredient” has the United States Food & Drug Administration-provided meaning of any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of a human body or an animal body. [0021] The term “ADCC” has the biomedical art-recognized meaning of antibody-dependent cell- mediated cytotoxicity, is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system kills a target cell, whose membrane-surface antigens were bound by specific antibodies. [0055] The term “ADCP” has the biomedical art-recognized meaning of antibody-dependent cell- mediated phagocytosis, an immunological mechanism of elimination whereby tumor cells are targeted with antibodies to promote their clearance from the body by phagocytic immune cells. [0022] The term “agent” has the biomedical art-recognized meaning of a composition of matter useful for performing a function. Several biomedically useful functions are described in this specification.
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[0056] The term “alleviate” has the biomedical art-recognized meaning of a process by which the severity of a sign or symptom of a disorder is reduced. A sign or symptom can be alleviated without being eliminated. The administration of compositions or pharmaceutical compositions of the invention may or can lead to the elimination of a sign or symptom, however, elimination is not required. Effective dosages should be expected to decrease the severity of a sign or symptom. [0057] The term “an effective amount” and the term “a therapeutically effective amount” has the biomedical art-recognized meaning of an amount effective to achieve its intended purpose. The effect can be detected by any assay method known in the art. The precise effective amount for a subject depends on the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a situation can be determined by routine experimentation that is within the skill and judgment of a clinician. In embodiments, the disease or condition to be treated is tendinopathy. [0058] The term “analogue” has the biomedical art-recognized meaning of a molecule that is not identical but has analogous functional or structural features. Biochemical modifications could increase the analogue's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. Insulin analogues are known in the biomedical art, such as the fast-working insulins called rapid-acting insulin. [0059] The term “anti-drug antibody (ADA)” has the biomedical art-recognized meaning. Many ADAs are IgG4 antibodies, such as those in Factor 8 hemophilia. [0060] The term “anti-insulin antibody” has the biomedical art-recognized meaning and includes insulin autoantibodies (IAA). 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 have similar affinities to mAb49. [0061] The term “antigen-binding fragment thereof” has the biomedical art-recognized meaning of (1) a fragment of an intact antibody that binds to the same antigen recognized by the full-length antibody, such as F(abʹ)2, F(ab)2, Fabʹ, Fab, Fv, sFv, or other fragments consisting of the variable regions, or (2) any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The term antigen-binding portion of an antibody encompasses single chain antibodies.
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[0062] The term “asialoglycoprotein receptor (ASGPR) binding group” has the biomedical art- recognized meaning of a binding group which binds to a hepatocyte asialoglycoprotein receptor. The ASGPR binding group selectively binds to hepatocyte asialoglycoprotein receptor on the surface of hepatocytes. In several embodiments of this specification, an ASGPR binding group is a component of a bifunctional agent as a cellular receptor-binding moiety which is covalently bound to the antibody binding moiety through a linker group or directly. It is through this ASGPR moiety that bifunctional agents complexed with a circulating protein, e.g., bind to hepatocytes. After the bifunctional agent complexed with a circulating protein is bound to a hepatocyte or other cell, the circulating protein is taken into the hepatocyte or other cell via a phagocytosis mechanism, wherein the circulating protein is degraded through lysosomal degradation. [0063] The term “asialoglycoprotein receptor” (ASGPR) has the biomedical art-recognized meaning of lectins which bind asialoglycoprotein and glycoproteins from which a sialic acid was removed to expose galactose residues. These cellular receptors are located on mammalian hepatocytes and other cells, such as glandular cells of the gallbladder and the stomach. ASGPR remove target glycoproteins from circulation. [0064] The term "at least one of," when preceding a list of elements, modifies the entire list of elements and does not modify the individual elements of the list. [0065] The term “AT” has the meaning described in this specification of an antibody moiety. In some embodiments of this specification, the AT binds to insulin. [0066] The term “autoantibody” has the biomedical art-recognized meaning. [0067] The term “binding moiety” has the biomedical art-recognized meaning a moiety on a binding protein, e.g., an antibody, an antibody variant, or an antigen-binding fragment thereof, that binds to insulin. [0068] The term “cellular receptor-binding moiety” has the biomedical art-recognized meaning. In several embodiments of this specification, the cellular receptor-binding moiety is an asialoglycoprotein receptor (ASGPR) binding group. [0069] The term “cellular receptor” has the biomedical art-recognized meaning of a protein on the surface of a cell that binds to a compound, e.g., a ligand, e.g., a protein, in solution or on another cell. Generally, ligand-receptor-binding induces one or more biological responses. In this specification, an asialoglycoprotein receptor (ASGPR) is a cellular receptor on the surface of hepatocytes or other cells that binds to an asialoglycoprotein or a derivative thereof.
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[0070] The term “chimerized” has the biomedical art-recognized meaning. Chimeric antibodies are made by fusing variable domains from one species, such as a mouse, with constant domains from another species, such as a human being. With such biotechnical manipulation, chimeric antibodies keep the foreign antibody’s antigen specificity and affinity. [0071] The term “combination therapy” and the “co-therapy” has the biomedical art-recognized meaning of the administration of a composition described in this specification and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co- action of these therapeutic agents. The beneficial effect of the combination may include, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time, usually minutes, hours, days, or weeks depending on the combination selected. The term combination therapy” includes the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies, e.g., surgery or radiation treatment. Where the combination therapy further comprises a non-drug treatment, the non- drug treatment may be conducted at any suitable time if a beneficial effect from the co-action of the combination of the therapeutic agents a is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks. [0072] The term “complementarity determining region (CDR)” has the biomedical art-recognized meaning of a polypeptide region of an antibody heavy chin or an antibody light chain that is a determinant of the antibody to antigen binding. Each antibody heavy chain contains three CDRs. Each antibody light chain contains three CDRs, usually different from the three CDRs on an antibody heavy chain. Persons having ordinary skill in the biomedical art can calculate the CDR structure using a standardized numbering method known as the Kabat numbering scheme. Other numbering schemes, such as the Chothia, IMGT, Martin (enhanced Chothia), and other standard numbering schemes, are also used by persons having ordinary skill in the biomedical art. [0073] The term "comprises," the term "comprising," the term "includes," and the term "including" specify stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof. [0074] The term “diabetes mellitus” has the biomedical art-recognized meaning. See Wikipedia, the free encyclopedia, Dipeptidyl peptidase-4 inhibitor (online, accessed July 3, 2024). In type 1 diabetes
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mellitus, the destruction of insulin-producing β-pancreatic cells results in an inability to control blood glucose levels since insulin signals glucose uptake into cells. Type 1 diabetes mellitus is a systemic disease that can lead to severe complications. Hyperglycemia is a symptom of diabetes. A human diabetic patient is someone whose blood sugar level is elevated or is expected to be elevated, and therefore someone who desires to inhibit from rising or to lower a blood sugar level. [0075] The term “diabetic macular edema” (DME) has the biomedical art-recognized meaning of a diabetic retinopathy. See Landry et al., Assay Drug Dev. Technol.11(5), 326-32 (June 2013). Tiny bulges, called microaneurysms, form in the blood vessels, leaking fluid into the retina. [0076] The term “EC50” has the biomedical art-recognized meaning of the ligand concentration required to achieve 50% of maximal receptor activation. [0077] The term “Fc-III-4c” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody. [0078] The term “Fc-M” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody. [0079] The term “Fc” has the biomedical art-recognized meaning. [0080] The term “FcB-1” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody. [0081] The term “FcB-2” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody. [0082] The term “first,” “second,” “third,” etc. have the plain meaning of describing several elements, components, regions, layers, and/or sections, these elements, components, regions, layers, and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. A first element, component, region, layer, or section could be called a second element, component, region, layer, or section without departing from the teachings of the present embodiments. [0083] The term “hepatocyte” has the biomedical art-recognized meaning of a cell of the main parenchymal tissue of the liver. Hepatocytes make up 55-65% of the liver's mass. [0084] The term “humanized” has the biomedical art-recognized meaning a protein, e.g., an antibody, is genetically engineered so it closely resembles the polypeptide structure of the human homologue. A variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, thus keeping the specificity of the rodent antibody. The human origin domain need not originate directly from a human because it is first synthesized in a human. Instead, human domains can
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be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially or completely humanized. In one approach, there are four general steps used to humanize a monoclonal antibody. These steps are (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process; (3) the actual humanizing methodologies/techniques; and (4) the transfection and expression of the humanized antibody. [0085] The term “IgG” antibody has the biomedical art-recognized meaning. Each IgG molecule consists of the basic four-chain immunoglobulin structure—two γ (gamma) heavy chains and two identical light chains (either kappa or lambda)—and carries two identical antigen-binding sites. There are four subclasses of IgG, each with minor differences in its H chains but with distinct biological properties. [0086] The term “IgG1” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-1 chain C region is a protein that in humans is encoded by the IGHG1 gene. [0087] The term “IgG2” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-2 chain C region is a protein that in humans is encoded by the IGHG2 gene. [0088] The term “IgG4” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-4 chain C region is a protein that in humans is encoded by the IGHG4 gene. IgG4 has little effector function. IgG4 cannot fix complement. [0089] The term “insulin efsitora alfa” (LY-3209590), means the selective agonist of insulin receptor (IR) that is a fusion protein composed of human IR agonists fused with the crystallizable (Fc) domain of human immunoglobulin G2 (lgG2) fragment, with a molecular weight of 64.1 kDa. Insulin efsitora alfa is being developed as a possible diabetes therapeutic. The protein may be used a tool compound or as a control compound for the insulin-Fc fusion protein and proinsulin FC fusion protein described in this specification. [0090] The term “insulin growth factor” (IGF) has the biomedical art-recognized meaning. Human IGF-1 means comprising the amino acid sequence corresponding to human IGF native to human tissue. Human IGF-1 may include fusion proteins. [0091] The term “insulin lispro” has the biomedical art-recognized meaning of a rapid-acting insulin analog, a synthetic version of insulin, used to lower blood sugar levels in people with diabetes, working faster 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
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positions of proline at position B28 and lysine at position B29 in the B chain have been reversed. In insulin lispro, these variations reduce the strength of the interactions that hold insulin dimers together, allowing for faster dissociation of hexamers and hence absorption of monomers. These analogs and formulations result in a quicker onset of action, a quicker time to peak activity, and a shorter duration of action compared to human regular insulin, making them more optimal for mealtime administration. [0092] The term “insulin receptor” has the biomedical art-recognized meaning. The insulin receptor has two main subunits: α and β. There is a single disulfide bridge between α and β subunits between Cys647 in the insert domain and Cys872 The α subunit contains five main domains, L1 (AA 28–174) CR (AA 182–339), and L2 (AA 340–497), and two fibronectin subunits: FnIII-1 (residue 624–726) and FnIII-2 (757–842). The two α-subunits are linked by four disulfide bonds. The insulin receptor β has different isoforms (IRA and IRB), depending on the gene splicing of exon 11. Insulin receptor B differs from insulin receptor A by including exon 11. The 12-amino acid sequence (residues 745–756) derived from exon 11 is present in the insulin receptor B isoform but absent in the insulin receptor A isoform. The isoforms have functionally different internalization and recycling. Insulin receptor A exhibits a greater internalization and recycling rate than insulin receptor B. Insulin receptor A exhibits a higher affinity for IGFs than IRB. Both isoforms have similar affinity for insulin Binding to these isoforms very different to proinsulin. Because of these differences, insulin receptor B is preferentially associated with metabolic and differentiating signals. Insulin receptor A mainly favors 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 biomedical art-recognized meaning of the well-characterized neutral peptide secreted by the liver to circulate in the blood. IGF-2 has growth- regulating, insulin-like and mitogenic activities. [0094] The term “insulin” has the biomedical art-recognized meaning. Insulin can be produced by chemical synthesis. [0095] The term “IVIG” has the biomedical art-recognized meaning of the administration of intravenous immunoglobulin. [0096] The term “linker moiety” has the biomedical art-recognized meaning of a moiety of a chemical compound that links one moiety of the chemical compound to another moiety of the same compound. [0097] The term “MoDE” has the proprietary meaning of molecular degraders. See International Patent Publications WO 2019/199634 (Yale University) and WO 2019/199621 (Yale University).
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[0098] The term “moiety” has the biomedical meaning of a defined chemical group or entity with a particular structure or activity. A moiety generally refers to a part of a molecule. In some embodiments, a binding moiety maintains one or more desired structural features, properties, functions, or properties, e.g., 3-dimension structure, antigen specificity, antigen-binding capacity, or immunological functions, etc. comparable to its corresponding binding protein, e.g., an antibody. In some embodiments, a moiety is monovalent. In some embodiments, a moiety is bivalent. In other embodiments, a moiety is polyvalent. [0099] The term “monotherapy” has the biomedical art-recognized meaning of the administration of a single active or therapeutic compound to a subject. Monotherapy usually is the administration of a therapeutically effective amount of an active composition. [00100] The term “Multimodal Antibody Therapy Enhancers (MATE or MATES)” has the proprietary meaning. See International Patent Publication WO 2021/102052 (Kleo Pharmaceuticals). [35] The term “nanobody” has the biomedical art-recognized meaning. the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains and a variety of forms including full length antibodies and antigen-binding portions thereof; including an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fabʹ, a F(abʹ)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a nanobody, a multi-specific, e.g., tri-specific, antibody, a dual specific antibody, a bispecific antibody, an anti-idiotypic antibody, a functionally active epitope-binding portion thereof, and/or bifunctional hybrid antibodies. [00101] The term "on" has the plain meaning. When an element is referred to as being on another element, it can be directly in contact with the other element or intervening elements may be present therebetween. When an element is referred to as being "directly on" another element, there are no intervening elements present. [00102] The term "or" as used in this specification means "and/or." The term "and/or" as used in this specification includes all combinations of one or more of the associated listed items. [00103] The term “other degrading cells” has the biomedical art-recognized meaning. Asialoglycoprotein receptors (ASGPRs) are on the glandular cells of the gallbladder and the stomach, as well as on hepatocytes.
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[00104] The term “partially humanized” has the biomedical art-recognized meaning a protein, e.g., an antibody, is genetically engineered so it more closely resembles the polypeptide structure of the human homologue. A variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, thus keeping the specificity of the rodent antibody. The domain of human origin need not originate directly from a human because it is first synthesized in a human. Instead, human domains can be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially or completely humanized. [00105] The term “peripheral blood mononuclear cell (PBMC)” has the biomedical art-recognized meaning of any peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. [00106] The term “pharmaceutically acceptable excipient” has the biomedical art-recognized meaning of an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use and human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient. A thorough discussion of pharmaceutically acceptable excipients is available in Remington’s, Pharmaceutical Sciences 23rd edition (Elsevier, 2020). [00107] The term “pharmaceutically acceptable” has the biomedical art-recognized meaning of those compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [00108] The term “proinsulin” has the biomedical art-recognized meaning, being a precursor to mature insulin. Proinsulin is commercially available from R+D systems. Proinsulin can also be produced by chemical synthesis. The chemically synthesized proinsulin is comparable to commercial proinsulin for the surface plasmon resonance (SPR) assay. Proinsulin does not bind to the insulin receptor. [00109] The term “protein binding moiety” has the biomedical art-recognized meaning of a region of a chemical composition, e.g., a polypeptide region of a chemical composition, that specifically binds to a protein, e.g., a specific protein. [00110] The term “ROC” has the biomedical art-recognized meaning of receiver operating characteristic curve.
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[00111] The term “subject” and the term “patient” have the biomedical art-recognized meanings. The term patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. [00112] The term “TBT” has the meaning described in this specification of target binding moiety, a cellular receptor-binding moiety. In some embodiments of this specification, the TBT binds to ASGPR. [00113] The term “TRAP” has the meaning described in this specification of a targeted removal of aberrant protein. A TRAP is a bifunctional degrader. [00114] The term “treating” and the “treat” has the biomedical art-recognized meaning of the management and care of a patient for combating a disease, condition, or disorder and includes the administration of a composition described in this specification to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. [00115] The term “Type 1 diabetes mellitus” has the biomedical art-recognized meaning of a chronic autoimmune disease that occurs when the immune system attacks the pancreas' insulin-producing cells, resulting in the autoimmune eradication of β-cells in pancreatic islets. Insulin can no longer be synthesized or be secreted into the blood. Other autoantibodies directed to islet cells may also be present in Type 1 diabetics. [00116] The term “Type 2 diabetes mellitus” has the biomedical art-recognized meaning of the progressive loss of insulin receptors Inducing hyperglycemia. Hyperglycemia induces more production of insulin by β-cells to compensate. The accumulation of amyloid in the pancreatic islets likely disrupts islet anatomy and physiology. Eventually, β-cells wear out leading to decreased insulin over time. [00117] The term “universal antibody binding moiety” has the biomedical art-recognized meaning of a polypeptide region of an antibody-binding protein that binds a class of antibodies, rather than a specific set of antibodies. [00118] Some embodiments are described below by referring to structures and schemes, to explain aspects of the description. [00119] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by persons having ordinary skill in the biomedical art. [00120] This specification does not concern a process for cloning humans, methods for modifying the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or procedures for modifying the genetic identity of animals likely to cause them suffering with no substantial medical benefit to humans or animals resulting from such processes.
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Methods of administration. [00121] The ideal mode of administration depends on where treatment is taking place, whether a hospital or outpatient. Methods of measuring the subject’s improvement after removal of anti-insulin antibodies. [00122] Many assays are commercially available to measure a subject’s insulin levels. As known by persons having ordinary skill in the biomedical art, a normal and stable insulin level indicates the health and continued health of a subject with diabetes or other insulin disorders. The chemical structure of the composition of matter (agent, TRAP). [00123] In an embodiment, the invention provides a composition of matter (agent) comprising: a binding moiety that can bind to 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. [00124] In some embodiments, the invention provides a composition of matter (an agent) having a structure selected from the Markush group of structures consisting of: RCN−(Xaa)y−RCC,
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or a pharmaceutically acceptable salt thereof. In these structures, a, b, and c may independently be an integer of 1 or greater. In some embodiments, each cellular receptor-binding moiety independently has the structure of −(RCN−(Xaa)y−RCC) or salt form thereof. [00125] In some embodiments, the agent has the structure of formula AGN101 or AGN103: , or a pharmaceutically acceptable salt thereof, wherein: each of a, b and c is independently 1-200; each AT is independently a binding moiety; L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety, wherein the binding moiety is an antigen-binding protein, an antigen-binding protein variant, or an antigen-binding fragment thereof. [00126] In some embodiments, an agent comprises one and no more than one binding moiety. In some embodiments, one or no more than one binding moiety is bound to a linker moiety. In some embodiments, a is 1. In some embodiments, a is 2 or more. In some embodiments, one and no more than one cellular receptor-binding moiety is bonded to a linker moiety. In some embodiments, b is 1. In some embodiments, two or more cellular receptor-binding moiety is bonded to a single linker moiety. In some embodiments, b is 2 or more. In some embodiments, an agent comprises one and no more than one cellular receptor-binding moiety. In some embodiments, c is 1. In some embodiments, b is 1 and c is 1. In some embodiments, a is 1, b is 1 and c is 1. In some embodiments, an agent comprises two or more
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target binding moieties. In some embodiments, b is 2 or more and c is 1. In some embodiments, b is 2 or more and c is 2 or more. In some embodiments, b is 1 and c is 2 or more. [00127] In some embodiments, each cellular receptor-binding moiety in an agent is the same. In some embodiments, each linker moiety connecting a cellular receptor-binding moiety to an antibody moiety is the same. In some embodiments, the TBT in agents is the same. In some embodiments, the −L−(TBT)b are the same. [00128] Persons having ordinary skill in the biomedical art know that several technologies can be used to conjugate antibody moieties with target binding moieties, e.g., certain technologies used for preparing antibody-drug conjugates in accordance with this specification. In some embodiments, target binding moieties are connected to antibody moieties through certain types of groups and/or amino acid residues. In some embodiments, target binding moieties are connected to lysine residues optionally through linker moieties. In some embodiments, target binding moieties are connected to cysteine residues optionally through linker moieties. In some embodiments, target binding moieties are connected to unnatural amino acid residues optionally through linker moieties. In some embodiments, the invention provides technologies for selectively linking target binding moieties to certain amino acid residues optionally through linker moieties. In some embodiments, provided technologies selectively connect target binding moieties to certain types of amino acid residues, e.g., lysine residues, optionally through linker moieties. In some embodiments, provided technologies selectively connect target binding moieties to sites of antibody moieties optionally through linker moieties. In some embodiments, provided technologies selectively connect target binding moieties to certain types of amino acid residues at sites optionally through linker moieties. In some embodiments, target binding moieties are connected to K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, target binding moieties are connected to K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, target binding moieties are connected to K239 and K241 of a heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, a cellular receptor-binding moiety is connected to a particular amino acid residue or site optionally through a linker. In some embodiments, each cellular receptor-binding moiety is independently connected to a particular amino acid residue or site optionally through a linker. As known by persons having ordinary skill in the biomedical art, an antibody agent may comprise more than one sites, e.g., one on each of the more than one chain, e.g., one or each heavy chain. In some embodiments, an antibody moiety comprises two heavy chains and one or both amino acid residues or
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amino acid residues corresponding thereto are each independently connected to a cellular receptor- binding moiety optionally through a linker. In some embodiments, one and no more than one is connected. In some embodiments, c is 1. In some embodiments, both are connected. In some embodiments, c is 2. In some embodiments, both target binding moieties and/or both linker moieties (if any) are the same. [00129] In some embodiments, the invention provides a composition of matter (an agent) of formula AGN105: or a pharmaceutically acceptable salt thereof, wherein: each Xaa is independently a residue of an amino acid or an amino acid analog; t is 0-50; z is 1-50; L is a linker moiety; TBT is a cellular receptor-binding moiety; each Rc is independently −La−R’; each of a and b is independently 1-200; each La is independently a covalent bond, or an optionally substituted bivalent group selected from a C1-C20 aliphatic group or a C1-C20 heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, or −C(O)O−; each −Cy− is independently an optionally substituted bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; each R’ is independently −R, −C(O)R, −CO 2R, or −SO2R; each R is independently −H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur,
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phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or two or more R groups on two or more atoms are optionally and independently taken with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. [00130] Further guidance is provided in International Patent Publication WO 2025/035052 (Biohaven Therapeutics Ltd.), the contents of which are incorporated by reference. TABLE 5
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TABLE 5 AGN302 Mutant insulin with GN3 N O NH O N O
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TABLE 5 AGN402 Synthetic C-peptide constructs (D-amino acids) with protease αGN3, no binding to O NH N O g
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TABLE 5 AGN404 βGN3, synthetic C-peptide constructs (D-amino acids), no binding to either of the C-
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TABLE 5 AGN303 Native proinsulin, lns-003, made by chemical synthesis conjugated to GN3 (triGalNAc) o
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TABLE 5 AGN310 Insulin TRAP. Target binding moiety (lns-004), made by chemical synthesis conjugated e-
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TABLE 5 AGN406 C-peptide with βGN3 (L-amino acids. C-peptide with protease site, synthetic C-
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TABLE 5 AGN407 C-peptide with protease βGN3 (L-amino acids). C-peptide without protease site, e =
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TABLE 5 AGN417 ABT410-TBT403. Native proinsulin TRAP. Native proinsulin-ASGPR ligand βGN3 (N- % d nt D
35 30125-WO-PCT TABLE 5 AGN8138 ABT410-TBT7988 e C-pept e ant o es. [00132] AGN404. β-GN3, synthetic C-peptide constructs (D-amino acids), no binding to either of the C-peptide antibodies.
[00133] AGN406. β-GN3, synthetic C-peptide constructs (L-amino acids). [0 nM. Recombinant mouse anti-C- peptide. KD = 15 µM. AGN406 C-peptide internalization IC50 = 4.2 nM.
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[00135] AGN407. β-GN3 (protease), synthetic C-peptide constructs (L-amino acids). [ . Recombinant mouse anti-C- peptide. KD = 7.9 µM. AGN407 C-peptide internalization IC50 = 7.9 nM.
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[00137] AGN412. Sequence: GIVEQ[CC*TSIC]SLYQLENYC**N β-GN-triazole-(pent-4-ynoic acid)- FVNQHLC*GSHLVEALYLVC**GERGAFYTPKT (SEQ ID NO: 43).
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[00138] AGN416. Sequence: αGN-triazole-(pent-4-ynoic acid)- GIVEQ[CC*TSIC]SLYQLENYC**FVNQHLC*GSHLVEALYLVC**GERGAFYTPKT (SEQ ID NO: 43). Target-binding moiety. [00139] In some embodiments, the target binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues. [00140] In some embodiments, each binding moiety in an agent may be of the same binding moiety or a pharmaceutically acceptable salt thereof. [00141] In some embodiments, the binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues.
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[00142] Several antibody binding moieties, including universal antibody binding moieties, can be used in accordance with the teachings of this specification. Certain antibody binding moieties and technologies for identifying or assessing antibody binding moieties are described in International Patent Publications WO 2019/023501 (Kleo Pharmaceuticals, Inc.) and WO 2019/136442 (Kleo Pharmaceuticals, Inc.), each of which is incorporated in this specification in its entirety by reference. Persons having ordinary skill in the biomedical art know that additional technologies in the biomedical art may be suitable for identifying or assessing antibody binding moieties in accordance with this specification. In some embodiments, an antibody binding moiety comprises one or more amino acid residues, each independently natural or unnatural. [00143] In some embodiments, a binding moiety, e.g., a protein binding moiety, e.g., an antibody binding moiety, e.g., a universal antibody binding moiety, has the structure of ABT101 or a salt form thereof, wherein: each of R1, R3 and R5 is independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or: R1 and R1’ are optionally taken with their intervening carbon atom to form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R3 and R3’ are optionally taken with their intervening carbon atom to form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; an R5 group and the R5’ group attached to the same carbon atom are optionally taken with their intervening carbon atom to form a 3-8 membered optionally substituted saturated or partially unsaturated spirocyclic carbocyclic ring or a 3-8 membered saturated or partially unsaturated spirocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or
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two R5 groups are optionally taken with their intervening atoms to form a C1-10 optionally substituted bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-3 methylene units of the chain are independently and optionally replaced with –S–, –SS–, –N(R)–, –O–, – C(O)–, –OC(O)–, –C(O)O–, –C(O)N(R)–, –N(R)C(O)–, –S(O)–, –S(O)2–, or –Cy1–, wherein each –Cy1– is independently a 5-6 membered heteroarylenyl with 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; each of R1’, R3’ and R5’ is independently hydrogen or optionally substituted C1-3 aliphatic; each of R2, R4 and R6 is independently hydrogen, or optionally substituted C1-4 aliphatic, or: R2 and R1 are optionally taken with their intervening atoms to form a 4-8 membered, optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R4 and R3 are optionally taken with their intervening atoms to form a 4-8 membered optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or an R6 group and its adjacent R5 group are optionally taken with their intervening atoms to form a 4-8 membered optionally substituted saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; L1 is a trivalent linker moiety; and each of m and n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. [00144] In some embodiments, L1 is an optionally substituted trivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, or −C(O)O−. In some embodiments L1 is –(CH2CH2O)2-4– or –(CH2CH2O)2–. [00145] Several antibody binding moieties, including universal antibody binding moieties, can be used in accordance with the teachings of this specification. Some antibody binding moieties and technologies for identifying or assessing antibody binding moieties are described in International Patent Publications WO 2019/023501 (Kleo Pharmaceuticals, Inc.) and WO 2019/136442 (Kleo Pharmaceuticals, Inc.), each of which is incorporated in this specification in its entirety by reference. Persons having ordinary skill in the biomedical art know that additional technologies in the biomedical art may be suitable for identifying or assessing antibody binding moieties in accordance with this specification. In
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some embodiments, an antibody binding moiety comprises one or more amino acid residues, each independently natural or unnatural. [00146] In some embodiments, selected locations of antibody moieties are used for conjugation. In some embodiments, K246 or K248 of an antibody agent (EU numbering, or corresponding residues) are conjugation locations. In some embodiments, a conjugation location is K246 of heavy chain (unless otherwise specified, locations in this specification include corresponding residues in, e.g., modified sequence, e.g., longer, shorter, rearranged, etc., sequences. In some embodiments, a location is K248 of heavy chain. In some embodiments, a location is K288 or K290 of heavy chain. In some embodiments, a location is K288 of heavy chain. In some embodiments, a location is K290 of heavy chain. In some embodiments, a location is K317. In some embodiments, an antibody moiety is a moiety of an IgG1 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG2 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an antibody or a fragment thereof. In some embodiments, a composition comprises a plurality of MATE agents, wherein antibody moieties of the plurality of MATE agents are independently an antibody moiety of an IgG1, IgG2, or IgG4 antibody, or a fragment thereof. [00147] In some embodiments, antibody heavy chains are selectively conjugated/labeled over light chains. [00148] In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target binding moieties, conjugated to antibody moieties of a particular type of antibodies, e.g., IgG1, or fragments thereof are conjugated to one or more particularly sites, typically one or two particularly sites, e.g., K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K239 and K241 of a heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., for a plurality of agents, for a composition, etc. are conjugated to antibody moieties of IgG1, IgG2, and/or IgG4 antibodies, or fragments thereof, e.g., for conjugation products with IgG1 antibodies or fragments thereof (antibody moieties being of IgG1 antibodies or fragments thereof), IgG2 antibodies or fragments thereof (antibody moieties being of IgG2 antibodies or fragments thereof), IgG4 antibodies or fragments thereof (antibody moieties being of IgG4 antibodies or
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fragments thereof), or for conjugation products with IVIG (when certain provided technologies described in this specification are used, selective conjugation with IgG1, IgG2 and IgG4). In some embodiments, a percentage is about 10% or more. In some embodiments, a percentage is about 20% or more. In some embodiments, a percentage is about 25% or more. In some embodiments, a percentage is about 30% or more. In some embodiments, a percentage is about 40% or more. In some embodiments, a percentage is about 50% or more. In some embodiments, a percentage is about 60% or more. In some embodiments, a percentage is about 65% or more. In some embodiments, a percentage is about 70% or more. In some embodiments, a percentage is about 75% or more. In some embodiments, a percentage is about 80% or more. In some embodiments, a percentage is about 85% or more. In some embodiments, a percentage is about 90% or more. In some embodiments, a percentage is about 95% or more. In some embodiments, a percentage is about 100%. [00149] Further guidance is provided in International Patent Publication WO 2025/035052 (Biohaven Therapeutics Ltd.), the contents of which are incorporated by reference. TABLE 6 ABT401 LALA i i F d i d i i b AGN301 I G =
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TABLE 6 ABT306 Alk-native insulin r, S s
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TABLE 6 ABT409 Anti-C-peptide antibody, mouse VH/VL-human IgG3 CH/CL monoclonal antibody. Linker Moieties [00150] In some embodiments, moieties are optionally connected to each other through linker moieties. In some embodiments, a reactive group, e.g., RG, is connected to a cellular receptor-binding moiety, e.g., TBT, through a linker, e.g., LRM. In some embodiments, a moiety, e.g., LG, may also comprise
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one or more linkers, e.g., LLG1, LLG2, LLG3, LLG4, etc., to link several portions. In some embodiments, LLG is a linker moiety described in this specification. In some embodiments, LLG1 is a linker moiety described in this specification. In some embodiments, LLG2 is a linker moiety described in this specification. In some embodiments, LLG3 is a linker moiety described in this specification. In some embodiments, LLG4 is a linker moiety described in this specification. In some embodiments, LRM is a linker moiety described in this specification. In some embodiments, LPM is L. In some embodiments, LPM is a linker moiety described in this specification. In some embodiments, LPM is L. [00151] Linker moieties of several types and/or for several purposes, e.g., those used in antibody- drug conjugates, etc., may be used in accordance with this specification. [00152] Linker moieties can be bivalent or polyvalent depending on how they are used. In some embodiments, a linker moiety is bivalent. In some embodiments, a linker is polyvalent and connecting more than two moieties. [00153] In some embodiments, a linker moiety, e.g., Lz, e.g., LPM, LRM, LLG, LLG1, etc. , is or comprises L. [00154] In some embodiments, the linker moiety has the structure of formula LNK101: NH(Ra1)−La1−C(Ra2)(Ra3)−La2−COOH [LNK101] or a pharmaceutically acceptable salt thereof, wherein: each of Ra1, Ra2, Ra3 is independently −La−R’; each of La1 and La2 is independently La; each La is independently a covalent bond, or an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with −C(R’)2−, −Cy−, −O−, −S−, −S−S−, −N(R’)−, −C(O)−, −C(S)−, −C(NR’)−, −C(O)N(R’)−, −N(R’)C(O)N(R’)−, −N(R’)C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R’)−, −C(O)S−, or −C(O)O−; each −Cy− is independently an optionally substituted bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; each R’ is independently −R, −C(O)R, −CO2R, or −SO2R; each R is independently −H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur,
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phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or two or more R groups on two or more atoms are optionally and independently taken with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. [00155] In some embodiments, an amino acid residue has the structure of −N(Ra1)−La1−C(Ra2)(Ra3)−La2−COO− or a salt form thereof. [00156] Further guidance is provided in International Patent Publication WO 2025/035052 (Biohaven Therapeutics Ltd.), the contents of which are incorporated by reference. MATES [00157] Persons having ordinary skill in the biomedical art can use MATES materials and methods as guidance to predictable results when making and using the invention. [00158] Such an agent may be called a MATE agent or MATE. The MATE agents are described, for example, in International Patent Publication WO 2021/102052 (Kleo Pharmaceuticals, Inc.), the contents of which are incorporated by reference. In some embodiments, an agent comprises an antibody moiety, a cellular receptor-binding moiety, and a linker moiety linking an antibody moiety and a cellular receptor-binding moiety. Cellular receptor-binding moiety. [00159] Several receptor-binding moieties, according to embodiments of present invention, are described in WO 2019/19962 (Bostal Drug Delivery Co., Ltd), WO 2019/199634 (Yale University), WO
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2021/072246 (Yale University), and WO 2021/072246 (Yale University), each of which is incorporated in this specification in its entirety by reference. [00160] In an embodiment, the cellular receptor-binding moiety may include an asialoglycoprotein receptor (ASGPR) binding group connected through an amine group to the linker moiety. [00161] The amine group may be a primary alkyl amine group or secondary alkyl amine group, each of which is optionally substituted on the amine group with a C1-C3 alkyl group. [00162] The cellular receptor-binding moiety may include an ASGPR binding group according to the chemical structure: , , wherein X is 1-4 atoms in length and comprises O, S, N(RN1) or C(RN1)(RN1) groups such that: when X is 1 atom in length, X is O, S, N(RN1) or C(RN1)(RN1), when X is 2 atoms in length, no more than 1 atom of X is O, S or N(RN1), when X is 3 or 4 atoms in length, no more than 2 atoms of X are O, S or N(RN1); wherein RN1 is H or a C1-C3 alkyl group optionally substituted with from 1-3 halo groups; R1 and R3 are each independently: H, -(CH2)KOH, -(CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -(CH2)K-vinyl, O- (CH2)K-vinyl, -(CH2)K-alkynyl, -(CH2)K-COOH,
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-(CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O-C(O)-C1- C4 alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, or R1 and R3 are each independently a group, which is optionally substituted with up to three halo groups, C1-C4 which alkyl group is optionally substituted with from one to three halo groups or one or two hydroxyl groups, or O-C1-C4 alkyl groups, each of which alkyl groups is optionally substituted with from one to three halo groups or one or two hydroxyl groups; and K is independently an integer of 0 to 4, or R1 and R3 are each independently a group according to the chemical structure: , wherein R7 is O-C1-C4 alkyl, which is optionally substituted dr 7 oxy groups, or R is a -NRN3RN4 group or a; or R1 and R3 are each independently a group according to the structure: , mical structure:
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R1 and R3 are each independentl group, where 8 saturated carbocyclic group; nt, H, C1-C4 alkyl optionally substituted with from 1-3 halo groups or 1-2 hydroxyl groups, or a group according to the structure: pendently, H, halo (F, Cl, B N1 N2 r, I), CN, NR R , -(CH2)KOH, -(CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C3 alkyl, which is optionally substituted with from 1-3 halo groups, -O-C1-C3-alkyl, which is optionally substituted with from 1-3 halo groups, -(CH2)KCOOH, -(CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O-C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, or a up, 30125-WO
wherein RN, RN1 and RN2 are each independently H or a C1-C3 alkyl group optionally substituted with from one to three halo groups or one or two hydroxyl groups; K is independently an integer of 0 to 4; K’ is an integer of 1 to 4; RN3 is H, or a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups; and RN4 is H, a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups, or RN4 is a group, where K is preferably 1; is a linker group which comprises at least one binding moiety and links the at least one binding moiety to the cellular receptor-binding moiety through the optional linker moiety, or is a linker group which contains at least one or more functional groups which can be used to covalently bond the linker group to at least one binding moiety or optional linker moiety; group wherein RN1 and K are the same as above; onally substituted with up to three halo groups and one or two hydroxyl groups, a -(CH2)KCOOH group, a -(CH2)KC(O)O-C1-C4 alkyl group optionally substituted with from 1-3 halo groups, a O-C(O)-C1-C4 alkyl group, which is optionally substituted with from 1-3 halo F groups, a -C(O)-C1-C4 alkyl group, which is optionally substituted with from 1-3 halo groups, a -(CH2)K- NRN3RN4 group where RN3 is H, or a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups; and RN4 is H, a C1-C3 alkyl group optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups, or a group, or group, , , , , CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups,
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-(CH2)KCOOH, -(CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O- C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, or RTA is a C3-C10 aryl or a three-to ten-membered heteroaryl group containing up to 5 heteroaryl atoms, each of said aryl or heteroaryl groups being optionally substituted with up to three (preferably 1) CN, NRN1RN2, -(CH2)KOH, -(CH2)KOC1-C4 alkyl, which is optionally substituted with from 1-3 halo groups, C1-C3 alkyl, which is optionally substituted with from 1-3 halo groups or 1 or 2 hydroxy groups, -O-C1-C3-alkyl, which is optionally substituted with from 1-3 halo groups, -(CH2)KCOOH, - (CH2)KC(O)O-C1-C4 alkyl optionally substituted with from 1-3 halo groups, O-C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo groups or -(CH2)KC(O)-C1-C4 alkyl optionally substituted with from 1-3 halo groups, or up ee halo groups, or RTA is a group, wherein RN, RN1 and RN2 are each independently H or a C1-C3 alkyl group optionally substituted with from one to three halo groups or one or two hydroxyl groups and each -(CH2)K group is optionally substituted with 1-4, preferably 1 or 2, C1-C3 alkyl groups optionally substituted with from 1-3 fluoro groups or 1-2 hydroxyl groups; and K is independently 0-4. [00163] Further guidance is provided in International Patent Publication WO 2025/035052 (Biohaven Therapeutics Ltd.), the contents of which are incorporated by reference.
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TABLE 7 TBT103.
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TABLE 7 TBT403 βGN3-GLPETGG-NH2. NH2
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TABLE 7 TBT6170 N-terminal modification of proinsulin. .
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TABLE 7 TBT7990 NH OH O
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TABLE 7 TBT7989 O OH econ agen . [00164] In another embodiment, the invention provides a composition including the agent and at least one additional agent comprising a moiety capable of binding to the antibody that forms the antibody moiety of the first compound. [00165] In some embodiments, a first composition is a composition comprising a first agent as described in this specification. In some embodiments, second agents independently comprise second reactive groups. In some embodiments, a second composition is a composition comprising a plurality of agents as described in this specification, wherein each cellular receptor-binding moiety is independently a reactive group as described in this specification. In some embodiments, a second composition is an antibody composition, wherein antibodies in the composition are not chemically modified. In some embodiments, a second composition is an IVIG preparation. In some embodiments, a product composition is a composition comprising a plurality of agents as described in this specification, wherein each cellular receptor-binding moiety is independently a cellular receptor-binding moiety as described in this specification. [00166] In some embodiments, a cellular receptor-binding moiety in a product agent is a cellular receptor-binding moiety in a first agent. In some embodiments, an antibody moiety in a product agent is an antibody moiety in a second agent. In some embodiments, a second agent is an antibody agent, e.g., a monoclonal antibody, an antibody in a polyclonal antibody, an antibody in an IVIG preparation, etc. In some embodiments, a second reactive group is a function group of an amino acid residue, e.g., −NH2 of
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lysine, −SH of cysteine, etc. In some embodiments, a second reactive group is −NH2 of a lysine residue, e.g., of a residue selected from K246 and K248 of IgG1 heavy chain amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of a heavy chain and amino acid residues corresponding thereto. In some embodiments, the invention provides selective reactions at amino acid residues of antibody moieties. [00167] In some embodiments, a second reactive group is installed to an antibody moiety optionally through a linker. In some embodiments, a second reactive group is installed to an antibody moiety through a linker. In some embodiments, a second reactive group is selectively linked to certain location(s) of an antibody moiety, e.g., certain location(s) selected from K246 and K248 of IgG1 heavy chain amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of a heavy chain and amino acid residues corresponding thereto. In some embodiments, the invention provides selective reactions at amino acid residues of antibody moieties. [00168] In some embodiments, the invention provides agents each independently comprising an antibody binding moiety that binds to an antibody agent, a reactive group, a cellular receptor-binding moiety, and optionally one or more linker moieties linking such groups/moieties. In some embodiments, such agents are useful as reaction partners, e.g., first agents) for conjugating moieties of interest, e.g., target binding moieties, reactive groups, e.g., second reactive groups) to agents comprising antibody moieties, e.g., second agents). In some embodiments, the invention provides agents for conjugating moieties of interest to antibody moieties in several agents or antibody agents, e.g., monoclonal antibody agents, polyclonal antibody agents, antibody agents of IVIG preparations, etc. In some embodiments, provided agents each comprise a cellular receptor-binding moiety, a reactive group, an antibody binding moiety, and optionally one or more linker moieties (linkers) linking such moieties. In some embodiments, an antibody binding moiety is part of a leaving group released after contacting this agent, e.g., a first agent, with an antibody moiety, e.g., of a second agent, and reacting a reactive group of this agent, e.g., a first reactive group of a first agent, with a reactive group of an antibody moiety, e.g., a second reactive group of a second agent, such as −NH2 of a Lys residue of an antibody protein. In some embodiments, provided technologies can provide improved conjugation efficiency, high selectivity, and/or fewer steps (sometimes, single step) to conjugation product agents. In some embodiments, a provided agent, e.g., a first agent, is a composition of matter of formula AGN110 or a salt thereof: LG−RG−LRM−TBT, [AGN110] or a salt thereof, wherein:
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LG is a group comprising an antibody binding moiety; RG is a reactive group; LRM is a linker; and TBT is a cellular receptor-binding moiety. [00169] In some embodiments, LG is or comprises an antibody binding moiety as described in this specification, and a linker which links an antibody binding moiety and RG. [00170] In some embodiments, LG is or comprises RLG−LLG−, wherein RLG is or comprises an antibody binding moiety, and LLG is a linker moiety as described in this specification. In some embodiments, LG is ABT-LLG−. In some embodiments, LLG is −LLG1−LLG2−, wherein each of LLG1 and LLG2 is independently a linker moiety as described in this specification. In some embodiments, LLG is −LLG1−LLG2−LLG3−, wherein each of LLG1, LLG2 and LLG3 is independently as linker moiety described in this specification. In some embodiments, LLG is −LLG1−LLG2−LLG3−LLG4−, wherein each of LLG1, LLG2, LLG3 and LLG4 is independently a linker moiety as described in this specification. In some embodiments, LLG1 is bonded to RLG. In some embodiments, LLG1 is bonded to cellular receptor-binding moiety. In some embodiments, LLG is −LLG1−, and a reactive group comprises LLG2, LLG3 and LLG4. In some embodiments, LLG is −LLG1−LLG2−, and a reactive group comprises LLG3 and LLG4. In some embodiments, LLG is −LLG1−LLG2−LLG3−, and a reactive group comprises LLG4. In some embodiments, each of LLG1, LLG2, LLG3 and LLG4 is independently L. [00171] In some embodiments, LLG4 is a covalent bond. In some embodiments, LLG4 is not a covalent bond. In some embodiments, LLG4 is −O−. In some embodiments, LLG4 is −N(R’)−. In some embodiments, LLG4 is −NH−. In some embodiments, LLG4 is −N(CH3)−. In some embodiments, LLG4 is −N(R’)−, and LLG3 is −O−. In some embodiments, R’ is optionally substituted C1-6 alkyl. In some embodiments, LLG4 is −S−. [00172] In some embodiments, RLG is or comprises an antibody binding moiety. In some embodiments, RLG is or comprises a protein binding moiety. In some embodiments, RLG is or comprises an antibody binding moiety. In some embodiments, RLG is an antibody binding moiety. In some embodiments, RLG is a protein binding moiety. In some embodiments, RLG is an antibody binding moiety. [00173] In some embodiments, RLG is ABT101, Rc−(Xaa)z−, a nucleic acid moiety, or a small molecule moiety. In some embodiments, RLG is or comprises ABT101. In some embodiments, RLG is or comprises Rc−(Xaa)z−. In some embodiments, RLG is or comprises a small molecule moiety. In some embodiments, RLG is or comprises a peptide agent. In some embodiments, RLG is or comprises a nucleic acid agent. In some embodiments, RLG is or comprises an aptamer agent. In some embodiments, an antibody binding moiety is or comprises ABT101. In some embodiments, a protein binding moiety is or comprises ABT101. In some embodiments, an antibody binding moiety is or comprises ABT101. In some
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embodiments, an antibody binding moiety is or comprises Rc−(Xaa)z−. In some embodiments, a protein binding moiety is or comprises Rc−(Xaa)z−. In some embodiments, an antibody binding moiety is or comprises Rc−(Xaa)z−. [00174] In some embodiments, target binding moieties may be conjugated to antibody moieties optionally through linker moieties using technologies described in published U.S. Patent Publication 2020/0190165. [00175] In some embodiments, where a particular protecting group (PG), leaving group (LG), or transformation condition is depicted, persons having ordinary skill in the biomedical art know that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated. Such groups and transformations are described in Greene’s Protecting Groups in Organic Synthesis (John Wiley & Sons, 2014), the entirety of which is incorporated in this specification by reference. [00176] In some embodiments, leaving groups include but are not limited to, halogens, e.g. fluoride, chloride, bromide, iodide, sulfonates, e.g. mesylate, tosylate, benzenesulfonate, brosylate, nosylate, triflate), diazonium, and the like. [00177] In some embodiments, an oxygen protecting group includes carbonyl protecting groups, hydroxyl protecting groups, etc. Hydroxyl protecting groups are well known in the biomedical art and include those described in Greene’s Protecting Groups in Organic Synthesis (John Wiley & Sons, 2014), the entirety of which is incorporated in this specification by reference. Examples of suitable hydroxyl protecting groups include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4- oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy- crotonate, benzoate, p-benylbenzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl, 9- fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p- methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, β-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl
63 30125-WO-PCT
ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, and 2-picolyl and 4-picolyl. [00178] Amino protecting groups are well known in the biomedical art and include those described in Greene’s Protecting Groups in Organic Synthesis (John Wiley & Sons, 2014), the entirety of which is incorporated in this specification by reference. Suitable amino protecting groups include, but are not limited to, aralkylamines, carbamates, cyclic imides, allyl amines, amides, and the like. Examples of such groups include t-butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl (CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, and the like. [00179] Persons having ordinary skill in the biomedical art know that provided agents may have one or more stereocenters and may be present as a racemic or diastereomeric mixture. Persons having ordinary skill in the biomedical art know there are many methods known in the biomedical art for the separation of isomers to obtain stereoenriched or stereopure isomers of those compounds, including but not limited to HPLC, chiral HPLC, fractional crystallization of diastereomeric salts, kinetic enzymatic resolution, e.g. by fungal-derived, bacterial-derived, or animal-derived lipases or esterases), and formation of covalent diastereomeric derivatives using an enantioenriched reagent. [00180] Persons having ordinary skill in the biomedical art know that several functional groups present in compounds of this specification such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles can be interconverted by techniques well known in the biomedical art including, but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (John Wiley & Sons, 2001), the entirety of which is incorporated in this specification by reference. Such interconversions may require one or more of the aforementioned techniques, and certain methods for synthesizing compounds of this specification are described below in the Exemplification. [00181] As known by persons having ordinary skill in the biomedical art, reaction partners are generally contacted with each other under conditions and for a time sufficient for production of the desired results, e.g., formation of product agents and compositions thereof to desired extents. Many reaction conditions/reaction times may be assessed and used if they are suitable for desired purposes in accordance with this specification; certain such conditions, reaction times, assessment, etc. are described in the Examples.
64 30125-WO-PCT
[00182] In some embodiments, an agent formed, e.g., a product MATE agent, has the structure of formula AGN101 or AGN102, or a salt thereof. In some embodiments, a cellular receptor-binding moiety in a product agent, e.g., a MATE agent, is the same as a cellular receptor-binding moiety in a reaction partner, e.g., a first agent comprising a cellular receptor-binding moiety) used to prepare a product agent. In some embodiments, an antibody moiety in a product agent, e.g., a MATE agent, is the same as an antibody moiety in a reaction partner, e.g., a second agent comprising an antibody moiety) used to prepare a product agent. [00183] In some embodiments, linker moieties or a part thereof connected to target binding moieties and/or antibody moieties may be transferred from reaction partners, e.g., LRM of formula AGN110 or a salt thereof. In some embodiments, a linker moiety in a product agent (may be called LPM; e.g., L in formula AGN101 or AGN102, is or comprises a linker moiety in a reaction partner, e.g., one between a reactive group and a cellular receptor-binding moiety, e.g., LRM. In some embodiments, LPM is or comprises LRM. In some embodiments, LPM is −LRM−LRG2−. In some embodiments, LRG2 is −C(O)−. In some embodiments, LRG2 is −C(O)−, and is bonded to −NH− of a target agent moiety, e.g., −NH− in a side chain of a lysine residue of a protein moiety, which in some embodiments, is an antibody moiety. [00184] In some embodiments, the invention provides products of provided processes, which have low levels of damage to antibody moieties compared to processes comprising steps performed for antibody binding moiety removal but not for substantial conjugation of moieties of interest, e.g. target binding moieties. In some embodiments, provided product agent compositions have high homogeneity, e.g., regarding the number of cellular receptor-binding moiety per antibody moiety, and/or positions of amino acid residues in antibody moieties conjugated to moieties of interest) compared to reference product compositions, e.g., those from technologies without using antibody binding moieties, or using extra step(s) for antibody binding moiety removal, e.g., not using reaction partners described in this specification which comprise a reactive group located between an antibody binding moiety and a cellular receptor-binding moiety. [00185] In some embodiments, the invention provides a product agent which is an agent comprising an antibody moiety, a cellular receptor-binding moiety and optionally a linker moiety linking an antibody binding moiety and a cellular receptor-binding moiety. In some embodiments, the invention provides compositions of such agents.
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Pharmaceutically acceptable excipients. [00186] Formulations suitable for parenteral administration, such as by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. [00187] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include these components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Plurality of agents. [00188] In some embodiments, the invention provides a composition comprising a plurality of agents, wherein each agent independently comprises: an antibody moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody binding moiety and a cellular receptor-binding moiety. [00189] In some embodiments, product agents are MATE agents. In some embodiments, an antibody agent moiety comprises IgG Fc region. In some embodiments, an antibody moiety is connected to a cellular receptor-binding moiety through an amino group optionally through a linker. In some embodiments, it is through a lysine residue wherein the amino group of the side chain is connected to a
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cellular receptor-binding moiety optionally through a linker, e.g., forming −NH−C(O)− as part of an amide group, a carbamate group, etc. [00190] In some embodiments, the invention provides a composition comprising a plurality of agents each of which independently comprising: an antibody moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking the antibody moiety and the cellular receptor-binding moiety; wherein antibody moieties of agents of the plurality comprise a common amino acid sequence, and agents of the plurality share a common cellular receptor-binding moiety independently at least one common amino acid residue of the common amino acid sequence; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprise the common amino acid sequence and the cellular receptor-binding moiety are agents of the plurality. [00191] In some embodiments, the invention provides a composition comprising a plurality of agents each of which independently comprising: an antibody moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody moiety and a cellular receptor-binding moiety; wherein agents of the plurality share the same or substantially the same antibody moiety, and a cellular receptor-binding moiety at least one common location; and wherein about 1%-100% of all agents that comprise the antibody moiety and the cellular receptor-binding moiety are agents of the plurality. [00192] Further guidance is provided in International Patent Publication WO 2025/035052 (Biohaven Therapeutics Ltd.), the contents of which are incorporated by reference. Methods of assessing the chemical structure and function of the agent. [00193] HDX-MS measures the exposure of hydrogen molecules on the surface of a protein or protein complex. The first step causes exposed hydrogen molecules to be readily exchanged with deuterium. Hydrogen molecules buried within the structure, for example at the binding site of an antibody-antigen complex, are not exchanged as readily. [00194] Comparing the deuterium uptake between the free and the complex states of proteins via mass spectrometry provides valuable information about the binding regions.
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[00195] HDX-MS is continuing to be performed. The inventors are modeling the antibody Fc-and bifunctional degrader interaction to predict paratope regions in parallel with HDX-MS. [00196] Protein sequencing. In one method of protein sequencing, eight digestions are prepared using five enzymes (pepsin, Lys C, trypsin, chymotrypsin, Asp N). The digestions for the sample are processed with disulfide reduction, cysteine blocking, and then enzyme digestion. Digestions were analyzed by LC-MS/MS using a Thermo-Fisher Orbitrap fusion™ mass spectrometer. Peptides are characterized from LC -MS/MS data using de nova peptide sequencing and then assembled into antibody sequences. Assays used to determine proof of concept. [00197] The inventors used these assays to determine proof-of-concept for the anti- insulin/proinsulin autoantibody bifunctional agents of the invention. [00198] Biophysical assay. Surface plasmon resonance assay (insulin receptor). The inventors produced mutant insulin conjugated with GN3 and is being tested for binding to the insulin receptor by surface plasmon resonance assay. Additional mutant insulins conjugated with GN3 are produced synthetically. [00199] Insulin receptors are also produced synthetically. qPCR method primer sequence IR-forward: GCAACATCACCCACTACCTGGT (SEQ ID NO: 39). qPCR method primer sequence IR-reverse: GAATGGTGGAGACCAGGTCCTC (SEQ ID NO: 40). [00200] Cellular Assay. iLite Insulin reporter assay (DT-40). Assay principle: Overexpression of insulin receptor and transfection of a non-canonical STAT5 recognition motif and a STAT5 expression vector. Thus, insulin signaling via the insulin receptor results in STAT5 phosphorylation, dimerization, and transcriptional activation of a promoter leading to luciferase production. [00201] Insulin receptor-binding to human insulin via surface plasmon resonance. Biophysical assessment of insulin/IGF1 binding to their respective receptors was demonstrated. Weak binding of proinsulin constructs to insulin receptor [00202] Nonobese diabetic (NOD) mouse model. The NOD mouse model is described by Jorns et al., Diabetologia, Volume 57, pages 512–521 (December 6, 2014). NOD mice are one of the best-studied mouse models for spontaneous type 1 diabetes. The immunogenic progression of diabetes in NOD mice is similar to that in humans. The disease progression is driven by immune cells (macrophages, dendritic cells, NK cells, B cells, T cells). Insulitis is established at twelve to fourteen weeks. T cells are the
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predominant cell infiltrating the islets, causing B cell destruction, and leading to overt diabetes/hyperglycemia. [00203] In humans, autoantibodies against several β-cell proteins are identified in more than 90% in newly diagnosed type 1 diabetes. The prevalence of most islet autoantibodies is associated with age at diagnosis. [00204] Humanized immunodeficient NOD mouse mode of type 1 diabetes. Several immunodeficient models were described in the scientific literature of the biomedical art. SCID mice reconstituted with PBMC from type 1 diabetic patients can produce autoantibodies against diabetes-associated antigens. See Petersen et al., Diabetes (1994). A genetic modification on the NOD/SCID background enables engraftment by human lymphoid cells. TABLE 8 Commercially available NOD/SCID mice e l n [00205] Pediatric patients are good pool of autoantibodies. PBMCs from type 1 diabetes patients and controls are commercially available. B cells in vitro generate a polyclonal pool of autoantibodies. The humanized mouse model can show degradation of patient autoantibodies. [00206] Canine diabetic patients. The inventors administer the bifunctional degrader to canine diabetic patients to treat their diabetes. Diabetes dogs are treated therapeutically with human insulin and commonly develop immune-neutralizing antibodies which decrease insulin concentrations and antidiabetic efficacy. The inventors can assay to confirm that the dog polyclonal response to human insulin likely mimics human autoantibody response. [00207] The inventors can also assay to confirm measurement of hypoglycemia in the dogs.
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[00208] MesoScale Discovery (MSD)-based assay for detecting low levels of human insulin autoantibodies. Detailed protocols are described in the scientific literature in the biomedical art. 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 the increased sensitivity and lower sample volume of serum required for the assay. With ELISA-based assays, the antigens are bound to the solid phase, i.e., the ELISA plate. This binding limits the conformational antigen epitope recognition by the antibodies tested. Only one antigen can be recognized per well. MesoScale Discovery-based assays can be developed for up to ten different autoantibodies. MesoScale Discovery-based assays enable a higher throughput capacity. TABLE 9 Tool antibodies and other biological agents y dy e ). -
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TABLE 9 Tool antibodies and other biological agents t. an a nt 1 -
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TABLE 10 Reagent kits , , s. TABLE 11 Detection of insulin/proinsulin L Detection of insulin/proinsulin Statistical analyses. Normal distribution quantitative variables can be expressed as means and SDs and compared by an independent-samples t test, as was done by Zhang et al. (October 2019). For non-normally distributed variables, persons having ordinary skill in the biomedical art can use median and interquartile range and analyze with the Mann-Whitney U test. Categorical data are summarized by percentages. A two-sided p
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value <0.05 is considered statistically significant. All statistical tests can be performed using SPSS version 16.0. Characterizing antibody binding moieties. [00210] Many technologies are available for identifying, assessing, or characterizing antibody binding moieties, including protein binding moieties, e.g., antibody binding moieties such as universal antibody binding moieties), and/or their use in provided technologies, e.g., those described in International Patent Publication WO 2019/023501 (Kleo Pharmaceuticals, Inc.), the technologies of which are incorporated in this specification by reference. In some embodiments, an antibody binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can selectively bind to IgG, and when used in provided technologies can provide and/or stimulate ADCC and/or ADCP. In some embodiments, peptide display technologies, e.g., phase display, non-cellular display, etc., can identify antibody binding moieties. In some embodiments, an antibody binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can bind to IgG and optionally can compete with known antibody binders, e.g., protein A, protein G, protein L, etc. [00211] Persons having ordinary skill in the biomedical art know that antibodies of several properties and activities, e.g., antibodies recognizing different antigens, having optional modifications, etc., may be targeted by antibody binding moieties described in this specification. In some embodiments, such antibodies include antibodies administered to a subject, e.g., for therapeutic purposes. In some embodiments, antibody binding moieties described in this specification may bind antibodies toward different antigens and are useful for conjugating moieties of interest with several antibodies. [00212] In some embodiments, an antibody binding moiety, e.g., an antibody binding moiety, is or comprises a Meditope agent moiety. See United States Patent Publication 2019/0111149 (Gardiner et al.). [00213] In some embodiments, an antibody binding moiety, e.g., an antibody binding moiety, can bind to human IgG. In some embodiments, an antibody binding moiety, e.g., an antibody binding moiety, can bind to an antibody selected from the Markush group of antibodies consisting of rabbit IgG, IgG1, IgG2, IgG3, and IgG4. In some embodiments, an antibody binding moiety, e.g., an antibody binding moiety, binds to IgG1, IgG2, and IgG4.
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[00214] In some embodiments is used in a reference technology as a non- antibody binding moiety. In some e mical group used in a reference technology as a non-antibody binding moiety is selected from the Markush group of chemical groups consisting of CH3−, CH3C(O)−, CH3C(O)NH−, CH3C(O)NHCH2−, CH3CH2−, CH3CH2NH−, and CH3CH2NHC(O)−. [00215] In some embodiments, antibody binding moieties, e.g., antibody binding moieties) bind to targets, e.g., antibody agents for antibody binding moieties) with a KD that is about 1 mM-1 pM or less. In some embodiments, a KD is about 1 mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, 0.01 mM, 0.005 mM, 0.002 mM, 0.001 mM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or less. In some embodiments, KD is an affinity selected from the Markush group of affinities consisting of about 1 mM or less, about 0.5 mM or less, about 0.1 mM or less, about 0.05 mM or less, about 0.01 mM or less, about 0.005 mM or less, about 0.001 mM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, and about 1 nM or less. In some embodiments, antibody binding moieties bind to IgG antibody agents with KD described in this specification. [00216] Persons having ordinary skill in the biomedical art know that antibodies of several properties and activities, e.g., antibodies recognizing different antigens, having optional modifications, etc., may be recruited by antibody binding moieties described in this specification. In some embodiments, such antibodies include antibodies administered to a subject, e.g., for therapeutic purposes. In some embodiments, antibodies recruited by antibody binding moieties comprise antibodies toward different antigens. In some embodiments, antibodies recruited by antibody binding moieties comprise antibodies whose antigens are not present on the surface or cell membrane of target cells. In some embodiments, antibodies recruited by antibody binding moieties comprise antibodies not targeting antigens present on surface or cell membrane of targets. In some embodiments, antigens on surface of target cells may interfere with the structure, conformation, and/or one or more properties and/or activities of recruited antibodies which bind such antigens. In some embodiments, recruited antibodies are those in IVIG. In some embodiments, IVIG may be administered before, concurrently with or subsequently to an agent or composition. Antibodies of several types of immunoglobulin structures may be recruited. In some embodiments, one or more subclasses of IgG are recruited. In some embodiments, recruited antibodies are selected from the Markush group of antibody classes consisting
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of IgG1, IgG2, IgG3, and IgG4. In some embodiments, recruited antibodies are or comprise IgG1 and IgG2. In some embodiments, recruited antibodies are or comprise IgG1, IgG2 and IgG4. In some embodiments, recruited antibodies are or comprise IgG1, IgG2, IgG3 and IgG4. Recruited antibodies may interact several types of receptors, e.g., those expressed by several types of immune cells. In some embodiments, recruited antibodies can effectively interact several types of Fc receptors and provide desired immune activities. In some embodiments, recruited antibodies can recruit immune cells. In some embodiments, recruited antibodies can effectively interact with human Fcγ receptor IIIA. In some embodiments, recruited antibodies can effectively interact with human Fcγ receptor IIIA on macrophages. In some embodiments, macrophages are recruited to provide ADCC or ADCP activities toward a virus, e.g., a SARS-CoV-2 virus, and/or cells infected thereby. In some embodiments, NK cells are recruited to provide immune activities. In some embodiments, recruited antibodies can effectively interact with human Fcγ receptor IIIA. In some embodiments, recruited antibodies can effectively interact with human Fcγ receptor IIIA on dendritic cells. In some embodiments, antibody moieties in agents of this specification comprise one or more properties, structures and/or activities of recruited antibodies described in this specification. Specific mutation strategies to reduce Fcγ receptor and c1q binding / effector function, and to enhance FcRn binding/exposure of antibodies. Fc mutation strategies to reduce Fcγ receptor and c1q binding. [00364] The inventors mutate an antibody, an antibody variant, or an antigen-binding fragment thereof in the Fc region to insert a LALA mutation using biomedical-art recognized methods first described by the Winter group in the 1990s. In this EXAMPLE, LALA = L234A/L235A. [00365] The inventors mutate an antibody, an antibody variant, or an antigen-binding fragment thereof in the Fc region to insert a LALA mutation using biomedical-art recognized methods first described by the Winter group in the 1990s, then inserted P mutations, such as by the biomedical-art recognized methods introduced by Roche team in 2016, such as the technology for adding P329G and P329G combined with LALA: In this EXAMPLE, LALA/PA = L234A/L235A/P329A. LALA/PG = L234A/L235A/P329G. See Tilman et al., Protein Engineering, Design and Selection, Volume 29, Issue 10, pages 457–466 (October 2016), which shows that even LALA itself abolishes c1q binding. P329A alone was tested, and abolishes c1q binding, and reduces FcgR binding. They show that P329G/LALA further reduces Fcγ receptor binding beyond LALA alone.
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[00366] The inventors also mutated an antibody, an antibody variant, or an antigen-binding fragment thereof in other regions. N297A/Q removes natural N-linked glycosylation site in the hinge region. Fc mutation strategies to enhanced FcRn binding to prolong exposure. [00367] There are many publicly available approaches and mutation sets to increase binding of an Fc to FcRn. TABLE 13 Examples of Fc sequence engineering for modification of half-life. F, [00368] An older summary is provided by Strohl, Optimization of Fc-mediated effector functions of monoclonal antibodies. Current Opinion in Biotechnology, 20(6), 685-691 (2009). [00369] For an example of introducing a YTE region in 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). Additional references with details of engineering also available. [00370] For an example of introducing a LS region in a protein (M428L/N434S), see Zalevsky et al., Nature Biotechnology, 28(2), 157-159 (2010). Structure of recombinant ligand constructs. [00371] GN3 sortase reagent. A basic protocol for sortase conjugation including the ASPGR binder for the sortase. C-terminal sortase tag (LPETGG) for conjugation (GN3/linker). [00372] GN3-maleimide reagent (maleimide-GN3/linker), with a C-terminal cysteine for conjugation.
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[00373] Biophysical/biochemical potency assay to quantify target-engagement. A biomolecule coupled to surface of sensor chip as ligand. As analyte is flowed in solution over immobilized ligand, binding to the sensor chip surface induces a change in refractive index proportional to bound mass. [00374] An assay was done using a Biacore S200 instrument. This machine has a high sensitivity, low- medium throughput. A ligand variant fragment is dosed 1 nM to 1 µM and characterized by single cycle kinetics. [00375] ASGPR-dependent uptake assay. A HEK293 bioluminescent cell-based assay was used to assess ternary complex formation uptake by cells in vitro. This on-mechanism endocytosis assay measures the accumulation of bifunctional degrader in HEK293 cells through the bifunctional degrader mechanism described in this specification. A component of this assay is a determination of the receptor expression on the surfaces of the assay’s HEK293 cells. [00376] Degradation assay. This on-mechanism degradation assay is a direct, low-throughput Western blot measurement or a high-throughput assay using an activatable fluorescence-quencher probe. [00377] Other off-target assays include measurement of cytotoxicity through In vitro toxicity with HepG2 cells, e.g., in a CellTiter-Glo assay, measurement of hemagglutination by red blood cell interactions, measurement of PBMC, and measurement of off-target bindings in a house C-type lectin panel. Signal is boosted in the HEK293 versus HepG2 cells when measuring signal accumulation not degradation. Western assays can measure the degradation in HEK cells and other cell lines. [00378] Next steps can include a competition assay with FC3 (qualitative). Manufacturing the agent. TABLE 14 P rifi ti n t H A 5 5
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TABLE 14 Purification steps 5 e, e, . Methods of making several agents. [00217] Agents of this specification may be prepared or isolated by synthetic and/or semi-synthetic methods or recombinant methods in accordance with this specification. In some embodiments, polypeptide agents, e.g., cellular receptor-binding moiety peptide agents, maybe be prepared using biological expression systems. In some embodiments, provided agents are prepared synthetically. In some embodiments, provided agents are prepared using certain technologies described in International Patent Publication WO 2019/023501 (Kleo Pharmaceuticals, Inc.), which is incorporated in this specification in its entirety by reference. [00218] Several technologies, e.g., those for preparing antibody-drug conjugates, may be used in preparation of MATE agents. In many such technologies, conjugation is not selective regarding amino acid residue sites, and product compositions usually contain several types of agents which may differ from each other regarding number of target binding moieties conjugated and/or conjugation sites. In some embodiments, the invention provides technologies that can be used for selective conjugation of target binding moieties at amino acid residue sites. [00219] In some embodiments, the invention provides a method of synthesis, comprising the steps of: contacting a first agent comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker with a second agent comprising an antibody moiety linked to a second reactive group optionally through a second linker, wherein the first reactive group reacts with a second reactive group, and
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forming a product agent comprising a cellular receptor-binding moiety and an antibody binding moiety optionally through a linker. [00220] In some embodiments, the invention provides a method of synthesis, comprising the steps of: contacting a first composition comprising a plurality of first agents each independently comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker moiety with a second composition comprising a plurality of second agents each independently comprising an antibody moiety optionally linked to a second reactive group optionally through a second linker moiety, wherein a product composition comprising a plurality of product agents each independently comprising a cellular receptor-binding moiety and an antibody binding moiety optionally through a linker is formed. EXAMPLES [00221] The invention is further illustrated by non-limited examples. EXAMPLE 1 Proinsulin and proinsulin-IgG1 Fc fusion protein sequences. [00380] The inventors produced His tagged and sortase tagged proteins. See SEQUENCE LISTING. [00381] The Fc fusion construct expression in the E. coli system is improved by using a fusion protein. The Fc fusion construct expression enables a highly purified material with a good yield. Conjugation of ASPGR via MATE reagent to yield degrader molecule. LALA and PA mutations to produce distinct chemical entities can attenuate the immune effector function. Quality control can address any issues with disulfide interactions, where insulin might misfold in with the Fc receptor and the cystines in the Fc to aggregates. Such misfolding can be detected by native protein gel assays and size exclusion assays. [00382] Persons having ordinary skill in the biomedical art understand how to make proinsulin-Fc fusion proteins. The prior art proinsulin-Fc fusion proteins can be tool compound and reference compounds. Persons having ordinary skill in the biomedical art can also make the insulin-Fc fusion proteins.
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TABLE 15 Protein Concentration Buffer , H H , antibodies bind or recognize Fc fusion protein via the proinsulin moiety). LALA mutation (in Fc domain) does not prevent recognition by anti-insulin antibodies. [00384] Both rabbit and mouse anti-insulin bind or recognize Fc fusion protein via the proinsulin moiety) EXAMPLE 2 Surface plasmon resonance assay – first results. [00385] binding ability of the binding moiety can be measured by surface plasmon resonance (Biacore™ and IBIS-MX96 systems) and bio-layer interferometry (ForteBio™ Octet™ systems). [00386] The screening cascade plans include surface plasmon resonance target engagement, ternary complex formation, the cell-based assays, and endocytosis assays. Additional bridging assays to confirm degradation. SPR/TR-FRET (ASGPR) à TR-FRET (ternary complex, biophysical) à Uptake à TR-FRET in cells (degradation). [00387] Binding confirmation by surface plasmon resonance. Fc is immobilized to CM5 chip via amine coupling immediately before the assays. binding moiety was dosed 1 nM to 1 µM and characterized by single cycle kinetics. First run, single cycle kinetics, steady state KD (nM): 22. Second run, single cycle kinetics, steady state KD (nM): 24. The inventors are validating commercial antibody that binds to human Fc of an . In n=2 characterization, KD of about20 nM was obtained. No binding response was observed for full-length IgG1-3 and IgG1-3 Fc regions at up to 1 µM concentrations. The theoretical Rmax indicates active surface (data QC metric and indicator of viable immobilization method).
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TABLE 16 Assay reagent MW Note Surface plasmon resonance assays
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TABLE 18 Summary Table Surface plasmon resonance assays for binding to Insulin [00388] The inventors tested tree anti-insulin autoantibodies mAb13, mAb48, and mAb49. Assay 1: (done) [00389] Sensor chip: CM5.
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[00390] Proteins: 1. Immobilize anti-insulin receptor α antibody [83-7] on a CM5 chip with amine coupling to 5000 RU. [00391] Proteins: 2. Capture human insulin receptor (isoform A) / CD220 (28-944) Protein, His Tag from Acro Biosystems to ~150, 300, and 600 RU. R&D Systems’ isoform B, recombinant Human Insulin R/CD220 (aa 28-956) Protein, CF. [00392] Analytes: Human insulin and Insulin lispro, 5 µM top, 2-fold dilution, MCK 1+10+1, 90s on, 180s off. [00393] Running buffer: 10 mM HEPES pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assay 2: (done) [00394] Sensor chip: CM5+Neutravidin. [00395] Proteins: Capture biotinylated human insulin receptor/CD220 (28-944) Protein, His, Avitag from Acro Biosystems on a SA chip at ~750, 1000, and 1500 RU. [00396] Analytes: Human insulin and Insulin lispro, 5 µM top, 2-fold dilution, MCK 1+10+1, 90s on, 180s off. [00397] Running buffer: 10 mM HEPES pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assay 3: (done) [00398] Sensor chip: CM5+Neutravidin. [00399] Proteins: Capture biotinylated human IGF-1 receptor/CD221 Protein, His, Avitag from Acro Biosystems on a SA chip at ~750 and 1250 RU. IGF-1 receptor can be produced synthetically. qPCR method primer sequence IGF1R-forward: CCTGCACAACTCCATCTTCGTG (SEQ ID NO: 41). qPCR method primer sequence IGF1R-reverse: CGGTGATGTTGTAGGTGTCTGC (SEQ ID NO: 42). [00400] Analytes: Human insulin and insulin lispro, 10 µM top, 2-fold dilution, MCK 1+10+1, 90s on, 180s off. [00401] Running buffer: 10 mM HEPES pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assay 4: [00402] Sensor chip: CM5. [00403] Proteins: Immobilize three anti-insulin autoantibodies on a CM5 chip with amine coupling at ~750 and 1250 RU. [00404] Analytes: Human insulin and Insulin lispro, 5 µM top, 2-fold dilution, MCK 1+10+1, 120s on, 600s off. [00405] Running buffer: 10 mM HEPES pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. Assay 5:
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[00406] Sensor chip: protein A. [00407] Proteins: Capture three anti-insulin autoantibodies on a protein A chip to ~750 and 1250 RU. [00408] Analytes: Human insulin and Insulin lispro, 5 µM top, 2-fold dilution, MCK 1+10+1, 120s on, 600s off. [00409] Running buffer: 10 mM HEPES, pH 7.4, 150 mM sodium chloride, 0.005% Tween-20. EXAMPLE 3 Pharmacodynamics. [00410] AGN310 study in non-obese diabetic (NOD) mice. Four mice per group. An additional two mice per group were excluded because they had very low levels of antibody to begin. The inventors observed 42% depletion by AGN310, based on area-under-the curve. There was a 50% depletion if the background signal is accounted for. [00411] In another analysis, AGN310 effectively removed 39% of anti-insulin antibodies (2 mg/kg) in nude mice. [00412] AGN310 was well-tolerated when administered at 3 mg/kg IV in nude mice, with no observed adverse events and changes in glucose levels when measured at twelve hours-post dose. EXAMPLE 4 Internalization and ternary complex formation assays. [00413] Assay conditions: Cell lines: HEK293-ASGPR (clone 3F7) Culture media: DMEM + 10% FBS + 1% P/S Antibodies: Constant at 100 nM (all labeled with AF647) Anti-insulin antibody, rabbit monoclonal, Sino, Cat#11038-CR007 Anti-insulin antibody, mouse monoclonal, Genscript, Cat#A01715 (clone 6E9F1) Agents: only AGN301 (constant at 50 nM) Readout: Ternary complex formation —> flow cytometry (IntelliCyt iQue Screener Plus) Internalization —> high content imager (Operetta) [00414] Molecules tested: [00415] AGN301: IgG degrader (reference molecule) [00416] ABT307: Proinsulin-IgG1 Fc fusion, G4S linker, hIgG1 hinge (C > A), LALA-PA. [00417] ABT308: Proinsulin-IgG1 Fc fusion, 5x G4S linker, hIgG1 hinge (C > A), LALA-PA.
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[00418] AGN303: Native insulin with GN3 [00419] AGN302: Mutant insulin with GN3 [00420] ABT306: Alk-Native Insulin without GN3 (just peptide) [00421] ABT304: Native Proinsulin [00422] ABT305: Mutant Proinsulin [00423] Testing that anti-insulin antibodies (rabbit versus. mouse) recognize proinsulin Fc fusion proteins. [00424] Conclusions. Anti-insulin agents working. Rabbit monoclonal antibody, but not mouse, can be used as surrogate with IgG degrader (AGN301). Non-GN3 peptide (ABT306) does not elicit internalization or ternary complex formation. Anti-proinsulin agents (ABT304/ABT305) do not internalize or complex with anti-insulin antibodies. EXAMPLE 5 Agents bind to insulin and proinsulin autoantibodies, resulting in uptake, hepatic degradation and correction of glucose homeostasis. [00425] Anti-insulin autoantibody MoDEs and anti-proinsulin autoantibody MoDEs form ternary complexes, show in vitro uptake and drive in vivo clearance without binding insulin receptor or IGF1 receptor. [00426] Robust and selective lowering of these autoantibodies was observed in mouse pharmacokinetics/pharmacodynamics assays. [00427] Efficacy studies and preliminary toxicology. Agents deplete naturally-occurring insulin autoantibodies in non-obese diabetic (NOD) mice. [00428] Conclusion: Lowering of neutralizing antibodies in NOD mice suggests potential for restoration of glucose homeostasis in Type 1 diabetes patients. EXAMPLE 6 Proinsulin production technical report [00429] Summary. ABT410 is a proinsulin molecule with N-terminal H27R leading sequence and TEV recognition site. ABT410 was expressed as inclusion bodies in BL21(DE3) strain. After properly refolding and buffer exchange, target protein was cleaved by TEV protease, then purified by SP HP resins. The purified protein was formulated in 50 mM Tris/HCl, 150 mM sodium chloride, 10% glycerol, pH 7.5 by
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UF/DF. The testing panel included protein concentration by Bradford, reducing and non-reducing SDS- PAGE, SEC-HPLC, endotoxin level and LC-MS. [00430] Host cell: E.coli BL21(DE3) competent cell [00431] Coding vector name: pET28a(+). [00432] Protein Sequence: [00433] ABT410 (before TEV digestion) MGMTMITNSPEISHHHHHHHHHHQLISEARENLYFQGGGGGFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAED LQVGQVELGGGPGAGSLQPLALEGSL QKRGIVEQCCTSICSLYQLENYCNG (SEQ ID NO: 19). [00434] ABT410 (after TEV digestion) GGGGGFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCT SICSLYQLENYCNG. [00435] Extinction coefficient: 0.651 after digestion. [00436] Molecular weight: 9736.98 Da (-SH, after digestion). [00437] pI: 5.20 after digestion. [00438] Transformation steps: [00439] (1) Add ~100 ng plasmids to 100 µl of BL21(DE3) competent cells. [00440] (2) Incubate thirty minutes on ice. [00441] (3) Heat shock forty-five seconds at 42℃ water-bath. [00442] (4) Incubate two minutes on ice. (5) Add 900 µl of LB media (BD-Cat.244620) and shake forty-five minutes at 37℃, 220rpm. [00443] (6) Spread the transformation into LB-kanamycin+ agar plate and incubate overnight at 37℃, upside down. [00444] Expression: [00445] (1) Pick colonies into 100 mL LB media with 50 µg/mL kanamycin + antibiotic. [00446] (2) Incubate at 30℃, 200 rpm for overnight as a seed. [00447] (2) Inoculate 10 mL seed into 1 L TB medium (BD-Cat.243820) containing 4 mL glycerol, culture at 37℃, 220 rpm. [00448] (3) When OD600 reaching 1.0~1.2 (~ 2.5 hours), add IPTG to a final concentration of 1 mM, and grow for additional sixteen hours at 18℃. [00449] (4) In one example, the cells were harvested when OD600 reached 8.69. Concentration of solubilized protein was 26.8 mg/mL. Volume of solubilized protein was 90.0 mL. Amount of solubilized protein was 2412.0 mg. The titer was 482.4 mg/L.
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[00450] (5) Harvest cells by centrifugation at 8000 g, fifteen minutes. [00451] (6) Cell pellet can be stored at -80℃. [00452] Preparation of inclusion bodies: [00453] (1) Thaw the expression pellet at room temperature for ten-twenty minutes. [00454] (2) Resuspend cells in freshly prepared lysis buffer at a ratio of 1:5 (WITH V). [00455] (3) In a magnetic stirrer, stir for thirty minutes at room temperature until the cell pellets solution is completely dissolved. [00456] (4) Homogenizer at 1000 bar for two-three times. [00457] (5) Centrifuge at 12,000 g for one hour at 4°C. [00458] (6) Remove supernatant carefully and resuspend the inclusion bodies with wash buffer to its original equal volume. Stir for thirty minutes at room temperature until the cell debris is completely dissolved. [00459] (7) Centrifuge at 12,000 g for one hour at 4°C. [00460] (8) Repeat step 6 and step 7 once more. [00461] (9) Discard the supernatant and the inclusion bodies was resuspended with solubilization buffer at a ratio of 1:10 (WITH V). Stir for overnight 4°C. [00462] (10) Centrifuge the solubilized inclusion bodies at 40,000 g for one hour at 4°C. [00463] (11) Collect the supernatant by 0.2 μm filter. [00464] (12) Determine protein concentration by Pierce Coomassie assay plus protein determination reagent following manufacturer instruction. [00465] Lysis buffer: 50 mM Tris/HCl, pH 8.0, 5mM EDTA, 1% Triton X-100. [00466] Wash buffer: 50 mM Tris/HCl, pH 8.0, 5mM EDTA. [00467] Solubilization buffer: 50 mM Tris/HCl, pH 8.0, 8 M Urea [00468] In one example, from 1 L pellets, 482.4 mg proteins were obtained. [00469] Refolding. For 1 L of refolding: [00470] (1) Dilute the denatured sample into 10 mg/mL by solubilization buffer. [00471] Refolding buffer: 20 mM Tris/HCl,10 mM Glycine, 1 mM EDTA,1 mM GSH, 5 mM GSSG, pH 10.0. [00472] (2) Add DTT to solubilized inclusion bodies to a final concentration of 10 mM and mix completely. [00473] (3) Incubate thirty minutes at 37°C to break mis-paired disulfide bonds before refolding.
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[00474] (4) Drop slowly 50 mL of reduced inclusion bodies (~500 mg protein) in 1 L of freshly made refolding buffer on magnetic stirrer (medium strength). [00475] (5) Incubate at 4°C for twenty-four hours with slow stirring. [00476] (6) Run SDS-PAGE and LC-MS to characterize the refolded protein. [00477] Based on the LC-MS profile, three pairs of disulfide bonds (-6Da/-6H) are formed after overnight refolding. The results also showed that the first ‘Met’ was successfully removed. [00478] Buffer exchange (UF/DF) and TEV digestion: [00479] (1) Refolded sample was filtered by 0.45 μm filter firstly to remove visible precipitation before ultrafiltration. [00480] (2) Sample concentration was concentrated to ~3 mg/mL by UF device. [00481] (3) Concentrated sample was exchanged into TEV digestion buffer for six-fold volume by DF device. [00482] TEV digestion buffer: 25 mM Tris/HCl, 100 mM sodium chloride, pH 7.5, 10% glycerol. [00483] Buffer exchange by UF/DF: SP HP pool sample can be exchanged into final formulation buffer with six-fold volume by UF/DF device. For small-scale batch production, SEC and desalting column was used to exchange buffer. [00484] (4) Add in-house produced TEV protease to protein at a ratio of 1:20(WITH W), and the mixtures were incubated at room temperature for overnight. [00485] (5) Analyze the digestion efficiency by SDS-PAGE. [00486] In one example, SDS-PAGE gel showed that most of target protein was cleaved by TEV protease. [00487] SP HP purification: [00488] (1) Add 10% acetic acid to adjust the sample pH to 4.5. [00489] (2) Dilute the sample with ultra-pure water to make loading conductivity around 7-8 mS/cm. [00490] (3) Filter the supernatant using 0.2 µm filter. [00491] (4) Load the protein on SP Sepharose HP (Cytiva) column with dynamic binding density around 20 mg/mL (residence time ~two -five minutes) using AKTA Pure system. [00492] (5) Wash the SP HP column with five column volumes (CV) buffer A. [00493] (6) Wash the SP HP column with five column volumes buffer C, assuring contact time 300 minutes. [00494] (7) Wash the SP HP column with ten column volumes buffer A.
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[00495] (8) Elute the protein in a gradient salt concentration.0-100% buffer B for twenty column volumes. [00496] (9) Determine protein concentration by Pierce Coomassie plus protein determination reagent following manufacture instruction. [00497] (10) Run the peak fractions in a non-reduced SDS-PAGE gel and pool the fractions that contain the purified target protein. [00498] SP HP buffer A: 50 mM NaAc-HAc, pH5.0, 10 mM sodium chloride, 10% glycerol. [00499] SP HP buffer B: 50 mM NaAc-HAc, pH5.0, 1M sodium chloride, 10% glycerol. [00500] SP HP buffer C: 50 mM NaAc, pH5.0, 10 mM sodium chloride, 10% glycerol, 0.1% Triton- X114. [00501] Peak 1 fractions with good purity of SP HP purification were pooled and exchanged into final formulation buffer. [00502] In one example, the purity of SEC-HPLC was 98.03%. [00503] Formulation buffer: 50 mM Tris/HCl, 150 mM sodium chloride, 10% glycerol, pH 7.5. [00504] Product quality analysis: The purified protein is subjected to quality control testing, including reducing and non-reducing SDS-PAGE, SEC-HPLC, Bradford assay, LC-MS, and endotoxin level. [00505] Bradford assay: The final protein concentration was determined based on Pierce Coomassie plus protein determination reagent following manufacturer instruction. The final concentration was controlled lees than 5 mg/mL. If higher concentration storage is required in the future, stability testing of highly concentrated proteins and screenings of buffer solutions can be conducted. [00506] 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 sample was eluted isocratically for 15 min at a flow rate of 1.0 mL/min. Protein elution was monitored by UV absorbance at 280 nm. The peaks corresponding to aggregates, monomer and low-molecular-weight (LMW) species were integrated to calculate the percentage of each species. [00507] SDS-PAGE. Non-reducing and reducing SDS-PAGE were performed using precast NuPAGE™ 4-12% Bis-Tris gel from ThermoFisher. Sample loading buffer (4X LDS) is from Invitrogen. Gel running buffer (20X MES) is from GenScript. Non-reducing samples were treated with 30 mM iodoacetamide and heated at 95°C for five minutes before analysis. Reducing samples were treated with 50 mM DTT and heated at 95°C for five minutes before analysis. Electrophoresis was carried out at a constant voltage of
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180 V for forty minutes. Gels were stained using Coomassie blue for thirty minutes and destained with water for one hour. [00508] Endotoxin level: Purified proteins were subjected to endotoxin test in Limulus Amoebocyte Lysate (LAL) based kinetic turbidimetric test. Purified proteins were diluted based on maximum valid dilution (MVD) in LAL reagent water (Charles River/W110), then sample was loaded in Charles River Laboratories EndoSafe LAL Cartridges (Charles River/ PTS55F) and detected by Charles River Laboratories Endosafe NexGen-MCS™ instrument. [00509] LC-MS: The LC-mass spectroscopy assays were performed with Agilent 6230 AdvanceBio LC/time-of-flight system with a PL1912-1502, PLRP-S 1000Å, 2.1 x 50 mm, 5 µm column (Agilent/ PL1912-1502). The mass spectroscopy data were analyzed by Bioconfirm 10.0 software and identify molecules based on molecular masses. For non-reduced mass spectroscopy, the sample was injected directly. For reduced mass spectroscopy, the sample was treated with 50 mM DTT for thirty minutes at room temperature before injection. [00510] The average yield of final sample was around 20-30 mg/L. EXAMPLE 7 AGN417 technical report [00511] AGN417 is a sortase-ligation based bifunctional degrader (TRAP). The molecule consists of one single chain (ABT410, peptide), which is conjugated to a pentapeptide motif LPETG of TBT403 by sortase-catalyzed ligation. The targeted antibody to conjugate ratio (BAR) is 1. [00512] AGN417 was successfully generated. All the results meet targeting quality. The sample was stable under three freeze-thaw cycles. [00513] TRAP purification method by AEX: [00514] The TRAP solution was purified by AEX to remove sortase A and linker-payload. [00515] (1) Instrument was AKTA pure 150 (Pure water generator Milli-Q Reference). [00516] (2) Column was Capto Q Impres, 500 mL. [00517] (3) Mobile phase A was 20 mM Tris, pH 8.3. Mobile phase B was 20 mM Tris,1M sodium chloride, pH 8.3. [00518] (4) Gradient was B from 0% to 45% in four column volumes. [00519] (5) Flow rate was 40 mL/min. [00520] (6) Wavelength was 280 nm and 220 nm.
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[00521] Purification method for TRAP by UF/DF: The UF/DF was preprocessed according to the following procedure: [00522] (1) Assemble the UF/DF system and install the cassette (Pellicon30.11m2 Cassette, Ultracel 10 kDa). [00523] (2) Flush the system with water, clean with the 0.1M sodium hydroxide for thirty minutes, and flush with water again. [00524] (3) Add phosphate-buffered saline, pH7.4 to the feed tank. Start the feed pump. Verify that the pH and conductivity in the system have been equilibrated to the level of the phosphate-buffered saline. [00525] (4) Add reaction mixture solution to the feed tank. Start the feed pump by partially closing the retentate valve and adjusting the pump speed. Diafilter the product with the phosphate-buffered saline, pH 7.4 for 15 DVs. [00526] (5) Open the retentate valve fully and collect all the product. Filter the product by 0.22 μm membrane. [00527] (6) Test the quality and concentration of product. [00528] Ultraviolet–visible spectroscopy platform by Nanodrop 2000 (ThermoFisher, 1294881) to determine protein concentration of in-process sample and final TRAP: [00529] (1) 750 nm was set up as baseline. [00530] (2) The ultraviolet absorption at 280 nm and 220 nm were measured respectively [00531] (3) Calculation method based on Beer-Lambert Law A=E*C*l [00532] A280=EmAb 280*C[mAb]*l, where E is the molar extinction coefficient; C is the molar concentration; and l is the light path (Nanodrop: 0.1 cm) [00533] BAR determination by LC-MS. LC-MS was performed using a combination of Agilent 1260 series high performance liquid chromatography system and time-of-flight mass spectrometry (Agilent Technologies, HPLC 1260 / 6224 TOF). BAR was calculated based on the peak abundance of the deconvoluted mass. [00534] Preparation of sample used in LC-MS analysis. An antibody-drug conjugate solution was diluted to approximately 1 mg/mL, 30 µL totally with water. The obtained sample was used in an LC-MS analysis. [00535] LC-MS/MS method. A high performance liquid chromatography analysis was carried out under the following measurement conditions. Equipment is HPLC Agilent 1260 (Agilent Technologies, HPLC 1260). Column: Agilent PLRP-S, 50 x 2.1 mm, 8 μm, 1000Å.Detection Wavelength: 280 nm, 214 nm.
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Column Oven Temp.: 80°C. Sampler temperature: 2~8℃.^Flow Rate: 0.05 mL/min. Injection Amount: 2 μg. Mobile phase: A was 0.025% trifluoroacetic acid+0.1%FA in water. Mobile phase B was 0.025% trifluoroacetic acid + 0.1% formic acid in acetonitrile. Gradient Program is for ten 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%. [00536] Aggregation determination by size exclusion chromatography-high performance liquid chromatography: Size-exclusion chromatography was performed using an Agilent 1260 series high performance liquid chromatography system with the TSK gel G3000SWXL Size-exclusion chromatography column (7.8×300 mm, 5 µm) at 25°C. The mobile phase was consisted of 78 mM KH2PO4, 122 mM K2HPO4, 250 mM potassium chloride, 15% isopropanol at pH 7.0±0.1. The flow rate was set at 0.75 mL/min. Sample loading was 40~50 μg per injection. Samples were detected at 280 nm and 370 nm with a UV detector. The retention time of the aggregation peak was recorded based on its relative molecular weight. The aggregation level was determined by the relative area of the peak at 280 nm. [00537] Residual free drug determination by LC-MS. The residual free drug level was determined by LC-MS. After protein precipitation, supernatant was loaded to InfinityLab Poroshell 120 SB-C18, 4.6 x 100 mm, 2.7 µm column, and eluted by a gradient of increasing the organic mobile phase. The percentage of residual free drug was quantified via peak area by comparing it to external standard curve. [00538] (1) Solvent I preparation: Weighed 10 g sodium chloride to the pre-mixed organic solvent of 30 mL methanol and 50 mL acetonitrile, mixed and stirred one hour at least, allowed the solution to stand for at least one hour before use. The supernatant was the saturated sodium chloride solution. [00539] (2) Preparation of standard curve: The stock standard linker-drug solution was firstly diluted to 40 µM with dimethyl sulfoxide for standard curve preparation. Then the standard curve and sample were prepared. MWmAb = 9731 Da. TRAP BAR value = 0.91 were used for calculation. [00540] (3) Preparation of sample. The sample were prepared as follows. (TRAP product concentration 22.45 mg/mL was used for calculation). TRAP concentration = 0.50 mg/mL. VTRAP 3.30 µL. Dimethyl sulfoxide volume fraction = 0.1. VDMSO 15.00 µL. VH2O was 31.70 µL. Solvent I was 100.00 µL. [00541] Liquid Chromatography parameters for free drug determination: [00542] Column: Agilent PLRP-S, 50 x 2.1 mm, 8 μm, 1000Å. [00543] Detection wavelength: 280 nm, 220 nM. [00544] Column oven temperature: 80°C. ^
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[00545] Sampler temperature: 2~8℃.^^ [00546] Flow rate was 0.4 mL/min. Stop time was at thirteen minutes. Maximum pressure was 350 bar. Injection Amount was 20 µL. [00547] Mobile phases. Mobile Phase A was 0.025% trifluoroacetic acid + 0.1% formic acid in water. Mobile Phase B was 0.025% trifluoroacetic acid + 0.1% formic acid in acetonitrile. The gradient was as follows: At time 2.00 minutes, A was 95.0%, and B was 5.0%. At time 5.00 minutes, A was 70.0% and B was 30.0%. At time 8.00 minutes, A was 20.0% and B was 80.0%. At time 8.50 minutes, A was 10.0% and B was 90.0%. At time 10.00 minutes, A was 10.0% and B was 90.0%. At time 10.01 minutes, A was 95.0% and B was 5.0%. At time 13.00 minutes, A was 95.0% and B was 5.0%. [00548] Quadrupole-time-of-flight parameters for free drug determination: Equipment was Agilent 6530 QTOF. Polarity was positive. Gas Temp. was 250oC. Drying Gas was 13 L/min. Nebulizer was 45 psig. VCap was 3500 V. Fragmentor was 125 V. Mass Range was 150 – 8000 m/z. Acquisition Rate was 1 spectra/s. [00549] Endotoxin determination. The endotoxin level was determined by Endosafe® NexGen-PTS™. Diluted TRAP sample with endo-free water, pipetted 25 µL of diluted sample into each of the four reservoirs of the Portable Test System (PTS) Cartridge. The reader drew and mixed the sample with the LAL reagent in the sample channels in addition to the LAL reagent plus positive product control in the spike channels. The sample was combined with the chromogenic substrate then incubated. After mixing, the optical density of the wells was measured and analyzed against an internally archived standard curve. [00550] Conjugation buffer and formulation buffer. [00551] AGN417 conjugation buffer is 50 mM Tris, 150mM sodium chloride, 10% glycerol, pH 7.5. [00552] AGN417 formulation buffer is phosphate-buffered saline, pH 7.4. [00553] ure for bulk conjugation. [00554] (1) To two 2000 mL bottle (Corning, 430281) containing 5971.2 mg of ABT410 in original buffer (50 mM Tris, 150 mM sodium chloride, 10% glycerol, pH 7.5) were added with 5.08 mg/mL sortase A (PT0008, 0.01 equivalents) solution, TBT404 (40 mM in dimethyl sulfoxide) (eighteen equivalents) into the reactions, mixed well and incubated at 4oC for overnight (seventeen hours). The protein concentration in the reaction was 3.21 mg/mL. [00555] (2) The crude conjugation was purified with AKTA pure 150 chromatography. The conjugate was bounded to the column in the loading sample process. The sortase A with His-tag and linker-payload
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were flowed through. Pooled flow through liquid together and performed buffer exchange to phosphate-buffered saline, pH 7.4 with UF/DF (3 kDa, 2*0.11 m2) for 15 DV. [00556] (3) The purified sample was filtered with 0.22 µm filter. [00557] (4) Perform the quality control test (including concentration, size exclusion chromatography- high performance liquid chromatography, HIC-high performance liquid chromatography, LC-MS, free drug, endotoxin level and osmolarity). [00558] Results of bulk conjugation. The reaction condition of bulk conjugation is summarized in the TABLE below. After conjugation, the reaction mixture is purified by AEX and exchange buffer to phosphate-buffered saline, pH 7.4 use UF/DF (3 kDa, 2*0.11 m2, 10 DV), then filter with 0.22 μm membrane. The molecular information of AGN417 was summarized in the TABLE below. The results of AKTA, LC-MS, free drug, endotoxin level and osmolarity were determined. [00559] Reaction conditions for bulk conjugation of AGN417 (ABT410-TBT403). Sortase A/monoclonal antibody ratio was 0.01. monoclonal antibody/conjugate ratio was 18. Conjugation concentration was 3.21 mg/mL. Conjugation temperature and time was 4oC, overnight. Conjugation pH was 7-7.5. [00560] Molecular information about AGN417. In one example, for AGN417 (ABT410-TBT403), the BAR1 molecular weight (amino acid,) was 11921.21 Da. Modification was BAR = 1. Measured mass was 11921.92 Da. [00561] Results for bulk conjugation. In one example, for AGN417 (ABT410-TBT403): Concentration measured by ultraviolet spectroscopy was 22.45 mg/mL, am was 5948.45 mg, MS-BAR was 0.91. Size-exclusion chromatography -purity was 97.40%, Free drug was <1.0%; endotoxin was <0.089 EU/mg; and osmolarity was 303 mOsm/kg. [00562] Results and discussion of stability tests. Store the sample under -70°C for one hour to ensure that material is frozen, then the material is allowed to thaw by letting the vial to sit at room temperature for sufficient amount of time until completed thawed. Collect the analytical characterization under different freeze-thaw cycles (1, 2, 3). The resulting ratios were BAR = 0.89-0.91. TABLE 19
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TABLE 19 Material name Material information .6 Procedure for preparation of TBT403 (Ins-011) [00563] Preparation of intermediate 2: methyltetrahydrofuran (450 mL) was added 1 (34.2 g, 200 mmol, 1.0 equivalent) in 2- methyltetrahydrofuran (160 mL) at 0°C. The mixture was stirred at 25°C for two hours. Thin layer chromatography (DCM: methanol = 20: 1, Rf = 0.70) showed the reaction was completed, one major new spot with lower polarity was detected. The reaction mixture was added HCl/EA (1 N, 27.0 mL) and stirred for thirty minutes. The white precipitate was removed by filtration, the filtrate was concentrated under reduced pressure to afford Intermediate 2 (crude, 105.0 g, 370.6 mmol) as yellow oil. LC-MS: retention time = 0.797 minutes, MS calculated: 283.14, mass observed: [M + Na]+ = 306.1.1H NMR (400 MHz, DMSO-d6) δ ppm 7.23 - 7.41 (m, 5 H), 5.01 (s, 2 H), 4.60 (br s, 1 H), 3.45 - 3.52 (m, 6 H), 3.38 - 3.43 (m, 5 H), 3.14 (q, J = 5.94 Hz, 2 H), 2.53 - 2.55 (m, 1 H).
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[00565] Preparation of intermediate 3: s added TMSOTf (85.6 g, 385 mmol, 1.5 equivalents) and stirred at 60°C for 2 h. The reaction was then cooled to room temperature (25°C) and stirred for another one hour. A mixture of Intermediate 2 (80.0 g, 282 mmol, 1.1 equivalents) and 4 Å powder molecular sieves (50.0 g) in DCE (500 mL) was added to the reaction. The resulting mixture was stirred for thirty minutes under nitrogen gas atmosphere. Then a solution of intermediate 2a (100.0 g, 257 mmol, 1.0 equivalent) in DCE was added dropwise to the mixture at 0°C. The mixture was stirred for sixteen hours at 25°C under nitrogen gas atmosphere. Thin layer chromatography (DCM: methanol = 10: 1, Rf = 0.42) indicated intermediate 2a was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was filtered and washed with sat. NaHCO3 (500 mL), water (500 mL) and brine (500 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PE: EA = 3: 1 to 1: 6, then DCM: methanol = 20: 1) to afford intermediate 3 (90.0 g, 146.9 mmol, 91.6% purity, 57.2% yield) as yellow oil. LC-MS: retention time = 0.860 minutes, MS calculated: 612.25, mass observed: [M + H]+ = 613.2.1H NMR (400 MHz, DMSO-d6) δ ppm 7.80 (d, J = 9.03 Hz, 1 H), 7.24 - 7.39 (m, 6 H), 5.22 (d, J = 3.51 Hz, 1 H), 4.95 - 5.05 (m, 3 H), 4.53 - 4.59 (m, 1 H), 3.99 - 4.06 (m, 3 H), 3.84 - 3.92 (m, 1 H), 3.73 - 3.82 (m, 1 H), 3.55 - 3.61 (m, 1 H), 3.45 - 3.53 (m, 7 H), 3.41 (t, J = 5.90 Hz, 2 H), 3.11 - 3.18 (m, 3 H), 2.10 (s, 3 H), 1.99 (s, 3 H), 1.89 (s, 3 H), 1.77 (s, 3 H). [00567] Preparation of intermediate 4: [00568] Pd/C (9.00 g, 10% purity) in reaction bottle (purged with argon gas for three times) was added THF (180 mL) slowly, then a solution of trifluoracetic acid (16.7 g, 147 mmol, 1.0 equivalent) and intermediate 3 (90.0 g, 147.0 mmol, 1.0 equivalent) in THF (720 mL) was added to the reaction slowly
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under nitrogen gas. The reaction was degassed and purged with nitrogen gas and hydrogen gas for three times, then stirred at 25°C for three hours under hydrogen gas atmosphere (40 psi). Thin layer chromatography (DCM: methanol = 10: 1, Rf = 0.20) indicated intermediate 3 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was dissolved in THF (100 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with THF (100 mL * 2), and the filtrate was concentrated under reduced pressure to get the residue. The residue was diluted with water (1000 mL), washed with DCM (300 mL * 3), the aqueous layer was lyophilized to afford intermediate 4 (80.0 g, 139.0 mmol, 95.1% purity, 91.8% yield, trifluoracetic acid salt) as a white solid. LC-MS: retention time = 0.484 minutes, MS calculated: 478.22, mass observed: [M + H]+ = 478.9.1H NMR (400 MHz, DMSO-d6) δ ppm 7.91 (br t, J = 9.03 Hz, 4 H), 5.21 (d, J = 3.26 Hz, 1 H), 4.96 (dd, J = 11.17, 3.39 Hz, 1 H), 4.54 (d, J = 8.53 Hz, 1 H), 3.98 - 4.08 (m, 3 H), 3.85 - 3.93 (m, 1 H), 3.75 - 3.84 (m, 1 H), 3.59 (br t, J = 5.14 Hz, 3 H), 3.50 - 3.56 (m, 6 H), 2.98 (br s, 2 H), 2.10 (s, 3 H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.78 (s, 3 H). [00569] Preparation of intermediate 6: [00570] To a mixture of intermediate 5 (60.0 g, 495.0 mmol, 1.0 equivalent) in DMSO (166 mL) was added aqueous NaOH (5.0 M, 9.91 mL, 0.1 equivalents) dropwise at 0-15°C for over five minutes. After addition, the mixture was stirred at 0-15°C for five minutes, then intermediate 5a (254.0 g, 1.98 mol, 287 mL, 4.0 equivalents) was added to the reaction mixture dropwise at 20°C. The resulting mixture was stirred at 25°C for sixteen hours. Thin layer chromatography (DCM: methanol = 10: 1, Rf = 0.7) indicated intermediate 5 was consumed completely, and one major new spot with lower polarity was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate (400 mL), quenched by addition of water (400 mL), and extracted with ethyl acetate (400 mL * 3). The combined organic layers were washed with brine (300 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to afford intermediate 6 (100.0 g, 197.8
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mmol, 96.0% purity, 40.0% yield) as colorless oil.1H NMR (400 MHz, DMSO-d6) δ ppm 3.51 - 3.61 (m, 7 H), 3.17 (s, 5 H), 2.39 (t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H). [00571] Preparation of intermediate 7: ) was added hydroxybenzotriazole (10.7 g, 79.1 mmol, 1.0 equivalent). Then 6a (16.5 g, 79.1 mmol, 1.0 equivalent) and DCC (16.3 g, 79.1 mmol, 1.0 equivalent) were added. The reaction was stirred at 25°C for sixteen hours. Thin layer chromatography (PE: EA = 1: 1, Rf = 0.80) indicated intermediate 6 was consumed completely. One major new spot with lower polarity was detected. Acetonitrile was evaporated to get the residue. The residue was purified by column chromatography (SiO2, PE: EA = 10: 1 to 1: 1) to afford intermediate 7 (40.0 g, 57.4 mmol, 82.9% purity, 72.5% yield) as a white solid. LC-MS: retention time = 1.151 minutes, MS calculated: 696.38, mass observed: [M + H]+ = 697.3, [M + Na]+ = 719.3.1H NMR (400 MHz, DMSO-d6) δ ppm 7.26 - 7.40 (m, 6 H), 7.06 (s, 1 H), 5.03 (s, 2 H), 3.49 - 3.61 (m, 14 H), 2.39 (br t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H). [00573] Preparation of intermediate 8: [00574] A solution of intermediate 7 (30.0 g, 43.0 mmol, 1.0 equivalent) in HCOOH (300 mL) was stirred at 25°C for sixteen hours. Thin layer chromatography (PE: EA = 1: 1, Rf = 0.04) indicated intermediate 7 was consumed completely, and one major new spot with larger polarity was detected. Solvent was evaporated under reduced pressure, then co-evaporated with toluene (50 mL * 3) under reduced pressure, and dried under reduced pressure to get the residue. The residue was purified by
98 30125-WO-PCT
prep-HPLC (A: 0.1% trifluoracetic acid/water, B: acetonitrile) to afford intermediate 8 (20.0 g, 37.8 mmol, 98.2% purity, 87.9% yield).1H NMR (400 MHz, DMSO-d6) δ ppm 12.17 (br s, 3 H), 7.26 - 7.43 (m, 6 H), 7.06 (s, 1 H), 5.02 (s, 2 H), 3.49 - 3.65 (m, 14 H), 2.42 (br t, J = 6.27 Hz, 6 H). LC-MS: retention time = 0.790 minutes, MS calculated: 528.20, mass observed: [M + H]+ = 529.2. [00575] Preparation of intermediate 9: iate 4 (78.5 g, 132 mmol, 3.5 equivalents, trifluoracetic acid salt) in DMF (400 mL) was added hydroxybenzotriazole (20.4 g, 151 mmol, 4.0 equivalents), EDCI (29.0 g, 151 mmol, 4.0 equivalents) and DIEA (22.0 g, 170 mmol, 4.5 equivalents) successively. The reaction was stirred at 25°C for two hours. Thin layer chromatography (DCM: methanol = 10: 1, Rf = 0.4) indicated intermediate 8 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was slowly poured into a stirring cold 0.5 mol/L hydrochloric acid solution (900 mL) and stirred for ten minutes. White precipitate was formed and filtered. The aqueous phase was extracted with DCM (600 mL* 2) twice. The combined organic layers were washed with 5% NaHCO3 (450 mL), dried over Na2SO4, and concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiO2, DCM: methanol = 100: 1 to 5: 1) to afford intermediate 9 (58.0 g, 30.4 mmol, 82.7% purity, 80.3% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) δ ppm 7.92 (br t, J = 5.14 Hz, 3 H), 7.81 (d, J = 9.03 Hz, 3 H), 7.28 - 7.39 (m, 6 H), 7.13 (s, 1 H), 5.21 (d, J = 3.26 Hz, 3 H), 5.02 (s, 2 H), 4.97 (dd, J = 11.17, 3.39 Hz, 3 H), 4.54 (d, J = 8.53 Hz, 3 H), 4.03 (s, 9 H), 3.84 - 3.92 (m, 3 H), 3.75 - 3.81 (m, 3 H), 3.45 - 3.61 (m, 37 H), 3.39 (br s, 3 H), 3.18 - 3.23 (m, 6 H), 2.30 (br t, J=6.15 Hz, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H). LC-MS: retention time = 3.455 minutes, MS calculated: 1908.81, mass observed: [M + 2H]2+ = 955.7.
99 30125-WO-PCT [00577] Preparation of intermediate 10: [00578] Pd/C (4.6 g, 25.13 mmol, 10% purity, 1.0 equivalent) in reaction bottle (purged with nitrogen gas for three times) was added methanol (230 mL) slowly, then a solution of trifluoracetic acid (2.75 g, 24.1 mmol, 1.0 equivalent) and intermediate 9 (46.0 g, 24.1 mmol, 1.0 equivalent) in methanol (230 mL) was added to the reaction slowly under nitrogen gas atmosphere. The reaction was degassed and purged with nitrogen gas and hydrogen gas for three times, stirred at 25°C for two hours under hydrogen gas atmosphere (15 psi). Thin layer chromatography (DCM: methanol = 10: 1, Rf = 0.3) indicated intermediate 9 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was dissolved in methanol (250 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with methanol (250 mL * 2), and the filtrate was concentrated under reduced pressure to afford intermediate 10 (crude, 42.0 g, 22.4 mmol, 94.1% purity, 92.2% yield, trifluoracetic acid salt) as a white solid.1H NMR (400 MHz, DMSO-d6) 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: retention time = 1.585 minutes, MS calculated: 1774.78, mass observed: [M + H]+ = 1775.7, [M + 2H]2+ = 888.7. [00579] Preparation of intermediate 12:
[00580] 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 of intermediate 10 (42.0 g, 22.2 mmol, 1.0 equivalent, trifluoracetic acid salt) and 11a (7.77 g, 33.3 mmol, 1.5 equivalents) in DMF (420 mL). The mixture was stirred at 25°C for 2.0 hours. LC-MS indicated intermediate 10 was consumed completely, several new peaks were shown on LC-MS and desired compound was detected. The reaction mixture was slowly poured into a stirring 0.5 mol/L hydrochloric acid solution (cold, 500 mL). The aqueous phase was extracted with DCM (500 mL * 3). The combined organic layers were washed with 5% NaHCO3 (500 mL), dried over Na2SO4, then concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiO2, DCM: methanol = 100: 1 to 5: 1) to afford intermediate 12 (34.0 g, 17.1 mmol, 93.3% purity, 77.0% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) 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: retention time = 0.501 minutes, MS calculated: 1989.87, mass observed: [M + 2H]2+ = 996.3. [00581] Preparation of Target A043: ) was added NaOMe (5.4 M in methanol, 9.91 mL, 4.2 equivalents) at 0°C. Then the solution was stirred at 0°C for 0.5 h. LC-MS indicated intermediate 12 was consumed completely, several new peaks were shown on LC-MS and desired compound was detected. The reaction mixture was adjusted pH to 6 with 1.0 M hydrochloric acid solution at 0°C and washed with DCM (250 mL * 3) for three times, the aqueous layer was lyophilized to afford Target A043 (crude, containing sodium chloride, 20.0 g, 12.4 mmol, 96.3% purity) as a white solid.1H NMR (400 MHz, DMSO-d6) 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),
101 30125-WO-PCT
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: retention time = 0.501 minutes, MS calculated: 1611.77, mass observed: [M + H]+ = 1612.8, [M + 2H]2+ = 807.2. [00583] Preparation of intermediate 13: [00585] (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) was bubbled with nitrogen gas for two hours at 25°C. Then 20% piperidine in DMF (200 mL) was added and the mixture was bubbled with nitrogen gas for thirty minutes at 25oC. The mixture was filtered and washed with DMF (200 mL) * 5 before proceeding to next step. [00586] (2) Coupling: A solution of Fmoc-G-G-OH (3.54 g, 10.00 mmol, 2.00 equivalents), HBTU (7.2 g, 9.5 mmol, 1.90 equivalents), DIEA (5.16 g, 40.00 mmol, 4.00 equivalents) in DMF (100 mL) was added to the resin with nitrogen gas bubbling for thirty minutes at 25°C. The coupling reaction was monitored by ninhydrin test. The resin was then washed with DMF (200 mL) * 5. [00587] (3) De-protection: 20% piperidine/DMF (300.0 mL) was added and mixed at room temperature (25°C) for thirty minutes. The de-protection reaction was monitored by ninhydrin test, if it showed blue or other brownish red, the reaction was completed. The resin was then washed with DMF (300.0 mL) *5. [00588] (4) Step 2 to 3 was repeated for all other amino acids: (#7 in TABLE below). At the last step, the resin was washed with methanol (300.0 mL) * 3 and dried by nitrogen gas bubbling for three hours. TABLE 20 Th li f i id d h di d PP
102 30125-WO-PCT
TABLE 20 The list of amino acids and the corresponding reagents used on SPPS. [ 00590] (1) Cleavage solution (TFA/TIS/water, 95/2.5/2.5, v/v/v, 220 mL) was added to the flask containing the side-chain protected resin-bound peptide (RINK AMIDE MBHA Resin, 22 g, 5.0 mmol) at 25°C and stirred for two hours. [00591] (2) After filtration, the filtrate was collected. [00592] (3) The filtrate was precipitated with cold isopropyl ether (1000 mL). After filtration, the solid was washed with isopropyl ether (500 mL) twice, and the crude peptide was dried under reduced pressure for 2 h to afford intermediate 1 (5 g, crude) as a white solid. The crude peptide was purified by prep-HPLC (A: 0.1% trifluoracetic acid/water, B: acetonitrile), followed by lyophilization to afford intermediate 13 (1.5 g, 90% purity, 42.3% yield) as a white solid. [00593] Preparation of TBT403 (Ins-011): [00594] To a solution of Target A043 (2.184 g, 1.354 mmol, 1.2 equivalents) and Intermediate 13 (0.8 g, 1.129 mmol, 1.0 equivalent) in DMF (1 mL) and water (10 mL) was added a solution of sodium ascorbate (670.827 mg, 3.386 mmol, 3.0 equivalents) and TBTA (Tris[(1-benzyl-1H-1,2,3-triazol-4- yl)methyl]amine, 490.430 mg, 1.129 mmol, one equivalent) and CuSO4-5H2O (0.4 M, 2.822 mL, 1.0 equivalent) in water (3 mL) at 0°C. Then the mixture was stirred at 25°C for one hour. LC-MS indicated Intermediate 13 was consumed completely, several new peaks were shown on LC-MS and desired compound was detected. The mixture was purified by prep-HPLC (A: 0.5% acetic acid/water, B: acetonitrile) followed by lyophilization to afford TBT403 (Ins-011) (1.42 g, 94% purity, 50.9% yield) as a white solid. LC-MS: retention time =0.15 minutes, MS calculated: Mav =2321.4, mass observed: [M + H]+ =2321.9.
103 30125-WO-PCT
EXAMPLE 9 Procedure for reparation of TBT405 (Ins-030) [00595] To a solution of Intermediate 15 (99.0 g, 52.4 mmol, 1.00 equivalent, trifluoracetic acid) in methanol (1.00 L) was added NaOMe (11.3 g, 210 mmol, 4.00 equivalents). The mixture was stirred at 25°C for one-half hour. LC-MS showed intermediate 15 was consumed completely and one main peak with desired mass. The reaction mixture was concentrated under reduced pressure to remove methanol. The residue was diluted with water (500 mL) and washed with DCM (500 mL *3), the aqueous layer was lyophilized to give a crude product. The crude product was purified by prep-HPLC (A: 0.5% acetic acid/water, B: acetonitrile) to afford Target A001A (36.0 g, 25.7 mmol, 49.1% yield, 97.5% purity) as 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: retention time = 1.103 minutes, MS calculated:1397.5, found: [M + H]+ = 1397.5, [(M + 2H)/2] 2+ = 699.6. [00596] Preparation of intermediate 15: added a solution of 2,3,5,6-tetrafluorophenol (18.406 g, 110.832 mmol, 2.5 equivalents) in DMF (50 mL) at 0°C. Then a solution of EDCI (21.247 g, 110.832 mmol, 2.5 equivalents) in DMF (50 mL) was added to the mixture and it was stirred at 25°C for two hours. LC-MS indicated intermediate 14 was consumed completely, several new peaks were shown on LC-MS and desired compound was detected. The crude solution was purified by prep-HPLC (A: 0.1% TFA/water, B: acetonitrile) followed by lyophilization to afford intermediate 15 (22 g, 97% purity, 75.8% yield) as a colorless oil. LC-MS: retention time = 0.724 minutes, MS calculated: Mav = 634.5, found: [M +H]+ = 635.3.1H NMR (400 MHz, DMSO-d6) 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).
104 30125-WO-PCT
[00598] Preparation of intermediate 16: mistry (CTC resin). [00600] (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 equivalent) in DCM (200 mL) was added DIEA (4.0 equivalents) dropwise and mixed for 2 h with nitrogen gas bubbling at 25°C. Then methanol (15.0 mL) was added and bubbled with nitrogen gas for another thirty minutes. The resin was washed with DMF (100 mL) * 5, followed by the addition of 20% piperidine in DMF (200 mL) and bubbled with nitrogen gas for thirty minutes at 25°C for Fmoc deprotection. The mixture was filtered. The resin was washed with DMF (100 mL) * 5 before proceeding to next step. [00601] (2) Coupling: A solution of Fmoc-Thr-OH (17.9 g, 45 mmol, 3.0 equivalents), HBTU (16.3 g, 42.75 mmol, 2.85 equivalents) in DMF (100 mL) was added to the resin with nitrogen gas bubbling. Then DIEA (6.0 equivalents) was added to the mixture dropwise and bubbled with nitrogen gas for thirty minutes at 25°C. The coupling reaction was monitored by ninhydrin test, if it showed colorless, the coupling was completed. The resin was then washed with DMF (200 mL) * 5. [00602] (3) Deprotection: 20% piperidine in DMF (200 mL) was added to the resin and the mixture was bubbled with nitrogen gas for thirty minutes at 25°C. The resin was then washed with DMF (200 mL) * 5. [00603] (4) Step 2 to 3 was repeated for all other amino acids: (#3-5 in TABLE below). At the last step, the resin was washed with methanol (200.0 mL) * 3 and dried by nitrogen gas bubbling for three hours. TABLE 21
105 30125-WO-PCT
TABLE 21 The list of amino acids and the corresponding reagents used on SPPS. [ 00605] (1) Cleavage solution (TFA/TIS/water, 95/2.5/2.5, v/v/v, 90 mL) was added to the flask containing the side-chain protected resin-bound peptide (CTC Resin, 30 g, 15.0 mmol) at 25°C and stirred for 2 h. [00606] (2) After filtration, the filtrate was collected. [00607] (3) The filtrate was precipitated with cold isopropyl ether (1500 mL). After filtration, the solid was washed with isopropyl ether (500 mL) twice, and the crude peptide was dried under reduced pressure for two hours to afford Intermediate 1 (10 g, crude) as a white solid. The crude peptide was purified by prep-HPLC (A: 0.1% TFA/water, B: acetonitrile) followed by lyophilization to afford intermediate 16 (6.0 g, 93% purity, 69% yield) as a white solid. LC-MS: retention time = 0.304 minutes, MS calculated: Mav =572.6, found: [M +H]+ = 573.4. [00608] Preparation of intermediate 17: 9 g, 20.957 mmol, 3.464 mL, 2.0 equivalents) in DMF (15 mL) was added intermediate 16 (6 g, 10.478 mmol, 1.0 equivalent) in DMF (15 mL) slowly at 25°C under nitrogen gas. The mixture was stirred at 25°C for one hour. LC-MS indicated intermediate 16 was consumed completely, several new peaks were shown on LC-MS and desired compound was detected. The mixture was adjusted to pH 5-6 with acetic acid. The crude solution was purified by prep-HPLC (A: 0.5% acetic acid/water, B: acetonitrile) followed by lyophilization to afford intermediate 17 (3.1 g, 95% purity, 28.4% yield) as a white solid. LC-MS: retention time = 0.491 minutes, MS calculated: Mav =1040.9, found: [M +H]+ = 1041.7.
106 30125-WO-PCT
[00610] Preparation of TBT405 (Ins-030): [ e 17 (548.89 mg, 500.906 μmol, 1.0 equivalent) in DMF (10 mL) was added DIEA (129.477 mg, 1.002 mmol, 2.0 equivalents) slowly at 0°C under nitrogen gas. The mixture was stirred at 25°C for one hour. LC-MS indicated intermediate 17 was consumed completely, several new peaks were shown on LC-MS and desired compound was detected. The mixture was adjusted to pH 5-6 with 1M hydrochloric acid at 0°C. The crude solution was purified by prep-HPLC (A: 0.5% acetic acid/water, B: acetonitrile) followed by lyophilization to afford TBT405 (Ins-030) (495.3 mg, 99.5% purity, 42.9% yield) as a white solid. LC-MS: retention time =0.328 minutes, MS calculated: Mav =2272.4, mass observed: [M + H]+/2 =1136.7. EXAMPLE 10 Biophysical assays. Surface plasmon resonance – second results. [00612] Surface plasmon resonance for insulin growth factor 1 receptor assay conditions: [00613] Buffer: HSB-N with 0.005% Tween-20, pH: 7.490 seconds on, 180 seconds off. [00614] Capture human insulin growth factor receptor (His-Avitag) from Acro on a SA chip at ~750, 1000, and 1500 RU. [00615] Assess binding of insulin to the receptor (10 µM top, 2-fold dilution). [00616] Surface plasmon resonance for insulin growth factor 1 receptor: Avi-tagged receptor captured on an SA chip. The inventors assayed insulin and insulin lispro. [00617] Surface shows better activity at higher density (50% versus 65% active) [00618] Traces were sufficient for a selectivity assay. [00619] Conclusion: A robust surface plasmon resonance assay was developed for insulin growth factor 1 receptor. Mutant insulin has significantly reduced insulin growth factor 1 receptor binding
107 30125-WO-PCT
TABLE 22 Surface plasmon resonance Surface plasmon resonance updates Sample Sample info IR-B KD IGF-1R KD mAb49 KD C-peptide Ab ASGPR IC50
108 30125-WO-PCT
EXAMPLE 11 Determination of total conjugate of AGN6567 in Rat K2EDTA plasma by LC-MS/MS [00620] Purpose. An LC-MS/MS method was developed for the determination of total antibody from AGN6567 in rat K2EDTA Plasma using SIL TTPP as the internal standards (IS). This method is used for non- GLP studies. AGN6567 is too large for direct analysis by LC-MS. Its signature peptide ELVAVITTDGSTNYADSVK (SEQ ID NO: 51) is used for the quantitation of AGN6567 after the immunoprecipitation and digestion step in rat plasma. TABLEX Method Summary ts equivalent, is utilized during the execution of the method: TABLE 23
109 30125-WO-PCT
TABLE 23 Equipment Vendor/Model als, or the equivalent, are utilized during the execution of the method: TABLE 24 R t G d
110 30125-WO-PCT [00623] HPLC conditions for Sciex 6500+: The instrument conditions may be adjusted to optimize the response. TABLE 25 Mobile Phase A 0.1% formic acid in water t onse. SIL ELVA peptide: EL*VAVITTDGSTNYADSVK (L*=Leu13C6,15N) TABLE 26
Detection Mode MRM Analyte ELVA SIL ELVA he SBA, Biotinylated Antibody™. The preparations described below may be scaled up or down to provide larger or smaller amounts of the reagents, mobile phases, etc. [00626] 1. phosphate-buffered saline buffer (wash buffer). Add 200 mL of phosphate-buffered saline buffer 10× to 1800 mL of purified water. Refrigerate the buffer. [00627] 2.1× phosphate-buffered saline with Tween (blocking buffer). Mix 50 mL of 20× phosphate- buffered saline with Tween solution with 950 mL of purified water. Mix well and store at room temperature. [00628] 3. Immobilize capturing reagent to Dynabeads M-280 Streptavidin beads. [00629] 4. Coat antibody to Dynabeads M-280 Streptavidin beads [00630] 5. Vortex and thoroughly re-suspend magnetic beads. [00631] 6. Transfer 5000 µL (equal to 10 mg of Dynabeads M-280 streptavidin beads) of Dynabeads M-280 streptavidin beads into a 5 mL Protein LoBind® tube.
112 30125-WO-PCT
[00632] 7. Apply magnet to engage beads to the side of the tube and discard the storage buffer. [00633] 8. Wash beads by adding 4000 µL of phosphate-buffered saline with Tween and vortex to re- suspend the beads [00634] 9. Apply magnet to engage beads to the side of the tube and remove the supernatant. [00635] 10. Repeat wash again. [00636] 11. Add 4000 µL of phosphate-buffered saline with Tween and 100 µL of SBA, Biotinylated Antibody (5 mg/mL) to the washed beads. [00637] 12. Mix thoroughly and incubate at room temperature while rotating for 60 minutes. Apply magnet and remove supernatant. [00638] 13. Wash beads two more times by phosphate-buffered saline with Tween and re-suspend beads with 5000 µL phosphate-buffered saline with Tween to a final concentration of 10 mg/mL in phosphate-buffered saline with Tween. [00639] 14. Beads can be kept in phosphate-buffered saline with Tween at 4°C. [00640] 15.10% acetonitrile in water (diluent). Add 10 mL acetonitrile to 90 mL purified water. Mix well. Store at room temperature. [00641] 16.300 mM calcium chloride (assay buffer). Dissolve 333 mg of calcium chloride in 10 mL of purified water. Mix well. Store at room temperature. Aliquot appropriately and store at -70°C. [00642] 17.50 mM ammonium bicarbonate (base buffer). Dissolve 1.98 g of ammonium bicarbonate with 500 mL purified water. Store at room temperature for one month. [00643] 18. RapiGest Solution 1mg/mL (denaturation solution). [00644] 19.50 mg (pre-weighed) of RapiGest powder is dissolved in 40 mL of 50 mM ammonia bicarbonate and 10 mL of acetonitrile. Make small portions into aliquots and store at -70°C for up to three months. [00645] 20.100 mM DTT (Reduction solution). Dissolve 154 mg of DTT (MW 154) in 10 mL of purified water. Aliquot appropriately and store at -70°C. [00646] 21.100 mM iodoacetamide (alkylation buffer). Dissolve 185 mg of iodoacetamide in 10 mL of 50 mM ammonium bicarbonate solution. Aliquot appropriately and store at -70°C. [00647] 22.10 % trifluoroacetic acid (acid buffer). Add 5 mL of trifluoroacetic acid to 45 mL of purified water. Vortex to mix well. Store at room temperature. [00648] 23. Mobile Phase A: 0.1% formic acid in water (Mobile Phase A). Add 2 mL formic acid in 2000 mL of purified water. Mix well. Store at room temperature.
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[00649] 24. Mobile Phase B: 0.1% formic acid in acetonitrile (Mobile Phase B). Add 2 mL formic acid in 2000 mL of acetonitrile. Mix well. Store at room temperature. [00650] 25. Needle Wash: 0.1% formic acid in isopropanol/water 50/50 (Needle Wash). Add 2 mL formic acid in 1000 mL of isopropanol and 1000 mL of purified water into a glass bottle. Store at room temperature for six months. [00651] 26. Preparation of Standard Stock Solutions [00652] Standard (STD) Stock 1: Freshly pipet 49 µL (8.16 mg/mL) of AGN6567 to 351 µL rat K2EDTA Plasma to give a final concentration of 1 mg/mL for total antibody in rat K2EDTA Plasma. [00653] Standard (STD) Stock 2: Pipet 100 µL STD stock 1 to 900 µL rat K2EDTA Plasma to give a final concentration of 100 µg/mL for total antibody in rat K2EDTA Plasma. [00654] Standard (STD) Stock 3: Pipet 100 µL STD stock 2 to 900 µL rat K2EDTA Plasma to give a final concentration of 10 µg/mL for total antibody in rat K2EDTA Plasma. [00655] Standard (STD) Stock 4: Pipet 100 µL STD stock 3 to 900 µL rat K2EDTA Plasma to give a final concentration of 1 µg/mL for total antibody in rat K2EDTA Plasma. [00656] The exact pipetting volume for stock solution should be calculated based on the actual antibody concentration of Reference Standard solutions. [00657] STD stock 1 and 2 are prepared freshly before use and discarded after use. [00658] Prepare the working standards in rat K2EDTA Plasma as detailed in the following TABLE: TABLE 27 Volume of source solution used (μL) t 114 30125-WO-PCT
[00659] Preparation of Quality Control Stock Solutions: The exact pipetting volume for stock solution should be calculated based on the actual antibody concentration of Reference Standard solutions. [00660] Quality Control (QC) Stock 1: Freshly pipet 49 µL (8.16 mg/mL) of AGN6567 to 351 µL rat K2EDTA Plasma to give a final concentration of 1 mg/mL for total antibody in rat K2EDTA Plasma. [00661] Quality Control (QC) Stock 2: Pipet 100 µL STD stock 1 to 900 µL rat K2EDTA Plasma to give a final concentration of 100 µg/mL for total antibody in rat K2EDTA Plasma. [00662] Quality Control (QC) Stock 3: Pipet 100 µL STD stock 2 to 900 µL rat K2EDTA Plasma to give a final concentration of 10 µg/mL for total antibody in rat K2EDTA Plasma. [00663] Quality Control (QC) Stock 4: Pipet 100 µL STD stock 3 to 900 µL rat K2EDTA Plasma to give a final concentration of 1 µg/mL for total antibody in rat K2EDTA Plasma. [00664] Preparation of Quality Control Samples. Quality control (QC) samples are prepared by spiking individual QC stock solutions into blank rat K2EDTA Plasma described in the TABLE below: TABLE 28 Volume of source solution used (μL) e [00665] SIL ELVA Stock: Dissolve 1 mg of SIL ELVA into 1 mL of diluent to give a concentration of 1 mg/mL for SIL ELVA and store at -70°C. [00666] IS Stock 1: Pipet 20 µL of SIL ELVA stock to 1980 µL diluent to give a final concentration of 10 µg/mL SIL ELVA. Store at -70oC.
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[00667] IS Spike: Pipet 50 µL of IS stock 2 toa total of 10 mL diluent to give a final concentration of 50 ng/mL SIL ELVA. Store at -20oC. [00668] Extraction procedures: Equilibrate the LC-MS/MS system with mobile phases for at least 60 minutes. Thaw calibration standard samples, QC samples, unknown samples and blank samples at ice- water bath. Vortex-mix the samples thoroughly. [00669] 1. Add 10 µL of each sample (SST and Standards) into a 96-well 2 mL plate. Add 10 µL of each sample (double blank, blank, QC and unknown samples). [00670] 2. Add 35 µL of 10 mg/mL of Anti-ID SBA-biotin coated beads and 15 µL 300mM CaCl2 solution to each well. [00671] 3. Add 150 µL of phosphate-buffered saline with Tween into each well. Cover the plate, centrifuge for about one minute at 500 rpm and vortex the plate for thirty seconds. [00672] 4. Incubate all samples at room temperature for sixty minutes in an incubation plate vortexer with a speed at 1000 RPM. [00673] 5. Process the plate with a KingFisher™ Flex Magnetic Particle Processor, wash beads two times with 300 µL of phosphate-buffered saline. Then transfer the beads to the “digestion plate” containing 100 µL of Rapi-Gest solution. [00674] 6. Add 10 µL 100 mM DTT to the digestion plate. Centrifuge for one minute at 1000 rpm and vortex for 20 s. [00675] 7. Cover the digestion plate with adhesive sealing film and incubate it on a shaker at 60°C, 900 rpm for one hour. Centrifuge the plate briefly at 800 rpm. [00676] 8. Add 12 µL of 100 mM iodoacetamide to each well. Cover the plate with an adhesive sealing film. Incubate the plate on a shaker in dark at room temperature for thirty minutes at 1000 rpm. [00677] 9. To prepare 0.1 mg/mL trypsin, thaw the required number of sequencing grade modified trypsin tubes. Add 150 µL of 50 mM ammonium to each tube (20 µg trypsin) and mix well. Combine the contents of all the tubes and mix well. This solution is prepared freshly. Approximately ten tubes of sequencing grade modified trypsin are required for a full 96-well plate. Add 20 µL of 0.1 mg/mL Trypsin to each well, Centrifuge for one minute at 1500 rpm. [00678] 10. Centrifuge the plate at 4000 rpm for three minutes. Stop the reaction by adding 10 µL of 10% trifluoroacetic acid. Centrifuge the plate at 1000 rpm for one minute, then shake at 1000 rpm for one minute. [00679] 11. Add 20 µL of the IS spike in each vial except for double blank. Add 20 µL diluent to double blank. Shake the plate at 1000 rpm for one minute, then centrifuge at 1000 rpm for one minute.
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[00680] 12. Inject 15 µL of total antibody digest into LC-MS/MS. [00681] Data evaluation. Retention time and peak area are determined by Analyst Data Acquisition/Processing software. Analyte concentrations are obtained from a calibration curve constructed by plotting peak area ratio versus concentration. Concentrations were calculated using linear regression according to the following equation: y = ax + b [00682] Where: y = peak area ratio of analyte/internal standard [00683] a = slope of the corresponding standard curve [00684] x = concentration of analyte (µg/mL) [00685] b = intercept of the corresponding standard curve (with 1/x2 weighting). EXAMPLE 12 Antibodies were labeled using an AF647 protein labeling kit [00686] Results of CR007 labeling. 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. Ratio of labeling = 79.8 µM/29.3 µM = 2.72. [00687] Results of 6E9F1 labeling. The yield is 60.6% = (0.13 ml*4.66 mg/ml)/1 mg.6E9F1 concentration = 4.66/150,000 = 31.1 µM. AF647=78.0 µM. Ratio of labeling=78.0 µM /31.1 µM = 2.50. [00688] Results of ABT404 labeling. The yield is 60.6%=(0.13 ml*4.66 mg/ml)/1 mg.6E9F1 concentration = 4.66/150,000 = 31.1 µM. AF647=78.0 µM. Ratio of labeling=78.0 µM/31.1 µM = 2.50. [00689] Results of ABT403 labeling: The yield was 58% = (0.25 ml*2.32 mg/ml)/1 mg. ABT403 concentration = 2.32/155,031 = 15.0 µM. AF647 = 31.0 µM. Ratio of labeling = 31.0 µM/15.0 µM = 2.1. [00690] Results of ABT404 labeling: The yield is 59.6% = (0.2 ml*2.98 mg/ml)/1 mg. ABT404 concentration = 2.98/144,685 = 20.6 µM. AF647 = 40.5 µM. Ratio of labeling = 40.5 µM/20.6 µM = 1.97. [00691] Results of ABT405 labeling: The yield is 62.2% = (0.2 ml*3.11 mg/ml)/1 mg. ABT405 concentration = 3.11/144,464 = 21.5 µM. AF647 = 35.8 µM. Ratio of labeling = 35.8 µM/21.5 µM = 1.67 [00692] Results of ABT410 labeling: The yield is 55.2% = (0.12 ml*4.60 mg/ml)/1 mg. ABT410 concentration = 4.60/144,680 = 31.8 µM. AF647 = 30.9 µM. Ratio of labeling = 30.9 µM/31.8 µM = 0.97. [00693] Results of ABT407 labeling: The yield is 63.1% = (0.19 ml*3.32 mg/ml)/1 mg. ABT407 concentration = 3.32/145,075 = 22.9 µM. AF647 = 27.4 µM. Ratio of labeling = 27.4 µM/22.9 µM = 1.2 [00694] Results of ABT408 labeling: The yield is 56.9% = (0.18 ml*3.16 mg/ml)/1 mg. ABT408 concentration = 3.16/144,855 = 21.8 µM. AF647 = 27.3 µM. Ratio of labeling = 88.1 µM/23.9 µM = 1.25. [00695] Results of ABT409 labeling: 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. Ratio of labeling = 16.5 µM/15.1 µM = 1.09.
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[00696] Results of 6E9F1 labeling: The yield is 65.1% = (0.37 ml*1.76 mg/ml)/1 mg. Mouse-6E9F1 concentration = 1.76/150,000 = 11.7 µM. AF647 = 48.6 µM. Ratio of labeling = 48.6 µM/11.7 µM = 4.15. [00697] Results of LS-C331895 labeling. The yield is 20%=(0.21 ml*0.95 mg/ml)/1 mg. ABT403 concentration = 0.95/150,000=6.3 µM. AF647=58.3 µM. Ratio of labeling = 58.3 µM/6.3 µM = 9.25. Compared to other antibodies, labeled yield is only 20% (compared to ~56-65% for other antibodies). Ratio of labeling is 9.25 (compared to ~0.97-4.15 for other antibodies). EXAMPLE 13 Biophysical assays. Surface plasmon resonance – third results. [00698] IGF1 receptor binding to IGF1 via surface plasmon resonance assay (Acro Biosystems) [00699] Biophysical assessment of insulin/IGF1 binding to their respective receptors has been demonstrated. EXAMPLE 14 Biophysical assays. Surface plasmon resonance – fourth results. [00700] Surface plasmon resonance assay can be used to measure anti-insulin autoantibodies and anti-proinsulin autoantibodies. Binding affinities of two agents to insulin receptors and human insulin antibody was assayed. [00701] Two TRAP agents were assayed in a TR-FRET/SPR assay (ASGPR1). AGN303 (native proinsulin) gave a result of IC50 = 1.2 mM. AGN310 (mutant proinsulin) gave a result of IC50 = 1.2 mM. TABLE 29 Surface lasmon resonance
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[00703] Cell-based FRET assay (AF488-labeled or AF647-labeled target) to assess ternary complex on cell surface. The inventors performed a FRET-based uptake/degradation of POI (DL650 and QC-1 labeled antibody). TABLE 30 TR-FRET Biophysical assays. Surface plasmon resonance – fifth results. [00704] Insulin receptor selectivity assays. Insulin receptor binding to human insulin can be measured via surface plasmon resonance assay. [00705] Native proinsulin was found to have lower affinity to insulin receptors but maintains similar affinity to insulin antibodies. [00706] Insulin receptor isoforms. Insulin receptor B differs from insulin receptor A by the inclusion of exon 11. Both isoforms have similar affinity for insulin. The insulin receptor isoforms show different functional features. Insulin receptor A exhibits a higher affinity for IGFs and a greater internalization and recycling rate than Insulin receptor B. Because of these differences, Insulin receptor B is preferentially associated with metabolic and differentiating signals. Insulin receptor A mainly favors cell growth, proliferation, and survival. TABLE 31 Surface plasmon resonance
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EXAMPLE 16 Biophysical assays. Surface plasmon resonance. [00708] Two TRAP agents were also assayed for binding affinities of TRAP agents to C-peptide antibodies (SPR). AGN406 (C-peptide with protease site, synthetic C-peptide degrader gave a result of EC50 = 9.8 nM. AGN407 (C-peptide without protease site, synthetic C-peptide degrader) gave a result of EC50 = 12.5 nM. EXAMPLE 17 Cellular In vitro internalization assays – first results [00709] The inventors performed cellular internalization of insulin antibodies in HEK293 cells. [00710] mAb49: Human anti-Insulin antibody. [00711] 6E9F1: Mouse anti-Insulin antibody. TABLE 32 Biochemistry selectivity (anti-insulin autoantibodies) ependent of insulin receptors. [00713] This finding provides flexibility with anti-human Insulin antibodies from different species in designing assays. [00714] Agents (TRAPs) assayed: [00715] AGN301: IgG degrader (reference IgG degrader control). Results: Continued good positive control for internalization with HEK293-ASGPR (clone 3F7) cells + rabbit anti-human insulin antibody. [00716] AGN303: Native insulin with GN3 Reference anti-insulin degrader control). Continued good positive control for internalization with HEK293-ASGPR (clone 3F7) cells + mouse anti-human insulin an. [00717] AGN411 (mutant construct) > AGN408 (native construct) for internalization, although minimal signal with either. Neither AGN411 (mutant construct) or AGN408 (native construct) induced internalization. [00718] AGN304: Proinsulin construct with C-terminal GN3 (Native) [00719] AGN305: Proinsulin construct with C-terminal GN3 (Mutant)
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[00720] ABT306: Alk-native insulin without GN3 (peptide only). [00721] ABT307: Proinsulin-IgG1 Fc fusion, G4S linker, hIgG1 hinge (C > A), LALA-PA. [00722] ABT308: Proinsulin-IgG1 Fc fusion, 5x G4S linker, hIgG1 hinge (C > A), LALA-PA. [00723] AGN411: Proinsulin construct with N-terminal αGN3 (mutant) [00724] AGN412: Synthetic Mutant insulin with βGN3. By comparison, AGN310 has αGN3. : (proinsulin Fc GN3) also low with no internalization. [00726] AGN414: Proinsulin-Fc conjugate (ABT307-TBT4438) [00727] AGN415: Proinsulin-Fc conjugate (ABT308-TBT4438) [00728] AGN416: Synthetic Mutant insulin with αGN3 on insulin A chain. By comparison, other synthetic insulin-GN3 compounds synthesized have GN3 attached to insulin B chain. [00729] AGN417: Native proinsulin with βGN3 on N-terminal. TABLE 33 AGN414 d 1 M t t ti 9 i t 3 DMSO 50 M AF647 l b l d d d d e recognized by either rabbit or mouse anti-insulin antibodies. [00731] (1) AGN301 + labeled rabbit Ab (CR007) should internalize (therefore positive signal) If add Proinsulin Fc protein, will compete with AGN301, and therefore have less signal [00732] (2) AGN301 + labeled mouse 6E9F1 Ab should internalize (therefore no signal). If add proinsulin Fc fusion protein, should get signal, since AGN301 binds to Fc portion of Fc fusion protein, and labeled mouse Ab will bind to proinsulin portion [00733] (3) LALA mutation on proinsulin Fc fusion protein should not interfere with IgG degrader binding, so the LALA mutation should not alter the results. [00734] Dose range with ABT307 and ABT308. [00735] Use both anti-insulin mouse antibody (clone 6E9F1) and rabbit antibody (clone CR007) at constant concentration.
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[00736] Test in HEK293-ASGPR (3F7) cells. [00737] Internalization assay conditions: [00738] Cell lines: HEPG2 and HEK293-ASGPR (clone 3F7, 3C6, 1D4). [00739] Culture media: DMEM + 10% fetal bovine serum + 1% P/S. [00740] Cell density: Constant at 7.5K cells/well. [00741] Antibodies: Constant at 100 nM (all labeled with AF647). Also tested different concentrations at final 25nM, 50nM, 100nM. Anti-insulin antibody, rabbit monoclonal, Sino, catalogue #11038-CR007. Anti-insulin antibody, mouse monoclonal, Genscript, catalogue #A01715 (clone 6E9F1) at ratio of labeling = about 2.9. [00742] Agents: AGN301 (constant at 50 nM). Based on results, antibody concentration at 50 nM is sufficient for internalization assay. Also tested AGN301 (constant at 370 nM). [00743] Incubation: Incubate at 37°C for eighteen hours. [00744] The inventors have also done incubation at 37°C for twenty-four hours. [00745] Readout: Internalization on High Content Imager (Operetta) [00746] When rabbit anti-insulin antibody (Sino, catalogue #11038-CR007) is used: Rabbit monoclonal antibody + AGN301 (IgG degrader) for internalization. AGN301 competes with IgG Fc-fusion protein in presence of rabbit monoclonal antibody. ABT307 and ABT308 compete with AGN301 at reduction in signal. [00747] When mouse anti-insulin antibody (Genscript, catalogue #A01715 (clone 6E9F1)) is used: Mouse monoclonal antibody + AGN301 (IgG degrader) for low internalization. AGN301 allows for complex formation of IgG Fc-fusion protein in presence of mouse monoclonal antibody. ABT307 and ABT308 binds to mouse antibody via proinsulin protein moiety. Then, AGN301 binds to Fc moiety of Fc protein at including in signal. [00748] Conclusion: Both rabbit and mouse anti-insulin antibodies recognize Fc fusion protein (via proinsulin moiety). [00749] Internalization assay: HEK293-ASGPR (clone 3F7) cells. Repeated four compounds employing AF647-labeled rabbit anti human insulin antibody on HEK293-ASGPR 3F7. [00750] AGN303 and AGN310 using AF647-labeled rabbit anti-human insulin antibody show great effect on HEK293-ASGPR 3F7. The results can be repeated compared to n = 1. [00751] Labeled Ab (100 nM). AGN301 (IgG degrader at 50 nM).
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TABLE 34 Internalization assay [ EK293+100nM CR007, HEK293-ASGPR 3 C6+100nM CR007 HepG2+100nM CR007, HEK293-ASGPR 1D4+100nM CR007 HEK293, and HepG2. TABLE 35 Internalization assay 3-ASGPR 3C6, HepG2, and HEK293-ASGPR 1D4. [00754] Conclusions: Anti-insulin agents results were as expected. Rabbit antibody (not mouse) can be used as surrogate with IgG control agent AGN301. Non-GN3 peptide (ABT306) does not elicit internalization. Anti-proinsulin agents (AGN304, AGN305) do not internalize. [00755] Subsequent actions: Assay anti-C-peptide monoclonal antibody. [00756] Time-course results. Clear internalization signal was observed for HEK293-ASGPR 3F7, 3C6, and HepG2 cells S/B for HEK293-ASGPR 3F7 and 3C6 peaked at six hours and persisted until eighteen hours. [00757] Based on the results, AGN418, AGN419, AGN420, and AGN7865 all show good effects on HEK293-ASGPR 3F7 employing CR007 and 6E9F1 but show some effects when using ABT404. [00758] Internalization: AGN310> AGN303 > AGN416. Weak signal with proinsulin constructs: AGN414, AGN415, AGN408, and AGN411. AGN310 induced strongest internalization. AGN301 did not induce internalization. [00759] Good signal with: AGN417 (proinsulin βGN3) and insulin constructs with GN3 (AGN303, AGN310, and AGN416). Weak or no signal with proinsulin N-terminal alpha constructs: AGN408 and AGN411. Weak or no signal βGN3 mutant insulin constructs: AGN412.
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TABLE 36 Internalization assay results 8 Cellular In vitro internalization assays – second results TABLE 37 Internalization assay
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TABLE 37 Internalization assay
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TABLE 37 Internalization assay
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TABLE 37 Internalization assay
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TABLE 37 Internalization assay 128 30125-WO-PCT
TABLE 37 Internalization assay
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TABLE 37 Internalization assay Ternary complex formation assay -first results. [00760] Ternary complex formation assay (Hep2) [00761] 1. Labeled two antibodies with AF647. Labelled antibody at fixed 100 nM. [00762] Anti-insulin antibody (IgG), Rabbit antibody to “T24”. Ratio of labeling = 0.49. [00763] Anti-insulin antibody (IgG), Rabbit monoclonal antibody was “CR007”. Ratio of labeling = 2.7. [00764] 2. For Ternary complex formation assay for assay development and validation, two IgG agents (AGN4006, AGN301) were tested as reference compounds with two labeled rabbit antibodies. [00765] Agents (TRAPs) tested: [00766] AGN301: IgG degrader (reference molecule). AGN301 + CR007 and AGN301 + T24. [00767] AGN4006: IgG degrader (reference molecule). AGN4006 + CR007 and AGN4006 + T24. [00768] AGN302: Mutant insulin with GN3. [00769] AGN303: Native insulin with GN3 [00770] AGN304: Native proinsulin [00771] AGN305: Mutant proinsulin [00772] ABT306: Alk-native insulin without GN3 (peptide only). [00773] Rationale. Test whether ABT307 and ABT308 (proinsulin portion) can be recognized by either rabbit or mouse anti-insulin antibodies [00774] (1) AGN301 + labeled rabbit Ab (CR007) form ternary complex formation, therefore positive signal. If add proinsulin Fc protein, will compete with AGN301, and therefore have less signal [00775] (2) AGN301 + labeled mouse 6E9F1 Ab will not form ternary complex formation, therefore no signal. If add proinsulin Fc fusion protein with above, should get signal, since AGN301 binds to Fc portion of Fc fusion protein, and labeled mouse Ab binds to proinsulin portion [00776] (3) LALA mutation on proinsulin Fc fusion protein should not interfere with IgG degrader binding, so the LALA mutation should not alter the results. [00777] Ternary Complex Formation assay conditions:
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[00778] Cell lines: HepG2 or HEK293-ASGPR (clone 3F7). [00779] Culture media: DMEM + 10% fetal bovine serum + 1% P/S. [00780] Antibodies: (all labeled with AF647). [00781] Anti-insulin antibody, rabbit monoclonal, Sino, Cat#11038-CR007. Ratio of labeling = 2.8 [00782] Anti-insulin antibody, mouse monoclonal, Genscript, Cat#A01715 (Clone 6E9F1). Ratio of labeling = about 2.9. [00783] Agents assayed: 10 µM high concentration, 3-fold dilutions, 9-point curve. In some assays, AGN301 was assayed, constant at 50 nM. [00784] Readout: Flow cytometry (IntelliCyt iQue Screener Plus). [00785] Results: AGN301 + CR007 worked well. [00786] 100 nM AGN301 with labeled CR007 is to be used as control in further assays [00787] Anti-insulin agents working as expected. [00788] Rabbit monoclonal antibody but not mouse monoclonal antibody can be used as surrogate with IgG control agent AGN301. [00789] Non-GN3 peptide (ABT306) does not elicit ternary complex formation or complex with anti- insulin antibodies. [00790] Rabbit monoclonal antibody + AGN301 (IgG degrader) showed strong ternary complex formation. ABT307 and ABT308 compete with AGN301 at reduction in signal. [00791] Mouse monoclonal antibody + AGN301 (IgG degrader) showed weak ternary complex formation. ABT307 and ABT308 binds to mouse Ab via proinsulin protein moiety. AGN301 then binds to Fc moiety of Fc protein [00792] Conclusion: Both rabbit and mouse anti-insulin antibody recognize Fc fusion protein via the proinsulin moiety. TABLE 38 T C l F ti H G2 ll
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TABLE 39 Ternary Complex Formation: HEK293-ASGPR (clone 3F7) on HepG2. [00794] AGN301 had effect with rabbit anti-human Insulin antibody but no effect with mouse anti- human insulin antibody on HepG2. [00795] AGN301 plus rabbit anti human insulin antibody had good ternary complex formation. [00796] AGN303 and AGN310 plus rabbit anti human insulin antibody had poor ternary complex formation. [00797] AGN303 and AGN310 plus mouse anti-human insulin antibody had good ternary complex formation. [00798] AGN301 plus mouse anti-human insulin antibody had poor ternary complex formation, which is consistent with mouse Ab and IgG degrader. [00799] The data were consistent with the results of internalization assay. [00800] AGN3764, AGN303, and AGN310 plus rabbit anti-human Insulin antibody had good ternary complex formation. [00801] The data consistent with the results of internalization assay. [00802] Of the compounds tested, none of the compounds showed effect using human or rabbit antibody on HEK293-ASGPR 3F7. AGN301 showed effect using rabbit antibody. [00803] Rabbit monoclonal antibody: AGN415 > AGN414 in ternary complex formation. Mouse monoclonal antibody: AGN415 > AGN414 in ternary complex formation. Both rabbit and mouse monoclonal antibodies work well with proinsulin-Fc conjugates. [00804] AGN414 and AGN415 show good effects in HEK293-ASGPR 3F7 using rabbit (CR007) and mouse antibody, which is consistent with the results of internalization assay.
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EXAMPLE 20 Ternary complex formation assays – second results. [00805] Biophysical assays. Ternary complex formation of TRAP agents was assayed using a method described above in this specification using HepG2 or HEK293 stable cells. TABLE 40 Ternary complex formation assay 133 30125-WO-PCT
TABLE 40 Ternary complex formation assay
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TABLE 40 Ternary complex formation assay 135 30125-WO-PCT
TABLE 40 Ternary complex formation assay 136 30125-WO-PCT
TABLE 40 Ternary complex formation assay
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EXAMPLE 21 Cellular In vitro internalization assays – second results [00806] Assay conditions: [00807] Cell lines: HepG2 vs. HEK293-ASGPR-high (Clone 3F7). [00808] Culture media: DMEM + 10% FBS + 1% P/S. [00809] Antibody: Antibodies all labeled with AF647. Anti-insulin antibody, rabbit monoclonal, Sino, Cat#11038-CR007 (labeled with AF647). Ratio of labeling = 2.8. Anti-insulin antibody, mouse monoclonal, Genscript, catalogue #A01715 (clone 6E9F1). Ratio of labeling = about 2.9. [00810] Agents: 10 µM high conc., 3-fold dilutions, 9-point curve [00811] Readout: High Content Imager (Operetta) [00812] Agents tested: [00813] AGN301: IgG degrader (reference molecule). [00814] AGN303: Native insulin with GN3. [00815] AGN302: Mutant insulin with GN3. [00816] ABT306: Alk-native Insulin without GN3 (just peptide). [00817] AGN304: Native proinsulin. [00818] AGN305: Mutant proinsulin. TABLE 41
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TABLE 41 AGN305 (10 µM top, 3 fold, 9 points).1% DMSO zation signal in ASGPR-transfected HEK293 cells, but not in HEPG2 cells. [00820] Anti-proinsulin degrader (AGN305) induces some internalization of anti-insulin antibody. Weak internalization with AGN304. [00821] C-peptide agents were assayed. No internalization of anti-insulin antibody, as expected, but this results shows the specificity of anti-insulin agents. [00822] AGN303 and AGN310 accompanied by mouse anti-human insulin antibody had an effect on HepG2, but these two compounds with rabbit anti-human insulin antibody have no obvious inhibitory effect on HepG2. [00823] AGN303 and AGN310 had a significant effects on HEK293-ASGPR 3F7 cells treated with mouse anti-human insulin antibody. Other data showed that rabbit anti-human insulin antibody has same effects. [00824] The effect of AGN305 with mouse anti-human insulin antibody show some weak effect as that of rabbit anti-human insulin antibody on HEK293-ASGPR 3F7. [00825] Internalization (although weak) of mouse anti-human insulin antibody was seen with AGN303 and AGN310 in HepG2 cells. [00826] Moderate internalization of mouse anti-human insulin antibody was seen with AGN303 and AGN310 in HEK293-ASGPR cells. [00827] Summary: Five compounds were tested in internalization assay employing AF647 labeled mouse anti-human insulin antibody. [00828] AGN303 and AGN310 show great effect on both HepG2 and HEK293-ASGPR 3F7. [00829] AGN301 plus rabbit anti-human insulin antibody showed great effect on HepG2.
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[00830] The compounds and proteins behave as follows: TABLE 42 Rabbit Ab HepG2 HEK293-ASGPR 3F7 Mouse Ab HepG2 HEK293-ASGPR 3F7 Agents tested in other assays [00831] High-Content Screening (HCS) is a high-throughput method used in drug discovery and cell biology that combines automated microscopy and image analysis to extract detailed cellular information. [00832] Fluorescence-Activated Cell Sorting (FACS) is a flow cytometry technique for sorting cells based on fluorescence. TABLE 44
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TABLE 44 mAb49(F AMAB- 0225WJ) Effects in HCS Effects in FACS Pharmacokinetics – first results. [00833] AGN303 (native proinsulin), synthetic Insulin degrader. Synthetic insulin MoDE has similar affinity to mAb49. AGN310 (mutant proinsulin), synthetic Insulin degrader. Synthetic insulin MoDE has similar affinity to mAb49. [00834] Insulin agents can be captured with GalNAc-binding lectin VVL and detected with an anti- insulin antibody. Proinsulin agents can be captured with an anti-insulin antibody and detected with anti- C-peptide antibody (provides higher sensitivity than VVL). [00835] Pharmacokinetics profile AGN310 in non-obese diabetic (NOD) mice (subcutaneous vs intravenous). [00836] 0.4 mg/kg AGN310 injection (~700 nM). [00837] Background is ~30 nM. Compound is cleared within one hour. [00838] Intravenous area under the curve = 170. [00839] Subcutaneous area under the curve = 131. TABLE 45 Stud desi n for NOD mice treated with anti-insulin a ents ek [00840] Urine glucose (optional) [00841] Blood glucose (once weekly) [00842] HbA1c. Keep whole blood.
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[00843] Pharmacodynamics: MesoScale Discovery/ELISA (autoantibody). Obtain serum. [00844] Blood draw for Insulin levels (MesoScale Discovery/ELISA, or LC/MS). Obtain the serum. Bank the serum to determine timepoints and readout. [00845] Blood draw for cytokines (MesoScale Discovery). Obtain the serum. Bank the serum to determine timepoints and readout. [00846] Full Biochemistry (baseline; after first dose; terminal) Obtain the serum. [00847] Full Hematology (baseline; after first dose; terminal). Keep the whole blood. [00848] Pancreatic islets (hematoxylin & eosin staining assay from formalin-fixed paraffin-embedded blocks). Save formalin-fixed paraffin-embedded blocks for follow-up staining. [00849] Pharmacokinetics: Multiple dose or single dose done. EXAMPLE 24 Pharmacokinetics – second results. [00850] Several agents (TARPs) were tested. [00851] AGN2640-P009 [00852] AGN301-P013 [00853] AGN4586-P007 Nonclinical protocol template. [00854] Pharmacokinetic evaluation of AGN417 in CD1 versus NOD mice: 1 mg/kg dosing via Intravenous or mg/kg subcutaneous. [00855] The objective was to compare the pharmacokinetic profiles of AGN417 administered at a dose of 1 mg/kg in two different mouse strains (CD1 or NOD), using intravenous or subcutaneous (SC) routes of administration. This study aims to evaluate any strain-based differences in absorption, distribution, metabolism, and elimination of AGN417, providing insights into the optimal mouse model and route of administration for future pharmacokinetic and pharmacodynamic studies. [00856] NOD mouse: NOD/ShiLtJ Strain Jackson #: 1976. [00857] CD1 female mouse: CD1 Strain CRL#: Crl:CD1(ICR). [00858] Age and weight range at start of study: Animals aged six-eight weeks with body weight of approximately 25-30 g. [00859] The mouse is a well characterized system for drug efficacy evaluation. [00860] Administration of agents [00861] Dose: 1 mg/kg or 8.8 mg/mL.
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[00862] Concentration dosing solution: 0.1 mg/ml [00863] Purity: AGN417-003 is 97.91%. [00864] Routes of administration: Intravenous or subcutaneous. [00865] Justification for route of administration: Intravenous and subcutaneous administration are acceptable routes of agent administration in small animals. [00866] Frequency and duration of dosing: See Tables 1 & 2 [00867] Administered doses: 1mg/kg intravenous or subcutaneous. [00868] Administered volume(s): 10 ml/kg. [00869] Assay design: Upon arrival at the facility, animals are acclimated for 7 days. On day 0, animals is weighed and randomly assigned to groups. All groups of animals receive their treatment of 1 mg/kg AGN417, as described in the TABLE below, either intravenously or subcutaneously. After a defined period, three animals from each group will undergo blood collection at two time points: the first time point involves collecting around 100 µl of blood from the submandibular vein, and the second will involve cardiac exsanguination following carbon dioxide asphyxiation. Blood samples from individual animals is collected in K3EDTA tubes, and plasma is separated. All samples are stored at -80°C until further analysis. [00870] Non-fasting blood glucose levels is monitored by collecting a small blood drop from a tail nick at the tip of the tail. The blood is applied to a One-Touch test strip and measured using a One-Touch Ultra 2 Glucometer in three mice per group at the following time points: pre-dose, five minutes, ten minutes, fifteen minutes, thirty minutes, one hour, and two hours post-dose. TABLE 46 . After dosing, weights is recorded daily.
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[00872] Tissue harvesting and sample analysis. Blood is harvested into K3EDTA containing tubes. Blood in plasma separator tubes is separated by spinning samples at 9 G at 4^C for 15 minutes. Plasma is collected and analyzed with our in-house MSD. Blood glucose levels is measured after a tail nick using One-Touch Ultra 2 Glucometer. [00873] Clinical observations/signs. During quarantine and the study period, a minimum of once daily cage-side or detailed observations and morbidity/mortality checks is performed on all animals by study personnel or a member of the animal care staff. Observations include but are not limited to the following: reactivity to general stimuli and description of any abnormal behaviors, lesions, or appearances, if applicable. [00874] Data analysis and reporting. Plasma levels of AGN417 are determined by in-house developed MSD. Data are presented in tabular form. A standard pharmacokinetic analysis is to be performed for each dose group. EXAMPLE 25 Pharmacodynamics. [00875] Study design: 13.3 nmol/kg anti-insulin autoantibodies – 6E9F1 intravenously (2 mg/kg).133 nmol/kg AGN310 intravenously – 1:10 ratio. [00876] An assay in mice showed a 39% depletion by AGN310, based on area under the curve. [00877] The inventors performed a pharmacodynamics evaluation of AGN310 in nude mice. Nude mouse plasma causes no interference in antibody detection. [00878] Degrader interference could be prevented by diluting samples to below 10 ng/mL antibody. EXAMPLE 26 Pharmacodynamics. NOD mouse insulin inhibition. TABLE 47 l f i
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TABLE 47 Rodent models for Type 1 diabetes o -o ese a e c ce we e assaye o u e s a e e ec o gucose eves. The assay used female seventeen weeks old NOD mice. The mice were checked for glucose level (>115 mg/dL). The assayed groups each group had five mice with glucose levels = 300 – 500 mg/dL and five mice with glucose levels = 115 – 300 mg/dL. The mice were regularly checked at day 0 and day 5. [00880] TRAP agents deplete naturally occurring insulin autoantibodies in non-obese diabetic (NOD) mice. Two TRAP agents were administered to NOD mice to assay NOD mouse insulin inhibition in vivo. AGN310 and AGN417 were administered, each both intravenously and subcutaneously. Along with this administration, NOD mice were assayed for body weight and glucose levels (PharmaLegacy). Body weight taken at each dosing, every five days. Non-fasting blood glucose taken every two weeks via tail vein bleed (9-10 AM). TABLE 48 i l l k i 145 30125-WO-PCT
TABLE 48 NOD mice IgG calculated based on background subtraction tibodies in NOD mice as they age. EXAMPLE 27 Chemical synthesis of proinsulin degrader [00882] Peptide 1. (1) Soft cleavage (leave protecting groups) except cysteine (trt). (2) Perform disulfide bond formation. (3) Orthogonal cysteine protection. [00883] Peptide 2. (1) Soft cleavage.(2) N-terminal Fmoc. (3) Fragment condensation (peptide 1 + peptide 2). [00884] Peptide 3. (1) Soft cleavage. (2) N-terminal Fmoc during condensation (peptide 1/2 + peptide 3). (3) Fmoc-deprotection and global deprotection, except Cys-Acm. [00885] Peptide 4. (1) C-terminal thioetser (Dbz resin). (2) In-solution. Coupling of sugar might be needed. (3) Deprotected except cysteine (Acm). (4) Perform NCL. [00886] See Dawson et al., Science, 266, 776-779 (1994). EXAMPLE 28 Tolerability in vivo in NOD mice. [00887] Fit for purpose tolerability assays were run with single intravenous 3 mg/kg dose to match concentration used in the PharmaLegacy assay. [00888] AGN303 (native proinsulin) and AGN310 (mutant proinsulin) Insulin agents were well- tolerated when administered at 3 mg/kg intravenously in pre-diabetic NOD mice, with no observed adverse events and no changes in glycemia when measured at twelve hours-post dose.
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EXAMPLE 29 AGN310 extended tolerability in NOD female mice. [00889] The objective of this EXAMPLE was to investigate the longer-term effects of multi-dosing IV AGN310 on NOD female mice. TABLE 49 Gr Nmbr Tr tmnt D Adminitrtin Bd Vlm of TABLE 51 Timlin f t,
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TABLE 51 Timeline [00890] Body weight: Weekly [00891] Blood glucose and urine glucose: Weekly pre-dose and post-dose. [00892] Behavioral assessments: Weekly for 20 minutes and side check at end of week. [00893] Biochemical assays: Blood glucose, Insulin. [00894] Histological analysis: Of tissues collected. EXAMPLE 30 Tolerability assay. [00895] Also assaying blood glucose levels (BGL). Intravenous administration resulted in a rapid and pronounced change in blood glucose levels, with an initial sharp decrease followed by a peak, indicating that intravenous administration may cause more dramatic blood glucose fluctuations due to faster absorption and clearance. Subcutaneous administration led to more stable blood glucose levels with less extreme fluctuations compared to intravenous administration. TABLE 52 h
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TABLE 52 Dose Mouse BW BGL Pre-dose BGL 5m BGL 15m BGL 30m BGL 1h BGL 1:30h BGL 2h One-week assay of AGN417 by subcutaneous Injection and intravenous infusion in Wistar Han IGS rats [00896] The objective of this EXAMPLE is to determine the potential toxicity of AGN417 given every once every 3 days via subcutaneous injection or intravenous injection for one week to Wistar Han IGS rats (available from Charles River Laboratories). The toxicokinetic characteristics of AGN417 are to be determined. [00897] The dose levels can be selected based on information provided in the specification above. Tolerability data associated with AGN417 is limited. In a previous pharmacokinetic study mice given AGN417, intravenous and subcutaneous doses up to 3 mg/kg were well-tolerated, but exposure correlated with transient reduction in blood glucose levels of approximately 50 mg/dL. At no point during pharmacokinetic studies did blood glucose fall below reference range intervals. Blood glucose levels returned to vehicle control animal levels when food was provided. The current study will evaluate the dose-response relationship of AGN417 in rats. The low dose of 15 mg/kg is a low multiple of previous doses in mice, and the mid and high doses of 50 and 150 mg/kg study will explore tolerability and attempt to build a dose-response relationship. The subcutaneous dose of 50 mg/kg is intended to understand the subcutaneous bioavailability and pharmacokinetic profile relative to intravenous administration. [00898] The Wistar Han IGS rat was chosen as the animal model for this assay because it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies. [00899] Assays in laboratory animals provide a basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
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TABLE 53 Preparation Details t t pH and osmolality to ensure matched and at physiologically appropriate levels. [00901] All dosing samples are stored on wet ice at the testing facility, before dosing. Collected samples stored at a temperature set to maintain -60-90°C. Dosing samples are evaluated for pH and osmolarity prior to dosing, All dose solutions are normalized to physiological osmolarity where possible, using sodium chloride. [00902] Animal Identification. Subcutaneously implanted electronic identification chip or other approved identification method such as indelible ink where required. [00903] Environmental Acclimation. A minimum acclimation period of four days is allowed between animal arrival and the start of treatment to accustom the animals to the laboratory environment. [00904] Selection and assignment of animals: Animals are randomly assigned to groups. Males and females is randomized separately. Animals in poor health or at extremes of body weight range will not be assigned to groups. Substitutions may be made after initial group assignment based on evaluation of data from individual animals collected before dosing. [00905] Replacement of animals: Before the initiation of dosing, any assigned animals considered unsuitable for use in the study are replaced by alternate animals. After initiation of dosing, study animals may be replaced during the replacement period with alternate animals in the event of accidental injury, non-test article-related health issues, or similar circumstances. The alternate animals may be used as replacements on the study within one to three days. [00906] The housing set-up is as specified in the United States Department of Agriculture Animal Welfare Act (9 C.F.R., Parts 1, 2 and 3) and as described in the Guide for the Care and Use of Laboratory Animals (National Research Council, Current edition; Office of Laboratory Animal Welfare, Current edition). Animals are separated during designated procedures/activities or separated as required for monitoring and/or health purposes, as deemed appropriate by Study Director and/or staff veterinarian.
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Animals may be individually housed due to incompatible behavior with a cage mates. Cages are arranged in group order. Control group animals are housed on a separate rack from the test article- treated animals. TABLE 54 Experimental design st TABLE 55 General in-life assessments r r r
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TABLE 55 General in-life assessments e Clinical pathology sample collection s m e
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TABLE 57 Hematology parameters Coagulation parameters Clinical chemistry parameters TABLE 60 Ui l i
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[00907] Bone marrow smear evaluation. Bone marrow smears are collected and prepared. Evaluation of stained smears may be performed. TABLE 61 Bioanalytical Sample Collection, Target Time Post-dose on Days 1 and 7 r ical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations, accuracy, and precision are performed. [00909] Toxicokinetic evaluation. TK parameters are generated using the concentration units provided by the bioanalytical lab. Concentration values below the limit of quantitation are treated as zero for the purposes of TK data analysis. TABLE 62 P b i d
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TABLE 62 Parameters to be estimated a r Secondary parameter abbreviations nly se. d. se values, may be derived and reported to aid interpretation. Descriptive statistics (e.g., number, arithmetic mean, median, standard deviation, standard error, coefficient of variation) are reported as deemed appropriate. Ratios for appropriate grouping and sorting variables may be generated when appropriate.
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TABLE 64 Ratio Description of Ratio s er appropriate samples obtained. Main animals are euthanized by exsanguination under deep isoflurane anesthesia (preferred) or by carbon dioxide asphyxiation. Main animals will undergo necropsy, and specified tissues are retained. If necessary, any animal found dead or unscheduled euthanasia the animal is refrigerated until necropsy to minimize autolysis. [00912] Scheduled euthanasia. Main animals surviving until scheduled euthanasia will have terminal body weights taken prior to euthanasia. At time of termination, appropriate samples are collected from the abdominal aorta under isoflurane anesthesia. Animals will then be euthanized by exsanguination under deep isoflurane anesthesia. When possible, the animals are euthanized rotating across dose groups such that similar numbers of animals from each group, including controls, are necropsied throughout the day. [00913] Necropsy. Animals are subjected to a complete necropsy examination, which includes evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. [00914] Organ weights. Organs are weighed at necropsy. Paired organs are weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair may be taken and entered as a tissue comment. Organ weight as a percent of body weight (using the terminal body weight) and organ weight as a percent of brain weight is calculated. [00915] Tissue collection and preservation. Representative samples of tissues are collected and preserved in 10% neutral buffered formalin, except for tissues requiring alternate fixatives as defined by standard operating procedures. [00916] Histology. Tissues are processed. Glass slides may be scanned at 20x/40x. [00917] Microscopic evaluation. Tissues are evaluated histopathologically by light microscopy or digitally using whole slide images (WSI). Special stains may be used at the discretion of the pathologist
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to further characterize lesions and changes identified during routine evaluation of individual animals. Any special stains are documented in the individual animal data. Any additional stains or evaluations, if deemed necessary by the pathologist, may be added. [00918] Statistical analysis. Any data collected during the pre-dose period are tabulated, summarized, or statistically analyzed. All statistical analyses are performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions are summarized and statistically analyzed as indicated below according to sex and occasion. [00919] Descriptive statistical analyses. Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), ratio, percentages, numbers, or incidences are reported as appropriate by dataset. [00920] Inferential statistical methods. All statistical tests are conducted at the 5% significance level. All pairwise comparisons are conducted using two sided tests and reported at the 1% and 5% levels, unless otherwise noted. Equivalent tests may be reported depending on the terminology defined in the software used in the statistical analysis. TABLE 65 Statistical matrix c variances. The groups are compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons are conducted using Dunnett’s or Dunn’s test, respectively. [00922] Non-parametric. The groups are compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the pairwise comparisons are conducted using Dunn’s test (equivalent to Wilcoxon Rank-Sum test in Nevis 2012 tables).
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[00923] Incidence. A Fisher’s exact test is used to conduct pairwise group comparisons of interest. [00924] This assay should comply with all applicable sections of the Final Rules of the Animal Welfare Act regulations (Code of Federal Regulations, Title 9), the Public Health Service Policy on Humane Care and Use of Laboratory Animals from the Office of Laboratory Animal Welfare, and the Guide for the Care and Use of Laboratory Animals from the National Research Council. [00925] If any animal is determined to be in overt pain/distress or appears moribund and is beyond the point where recovery appears reasonable, the animal is euthanized for humane reasons in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. EXAMPLE 32 Primate assay [00926] Ageing chimpanzees develop aging-related illnesses and thus provide a good model for aging-related diseases associated with increased anti-insulin autoantibodies or ant-proinsulin autoantibodies. EXAMPLE 33 Guidance for starting dose and human dose. [00927] The inventors’ rationale for starting dose is guided by the United States Food & Drug Administration Guidance for Industry: Estimating the Maximum Safe Starting Dose (2005) and any subsequent updates. The maximum recommended starting dose (MRSD) for first-in-human (FIH) clinical trials of new molecular entities in adult healthy volunteers based on human equivalent dose (HED) based on the no observed effect level (NOEAL) in the most sensitive species. [00928] The inventors’ rationale for starting dose is guided by the EMA Guideline on strategies to identify and mitigate risks for first-in-humans (FIH) and early clinical trials (2017) and any subsequent updates. [00929] The starting dose is expected to be the lower of the following values: (1) NOAEL in the most sensitive species adjusted for HED, (2) minimal anticipated biological effect level (MABEL), or (3) pharmacologically active dose (PAD). [00930] The human dose projections for the insulin TRAP proinsulin degrader or the proinsulin TRAP degrader program is expected to further depend on the preclinical data, the in-vitro data, and the
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understanding of the pharmacokinetics/pharmacodynamics relationship, some of which is disclosed in the EXAMPLES above. [00931] Model independent HED-type of approach to determine MRSD and scale effective animal model exposures/doses to human. Scaling using body weight-based approach known to persons having ordinary skill in the biomedical arts from preclinical species using standard allometric principles. [00932] Model dependent approach. Leverage animal pharmacokinetics models that can be scaled to humans and use information about pharmacokinetics/pharmacodynamics relationship to understand effective exposure and perform translational simulation. [00933] Given the potential agonistic activity, i.e., hypoglycemia, a pharmacologically active dose (PAD) approach can be informed by either dose-response, exposure-response and/or pharmacokinetics/pharmacodynamics modeling using mouse, rat, or non-human primate data. [00934] Quantitative systems pharmacology approaches can also be evaluated and may be beneficial for further clinical development. EQUIVALENTS [00935] Persons having ordinary skill in the biomedical art will recognize or be able to determine using no more than routine experimentation many equivalents to the specific procedures described in this specification. Such equivalents are within the scope of this invention and are covered by these claims. For example, pharmaceutically acceptable salts other than those specifically disclosed in the description and Examples in this specification can be employed. Furthermore, it is intended that specific items within lists of items, or subset groups of items within larger groups of items, can be combined with other specific items, subset groups of items or larger groups of items whether there is a specific disclosure in this specification identifying this combination. [00936] Some embodiments of the invention can be practiced according to the following numbered paragraphs: [00937] 1. A composition of matter comprising: a binding moiety, a cellular receptor binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on surface degrading cells, and a linker moiety (optionally a single peptide linkage) connecting the binding moiety and the cellular receptor binding moiety. [00938] 2. The composition of matter of Embodiment 1, wherein the binding moiety is an antigen- binding protein, an antigen-binding protein variant, or an antigen-binding fragment thereof.
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[00939] 3. The composition of matter of Embodiment 1, wherein the binding moiety is an insulin, an insulin variant, an insulin analogue, or an insulin fragment thereof. [00940] 4. The composition of matter of Embodiment 1, wherein the binding moiety is a proinsulin, a proinsulin variant, a proinsulin analogue, or a proinsulin fragment thereof. [00941] 5. The composition of matter of Embodiment 1, wherein the binding moiety is an insulin Fc fusion protein. [00942] 6. The composition of matter of Embodiment 1, wherein the binding moiety is a proinsulin Fc fusion protein. [00943] 7. The composition of matter of Embodiment 1, having a structure of: RCN−(Xaa)y−RCC, [AGN103], or [AGN104] or a pharmaceutically acceptable salt thereof. [00944] 8 f matter of Embodiment 1, wherein the agent has the structure of formula [AGN102] , or a pharmaceutically acceptable salt thereof, wherein: each of a and b is indepen 1 or greater; each AT is a binding moiety or a fragment thereof; L is a linker moiety; and each TBT is independently a cellular receptor binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, wherein the binding moiety is a ligand variant or an antigen-binding fragment thereof. [00945] 9. The composition of matter of Embodiment 1, wherein the agent has the structure of formula a salt thereof, wherein the composition of matter has add n.
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[00946] 10. The composition of matter of Embodiment 1, wherein the agent has the structure of formula r a salt thereof, wherein the composition of matter ation. [00947] 16. The composition of matter of Embodiment 15, wherein the cellular receptor binding moiety has the following structure: ; o) 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) where IM and RM are the same as above; and ZB is absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)IM-NRM, where IM and RM are the same as above. [00950] 17. The composition of matter of Embodiment 16, wherein RA is a methyl or ethyl group optionally substituted with from 1-3 fluoro groups. [00951] 18. The composition of matter of Embodiment 17, wherein ZA is a PEG group containing from 1 to 4 ethylene glycol residues. [00952] 19. The composition of matter of Embodiment 18, wherein the methyl or ethyl group is substituted with from 1-3 fluoro groups. [00953] 20. The composition of matter of Embodiment 17, wherein the ASGPR binding group is N- acetyl-D-galactosamine.
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[00954] 21. The composition of matter of Embodiment 1, wherein the cellular receptor binding moiety is a low-density lipoprotein receptor-related protein 1 (LRP1), a low-density lipoprotein receptor (LDLR), a Fcγ receptor I-binding group, a FcRn binding group, a transferrin receptor binding group, or a macrophage scavenger receptor binding group. [00955] 22. A pharmaceutical composition comprising composition of matter of any of the preceding claims and a pharmaceutically acceptable excipient. [00956] 23. The composition of matter of Embodiment 1, wherein the binding moiety is a ligand variant or an antigen-binding fragment thereof. [00957] 24. The composition of matter of Embodiment 1, wherein the agent has the structure of formula [AGN102] , or a pharmaceutically acceptable salt thereof, wherein: each of a and b is indepen 1 or greater; each AT is a binding moiety; L is a linker moiety; and each TBT is independently a cellular receptor binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject. [00958] 25. The composition of matter of Embodiment 24, wherein a is 1 and b is 3. [00959] 26. The composition of matter of Embodiment 25, wherein binding moiety comprises a peptide moiety that binds to a specific amino acid residue of the glycan-specific IgG antibody. [00960] 27. A composition comprising: a first composition of matter comprising: an antibody moiety, a cellular receptor binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, and a linker moiety linking the antibody moiety and the cellular receptor binding moiety, and at 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. [00961] 28. The composition of Embodiment 27, wherein the at least one additional agent has the structure of formula AGN302, MAT302, or a combination thereof: LG−RG−LRM(−TBT)b, [AGN302]; and LG−RG−H [MAT302]. [00962] 29. The composition of Embodiment 43, wherein AT is an anti-autoantibody moiety, TBT is N-acetylgalactosamine (GalNAc), a is 1, and b is 3. [00963] 30. A method of removing in a patient or subject in need comprising administering to the mammal the agent of any of Embodiment 1-29.
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[00964] 31. A method of treating a disease state and/or condition associated with the upregulation of in a patient or subject in need administering to the mammal an effective amount of the agent of any of Embodiment 1-29. [00965] 32. A method of treating IMN in a patient in need comprising administering to the mammal an effective amount of the agent of any of Embodiment 1-29. REFERENCES [00966] Persons having ordinary skill in the biomedical art can use these patents, patent applications, and scientific references as guidance to predictable results when making and using the invention. Patent literature [00967] U.S. Pat. No.7,083,784 (Dall'Acqua et al.), Molecules with extended half-lives, compositions and uses thereof. [00968] U.S. Pat. No.7,658,921 (Dall'Acqua et al.), Molecules with extended half-lives, compositions and uses thereof. [00969] U.S. Pat. No.8,088,376 (Chamberlain et al.), Fc variants with altered binding to FcRn. [00970] U.S. Pat. No.8,969,526 (Baehner), Antibody Fc variants. [00971] U.S. Pat. No.9,562,100 (Dall'Acqua et al.), Molecules with extended half-lives, compositions and uses thereof. [00972] U.S. Pat. Publ.2019/0111149 (Gardiner et al.). [00973] European Pat. No. EP1355919 (MedImmune LLC et al.), Molecules with extended half-lives, compositions and uses thereof. [00974] Intl. Pat. Publ. WO 2002/060919 (MedImmune LLC et al.), Molecules with extended half- lives, compositions and uses thereof. [00975] Intl. Pat. Publ. WO 2012/017021 (Graffinity Pharmaceuticals GmbH), Ligands for antibody and Fc-fusion protein purification by affinity chromatography. [00976] Intl. Pat. Publ. WO 2017/125885 (Biocon Limited), Bio-analytical method for insulin analogues (2017) [00977] Intl. Pat. Publ. WO 2019/023501 (Kleo Pharmaceuticals, Inc.). Universal ABT compounds and uses thereof. [00978] Intl. Pat. Publ. WO 2019/136442 (Kleo Pharmaceuticals, Inc.). Cd16a binding agents and uses thereof.
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[00979] Intl. Pat. Publ. WO 2019/199621 (Yale University). Bi-functional molecules to degrade circulating proteins. [00980] Intl. Pat. Publ. WO 2019/199634 (Yale University). Bifunctional small molecules to target the selective degradation of circulating proteins. [00981] Intl. Pat. Publ. WO 2021/102052 (Kleo Pharmaceuticals). Directed conjugation technologies. [00982] Intl. Pat. Publ. WO 2025/035052 (Biohaven Therapeutics Ltd.). Molecular agents for the treatment of IgA nephropathy. Non-patent literature [00983] Abhinandan & Martin, Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Molecular Immunology, 45(14), 3832–3839 (2008)., providing the Martin (enhanced Chothia) CDR definitions and numbering used for biologics. [00984] Acqua et al., Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences. The Journal of Immunology, 169(9), 5171-5180 (2002). [00985] Aguirre, et.al., Insulin-like growth factor 1 in the cardiovascular system. Rev. Physiol. Biochem. Pharmacol., Vol.175, pages 1-45 (2018). This publication shows the schematic structure of insulin, IGFs, and their receptors. Resemblances between insulin and IGFs allow them to cross-interact with each other’s receptors. IGF2 can also interact with IGF1R, hybrid receptors, and the insulin receptor, with a lower affinity. IGF1 can also bind to IGF2R, albeit with less affinity to that of its putative receptor. [00986] Akashi et al., Inter. Immu. (1997). In NOD mouse model, insulin autoreactive B cells induce diabetes, while B cell deficiency or depletion protects from Type 1 diabetes and allows for normoglycemia. [00987] Al-Lazikani, Lesk, & Chothia, C. (1997). Standard conformations for the canonical structures of immunoglobulins. Journal of Molecular Biology, 273(4), 927–948. The Chothia CDR definitions and numbering vary from publication to publication, and to also vary within the figures of papers themselves. For consistency, persons having ordinary skill in the biomedical can use the definitions described in this 1997 publication. [00988] Alleva et al., An antigen-specific immunotherapeutic, AKS-107, deletes insulin-specific B cells and prevents murine autoimmune diabetes. Frontiers in Immunology, 15, 1367514 (2024). In NOD mouse model, insulin autoreactive B cells induce diabetes, while B cell deficiency or depletion protects from Type 1 diabetes and allows for normoglycemia.
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[00989] Alves, Kiziltepe, & Bilgicer, Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection. Langmuir, 28, 9640−9648 (2012). [00990] Asadi, Bruin, & Kieffer, Characterization of antibodies to products of proinsulin processing using immunofluorescence staining of pancreas in multiple species. Journal of Histochemistry & Cytochemistry, 63(8), 646–662 (2015). [00991] Belfiore et al., Insulin receptor isoforms in physiology and disease: An updated view. Endocrine Reviews, Volume 38, Issue 5, 1 pages 379–431 (October 2017), describing insulin receptor affinities. [00992] Beneit et al., Expression of insulin receptor (IR) A and B isoforms, IGF-IR, and IR/IGF-IR hybrid receptors in vascular smooth muscle cells and their role in cell migration in atherosclerosis. Cardiovasc. Diabetol., 15, 161 (2016). [00993] Berman et al., Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus. EMBO J., 7, 727 (1988). [00994] Berry et al., J. Visu. Exp. (2013). As demonstrated that adoptive transfer of T cells from diabetic NOD mice can accelerate Type 1 diabetes in immunocompromised non-diabetic NOD.SCID mice. [00995] Böhmer et al., Proinsulin autoantibodies are more closely associated with type 1 (insulin- dependent) diabetes mellitus than insulin autoantibodies. Diabetologia, 34, 830-4 (November 1991). Prevalence of anti-Insulin autoantibodies (IAAs) and anti-proinsulin autoantibodies (PAAs) in newly diagnosed Type 1 diabetes patients versus controls. Anti-proinsulin autoantibodies are also prevalent in newly diagnosed T1D patients. [00996] Brandt et al., Controlled intramolecular antagonism as a regulator of insulin receptor maximal activity. Peptides 100, 18–23 (2018) (Novo Nordisk). [00997] Bright et al., Stability of proinsulin in whole blood. Clinical Biochemistry, 52,153-5 (February 1, 2018). The aim was to assess the stability of proinsulin across a wide concentration range (3–882 and 2–187 pmol/L; total and intact proinsulin respectively) in whole blood to determine whether it could be used in routine clinical care .The samples were kept at room temperature (~20°C) with aliquots taken, centrifuged and frozen at 0, 24, 48, and 72 hours. Based on stability, one can assume plasma stability studies should be more than 72 hours. [00998] Burrack, Martinov, & Fife, T cell-mediated beta cell destruction: Autoimmunity and alloimmunity in the context of Type 1 diabetes. Front Endocrinol (Lausanne), 8, 343 (December 5, 2017). Type 1 diabetes results from destruction of pancreatic β cells by T cells of the immune system. Despite improvements in insulin analogs and continuous blood glucose level monitoring, there is no cure for
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Type 1 diabetes, and some individuals develop life-threatening complications. Pancreas and islet transplantation have been attractive therapeutic approaches. Transplants containing insulin-producing cells are vulnerable to both recurrent autoimmunity and conventional allograft rejection. Current immune suppression treatments subdue the immune system, but not without complications. Ideally a successful approach would target only the destructive immune cells and leave the remaining immune system intact to fight foreign pathogens. This review discusses the autoimmune diabetes disease process, diabetic complications that warrant a transplant, and alloimmunity. [00999] Casali et al., Frequency of B cells committed to the production of antibodies to insulin in newly diagnosed patients with insulin-dependent diabetes mellitus and generation of high affinity human monoclonal IgG to insulin. Journal of immunology (Baltimore, Md.: 1950), 144(10), 3741-7 (May 15, 1990). In Type 1 diabetes, B cells committed to insulin autoantibody production are more frequent than in non-diabetic children. [001000] Chen et.al., Effects of TCM on polycystic ovary syndrome and its cellular endocrine mechanism. Frontiers in Endocrinology, 14, 956772 (May 16, 2023). [001001] Choe, Durgannavar, & Chung, Fc-binding ligands of immunoglobulin G: An overview of high affinity proteins and peptides. Materials, 9(12) (2016). [001002] Christianson, Shultz, & Leiter, Adoptive transfer of diabetes into immunodeficient NOD- scid/scid mice: relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD. NON-Thy-1a donors. Diabetes, 42(1),.44-55 (1993). As demonstrated that adoptive transfer of T cells from diabetic NOD mice can accelerate Type 1 diabetes in immunocompromised non-diabetic NOD.SCID mice. [001003] Clark et al., Assays for insulin, proinsulin(s) and C-peptide (1999). This publication describes various methodologies to detect proinsulin (i.e. immunoassays), so maybe not relevant here. [001004] Cobb et al., A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in rhesus macaques. bioRxiv, 2021-09 (2021). [001005] Cong et al., MicroRNA-218 promotes prostaglandin E2 to inhibit osteogenic differentiation in synovial mesenchymal stem cells by targeting 15-hydroxyprostaglandin dehydrogenase [NAD(+)]. Mol. Med. Rep.16, 9347-9354 (2017). [001006] DeLano et al., Convergent solutions to binding at a protein–protein interface. Science, 287, 1279-1283 (2000). [001007] DiMeglio et al., Type 1 diabetes. Lancet, 391(10138), 2449-2462 (June 16, 2018)
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[001008] Dludla et al., Pancreatic β-cell dysfunction in type 2 diabetes: Implications of inflammation and oxidative stress. World J Diabetes, 14(3),130-146 (March 15, 2023). [001009] Elso et al., Replacing murine insulin 1 with human insulin protects NOD mice from diabetes. PLoS One, 14(12), e0225021 (December 10, 2019). [001010] Fang et al., Ann. Intern. Med. (2023). Prevalence and management of obesity in US adults with type 1 diabetes. Annals of Internal Medicine.176(3), 427-9 (March 2023). [001011] Fathallah et al., Effects of hypertonic buffer composition on lymph node uptake and bioavailability of rituximab, after subcutaneous administration. Biopharm. Drug Dispos., 36(2), 115-25 (March 2015). Illustration of the proposed physiological changes in the subcutaneous space in response to hypertonic buffers. Left side represents normal physiological conditions. Oncotic/osmotic forces in the subcutaneous space favor blood filtration at the arteriolar end of the capillary bed, as blood flows to the venular end of the capillary bed, oncotic forces within the blood capillaries favors reabsorption of filtrate. Excess fluid is taken up by the initial lymphatic to maintain fluid homeostasis. Right side represents the proposed changes to this process in response to hypertonic buffers. The increased interstitial osmolarity will result in increased blood filtration at the arteriolar end while hindering reabsorption at the venular end. The excess volume is removed by the initial lymphatic to maintain fluid homeostasis in the intestinal space. [001012] Foreman et al., LC-MS/MS based detection of circulating proinsulin derived peptides in patients with altered pancreatic beta cell function. MS Based Analysis of Circulating Proinsulin Derived Peptides: New Opportunities for Precision Diagnosis and Management of Diabetes. (2022). This is a paper on hyperosmolar, i.e., hypertonic, solutions driving subcutaneous bioavailability. Established LC- MS/MS assays for insulin-related peptides. See Table 1. The authors spiked human plasma with insulin and C-peptide for method development at 50-15,000 pg/mL. The proinsulin reference solution (70 ng/mL) was used to confirm retention time. [001013] Frommer & Kahaly, Type 1 diabetes and associated autoimmune diseases. World Journal of Diabetes, 11(11), 527-539 (November 15, 2020). Autoantibodies against several β-cell proteins are identified in over 90% in newly diagnosed type 1 diabetes. Most prominent autoantibodies measured in children that progress to Type 1 diabetes (TEDDY study, n=100 children); virtually no insulin autoantibodies in normal controls. [001014] Galal et al., Insulin Receptor isoforms and insulin growth factor-like receptors: Implications in cell signaling, carcinogenesis, and chemoresistance. International Journal of Molecular Sciences, 24(19), 15006 (October 9, 2023).
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[001015] Goy et al., Development and validation of an LC-MS/MS based quantitative assay for marmoset insulin in serum (2022). The marmoset insulin peptide is not homologous with human insulin and therefore commonly available assays do not work for this species. This study aimed to develop and validate a bottom-up proteomic workflow with trypsin digestion and analysis using LC coupled with triple quadrupole mass spectrometry (LC-MS/MS). [001016] Griffeth, Bianda, and Nef, The emerging role of insulin-like growth factors in testis development and function. Basic & Clinical Andrology, 24, 1-10 (December 2014). This publication shows the receptors for insulin, IGF1 and IGF2. INSR and IGF1R are composed of two αβ dimers which associate to form heterotetrameric complexes. The αβ dimers are linked together by disulfide bonds and two dimers are also linked by disulfide bonds to form the tetramer. The α subunit is the extracellular portion of the receptor while the β subunit spans the membrane and its cytoplasmic portion interacts with IRS proteins which are key intracellular mediators of insulin/IGF signaling. Single αβ dimers are derived from separate genes and the INSR has two splice variants, INSR-B and INSR-A. Each variant shares the same membrane-spanning β subunit (dark blue) but differs in the extracellular α subunit (light pink or dark pink, respectively). The IGF1R has different α and β subunits compared to the INSR (dark green). These combinations of αβ dimers allow for hybrid receptors, which bind insulin, IGF1, and IGF2 with differing affinities. [001017] Gu et.al., Islet autoantibody detection by electrochemiluminescence (ECL) assay, J. Clin. Cellular Immunology, 8(6):1000531 (2017). [001018] Gupta et al., Computationally designed antibody–drug conjugates self-assembled via affinity ligands. Nature Biomedical Engineering, 3, 917–929 (2019). [001019] Haskins & McDuffie, Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone. Science, 249(4975), 1433-1436 (1990). As demonstrated that adoptive transfer of T cells from diabetic NOD mice can accelerate Type 1 diabetes in immunocompromised non-diabetic NOD.SCID mice. [001020] Hilgert et al., A monoclonal antibody applicable for determination of C-peptide of human proinsulin by RIA. Hybridoma 10(3) 379-386 (1991). [001021] Hinman et al., Curr. Diab. Rep. (2014). Immune cell infiltration into pancreatic islets involves mostly CD4+ & cytotoxic CD8+ T cells (leading to antigen spreading), but growing evidence of B cell infiltration & role in Type 1 diabetes pathogenesis. [001022] Hu et al., Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor–related protein 1–mediated integrin signaling. The Journal of Clinical
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Investigation, 117(12), 3821-3832. (2007). Adoptive transfer of plasmablasts also accelerate diabetes in non-diabetic animals, suggesting their function as APCs & promoting proinflammatory T cell response. [001023] Ikematsu et al., VH and Vκ segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus: Evidence for somatic selection. Journal of immunology (Baltimore, Md.: 1950) 152(3), 1430-1441 (February 1, 1994). The mAb49 antibody and hybridoma were described in this scientific publication back. [001024] Jorns et.al., Islet infiltration, cytokine expression and beta cell death in the NOD mouse, BB rat, Komeda rat, LEW.1AR1-iddm rat and humans with type 1 diabetes. Diabetologia, Volume 57, pages 512–521 (December 6, 2014). [001025] Kabat et al., Sequences of Proteins of Immunological Interest. (U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, 1991), providing the Kabat CDR definitions and numbering used for biologics. [001026] Kruljec et al., Alternative affinity ligands for immunoglobulins. Bioconjugate Chem., 28(8): 2009-2030 (2017). [001027] Kruljec et al., Development and characterization of peptide ligands of immunoglobulin G Fc region. Bioconjugate Chem., 29(8), 2763-2775 (2018). [001028] Kuglin et al., Proinsulin autoantibodies: association with type I diabetes but not with islet cell antibodies, insulin autoantibodies or HLA-DR type. J. Autoimmun., 3(5), 573-7 (October 1, 1990). Anti- proinsulin autoantibodies are also prevalent in newly diagnosed Type 1 diabetes patients. [001029] Kuglin, Gries, & Kolb, Evidence of IgG autoantibodies against human proinsulin in patients with IDDM before insulin treatment. Diabetes, 37(1), 130-2 (1988). Prevalence of anti-insulin autoantibodies (IAAs) and anti-proinsulin autoantibodies (PAAs) in newly diagnosed Type 1 diabetes patients versus controls. Anti-proinsulin autoantibodies are also prevalent in newly diagnosed Type 1 diabetes patients. [001030] Larsson et al., TEDDY Study Group. Children followed in the TEDDY study are diagnosed with type 1 diabetes at an early stage of disease. Pediatr. Diabetes, 15(2):118-26 (March 2013). First positive autoantibodies (TEDDY study). Most prominent autoantibodies measured in children that progress to Type 1 diabetes (TEDDY study, n=100 children); virtually no insulin autoantibodies in normal controls. [001031] Leete et al., Frontiers in Immunology (2021). Immune cell infiltration into pancreatic islets involves mostly CD4+ & cytotoxic CD8+ T cells, leading to antigen spreading, but growing evidence of B cell infiltration & role in Type 1 diabetes pathogenesis.
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[001032] Lefrancet al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental & Comparative Immunology, 27(1), 55–77 (2003), providing the IMGT CDR definitions and numbering used for biologics. [001033] Ling et al., Increased plasmablasts enhance T cell-mediated beta cell destruction and promote the development of type 1 diabetes. Molecular Medicine, 28(1), 18 (2022) Adoptive transfer of plasmablasts also accelerate diabetes in non-diabetic animals, suggesting their function as APCs & promoting proinflammatory T cell response. [001034] Liu et al., Immune and metabolic effects of antigen-specific immunotherapy using multiple β-cell peptides in Type 1 diabetes. Diabetes, 71(4), 722-732 (April 1, 2022). [001035] Luong et al., Analytical and biosensing platforms for insulin: A review. Sensors and Actuators Reports, Vol.3, p.100028. (2021). Review focuses on the biosensing of insulin based on monoclonal antibodies and aptamers. Describes method using High-performance liquid chromatography (HPLC). [001036] Mannering & Bhattacharjee, Insulin’s other life: An autoantigen in type 1 diabetes. Immunology & Cell Biology, 99(5), 448–460 (May/June 2021). Insulin and insulin precursors (PPI, PI) play role in pathogenesis of Type 1 diabetes, as mediated by CD4+ and CD8+ T cell responses to autoantigens. [001037] Michel, Boitard, & Bach. Insulin autoantibodies in non-obese diabetic (NOD) mice. Clinical and Experimental Immunology, 75(3), 457 (1989). [001038] Moyers et al., Preclinical characterization of LY3209590, a novel weekly basal insulin Fc- fusion protein. J. Pharmacol. Exp. Ther., 382, 346-355 (September 2022) (Eli Lilly). LY320959 is produced by Eli Lilly and Company, Indianapolis, Indiana and San Diego, California. [001039] Muguruma et al., Kinetics-based structural requirements of human immunoglobulin G binding peptides. ACS Omega, 4, 14390−14397 (2019). [001040] Mustafaoglu et al., Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule, Analyst, 141(24), 6571–6582 (November 28, 2016). [001041] Narendran, Mannering, & Harrison, Proinsulin—a pathogenic autoantigen in type 1 diabetes. Autoimmune Reviews, 2(4), 204-10 (June 1, 2003). [001042] Nath, Godat, Flemming &, Urh, Deciphering the interaction between neonatal fc receptor and antibodies using a homogeneous bioluminescent immunoassay. The Journal of Immunology, 207(4), 1211–1221 (2021).
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[001043] Neyrinck et.al., Oxidative stress in the pathogenesis and evolution of chronic kidney disease: untangling Ariadne’s thread. International Journal of Molecular Sciences, 20(15), 3711 (July 29, 2019). This publication shows the insulin/insulin-like growth factors system of ligand–receptor binding affinities. Insulin receptor and insulin-like growth factor receptor receptors are each composed of two dimers. [001044] Nishi & Nanjo, Insulin gene mutations and diabetes. J. Diabetes Investig., 2(2), 92-100 (April 7, 2011). Insulin gene mutations. [001045] O’Kell et al., Exploration of autoantibody responses in canine diabetes using protein arrays. Scientific Reports, 12(1), 2490 (2022). [001046] Petersen et al., Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay, Diabetes, Volume 43, Issue 3 (March 1, 1994). [001047] Pettersson, M., & Crews, C. M. (2019). PROteolysis TArgeting Chimeras (PROTACs) — Past, present and future. In Drug Discovery Today: Technologies (Vol.31, pp.15–27). Elsevier BV. [001048] Pissarnitski et al., Discovery of insulin receptor partial agonists MK-5160 and MK-1092 as novel basal insulins with potential to improve therapeutic index. J. Med. Chem., 65, 5593−5605 (Merck) (April 14, 2022). [001049] Sabljic et al., Novel flow cytometric immunoassay for detection of proinsulin autoantibodies in diabetes mellitus employing a recombinant autoantigen expressed in E. coli. Front. Immunol., 12, 648021 (April 6, 2021). Many publications with validated specific diagnostics have not demonstrated anti-proinsulin antibodies in the general population, even with the most sensitive MSD and radioimmune assay platforms. [001050] Saxena & Wu, Advances in therapeutic Fc engineering–modulation of IgG-associated effector functions and serum half-life. Frontiers in immunology, 7, 580 (2016) (review). [001051] Saxon, McDermott, & Michels, Novel management of insulin autoimmune syndrome with rituximab and continuous glucose monitoring. The Journal of Clinical Endocrinology & Metabolism, 101(5), 1931-4 (May 1, 2016). In clinical setting, depletion of autoantibody-producing β-cells by anti- CD20 rituximab can suppress insulin autoantibody titers. [001052] Shields et al., High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. Journal of Biological Chemistry 276(9), 6591-6604 (2001). [001053] Smith & Peakman, Peptide immunotherapy for Type 1 diabetes-clinical advances. Front Immunol., 9, 392 (February 28, 2018). Peptide therapy for Type 1 diabetes.
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[001054] Strohl, Optimization of Fc-mediated effector functions of monoclonal antibodies. Current Opinion in Biotechnology, 20(6), 685-691 (2009). [001055] Subramanian et al., Insulin receptor-insulin interaction kinetics using multiplex surface plasmon resonance. Journal of Molecular Recognition, 26(12), 643-52. (December 2013). [001056] Tamm & Schmidt, IgG binding sites on human Fcγ receptors. International reviews of immunology 16(1-2), 57-85 (1997). [001057] Teng et al., A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand, J. Mol. Recognition, 12, 67– 75 (1999). [001058] Tilman et al., Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Engineering, Design and Selection, Volume 29, Issue 10, pages 457–466 (October 2016). This paper shows that even LALA itself abolishes c1q binding. P329A alone is tested, and abolishes c1q binding, and reduces FcgR binding. They show that P329G/LALA further reduces FcgR binding beyond LALA alone. [001059] Uttamchandani et al., Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG, J. Comb. Chem., 6(6), 862-8 (November-December 2004). [001060] Valdez et al., E. A radioligand-binding assay for detecting antibodies specific for proinsulin and insulin using 35S-proinsulin. J. Immunol. Methods, 279(1-2), 173-81 (August 2003). Many publications with validated specific diagnostics have not demonstrated anti-proinsulin antibodies in the general population, even with the most sensitive MSD and radioimmune assay platforms. [001061] Watanabe et al., Human soluble phospholipase A2 receptor is an inhibitor of the integrin- mediated cell migratory response to collagen. Am. J. Physiol. Cell Physiol., 315, C398-C408 (2018). [001062] Weiss et. al. Evolution of insulin at the edge of foldability and its medical implications. Proceedings of the National Academy of Sciences (PNAS), vol.117, no.47 (2020). [001063] Williams et al., An artificial intelligence-based deep learning algorithm for the diagnosis of diabetic neuropathy using corneal confocal microscopy: A development and validation study. Diabetologia, 63, 419-30 (February 2020). [001064] Williams et al., Racial differences in the associations between adiposity, placental growth hormone and inflammatory cytokines in pregnant women. Frontiers in Endocrinology, 14, 1100724 (March 17, 2023). Most prominent autoantibodies measured in children that progress to Type 1 diabetes (TEDDY study, n=100 children); virtually no anti-insulin autoantibodies in normal controls.
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[001065] Williams et.al., The measurement of autoantibodies to insulin informs diagnosis of diabetes in a childhood population negative for other autoantibodies. Frontiers in Endocrinology, 39(12), e14979 (December 2022). Autoantibodies against several β-cell proteins are identified in more than 90% in newly diagnosed type 1 diabetes. [001066] Yamada et al., Angew Chem. Int., Ed Engl.; 58(17), 5592-5597 (April 16, 2019). [001067] Yamada et al., Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J. Exp. Med., 173, 395 (1991). [001068] Yeh et al. Pathogenic human monoclonal antibody against desmoglein 3. Clinical Immunology.120(1), 68-75(2006). [001069] Yu et al., APRIL and TALL-1 and receptors BCMA and TACI: system for regulating humoral immunity. Nature immunology.1(3), 252-6 (September 2000). Most prominent autoantibodies measured in children that progress to Type 1 diabetes (TEDDY study, n=100 children); virtually no anti- insulin autoantibodies in normal controls. [001070] Yu et al., Distinguishing persistent insulin autoantibodies with differential risk: Nonradioactive bivalent proinsulin/insulin autoantibody assay. Diabetes, 61(1), 179-86 (January 2012). Many publications with validated specific diagnostics have not demonstrated anti-proinsulin antibodies in the general population, even with the most sensitive MSD and radioimmune assay platforms. [001071] Yu, Islet autoantibody detection by electrochemiluminescence (ECL) assay, Methods in Molecular Biology, 1433, 85-91 (2016). [001072] Zalevsky et al. Enhanced antibody half-life improves in vivo activity. Nature Biotechnology, 28(2),157-159 (2010). Textbooks and technical references [001073] Janeway's Immunobiology, (2014). Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, (ISBN 0815345305, 9780815345305). [001074] Laboratory Methods in Enzymology: DNA, (2013). Jon Lorsch (ed.) Elsevier (ISBN 0124199542). [001075] The Merck Manual of Diagnosis and Therapy, 19th edition (Merck Sharp & Dohme Corp., 2018). [001076] Remington’s, Pharmaceutical Sciences 23rd edition (Elsevier, 2020). [001077] Greene's Protective Groups in Organic Synthesis, Wuts (editor) (John Wiley & Sons, first published August 11, 2014).
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[001078] American Veterinary Medical Association. AVMA Guidelines for the Euthanasia of Animals. Current edition. [001079] National Research Council. Guide for the Care and Use of Laboratory Animals.8th edition. Washington, DC: National Academy Press. [001080] Office of Laboratory Animal Welfare. Public Health Services Policy on Humane Care and Use of Laboratory Animals. Bethesda, MD: National Institutes of Health. [001081] Throughout this application, several publications are referenced by author name and date, or by patent number or patent publication number. The disclosures of these publications are incorporated in their entireties by reference into this application to describe the state of the art more fully as known to persons having ordinary skill in the art as of the date of the invention described and claimed in this specification. However, the citation of a reference in this specification should not be construed as an acknowledgement that this reference is prior art to the present invention. [001082] All patents and publications cited throughout this specification are incorporated by reference to disclose and describe the materials and methods that might be used with the technologies described in this specification. The publications discussed are provided only for their disclosure before the filing date. They should not be construed as an admission that the inventors may not antedate such disclosure under prior invention or for any other reason. If there is an apparent discrepancy between a prior patent or publication and the description provided in this specification, the specification (including any definitions) and claims shall control. All statements about the date or contents of these documents are based on the information available to the applicants. These statements constitute no admission to the correctness of the dates or contents of these documents. The publication dates provided in this specification may differ from the actual publication dates. If there is an apparent discrepancy between a publication date in this specification and the actual publication date supplied by the publisher, the actual publication date shall control.
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SEQUENCE LISTING Sequence Number (ID): 1 Length: 330 Molecule Type: AA Features Location/Qualifiers: - source, 1..330 > mol_type, protein > organism, Homo sapiens Residues: ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 120 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 180 STYRVVSVLT VLHQDWINGK EYKCRVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 240 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEATHNHYT QKSLSLSPGK 330 Sequence Number (ID): 2 Length: 326 Molecule Type: AA Features Location/Qualifiers: - source, 1..326 > mol_type, protein > organism, Homo sapiens Residues: ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP APPVAGPSVF 120 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP REBQFNSTFR 180 VVSVLTVLHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG OPREPQVYTL PPSREEMTKN 240 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN 300 VFSCSVMHEA THNHYTQKSL SLSPGK 326 Sequence Number (ID): 3 Length: 327 Molecule Type: AA Features Location/Qualifiers: - source, 1..327 > mol_type, protein
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> organism, Homo sapiens Residues: ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KTGPPCPSCP APEFLGGPSV 120 FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY 180 RVVSVLTVLH QDWINGKEYK CKVSNKGLPS SIEKTISKAK GOPREPQVYT LPPSQEEMTK 240 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 300 NVFSCSVMHE ATHNHYTQKS LSLSLGK 327 Sequence Number (ID): 4 Length: 110 Molecule Type: AA Features Location/Qualifiers: - source, 1..110 > mol_type, protein > organism, Homo sapiens - PROPEP, 1..24 > note, Proinsulin leader sequence - REGION, 25..54 > note, B chain of insulin - REGION, 55..89 > note, Connecting peptide - REGION, 90..110 > note, A chain of insulin Residues: MALWMRILPL LAILALWGPD PAAAFVNQHI CGSHLVEALY LVCGERGFFY TPKTRREAED 60 LQVGQVEIGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN 110 Sequence Number (ID): 5 Length: 98 Molecule Type: AA Features Location/Qualifiers: - source, 1..98 > mol_type, protein > organism, Homo sapiens - PROPEP, 1..12 > note, Proinsulin leader sequence - REGION, 13..42
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> note, Insulin B-chain - REGION, 43..77 > note, Connecting peptide - REGION, 78..98 > note, Insulin A chain Residues: MNHKVHHHHH HMFVNQHLCG SHLVEALYLV CGERGFFYTP KTRREAEDLQ VGEVELGGGP 60 GAGSLQPLAL EGSLQKRGIV EQCCTSICSL YQLENYCN 98 Sequence Number (ID): 6 Length: 106 Molecule Type: AA Features Location/Qualifiers: - source, 1..106 > mol_type, protein > organism, Homo sapiens - REGION, 6..35 > note, Insulin B chain - REGION, 36..70 > note, Connecting peptide - REGION, 71..91 > note, Insulin A chain - BINDING, 92..100 > note, Sortase tag - BINDING, 101..106 > note, His tag - VARIANT, 30 > note, F25A modification Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFA YTPKTRREAE DLQVGEVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGS HHHHHH 106 Sequence Number (ID): 7 Length: 101 Molecule Type: AA Features Location/Qualifiers: - source, 1..101 > mol_type, protein
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> organism, Homo sapiens - REGION, 96..101 > note, His6 tag - REGION, 92..95 > note, GGGS linker - REGION, 87..91 > note, GGGGS - PEPTIDE, 1..101 > note, Native proinsulin with C-terminal His tag - VARIANT, 25 > note, F sequence - VARIANT, 31..32 > note, RR sequence - VARIANT, 64..65 > note, KR sequence Residues: FVNQHLCGSH LVEALYLVCG ERGFFYTPKT RREAEDLQVG EVELGGGPGA GSLQPLALEG 60 SLQKRGIVEQ CCTSICSLYQ LENYCNGGGG SGGGSHHHHH H 101 Sequence Number (ID): 8 Length: 107 Molecule Type: AA Features Location/Qualifiers: - source, 1..107 > mol_type, protein > organism, synthetic construct - REGION, 102..107 > note, His6 tag - REGION, 96..101 > note, Sortase tag - REGION, 92..95 > note, GGGS linker - REGION, 87..91 > note, GGGGS linker - PEPTIDE, 1..107 > note, Native proinsulin with C-terminal sortase - VARIANT, 25 > note, F sequence
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- VARIANT, 31..32 > note, RR sequence - VARIANT, 64..65 > note, KR sequence Residues: FVNQHLCGSH LVEALYLVCG ERGFFYTPKT RREAEDLQVG EVELGGGPGA GSLQPLALEG 60 SLQKRGIVEQ CCTSICSLYQ LENYCNGGGG SGGGSLPETG GHHHHHH 107 Sequence Number (ID): 9 Length: 102 Molecule Type: AA Features Location/Qualifiers: - source, 1..102 > mol_type, protein > organism, synthetic construct - REGION, 1..6 > note, His6 tag - REGION, 7..11 > note, SGGGG linker - REGION, 12..16 > note, SGGGG linker - REGION, 17..46 > note, B chain - VARIANT, 47..48 > note, RR sequence - REGION, 47..81 > note, C-peptide in the middle - VARIANT, 80..81 > note, KR sequence - REGION, 82..102 > note, A chain Residues: HHHHHHSGGG GSGGGGFVNQ HLCGSHLVEA LYLVCGERGF FYTPKTRREA EDLQVGQVEL 60 GGGPGAGSLQ PLALEGSLQK RGIVEQCCTS ICSLYQLENY CN 102 Sequence Number (ID): 10 Length: 107 Molecule Type: AA
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Features Location/Qualifiers: - source, 1..107 > mol_type, protein > organism, synthetic construct - REGION, 1..5 > note, Sortase tag - REGION, 102..107 > note, His6 tag - REGION, 97..101 > note, GGGGS linker - REGION, 92..96 > note, GGGGS linker - REGION, 71..91 > note, A chain - VARIANT, 69..70 > note, KR sequence - REGION, 6..35 > note, B chain - REGION, 36..70 > note, C-peptide in the middle - VARIANT, 36..37 > note, RR sequence Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFF YTPKTRREAE DLQVGQVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGG SHHHHHH 107 Sequence Number (ID): 11 Length: 107 Molecule Type: AA Features Location/Qualifiers: - source, 1..107 > mol_type, protein > organism, Homo sapiens - REGION, 1..5 > note, Sortase Tag - REGION, 6..35 > note, B chain - VARIANT, 30
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> note, A sequence - REGION, 36..70 > note, C-peptide in the middle - VARIANT, 69..70 > note, KR sequence - REGION, 71..91 > note, A chain - REGION, 102..107 > note, His6 tag - REGION, 97..101 > note, GGGGS linker - REGION, 92..96 > note, GGGGS linker - VARIANT, 36..37 > note, RR sequence Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFA YTPKTRREAE DLQVGQVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGG SHHHHHH 107 Sequence Number (ID): 12 Length: 107 Molecule Type: AA Features Location/Qualifiers: - source, 1..107 > mol_type, protein > organism, Homo sapiens - VARIANT, 47 > note, I sequence Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFF YTPKTRREAE DLQVGQIELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGG SHHHHHH 107 Sequence Number (ID): 13 Length: 107 Molecule Type: AA Features Location/Qualifiers: - source, 1..107 > mol_type, protein
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> organism, Homo sapiens Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFA YTPKTRREAE DLQVGQVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGG SHHHHHH 107 Sequence Number (ID): 14 Length: 107 Molecule Type: AA Features Location/Qualifiers: - source, 1..107 > mol_type, protein > organism, Homo sapiens Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFF YTPKTRREAE DLQVGQVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGG SHHHHHH 107 Sequence Number (ID): 15 Length: 106 Molecule Type: AA Features Location/Qualifiers: - source, 1..106 > mol_type, protein > organism, synthetic construct - REGION, 43..77 > note, Connecting peptide - REGION, 13..42 > note, B-chain - REGION, 78..98 > note, A chain - REGION, 101..106 > note, His6 tag - REGION, 92..100 > note, GGGGSGGGS linker - VARIANT, 36..37 > note, RR sequence - VARIANT, 69..70 > note, KR sequence Residues:
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GGGGGFVNQH LCGSHLVEAL YLVCGERGFA YTPKTRREAE DLQVGEVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGS HHHHHH 106 Sequence Number (ID): 16 Length: 298 Molecule Type: AA Features Location/Qualifiers: - source, 1..298 > mol_type, protein > organism, Homo sapiens - REGION, 1..26 > note, B chain of insulin - REGION, 78..298 > note, IgG Fc region - REGION, 39..47 > note, A chain of insulin Residues: FVNQHLCGSH LVEALELVCG ERGFHYGGGG GGSGGGGGLV EQCCTSTCSL DQLENYCGGG 60 GGQGGGGQGG GGQGGGGGEC PPCPAPPVAG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 120 HEDPEVQFNW YVDGVEVHNA KTKPREEQFN STFRVVSVLT WHQDWLNGKE YKCKVSNKGL 180 PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE 240 NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG 298 Sequence Number (ID): 17 Length: 323 Molecule Type: AA Features Location/Qualifiers: - source, 1..323 > mol_type, protein > organism, synthetic construct Residues: FVNQHLCGSH LVEALYLVCG ERGFFYTPKT RREAEDLQVG QVELGGGPGA GSLQPLALEG 60 SLQKRGIVEQ CCTSICSLYQ LENYCNGGGG SEPKSADKTH TCPPCPAPEA AGGPSVFLFP 120 PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 180 VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS 240 LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS 300 CSVMHEALHN HYTQKSLSLS PGK 323
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Sequence Number (ID): 18 Length: 343 Molecule Type: AA Features Location/Qualifiers: - source, 1..343 > mol_type, protein > organism, synthetic construct Residues: FVNQHLCGSH LVEALYLVCG ERGFFYTPKT RREAEDLQVG QVELGGGPGA GSLQPLALEG 60 SLQKRGIVEQ CCTSICSLYQ LENYCNGGGG SGGGGSGGGG SGGGGSGGGG SEPKSADKTH 120 TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV 180 HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR 240 EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 300 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 343 Sequence Number (ID): 19 Length: 92 Molecule Type: AA Features Location/Qualifiers: - source, 1..92 > mol_type, protein > organism, synthetic construct Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFF YTPKTRREAE DLQVGQVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NG 92 Sequence Number (ID): 20 Length: 22 Molecule Type: AA Features Location/Qualifiers: - source, 1..22 > mol_type, protein > organism, synthetic construct - PEPTIDE, 1..22 > note, C-terminal sortase tag with His6 tag - REGION, 17..22 > note, His6 tag Residues:
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GGGGSGGGGS LPETGGHHHH HH 22 Sequence Number (ID): 21 Length: 21 Molecule Type: AA Features Location/Qualifiers: - source, 1..21 > mol_type, protein > organism, synthetic construct Residues: HHHHHHSPST PPTPSPSTPP C 21 Sequence Number (ID): 22 Length: 106 Molecule Type: AA Features Location/Qualifiers: - source, 1..106 > mol_type, protein > organism, synthetic construct - REGION, 1..5 > note, Sortase tag - VARIANT, 30 > note, A sequence - REGION, 5..35 > note, B chain - VARIANT, 36..37 > note, RR sequence - REGION, 36..70 > note, C-peptide in the middle - VARIANT, 69..70 > note, KR sequence - REGION, 71..81 > note, A chain - REGION, 82..86 > note, GGGGS linker - REGION, 87..90 > note, GGGS linker - REGION, 91..96
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> note, HHHHHH Residues: GGGGGFVNQH LCGSHLVEAL YLVCGERGFA YTPKTRREAE DLQVGEVELG GGPGAGSLQP 60 LALEGSLQKR GIVEQCCTSI CSLYQLENYC NGGGGSGGGS HHHHHH 106 Sequence Number (ID): 23 Length: 299 Molecule Type: AA Features Location/Qualifiers: - source, 1..299 > mol_type, protein > organism, synthetic construct - REGION, 1..26 > note, B chain - VARIANT, 16 > note, E sequence - REGION, 38..58 > note, A chain - VARIANT, 47 > note, T sequence - VARIANT, 51 > note, D sequence - VARIANT, 58 > note, G sequence - REGION, 79..299 > note, Fc region Residues: FVNQHLCGSH LVEALELVCG ERGFHYGGGG GGSGGGGGIV EQCCTSTCSL DQLENYCGGG 60 GGQGGGGQGG GGQGGGGGEC PPCPAPPVAG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 120 HEDPEVQFNW YVDGVEVHNA KTKPREEQFN STFRVVSVLT VVHQDWLNGK EYKCKVSNKG 180 LPAPIEKTIS KTKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP 240 ENNYKTTPPM LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 299 Sequence Number (ID): 24 Length: 86 Molecule Type: AA Features Location/Qualifiers: - source, 1..86
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> mol_type, protein > organism, Canis lupus Residues: PVNQHLCGSH LVEALYLVCG ERGFFYTFKA RREVEDLOVR DVZLAGAPGE GGLQPLALEG 60 ALQKRGIVEQ CCTSICSLYQ LENYCN 86 Sequence Number (ID): 25 Length: 84 Molecule Type: AA Features Location/Qualifiers: - source, 1..84 > mol_type, protein > organism, Sus scrofa Residues: PVNQHLCGSH LVEALYLVCG ERGFFYTFKA RREAENPPAG AVELGOGLGG LQALALEGPP 60 OKRGIVEQCC TSICSLYQLE NYCN 84 Sequence Number (ID): 26 Length: 86 Molecule Type: AA Features Location/Qualifiers: - source, 1..86 > mol_type, protein > organism, Mus musculus Residues: FVKQHLCGSH LVEALYLVCG ERGFFYTFMS RREVEDPDVA QLELGSGPGA GDLQTLALEV 60 AQDKRGIVDQ CCTSICSLYQ LENXCN 86 Sequence Number (ID): 27 Length: 84 Molecule Type: AA Features Location/Qualifiers: - source, 1..84 > mol_type, protein > organism, Mus musculus Residues: FVKDHLCGPH LVEALYLVCG ERGFFYTFKS RREVEDPOVE QLELGSSPGD LOTLALEVAR 60 DKRGIVDQCC TSICSLYQLE NXCN 84
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Sequence Number (ID): 28 Length: 86 Molecule Type: AA Features Location/Qualifiers: - source, 1..86 > mol_type, protein > organism, Rattus norvegicus Residues: FVKQHLCGSH LVEALYLVCG ERGFFYTFMS RREVEDPOVA QLSLGSGPGA GDLOTLALEV 60 ARQKRGIVDQ CCTSICSLYQ LENYCN 86 Sequence Number (ID): 29 Length: 86 Molecule Type: AA Features Location/Qualifiers: - source, 1..86 > mol_type, protein > organism, Rattus norvegicus Residues: PVKQHLCCPH LVEALYLVCG ERGFPYTFKS RREVEDPOVP QLELGSGPEA GDLQTLALEV 60 AROKRGIVDQ CCTSICSLYQ LENYCN 86 Sequence Number (ID): 30 Length: 344 Molecule Type: AA Features Location/Qualifiers: - source, 1..344 > mol_type, protein > organism, synthetic construct - PROPEP, 1..21 > note, Mouse Ig kappa signal - VARIANT, 117 > note, C>A - REGION, 108..112 > note, G4S linker - VARIANT, 131..132 > note, LALA modification
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- VARIANT, 226 > note, PA modification Residues: METDTLLLWV LLLWVPGSTG DFVNQHLCGS HLVEALYLVC GERGFFYTPK TRREAEDLQV 60 GQVELGGGPG AGSLQPLALE GSLQKRGIVE QCCTSICSLY QLENYCNGGG GSEPKSADKT 120 HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE 180 VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALAAPIE KTISKAKGQP 240 REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 300 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 344 Sequence Number (ID): 31 Length: 1382 Molecule Type: AA Features Location/Qualifiers: - source, 1..1382 > mol_type, protein > organism, Homo sapiens Residues: MATGGRRGAA AAPLLVAVAA LLLGAAGHLY PGEVCPGMDI RNNLTRLHEL ENCSVIEGHL 60 QILLMFKTRP EDFRDLSFPK LIMITDYLLL FRVYGLESLK DLFPNLTVIR GSRLFFNYAL 120 VIFEMVHLKE LGLYNLMNIT RGSVRIEKNN ELCYLATIDW SRILDSVEDN YIVLNKDDNE 180 ECGDICPGTA KGKTNCPATV INGQFVERCW THSHCQKVCP TICKSHGCTA EGLCCHSECL 240 GNCSQPDDPT KCVACRNFYL DGRCVETCPP PYYHFQDWRC VNFSFCQDLH HKCKNSRRQG 300 CHQYVIHNNK CIPECPSGYT MNSSNLLCTP CLGPCPKVCH LLEGEKTIDS VTSAQELRGC 360 TVINGSLIIN IRGGNNLAAE LEANLGLIEE ISGYLKIRRS YALVSLSFFR KLRLIRGETL 420 EIGNYSFYAL DNQNLRQLWD WSKHNLTITQ GKLFFHYNPK LCLSEIHKME EVSGTKGRQE 480 RNDIALKTNG DQASCENELL KFSYIRTSFD KILLRWEPYW PPDFRDLLGF MLFYKEAPYQ 540 NVTEFDGQDA CGSNSWTVVD IDPPLRSNDP KSQNHPGWLM RGLKPWTQYA IFVKTLVTFS 600 DERRTYGAKS DIIYVQTDAT NPSVPLDPIS VSNSSSQIIL KWKPPSDPNG NITHYLVFWE 660 RQAEDSELFE LDYCLKGLKL PSRTWSPPFE SEDSQKHNQS EYEDSAGECC SCPKTDSQIL 720 KELEESSFRK TFEDYLHNVV FVPRKTSSGT GAEDPRPSRK RRSLGDVGNV TVAVPTVAAF 780 PNTSSTSVPT SPEEHRPFEK VVNKESLVIS GLRHFTGYRI ELQACNQDTP EERCSVAAYV 840 SARTMPEAKA DDIVGPVTHE IFENNVVHLM WQEPKEPNGL IVLYEVSYRR YGDEELHLCV 900 SRKHFALERG CRLRGLSPGN YSVRIRATSL AGNGSWTEPT YFYVTDYLDV PSNIAKIIIG 960 PLIFVFLFSV VIGSIYLFLR KRQPDGPLGP LYASSNPEYL SASDVFPCSV YVPDEWEVSR 1020 EKITLLRELG QGSFGMVYEG NARDIIKGEA ETRVAVKTVN ESASLRERIE FLNEASVMKG 1080 FTCHHVVRLL GVVSKGQPTL VVMELMAHGD LKSYLRSLRP EAENNPGRPP PTLQEMIQMA 1140 AEIADGMAYL NAKKFVHRDL AARNCMVAHD FTVKIGDFGM TRDIYETDYY RKGGKGLLPV 1200
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RWMAPESLKD GVFTTSSDMW SFGVVLWEIT SLAEQPYQGL SNEQVLKFVM DGGYLDQPDN 1260 CPERVTDLMR MCWQFNPKMR PTFLEIVNLL KDDLHPSFPE VSFFHSEENK APESEELEME 1320 FEDMENVPLD RSSHCQREEA GGRDGGSSLG FKRSYEEHIP YTHMNGGKKN GRILTLPRSN 1380 PS 1382 Sequence Number (ID): 32 Length: 1370 Molecule Type: AA Features Location/Qualifiers: - source, 1..1370 > mol_type, protein > organism, Homo sapiens Residues: MATGGRRGAA AAPLLVAVAA LLLGAAGHLY PGEVCPGMDI RNNLTRLHEL ENCSVIEGHL 60 QILLMFKTRP EDFRDLSFPK LIMITDYLLL FRVYGLESLK DLFPNLTVIR GSRLFFNYAL 120 VIFEMVHLKE LGLYNLMNIT RGSVRIEKNN ELCYLATIDW SRILDSVEDN YIVLNKDDNE 180 ECGDICPGTA KGKTNCPATV INGQFVERCW THSHCQKVCP TICKSHGCTA EGLCCHSECL 240 GNCSQPDDPT KCVACRNFYL DGRCVETCPP PYYHFQDWRC VNFSFCQDLH HKCKNSRRQG 300 CHQYVIHNNK CIPECPSGYT MNSSNLLCTP CLGPCPKVCH LLEGEKTIDS VTSAQELRGC 360 TVINGSLIIN IRGGNNLAAE LEANLGLIEE ISGYLKIRRS YALVSLSFFR KLRLIRGETL 420 EIGNYSFYAL DNQNLRQLWD WSKHNLTITQ GKLFFHYNPK LCLSEIHKME EVSGTKGRQE 480 RNDIALKTNG DQASCENELL KFSYIRTSFD KILLRWEPYW PPDFRDLLGF MLFYKEAPYQ 540 NVTEFDGQDA CGSNSWTVVD IDPPLRSNDP KSQNHPGWLM RGLKPWTQYA IFVKTLVTFS 600 DERRTYGAKS DIIYVQTDAT NPSVPLDPIS VSNSSSQIIL KWKPPSDPNG NITHYLVFWE 660 RQAEDSELFE LDYCLKGLKL PSRTWSPPFE SEDSQKHNQS EYEDSAGECC SCPKTDSQIL 720 KELEESSFRK TFEDYLHNVV FVPRPSRKRR SLGDVGNVTV AVPTVAAFPN TSSTSVPTSP 780 EEHRPFEKVV NKESLVISGL RHFTGYRIEL QACNQDTPEE RCSVAAYVSA RTMPEAKADD 840 IVGPVTHEIF ENNVVHLMWQ EPKEPNGLIV LYEVSYRRYG DEELHLCVSR KHFALERGCR 900 LRGLSPGNYS VRIRATSLAG NGSWTEPTYF YVTDYLDVPS NIAKIIIGPL IFVFLFSVVI 960 GSIYLFLRKR QPDGPLGPLY ASSNPEYLSA SDVFPCSVYV PDEWEVSREK ITLLRELGQG 1020 SFGMVYEGNA RDIIKGEAET RVAVKTVNES ASLRERIEFL NEASVMKGFT CHHVVRLLGV 1080 VSKGQPTLVV MELMAHGDLK SYLRSLRPEA ENNPGRPPPT LQEMIQMAAE IADGMAYLNA 1140 KKFVHRDLAA RNCMVAHDFT VKIGDFGMTR DIYETDYYRK GGKGLLPVRW MAPESLKDGV 1200 FTTSSDMWSF GVVLWEITSL AEQPYQGLSN EQVLKFVMDG GYLDQPDNCP ERVTDLMRMC 1260 WQFNPKMRPT FLEIVNLLKD DLHPSFPEVS FFHSEENKAP ESEELEMEFE DMENVPLDRS 1320 SHCQREEAGG RDGGSSLGFK RSYEEHIPYT HMNGGKKNGR ILTLPRSNPS 1370 Sequence Number (ID): 33
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Length: 215 Molecule Type: AA Features Location/Qualifiers: - source, 1..215 > mol_type, protein > organism, Homo sapiens Residues: QIVLTQSPTI MSASLGERVT MTCTASSSVS SSYLHWYQQK PGSSPKLWIY STSNLASGVP 60 ARFSGSGSGT SYSLTISSME AEDAATYYCH QYHRSPPTFG AGTKLELKRT VAAPSVFIFP 120 PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL 180 TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 215 Sequence Number (ID): 34 Length: 492 Molecule Type: AA Features Location/Qualifiers: - source, 1..492 > mol_type, protein > organism, Homo sapiens - MOD_RES, 345 > note, N-glycosylation - MOD_RES, 440 > note, N-glycosylation Residues: QIQLVQSGPE LKKPGETVKI SCKASGYTFT DYSMHWVKQA PGKGLKWMDW INTETGVPTY 60 ADDFKGRFAF SLETSASTAY LQINDLKNED TATYFCTRGY GKGYFDVWGA GTTVTVSSAS 120 TKGPSVFPLA PCSRSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180 YSLSSVTVPS SSLGTQTYTC NVNHKPSNTK VDKRVELKTP LGDTTHTCPR CPEPKSCDTP 240 PPCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPAPE LLGGPSFLFP PKPKDTLMIS 300 RTPEVTCVVV DVSHEDPEVQ FKWYVDGVEV HNAKTKPREE QYNSTFRVVS VLTVLHQDWL 360 NGKEYKCKVS KALPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS 420 DIAVEWESSG QPENNYNTTP PMLDSDGSFF LYSKLTVDKS RWQQGNIFSC SVMHEALHNR 480 FTQKSLSLSP GK 492 Sequence Number (ID): 35 Length: 218 Molecule Type: AA Features Location/Qualifiers:
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- source, 1..218 > mol_type, protein > organism, Mus musculus Residues: DVVMTQTPLS LPVSLGDQAS ISCRSSQSLV HNNGNTFLQW YLQKPGQSPK LLIYKLSNRF 60 SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHEP YTFGGGTKLE IKRADAAPTV 120 SIFPPSSEQL TSGGASWCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS 180 STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC 218 Sequence Number (ID): 36 Length: 434 Molecule Type: AA Features Location/Qualifiers: - source, 1..434 > mol_type, protein > organism, Mus musculus - MOD_RES, 55 > note, N-linked glycosylation - MOD_RES, 287 > note, N-linked glycosylation Residues: EVQLQQSGPE LVRPGASVKI SCKASGYTFA DYVMHWVKQS HGKSLEWIGY FYPHNDSFGY 60 NQKFKSKATL TVDTSSSTAY MELRSTFEDS AVYYCTSGLH SWGQGTTLTV SSAKTTPPSV 120 YPLAPGSAAQ TNSMVTLGCL VKGYFPEPVT VWNSGSLSSG VHTFPAVLQS DLYTLSSSVT 180 VPSSTWPSET VTCNVAHPAS STKVDKKIVP RDCGCKPCIC TVPEVSSVFI FPPKPKDVLT 240 ITLTPKVTCV VDISKDDPEV QFSWFVDDVE VHTAQTQPRE EQFNSTFRSV SELPIMHQDW 300 LNGKEFKCRV NSAAFPAPIE KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITDFF 360 PEDITVEWQW NGQPAENYKN TQPIMDTDGS YFVYSKLNVQ KSNWEAGNTF TCSVLHEGLH 420 NHHTEKSLSH SPGK 434 Sequence Number (ID): 37 Length: 21 Molecule Type: AA Features Location/Qualifiers: - source, 1..21 > mol_type, protein > organism, synthetic construct Residues:
192 30125-WO-PCT
HHHHHHSPST PPTPSPSTPP C 21 Sequence Number (ID): 38 Length: 96 Molecule Type: AA Features Location/Qualifiers: - source, 1..96 > mol_type, protein > organism, synthetic construct - REGION, 1..5 > note, Sortase tag - REGION, 10..38 > note, B chain - VARIANT, 33 > note, B24 F to A mutation - PEPTIDE, 40..72 > note, C peptide - REGION, 74..95 > note, A chain Residues: LPETGGGGGF VNQHLCGSHL VEALYLVCGE RGAFYTPKTR REAEDLQVGQ VELGGGPGAG 60 SLQPLALEGS LQKRGILEQC CTSICSLYQL ENYCNA 96 Sequence Number (ID): 39 Length: 22 Molecule Type: AA Features Location/Qualifiers: - source, 1..22 > mol_type, protein > organism, Homo sapiens Residues: GCAACATCAC CCACTACCTG GT 22 Sequence Number (ID): 40 Length: 22 Molecule Type: AA Features Location/Qualifiers: - source, 1..22
193 30125-WO-PCT
> mol_type, protein > organism, Homo sapiens Residues: GAATGGTGGA GACCAGGTCC TC 22 Sequence Number (ID): 41 Length: 22 Molecule Type: AA Features Location/Qualifiers: - source, 1..22 > mol_type, protein > organism, Homo sapiens Residues: CCTGCACAAC TCCATCTTCG TG 22 Sequence Number (ID): 42 Length: 22 Molecule Type: AA Features Location/Qualifiers: - source, 1..22 > mol_type, protein > organism, Homo sapiens Residues: CGGTGATGTT GTAGGTGTCT GC 22 Sequence Number (ID): 43 Length: 50 Molecule Type: AA Features Location/Qualifiers: - source, 1..50 > mol_type, protein > organism, synthetic construct Residues: GIVEQCCTSI CSLYQLENYC FVNQHLCGSH LVEALYLVCG ERGAFYTPKT 50 Sequence Number (ID): 44 Length: 87 Molecule Type: AA
194 30125-WO-PCT
Features Location/Qualifiers: - source, 1..87 > mol_type, protein > organism, Homo sapiens Residues: FVNQHLCGSH LVEALYLVCG ERGFFYTPKT RREAEDLQVG QVELGGGPGA GSLQPLALEG 60 SLQKRGIVEQ CCTSICSLYQ LENYCNG 87 Sequence Number (ID): 45 Length: 18 Molecule Type: AA Features Location/Qualifiers: - source, 1..18 > mol_type, protein > organism, Homo sapiens Residues: GSLIYSFSEC AFTGPLRP 18 Sequence Number (ID): 46 Length: 18 Molecule Type: AA Features Location/Qualifiers: - source, 1..18 > mol_type, protein > organism, Homo sapiens Residues: YNITDHGSLI YSFSECAF 18 Sequence Number (ID): 47 Length: 18 Molecule Type: AA Features Location/Qualifiers: - source, 1..18 > mol_type, protein > organism, Homo sapiens Residues: FSECAFTGPL RPFFSPGF 18
195 30125-WO-PCT
Sequence Number (ID): 48 Length: 322 Molecule Type: AA Features Location/Qualifiers: - source, 1..322 > mol_type, protein > organism, synthetic construct Residues: FVNQHLCGSH LVEALYLVCG ERGFFYTPKT RREAEDLQVG QVELGGGPGA GSLQPLALEG 60 SLQKRGIVEQ CCTSICSLYQ LENYCNGGGG SEPKSADKTH TCPPCPAPEA AGGPSVFLFP 120 PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 180 VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS 240 LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS 300 CSVMHEALHN HYTQKSLSLS PG 322 Sequence Number (ID): 49 Length: 87 Molecule Type: AA Features Location/Qualifiers: - source, 1..87 > mol_type, protein > organism, Homo sapiens Residues: FVNQHLCGSH LVEALYLVCG ERGFFYTPKT RREAEDLQVG QVELGGGPGA GSLQPLALEG 60 SLQKRGIVEQ CCTSICSLYQ LENYCNG 87 Sequence Number (ID): 50 Length: 7 Molecule Type: AA Features Location/Qualifiers: - source, 1..7 > mol_type, protein > organism, synthetic construct Residues: GLPETGG 7 Sequence Number (ID): 51 Length: 19
196 30125-WO-PCT
Molecule Type: AA Features Location/Qualifiers: - source, 1..19 > mol_type, protein > organism, synthetic construct Residues: ELVAVITTDG STNYADSVK 19
197 30125-WO-PCT
In another embodiment, the composition of matter (the agent, the TRAP) has one of the structures:
198 30125-WO-PCT
In the ab ulae, “Extracellular Protein Targeting Ligand” refers to a target-binding moiety that can bind to an anti-proinsulin or anti-insulin antibody. In the above formulae,
212 30125-WO-PCT X1 is 1 to 5 groups independently selected from O, S. N(R6), arid C(R4XR4), wherein if X! group then X1 is O, S, N(R”), or C(R4)(R4), if X1 is 2 groups then no more than 1 group of X1, S. or N(R6), if X1 is 3, 4, or 5 groups then no more than 2 groups of X1 are O, S, orN(R&);
R* is selected from
(i) aryl, heterocycle, and heteroaryl containing ' or 2. heteroatoms independently selected from N. O, and S, each of which aryl, heterocycle., and heteroaryl is optionally substituted with 1, 2, 3, or 4 substituents;
(iii) -N.Rs-StO)~R:\ -NR*-C(S)-R\ -NR*-S(OXNR6)-R\ -N-S(0XR:?)2, -NRXC(O)NR’S(O)2R3, -NR^-SCOJ.J-R10, and -NR8-C(NR§)-R3 each of which is optionally substituted with 1, 2, 3, or 4 substituents, and
Civ) hydrogen, Ru*, alkyl-CXP)-^. -C(O)-R\ alkyl, haioalkyl, “()C(O)R\ and ^NRS-CCOJR’0;
R10 is selected from aryl, alkyl-NRs-C(O)-R alkyl-aryl, alkyl -heteroaryl with 1 , 2, or 4 heteroatoms, alkyl -cyano, alkyLOR6, alkyl-NRV, NlV-hiR6-C(O)R\ NRM(O>R3, alkenyl, ally 1, alkynyl, -N'R('-a1kenyi, -O-alkenyl, -NR^-alkynyL -NRMieteroatyl, ■■NR/’-aryl, -O-heteroaryL -O-ary! , and -O-alkynyl. each of which R’" is optionally substituted with 1 , 2, 3, or 4 substituents;
R* and R’ are independently selected from hydrogen, heteroalkyl, Ce-Qalkyl-cyano, alkyl, alkenyl, alkynyl, haioalkyl, F, Cl, Br, 1, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hcterocycloalkyl, haloalkoxy, -O-alkenyl, -O-alkynyl, Cn-GalkyL OR*', Co-Qalkyl-SR6, Co-Qalkyl-NR’R7, Co-C6aJkyl-C(0)R3, Cfi-Qalkyl-S(p)R:i, Co-Csalkyl- C(S)R\ C<>-Galkyl-S(O)2R5, €o-C6alkyi-N(RG-C(0)R\ Co-C^alkyl-N(Rs)-S(0)R3, Co-Coalkyl- N(R»K(S)R3, Co-C^alkyl-aNCR^t-SCOhR3 C«-C6alkyl-O-C(O)R3, Cfl-C6alkyl-O-S(O)R-’, Q- Csaikyl-O-€(S)R’, -N=S(O)(R>, C0-C6alkylN3, and CwCsaikyl-O-SfOhlV, each of which is optionally substituted with 1 , 2, 3, or 4 substituents;
R ' at each occurrence is independently selected from hydrogen, alkyl, heteroalkyl, haioalkyl (including -€Fg -CHF;, -CH>F, -ClfaCF?, -CH2CH2F, and -CF?CF-)> arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, hetcroaryl. heterocycle, -PR*8, and -NRSR9;
R'' is independently selected at each occurrence from hydrogen, heteroalkyi, alkyl, haioalkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycle, -OR6, -NIV’R', C(0)R3, S(O)R3, C(S)R3, and S(O)’R3,
R* andR' are independently selected at each occurrence from hydrogen, heteroalkyl, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, haioalkyl, heteroatyl, heterocycle, -alkyl-OR8, - alkyl-MVR9, C(0)R3, S(O)R3, C(S)R3, and S(O) -RA
R* and K;‘ are independently selected at each occurrence from hydrogen, beteroalkyl. alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroatyl, and heterocycle;
Cycle is a 3-8 membered fused cyclic group optionally substituted with 1 , 2. 3, or 4 substituents, each Linker' is a bond or a moiety that covalently links the ASGPR ligand to Linker’3;
Linker0 is a bond or a moiety that covalentfy links Linker* to an Exlracellular Protein Targeting Ligand;
Various moieties [LinkerA], [LinkerB], [LinkerC], [LinkeDC], [Cycle], and X1 are known in the art and described, for example, in International Publication No. WO 2021/155317 published August 5, 2021 and International Publication No. WO 2022/235699 published November 10, 2022, the contents of which publications are incorporated herein in their entireties by reference. Throughout this application, various publications are referenced by author name and date, or by patent number or patent publication number. The disclosures of these publications are hereby incorporated in their entireties by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. For example, pharmaceutically acceptable salts other than those specifically disclosed in the description and Examples herein can be employed. Furthermore, it is intended that specific items within lists of items, or subset groups of items within larger groups of items, can be combined with other specific items, subset groups of items or larger groups of items whether or not there is a specific disclosure herein identifying such a combination.
215 30125-WO-PCT

Claims

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, [AGN104] or a pharmaceutically acceptable salt thereof.
216 30125-WO-PCT
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. 4. 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.
217 30125-WO-PCT
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: ; bly 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); and ZB 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.
218 30125-WO-PCT
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, and at 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.
219 30125-WO-PCT
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