Conjugation of inhibitory receptor ligands to induce anergy in insulin-binding B cells

Abstract

The present disclosure provides a conjugate, a method for making the conjugate, and uses thereof. The conjugate of the present technology is useful for inducing anergy in insulin-binding B cells. TIFF2024505002000026.tif38128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 142,224, filed January 27, 2021, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present technology generally relates to conjugates, methods of making said conjugates, and uses thereof. The conjugates of the present technology are useful for inducing anergy in insulin-binding B cells. [Background technology]

[0003] background The following description of the background of the present technology is provided merely as an aid for understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Type 1 diabetes (T1D) is an autoimmune disease caused by the loss of self-tolerance to islet antigens and the subsequent T cell-mediated destruction of pancreatic islets, which contain the beta cells responsible for the production of insulin and the regulation of blood glucose. Self-tolerance is the ability of the immune system to recognize self-tissues but not respond inflammatoryly, and is coordinated both centrally and peripherally. Impairment of central tolerance in T1D is indicated by genetic risk factors for certain human leukocyte antigen (HLA) alleles that code for major histocompatibility complex class II (MHC II). These alleles allow MHC II to bind weakly to the insulin T cell epitope, a key antigen, and allow some insulin-responsive T cells to avoid negative selection and clonal deletion within central tolerance. After entering the periphery, autoreactive T cells can be activated to mount an immune response against insulin and attack β-cells (Stadinski, BD et al., Proceedings of the National Academy of Sciences 2010, 107 (24), 10978).

[0005] Failure to maintain peripheral tolerance has also been noted in the pathogenesis of T1D. Although T1D is T cell mediated, antigen presenting cells (APCs) such as B cells play a role in pathogenesis through T cell activation (Creusot, RJ et al., Diabetes 2018, 67 (8), 1481). Autoreactive B cells, although present in the periphery, still help maintain peripheral tolerance by remaining in an anergic or non-responsive state. Transient reduction of insulin-binding anergic B cells correlates with the development of T1D, suggesting that loss of anergic B cells is responsible for the activation and pathogenesis of autoreactive T cells (Smith, MJ et al., Diabetes 2015, 64 (5), 1703). Preservation of peripheral tolerance via B cell anergy has been proposed as a preventative treatment for individuals at high risk of developing T1D.

[0006] Thus, there is a need for treatments and preventions for autoimmune diseases, such as T1D, that avoid the deleterious effects caused by the destruction of healthy cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Stadinski, BD et al., Proceedings of the National Academy of Sciences 2010, 107 (24), 10978 [Non-Patent Document 2] Creusot, RJ et al., Diabetes 2018, 67 (8), 1481 [Non-Patent Document 3] Smith, MJ et al., Diabetes 2015, 64 (5), 1703 Summary of the Invention

[0008] Overview of this technology In one aspect, the present disclosure provides a compound of formula I TIFF2024505002000002.tif69128, or a pharma- ceutically acceptable salt and / or solvate thereof; During the ceremony, R 1 , R 2 , R 3 , and R 4 are each independently TIFF2024505002000003.tif93145; R 5 is independently at each occurrence aryl or aralkyl; R 6 is independently at each occurrence halo, hydroxyl, aryl, or heteroaryl; X 1is independently, at each occurrence, the epsilon nitrogen atom of a lysine of SEQ ID NO:2, or a substitution variant thereof in the first proinsulin polypeptide; X 2 is independently, in each occurrence, the carbonyl carbon of the nitrogen atom of the C-terminal glycine of the sortase moiety, where the sortase moiety is fused to a second proinsulin polypeptide at the C-terminus of the second proinsulin polypeptide; x is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; m is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; n is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and p is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0009] In one aspect, the present disclosure provides compositions comprising a conjugate of any embodiment disclosed herein as well as a pharma- ceutically acceptable carrier or one or more excipients, bulking agents or agents (hereinafter collectively referred to as "pharmaceutically acceptable carriers" unless otherwise indicated and / or detailed).

[0010] In a related aspect, a medicament for treating or preventing autoimmune diabetes in a subject is provided, the medicament comprising a conjugate of any embodiment disclosed herein and, optionally, a pharma- ceutically acceptable carrier.

[0011] In a related aspect, a pharmaceutical composition is provided that includes a pharma- ceutically acceptable carrier and an effective amount of a conjugate of any of the embodiments disclosed herein.

[0012] In one aspect, a method of treating or preventing autoimmune diabetes in a subject in need thereof is provided, comprising administering to the subject an effective amount of a conjugate of any embodiment disclosed herein. [Brief description of the drawings]

[0013] [Figure 1] B cell activation signaling is evoked by antigen binding to the B cell receptor (BCR) and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). The B cell activation inhibitory signaling pathway can be evoked by simultaneous ligation of the BCR together with α2,6-linked sialic acid binding to CD22. This inhibitory signaling pathway blocks B cell activation by dephosphorylating inflammatory signaling proteins. [Diagram 2] Figure 2A: General design of the proinsulin-CD22L conjugate. Figure 2B: Co-ligation of the B cell receptor (BCR) with the immunoinhibitory receptor CD22 to block B cell activation. [Diagram 3]3A-3B show human insulin and human proinsulin variants, respectively. FIG. 3A-3B show the primary, secondary, and tertiary structures and sequences of human insulin and proinsulin variants. Mutations and modifications to human insulin and proinsulin are noted along with residues involved in binding of the hormone insulin receptor (IR-B), T cell epitopes, and important B cell epitopes. The B cell epitopes include amino acid residue 3 of the insulin B chain and amino acid residues 4 and 8-10 of the insulin A chain. The T cell epitopes include amino acid residues 9-23 of the insulin B chain and residues of the C-peptide fragment having QVELGGGPGAGSLQPLALEGSLQKR (SEQ ID NO:7). The proinsulin variants contain substitutions F25D in the insulin B chain and Y19A in the insulin A chain, which reduce affinity for the insulin receptor (IR). Proinsulin variants may further contain substitutions P28K and K29P (lispro), or P28K, K29P and H10D (DKP) in the insulin B chain, which reduce self-association. The sequence of the sortase tag is LPETGGHG (SEQ ID NO:6). Native proinsulin excludes the red eSrtA tag and the F25D mutation. Insulin does not contain a C-peptide. [Figure 4] Figures 4A-4C: Analysis of G3-proinsulin. Similar SDS-PAGE and HPLC analysis results obtained for all proinsulin variants, but omitted for brevity. Figure 4A: SDS-PAGE gel of standard ladder (lane 1), low and high concentration of purified G3-proinsulin (lanes 2, 4), and low and high concentration of unpurified fusion-precursor FP-G3-proinsulin (lanes 3, 5). Figure 4B: HPLC chromatogram of purified G3-proinsulin using a Waters reversed-phase BEH C4 column with a 20 min gradient of 27_40% ACN. The main peak accounts for >90% of the AUC. Figure 4C: Mass spectrum from LC ESI TOF mass spectrometer. The main peak at 9559.7 m / z matches the calculated mass for the [M+H]+ ion of G3-proinsulin. [Diagram 5] Figures 5A-5C: PEGylation of proinsulin via eSrtA ligation and CuAAC to give an alkyne-functionalized species for CuAAC conjugation with azide-functionalized CD22L. Figure 5A: Synthesis of alkyne-PEGn-G3 proinsulin, n=10 kDa or 20 kDa. Figure 5B: Synthesis of alkyne-G3 proinsulin. Figure 5C: Synthesis of alkyne-PEGn-(proinsulin LPETG), n=10 kDa or 20 kDa. [Figure 6] Figures 6A-6C: Proposed synthesis of CD22L-proinsulin conjugates. Figure 6A: Structure of azide-functionalized high affinity, high specificity CD22 ligand. Figure 6B: Synthesis of CD22L-G3 proinsulin. Figure 6C: Synthesis of CD22L-PEGn-G3 proinsulin, where n=10 kDa or 20 kDa. Alternatively, CD22L-PEGn-(proinsulin LPETG) can be similarly prepared by replacing alkyne-PEGn-G3 proinsulin with alkyne-PEGn-(proinsulin LPETG). [Figure 7] Determination of insulin variants binding with anti-insulin antibodies. Binding kinetics data from Biolayer Interferometry (BLI) of insulin variants binding with anti-insulin antibody 125 mAb. [Figure 8] Determination of insulin variant hormone activity. Activation data of the hormone insulin receptor (CD220) from a luciferase-reporter cell line. [Figure 9] Conjugation of insulin variants with functionalized oligoglycines. Site-selective conjugation of insulin variants with functionalized oligoglycines using sortase-mediated ligation for the synthesis of distinct multivalent conjugates. [Figure 10] Determination of 4-arm PEG-insulin binding with anti-insulin antibody. Binding kinetics data from biolayer interferometry (BLI) of 4-arm PEG-insulin, where one to four insulins were conjugated to the 4-arm PEG scaffold. [Figure 11-1]Figure 11: Determination of insulin self-association within 4-arm PEG-insulin. (Top) Representation of the effect of insulin self-association on the near-UV circular dichroism (CD) spectra and the self-association potential of 4-arm PEG-insulin (4). (Bottom) Near-UV CD data for insulin, synthetic intermediates, 4-arm PEG-insulin (1), and 4-arm PEG-insulin (4). [Figure 11-2] See description of Figure 11-1. [Figure 12] Determination of hydrodynamic radius of insulin variants and 4-arm PEG-insulin variants. Hydrodynamic radius data from dynamic light scattering for insulin variants and 4-arm PEG-insulin variants. [Figure 13] Secondary structure determination of insulin variants and 4-arm PEG-insulin variants. Secondary structure data from far-UV circular dichroism (CD) of insulin variants and 4-arm PEG-insulin variants. [Figure 14] Size and heterogeneity determination of insulin variants and 4-arm PEG-insulin variants. Molecular weight estimation and heterogeneity data from size exclusion chromatography (SEC) for insulin variants and 4-arm PEG-insulin variant conjugates. [Figure 15-1] FIG. 15: Determination of insulin variant binding to anti-insulin B cells. Anti-insulin VH125 non-obese diabetic mouse splenocyte binding data from flow cytometry for insulin variants labeled with FITC by site-selective sortase-mediated ligation. [Figure 15-2] See description of Figure 15-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description It should be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in varying levels of detail in order to provide a substantial understanding of the present technology.

[0015] The practice of the subject methods employs many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques (see also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0016] Without wishing to be bound by theory, it appears that reducing the hormonal activity of proinsulin is important to minimize the risk of hypoglycemia while maintaining recognition of proinsulin epitopes by immune receptors. Insulin receptor B is responsible for metabolic regulation and has a 20-30 fold lower affinity for proinsulin than insulin (Malaguarnera, R. et al., Endocrinology 2012, 153, 2152-2163; Belfiore, A. et al., Endocrine Reviews 2017, 38, 379-431). As is evident from Figures 3A-3B, the apparent co-dependence of immune and hormonal recognition on similar regions presents a challenge to selectively reduce hormonal function without adversely affecting epitope recognition.

[0017] The conjugate of the present technology shows reduced hormone function while maintaining the binding kinetics and affinity (<10nM) that was comparable to native human insulin to anti-insulin antibodies.Therefore, the conjugate disclosed herein is useful in methods for inducing anergy in insulin-binding B cells and / or for preventing and / or treating autoimmune diseases (e.g., autoimmune diabetes, e.g., type 1 diabetes).

[0018] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the testing procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are well known and commonly employed in the art. Recitation of ranges of values ​​herein is only intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better clarify the aspects and does not impose limitations on the claims, unless specifically stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0019] As used herein, IC 50 refers to the concentration of a conjugate at which a given biological function or biochemical process (e.g., binds to or induces anergy in insulin-specific B cells) is half-inhibited.

[0020] As used herein, "about" is understood by those of ordinary skill in the art and varies somewhat depending on the context in which it is used. If there are uses of the term that are not clear to those of ordinary skill in the art, given the context in which it is used, "about" means up to plus or minus 10% from the particular term. For example, "about 10 wt.%" is understood to mean "9 wt.% to 11 wt.%". It should be understood that when "about" precedes a term, the term should be interpreted as disclosing not only the term with "about" attached, but also the term unmodified with "about". For example, "about 10 wt.%" not only discloses "9 wt.% to 11 wt.%", but also discloses "10 wt.%".

[0021] The term "and / or" as used in this disclosure is understood to mean any one of the listed members individually or any two or more combinations thereof. For example, "A, B, and / or C" means "A, B, C, A and B, A and C, B and C, or A, B, and C."

[0022] In general, a reference to a particular element, such as hydrogen or H, is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Thus, tritium, C 14 , P 32 and S 35 Compounds containing radioisotopes such as are within the scope of the present technology. Procedures for incorporating such labels into the compounds of the present technology will be readily apparent to those of skill in the art based on the disclosure herein.

[0023] Generally, "substituted" refers to an organic group (e.g., an alkyl group) as defined below in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to a non-hydrogen or non-carbon atom. Substituted groups also include groups in which one or more bonds to a carbon or hydrogen atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents. Examples of substituents include halogens (i.e., F, Cl, Br, and I); hydroxyl; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyl (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e., SF5), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; and nitriles (i.e., CN).

[0024] Substituted ring groups, such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups, also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Thus, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

[0025] Alkyl groups include straight and branched chain alkyl groups having 1-12 carbon atoms, typically 1-10 carbons, or in some embodiments 1-8, 1-6, or 1-4 carbon atoms. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents as described above, including, but not limited to, haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

[0026] Cycloalkyl groups include monocyclic, bicyclic, or tricyclic alkyl groups having 3 to 12 carbon atoms in the ring, or in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Bicyclic and tricyclic systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Substituted cycloalkyl groups may be substituted one or more times with non-hydrogen and non-carbon groups as described above. However, substituted cycloalkyl groups also include rings substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups can be mono- or more than one substituted, for example, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstituted cyclohexyl groups, which can be substituted, but not limited to, with substituents such as those listed above.

[0027] Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced by a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups can be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups can be substituted on the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups can be mono-substituted or substituted more than once, for example, mono-, di-, or tri-substituted with substituents such as, but not limited to, those listed above.

[0028] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond is present between two carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups have 2-12 carbon atoms, typically 2-10 carbons, or in some embodiments, 2-8, 2-6, or 2-4 carbon atoms. In some embodiments, alkenyl groups have 1, 2, or 3 carbon-carbon double bonds. Examples include, but are not limited to, vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, among others. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, for example, mono-, di-, or tri-substituted with substituents such as, but not limited to, those listed above.

[0029] Cycloalkenyl groups include cycloalkyl groups as defined above that have at least one double bond between two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments, cycloalkenyl groups may have one, two or three double bonds, but do not contain aromatic compounds. Cycloalkenyl groups have 4 to 14 carbon atoms, or in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.

[0030] Cycloalkenylalkyl groups are alkyl groups as defined above, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, cycloalkenyl, or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with the substituents as described above.

[0031] Alkynyl groups include straight and branched chain alkyl groups as defined above, except that there is at least one triple bond between two carbon atoms. Alkynyl groups may be substituted or unsubstituted. Alkynyl groups have 2-12 carbon atoms, typically 2-10 carbons, or in some embodiments, 2-8, 2-6, or 2-4 carbon atoms. In some embodiments, alkynyl groups have 1, 2, or 3 carbon-carbon triple bonds. Examples include, but are not limited to, -C≡CH, -C≡CCH3, -CH2C≡CCH3, and -C≡CCH2CH(CH2CH3)2, among others. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted, with substituents such as, but not limited to, those listed above.

[0032] An aryl group is a cyclic aromatic hydrocarbon that does not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic, and tricyclic ring systems. Aryl groups may be substituted or unsubstituted. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons in the ring portion of the group, and in others 6-12 or even 6-10 carbon atoms. In some embodiments, the aryl group is phenyl or naphthyl. The phrase "aryl group" includes groups containing fused rings, such as fused aromatic aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.). Representative substituted aryl groups may be monosubstituted (e.g., tolyl) or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

[0033] An aralkyl group is an alkyl group, as defined above, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to an aryl group, as defined above. An aralkyl group may be substituted or unsubstituted. In some embodiments, an aralkyl group contains 7-16 carbon atoms, 7-14 carbon atoms, or 7-10 carbon atoms. Substituted aralkyl groups may be substituted on the alkyl, aryl, or both the alkyl and aryl portions of the group. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl groups, and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents as described above.

[0034] Heterocyclyl groups include aromatic (also referred to as heteroaryl) and non-aromatic ring compounds containing three or more ring members, one or more of which is a heteroatom, such as, but not limited to, N, O, and S. Heterocyclyl groups may be substituted or unsubstituted. In some embodiments, heterocyclyl groups contain one, two, three, or four heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi-, and tricyclic rings having 3-16 ring members, while other such groups have 3-6, 3-10, 3-12, or 3-14 ring members. Heterocyclyl groups encompass aromatic partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl, and imidazolidinyl groups. The phrase "heterocyclyl group" includes fused ring species, including those containing fused aromatic and nonaromatic groups, such as, for example, benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing heteroatoms, such as, but not limited to, quinuclidyl. The phrase includes heterocyclyl groups having other groups, such as alkyl, oxo, or halo groups, attached to one of the ring members, referred to as "substituted heterocyclyl groups."Heterocyclyl groups include aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, and morpholinyl. , thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, Benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl , naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclyl groups can be 2-, 3-, 4-, 5-, or 6-substituted or disubstituted with a variety of substituents such as those listed above, and can be mono- or more than once substituted, such as, but not limited to, pyridyl or morpholinyl groups.

[0035] Heteroaryl groups are aromatic cyclic compounds containing five or more ring members, one or more of which are heteroatoms, including but not limited to N, O, and S. Heteroaryl groups can be substituted or unsubstituted. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused ring compounds in which all rings are aromatic, such as indolyl groups, and fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydroindolyl groups. Representative substituted heteroaryl groups may be substituted one or more times with a variety of substituents as described above.

[0036] Heterocyclylalkyl groups are alkyl groups as defined above, where a hydrogen or carbon bond of the alkyl group is replaced with a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted on the alkyl, heterocyclyl, or both the alkyl and heterocyclyl portions of the group. Representative heterocyclylalkyl groups include, but are not limited to, morpholin-4-yl-ethyl, furan-2-yl-methyl, imidazol-4-yl-methyl, pyridin-3-yl-methyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those described above.

[0037] Heteroaralkyl groups are alkyl groups as defined above, where a hydrogen or carbon bond of the alkyl group is replaced with a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted at the alkyl, heteroaryl, or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those described above.

[0038] Within the compounds of the present technology, groups described herein that have two or more attachment points (i.e., divalent, trivalent, or polyvalent) are named by using the suffix "ene". For example, a divalent alkyl group is an alkylene group, a divalent aryl group is an arylene group, a divalent heteroaryl group is a divalent heteroarylene group, and so on. Substituted groups that have a single attachment point to the compounds of the present technology are named without using the name "ene". Thus, for example, chloroethyl is not referred to herein as chloroethylene.

[0039] An alkoxy group is a hydroxyl group (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. An alkoxy group may be substituted or unsubstituted. Examples of straight-chain alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Representative substituted alkoxy groups may be substituted one or more times with substituents as described above.

[0040] The terms "alkanoyl" and "alkanoyloxy" as used herein can refer to -C(O)-alkyl and -OC(O)-alkyl groups, respectively, containing 2 to 5 carbon atoms. Similarly, "aryloyl" and "aryloyloxy" refer to -C(O)-aryl and -OC(O)-aryl groups.

[0041] The terms "aryloxy" and "arylalkoxy" refer to a substituted or unsubstituted aryl group bonded to an oxygen atom, and a substituted or unsubstituted aralkyl group bonded to an oxygen atom at an alkyl, respectively. Examples include, but are not limited to, phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.

[0042] As used herein, the term "carboxylate" refers to a -COOH group.

[0043] As used herein, the term "ester" refers to an ester of -COOR 70and the -C(O)OG group. 70 is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. G is a carboxylate protecting group. Carboxylate protecting groups are well known to those skilled in the art. An extensive list of protecting groups for the carboxylate functionality can be found in Protective Groups in Organic Synthesis, Greene, TW; Wuts, PGM, John Wiley & Sons, New York, NY, (3rd Edition, 1999), which can be added or removed using the procedures set forth therein, and which is incorporated herein by reference in its entirety for all purposes as if set forth herein in its entirety.

[0044] The term "amide" (or "amido") includes C- and N-amide groups, i.e., -C(O)NR, respectively. 71 R 72 , and -NR 71 C(O)R 72 R 71 and R 72 are independently hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Amide groups thus include, but are not limited to, carbamoyl groups (-C(O)NH2) and formamide groups (-NHC(O)H). In some embodiments, an amide is -NR 71 C(O)-(C 1~5 In some cases the amide is --NHC(O)-alkyl, and the group is termed "carbonylamino", in others the amide is --NHC(O)-alkyl, and the group is termed "alkanoylamino".

[0045] As used herein, the term "nitrile" or "cyano" refers to a --CN group.

[0046] The urethane group includes N- and O-urethane groups, i.e., -NR 73 C(O)OR 74 and -OC(O)NR 73 R 74 R 73 and R 74 R is independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. 73 can also be H.

[0047] As used herein, the term "amine" (or "amino") refers to -NR 75 R 76 In this case, R 75 and R 76 are independently hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl; or heterocyclyl group as defined herein. In some embodiments, the amine is an alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.

[0048] The term "sulfonamide" includes S- and N-sulfonamide groups, i.e., -SONR, respectively. 78 R 79 and -NR 78 SO2R 79 R 78 and R 79are independently hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Thus, sulfonamide groups include, but are not limited to, sulfamoyl groups (-SO2NH2). In some embodiments herein, the sulfonamide is -NHSO2-alkyl, and is referred to as an "alkylsulfonylamino" group.

[0049] The term "thiol" refers to the -SH group, whereas "sulfide" has the -SR 80 groups, and "sulfoxide" includes the -S(O)R 81 The term "sulfone" includes the group -SO2R 82 groups, and "sulfonyl" includes -SOOR 83 Contains: R 80 , R 81 , R 82 , and R 83 is each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein. In some embodiments, the sulfide is an alkylthio group, -S-alkyl.

[0050] The term "urea" means -NR 84 -C(O)-NR 85 R 86 R 84 , R 85 , and R 86 The groups are independently hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0051] The term "amidine" refers to -C(NR 87 )NR 88 R 89 and -NR 87 C(NR 88 )R89 In this case, R 87 , R 88 , and R 89 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0052] The term "guanidine" means -NR 90 C(NR 91 )NR 92 R 93 In this case, R 90 , R 91 , R 92 and R 93 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0053] The term "enamine" refers to -C(R 94 )=C(R 95 )NR 96 R 97 and -NR 94 C(R 95 )=C(R 96 )R 97 In this case, R 94 , R 95 , R 96 and R 97 are each independently hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0054] As used herein, the term "halogen" or "halo" refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0055] As used herein, the term "hydroxyl" refers to either -OH or its ionized form, -O - A "hydroxyalkyl" group is a hydroxyl-substituted alkyl group such as HO-CH2-.

[0056] The term "imide" means -C(O)NR 98 C(O)R 99 In this case, R 98 and R 99 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0057] The term "imine" means -CR 100 (NR 101 ) and -N(CR 100 R 101 ) group, where R 100 and R 101 are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein, with the proviso that R 100 and R 101 can never both be hydrogen at the same time.

[0058] As used herein, the term "nitro" refers to the group --NO.sub.2.

[0059] As used herein, the term "trifluoromethyl" refers to --CF.

[0060] As used herein, the term "trifluoromethoxy" refers to --OCF.

[0061] The term "azido" refers to -N3.

[0062] The term "trialkylammonium" refers to a -N(alkyl)3 group. Trialkylammonium groups are positively charged and therefore typically have an associated anion, such as a halogen anion.

[0063] The term "isocyano" refers to --NC.

[0064] The term "isothiocyano" refers to --NCS.

[0065] The term "pentafluorosulfanyl" refers to -SF5.

[0066] As will be understood by those skilled in the art, virtually all ranges disclosed herein, especially with respect to the provision of a written description, also encompass any and all possible subranges and combinations of subranges thereof. It can be easily recognized that any range described fully describes and allows for the same range to be equally divided into at least one half, one third, one quarter, one fifth, one tenth, etc. As a non-limiting example, each range described herein can be easily divided into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all phrases such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited numbers and that can be subsequently divided into the subranges described above. Finally, as will be understood by those skilled in the art, ranges include individual members. Thus, for example, a group having 1-3 atoms refers to a group having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to a group having 1, 2, 3, 4, or 5 atoms, etc.

[0067] As will be appreciated by those of skill in the art, "molecular weight" (also known as "relative molar mass") is a dimensionless quantity, but is converted to molecular mass by multiplying by 1 gram / mole or by multiplying by 1 Da, e.g., a compound with a weight average molecular weight of 5,000 has a weight average molar mass of 5,000 g / mol and a weight average molar mass of 5,000 Da.

[0068] Pharmaceutically acceptable salts of the compounds described herein are within the scope of the present technology, including acid or base addition salts that retain the desired pharmacological activity and are not biologically undesirable (e.g., the salts are not overly toxic, allergenic, or irritating and are bioavailable). When the compound of the present technology has a basic group, such as an amino group, the pharma- ceutically acceptable salt can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (such as alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as a carboxylic acid group, it can be formed with metals, such as alkali metals and alkaline earth metals (e.g., Na + , Li + , K + , Ca 2+ , Mg 2+ , Zn 2+ ), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during the isolation and purification of the compounds, or by separately reacting the purified compounds in their free base or free acid form with the appropriate acid or base, respectively, and isolating the salt thus formed.

[0069] Those skilled in the art recognize that the compounds of the present technology may exhibit the phenomenon of tautomerism, conformational isomerism, geometric isomerism, and / or stereoisomerism.The formula drawings in the specification and claims can only represent one of possible tautomerism, conformational isomerism, stereochemistry or geometric isomerism, so it should be understood that the present technology encompasses not only any tautomerism, conformational isomerism, stereochemistry and / or geometric isomerism of the compounds having one or more of the utilities described herein, but also the mixture of these various different forms.

[0070] "Tautomer" refers to isomeric forms of a compound that are in equilibrium with each other. The presence and / or concentration of isomeric forms depends on the environment in which the compound is found, and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in an aqueous solution, quinazolinone can exist in the following isomeric forms, referred to as tautomers with each other: In another example, guanidine can exist in protic organic solutions in the following isomeric forms, also referred to as tautomers: TIFF2024505002000005.tif30128. Due to the limitations of representing compounds as structural formulas, it should be understood that all chemical formulas of compounds described herein represent all tautomeric forms of the compounds and are within the scope of the present technology.

[0071] Stereoisomers (also known as optical isomers) of a compound include all chiral, diastereomeric, and racemic forms of a structure unless a specific stereochemistry is explicitly indicated. Thus, as is clear from the depiction, the compounds used in the present technology include enriched or resolved optical isomers at any and all asymmetric atoms. Both racemic and diastereomeric mixtures, as well as individual optical isomers, can be isolated or synthesized substantially free of their enantiomeric or diastereomeric partners, and all of these stereoisomers are within the scope of the present technology.

[0072] The compounds of the present technology can exist as solvates, particularly hydrates.Hydrates may form during the preparation of the compounds or compositions containing the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds.The compounds of the present technology can also exist as organic solvent solvates, including DMF, ether, and alcohol solvates, among others.The identification and preparation of any particular solvate is within the skill of those skilled in the art of synthetic organic chemistry or medicinal chemistry.

[0073] As used herein, "administration" of an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, or topical. Administration includes self-administration and administration by another.

[0074] As used herein, "anergy" refers to the lack of response by the body's defense mechanisms to foreign substances, with the direct induction of peripheral lymphocyte tolerance. Individuals in an anergic state often exhibit an inability of the immune system to mount a normal immune response to a specific antigen, usually a self-antigen. Lymphocytes are said to be anergic when they do not respond to their specific antigen. Anergy thus induces tolerance by modifying the immune system to prevent self-destruction.

[0075] As used herein, the term "autoantibody" refers to an antibody that targets and / or reacts with one or more of an individual's own proteins, cells, tissues, or organs. As used herein, the term "autoantigen" refers to an antigen that is composed of normal tissues, cells, proteins, peptides, or DNA that is the target of an immune response (e.g., humoral or cellular immune response). Autoantigens may be targeted or reacted with by autoantibodies in the case of autoimmune diseases.

[0076] As used herein, "autoimmune diabetes" refers to diabetes characterized by the destruction of insulin-producing beta cells of the pancreas.

[0077] As used herein, the term "biological sample" refers to sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from eye, breast tissue, kidney tissue, cervix, endometrium, head and neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsils, thymus, blood, hair, oral cavity, skin, serum, plasma, CSF, semen, prostatic fluid, seminal plasma, urine, stool, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsy of internal organs. Biological samples can be obtained from subjects for diagnosis or research, or from non-diseased individuals as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, biological samples are tissue samples obtained by needle biopsy.

[0078] As used herein, the term "cell surface receptor" refers to a molecule, such as a protein, that is generally found on the outer surface of a cell membrane and interacts with soluble molecules circulating, for example, in the blood supply. Cell surface receptors may also be secreted in soluble form into the extracellular space or may be shed from the outer surface of the cell. In some embodiments, cell surface receptors may include antigens or antigen receptors. In other embodiments, B lymphocytes, also named B cells, have a cell surface receptor called the "B cell receptor", or "BCR", or sometimes the "IgM" receptor.

[0079] As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive treatment or receives a placebo) are typically employed.

[0080] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount resulting in the prevention or reduction of a disease or condition described herein, or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics such as general health, age, sex, weight, and drug tolerance. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition can be administered to a subject having one or more signs or symptoms of a disease or condition described herein (e.g., autoimmune diabetes, e.g., type 1 diabetes). As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. The therapeutically effective amount can be given in one or more administrations. As used herein, a "prophylactically effective amount" of a composition refers to a level of the composition that prevents or delays the onset of at least one symptom of a disease or condition described herein. A prophylactically effective amount can be given in one or more administrations.

[0081] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce a mature mRNA; mRNA stability; translation of mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if necessary for proper expression and function.

[0082] As used herein, the terms "individual," "patient," or "subject" can be an individual organism, vertebrate, mammal, or human. In some embodiments, the individual, patient, or subject is a human.

[0083] As used herein, the term "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption retarding compounds, etc., that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Examples of pharmaceutical acceptable carriers include liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, talc, magnesium stearate, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulating materials that are useful for introducing active agents into the body.

[0084] As used herein, "prevention", "preventing" or "preventing" of a disorder or condition refers to one or more compounds that reduce the occurrence of a disorder or condition in a treated sample compared to an untreated control sample, or delay the occurrence of one or more symptoms of a disorder or condition in a statistical sample compared to an untreated control sample. As used herein, preventing an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) includes preventing or delaying the onset of symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes). As used herein, preventing an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) also includes preventing the recurrence of one or more signs or symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes).

[0085] As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes.

[0086] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration route is the same or different.More specifically, sequential use refers to the full administration of one of the active ingredients before the administration of one or more other active ingredients begins.Therefore, it is possible to administer one of the active ingredients for several minutes, several hours, or several days before administering the other active ingredient or ingredients.In this case, there is no simultaneous treatment.

[0087] As used herein, the term "concurrent" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by the same route.

[0088] As used herein, "treating" or "treatment" refers to the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., preventing its occurrence; (ii) relieving the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in remission.

[0089] It should also be appreciated that the various modes of treatment of disorders described herein are intended to mean "substantially," including not only total treatment but also less than total treatment, in which some biologically or medically relevant result is achieved. Treatment can be continuous long-term treatment for chronic diseases, or single or few doses for treatment of acute conditions.

[0090] Insulin Insulin is a peptide hormone produced by beta cells in the islets of Langerhans in the pancreas. Insulin functions by regulating the absorption of glucose from the blood. Upon exposure to stimuli such as increased protein and glucose levels, insulin is released from beta cells, binds to the insulin receptor, and initiates a signaling cascade that affects many aspects of human metabolism. Disruption of this process is directly related to several diseases, autoimmune diabetes (e.g., type 1 diabetes), insulinoma, insulin resistance, metabolic syndrome, and polycystic ovarian syndrome. The amino acid sequence of insulin is highly conserved throughout evolution, especially in vertebrates, and consists of two polypeptide chains, designated A and B chains, linked via disulfide bonds.

[0091] Insulin is initially synthesized as an inactive precursor called preproinsulin. Through a series of highly coordinated enzymatic regulatory steps, preproinsulin is converted to mature insulin. Cleavage of the signal peptide of preproinsulin in the endoplasmic reticulum, followed by oxidation and chaperone-mediated folding, yields proinsulin, which is transported to the trans-Golgi network. Proinsulin then undergoes proteolytic processing steps that result in the release of a fragment called the C-peptide and the formation of mature insulin, which is converted to insulin by the action of zinc (Zn 2+ ) and calcium (Ca 2+ After exposure to a stimulus, the secretory vesicles fuse with the plasma membrane, releasing insulin and promoting the dissociation of the hexamers into active insulin monomers.

[0092] Throughout this disclosure, various publications, patents and published patent specifications are referenced by identifying citations. Also within this disclosure are Arabic numerals that refer to the referenced citations, the full bibliographic details of which are provided after the Examples section. The disclosures of these publications, patents and published patent specifications are incorporated by reference into this disclosure to more fully describe the present technology.

[0093] type 1 diabetes Lifelong insulin replacement therapy is the current treatment for T1D and requires consistent blood glucose monitoring. Unfortunately, this approach fails to fully regulate blood glucose, resulting in chronic hyperglycemia that increases the risk of renal, neurological and cardiac disease. Large-scale clinical trials have demonstrated that the implementation of intensive blood glucose regulation that minimizes chronic hyperglycemia reduced the risk of these comorbidities (Group, TDC a. CTR, New England Journal of Medicine 1993, 329 (14), 977-986). Thus, treatments that can maintain or restore remaining beta cell function to help regulate blood glucose should improve patient outcomes.

[0094] Immunotherapies are being evaluated in T1D clinical trials with the goal of maintaining or restoring β-cell function. The ideal immunotherapy for T1D would be antigen-specific to anergize or delete autoreactive B and T cells without compromising infection defenses. Preserving infection defenses is particularly relevant for T1D because the disease begins primarily in children and insulin replacement therapy is relatively safe. Thus, treatments that rely on comprehensive immunosuppression have a higher level of risk, especially if they require chronic administration. Unfortunately, the most practical antigen-specific approach, antigen administration at mucosal surfaces, has failed to induce sufficient tolerance to preserve β-cell function in numerous clinical trials (Jacobsen, L. M et al., Current Diabetes Reports 2018, 18 (10), 90). Therefore, investigating new approaches to restore tolerance in an antigen-specific manner is justified.

[0095] Although not ideal, a number of comprehensive immunosuppressive therapies are being studied in clinical trials, providing insight into potential targets for antigen-specific approaches. The B-cell-depleting anti-CD20 antibody rituximab demonstrated beta-cell preservation with increased endogenous insulin production, reduced need for exogenous insulin, and reduced hemoglobin A1C at 1 year (Bluestone, JA et al. Nature 2010, 464 (7293), 1293-1300). It is hypothesized that this outcome results in the loss of autoreactive B cells that promote the disease, either by secreting inflammatory cytokines into the pancreas or by presenting self-antigens to T cells.

[0096] Conjugates of this technology The conjugates of the present technology are based on the premise that B cells actively contribute to T1D and therefore may be targeted to induce antigen-specific tolerance.

[0097] Antigen specificity of B cells is determined by the membrane-anchored antibody-like B cell receptor (BCR). A unique receptor on B cells has been identified that shuts down inflammatory signaling pathways and promotes B cell anergy when colocalized with the BCR (Figure 1). This immune response inhibitory receptor is CD22, a sialic acid-binding Ig-like lectin (siglec) receptor with binding preference for α2,6-linked sialylated glycans. This receptor has been implicated as functioning as a self-versus-nonself recognition mechanism, since many mammalian proteins, unlike bacterial and yeast proteins, have sialic acid-terminal glycans. The inflammatory inhibitory response from CD22 is generated by its intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Conjugation of both BCR and CD22 with dinitrophenol (DNP) and multivalent polymers displaying high affinity CD22 ligands has been shown to prevent increases in calcium flux, reduce phosphorylation of immune activation response pathway proteins, and stimulate endocytosis of BCR in DNP-specific B cell lines and mice.

[0098] Proinsulin variants Native proinsulin comprises the insulin B chain, insulin C chain, and insulin A chain. As an example, the complete amino acid sequence of native human proinsulin is: The file is TIFF2024505002000006.tif12158.

[0099] The native amino acid sequences of each of these chains are provided below. Insulin B chain: TIFF2024505002000007.tif4128Insulin C-chain: TIFF2024505002000008.tif4128Insulin A chain: TIFF2024505002000009.tif4128

[0100] In one aspect, the present disclosure provides a compound of formula I TIFF2024505002000010.tif69128, or a pharma- ceutically acceptable salt and / or solvate thereof; During the ceremony, R 1 , R 2 , R 3 , and R 4 are each independently TIFF2024505002000011.tif94146; R 5 is independently at each occurrence aryl or aralkyl; R 6 is independently at each occurrence halo, hydroxyl, aryl, or heteroaryl; X 1 is independently, at each occurrence, the epsilon nitrogen atom of a lysine of SEQ ID NO:2, or a substitution variant thereof in the first proinsulin polypeptide; X 2is independently, in each occurrence, the carbonyl carbon of the nitrogen atom of the C-terminal glycine of the sortase moiety, where the sortase moiety is fused to a second proinsulin polypeptide at the C-terminus of the second proinsulin polypeptide; x is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; m is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; n is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and p is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. For clarity, a representation of the amino acid L-lysine is provided below with the ε-nitrogen atom displayed. TIFF2024505002000012.tif35128

[0101] The conjugates of the present technology may comprise a proinsulin polypeptide fused to a sortase moiety, where the proinsulin polypeptide comprises an insulin B chain, an insulin C chain, and an insulin A chain. The sortase moiety may be located at the N-terminus or C-terminus of the proinsulin polypeptide. Additionally or alternatively, in some embodiments of the conjugates disclosed herein, the sequence of the sortase moiety is LPETGGHG (SEQ ID NO:6).

[0102] Additionally or alternatively, in some embodiments of the conjugates disclosed herein, the proinsulin polypeptide comprises an F25D substitution in the insulin B chain (SEQ ID NO:2) and / or a Y19A or Y19L substitution in the insulin A chain (SEQ ID NO:4).

[0103] Additionally or alternatively, in some embodiments of the conjugates disclosed herein, the proinsulin polypeptide comprises an H10D substitution in the insulin B chain (SEQ ID NO:2). Additionally or alternatively, in certain embodiments of the conjugates disclosed herein, the proinsulin polypeptide comprises both a P28K and a K29P substitution in the insulin B chain (SEQ ID NO:2). In some embodiments of the conjugates disclosed herein, the proinsulin polypeptide comprises a P28K, a K29P, and an H10D substitution in the insulin B chain (SEQ ID NO:2). The proinsulin polypeptide may or may not comprise a signal peptide sequence. The signal peptide sequence may be a native or engineered signal peptide sequence. In certain embodiments, the signal peptide sequence is MGSSHHHHHHSSFLDPVLM (SEQ ID NO:5).

[0104] Additionally or alternatively, in some embodiments of the conjugates disclosed herein, the proinsulin polypeptide is The native proinsulin sequence ( β , C-, Alpha Peptide )(SEQ ID NO:8) TIFF2024505002000013.tif19144X38(B25-Asp) Proinsulin ( β , C-, Alpha Peptide , positional variants in bold) (SEQ ID NO:9) TIFF2024505002000014.tif19145A19-LeuProinsulin( β , C-, Alpha Peptide , positional variants in bold) (SEQ ID NO:10) It comprises an amino acid sequence selected from TIFF2024505002000015.tif19144.

[0105] Additionally or alternatively, in some embodiments of the conjugates disclosed herein, the proinsulin polypeptide exhibits reduced activation of the hormone insulin receptor (IR) compared to native human insulin.

[0106] In any embodiment disclosed herein, the first proinsulin polypeptide may be fused to a sortase moiety, wherein the first proinsulin polypeptide comprises an insulin B chain of SEQ ID NO:2, an insulin C chain of SEQ ID NO:3, and an insulin A chain of SEQ ID NO:4. In any embodiment disclosed herein, the sortase moiety may be located at the N-terminus or C-terminus of the first proinsulin polypeptide. In any embodiment disclosed herein, the sequence of the sortase moiety may be SEQ ID NO:6. In any embodiment disclosed herein, the first proinsulin polypeptide may comprise an F25D substitution in the insulin B chain of SEQ ID NO:2 and / or an Y19A or Y19L substitution in the insulin A chain of SEQ ID NO:4. In any embodiment disclosed herein, the first proinsulin polypeptide may comprise an H10D substitution in the insulin B chain of SEQ ID NO:2. In any embodiment disclosed herein, the first proinsulin polypeptide may comprise both P28K and K29P substitutions in the insulin B chain of SEQ ID NO:2. In any embodiment disclosed herein, the first proinsulin polypeptide may comprise P28K, K29P, and H10D substitutions in the insulin B chain of SEQ ID NO:2. In any embodiment disclosed herein, the first proinsulin polypeptide may comprise a signal peptide sequence. In any embodiment disclosed herein, the signal peptide sequence may be a native or engineered signal peptide sequence. In any embodiment disclosed herein, the signal peptide sequence may be SEQ ID NO:5.

[0107] In any embodiment disclosed herein, the second proinsulin polypeptide may comprise an F25D substitution in the insulin B chain of SEQ ID NO:2 and / or a Y19A or Y19L substitution in the insulin A chain of SEQ ID NO:4. In any embodiment disclosed herein, the second proinsulin polypeptide may comprise an H10D substitution in the insulin B chain of SEQ ID NO:2. In any embodiment disclosed herein, the second proinsulin polypeptide may comprise both a P28K and a K29P substitution in the insulin B chain of SEQ ID NO:2. In any embodiment disclosed herein, the second proinsulin polypeptide may comprise a P28K, a K29P, and a H10D substitution in the insulin B chain of SEQ ID NO:2. In any embodiment disclosed herein, the second proinsulin polypeptide may comprise a signal peptide sequence. In any embodiment disclosed herein, the signal peptide sequence may be a native or engineered signal peptide sequence. In any of the embodiments disclosed herein, the signal peptide sequence can be SEQ ID NO:5. In any of the embodiments disclosed herein, the first proinsulin polypeptide and / or the second proinsulin polypeptide can comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:8-10. In any of the embodiments disclosed herein, the first proinsulin polypeptide and the second proinsulin polypeptide can be the same sequence.

[0108] In any embodiment disclosed herein, the conjugate may have a volume weighted mean diameter of about 2 nm to about 15 nm as measured by dynamic light scattering. Thus, in any embodiment disclosed herein, the conjugate may have a volume weighted mean diameter of about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, or any range including and / or between any two of these values ​​as measured by dynamic light scattering. For example, in any embodiment disclosed herein, the conjugate may have a volume weighted mean diameter of about 4 nm to about 8 nm as measured by dynamic light scattering.

[0109] In any embodiment disclosed herein, the conjugate may have a number average molecular weight of about 10,000 to about 150,000. Thus, in any embodiment disclosed herein, the conjugate may have a number average molecular weight of about 10,000, about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, or any range including and / or between any two of these values. For example, in any embodiment disclosed herein, the conjugate may have a number average molecular weight of about 40,000 to about 80,000.

[0110] As mentioned above, R 5 is independently, at each occurrence, aryl or aralkyl; R 6 is independently at each occurrence halo, hydroxyl, aryl, or heteroaryl. See, e.g., Collins, BE et al. J Immunol 2006, 177, 2994-3003; Rillahan, CD et al. Chem. Sci. 2014, 5, 2398-2406; U.S. Patent No. 9,981,023. In any embodiment disclosed herein, R 6may independently at each occurrence be halo or hydroxyl. In any embodiment disclosed herein, R 5 is, independently in each case, It could be TIFF2024505002000016.tif20128.

[0111] Therapeutic and preventive methods of the present technology The following description is offered by way of example only and is not intended to be limiting.

[0112] Described herein is a method for treating or preventing an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) and / or inducing anergy in insulin-binding B cells, comprising administering to a subject a conjugate described herein. In some embodiments of the methods disclosed herein, the autoimmune disease is autoimmune diabetes, e.g., diabetes type 1 (i.e., type 1 diabetes (T1D), juvenile diabetes, or insulin-dependent diabetes), or latent autoimmune diabetes in adults (LADA). LADA, also referred to as slow onset type 1 diabetes, is a form of diabetes type 1 that occurs in adults and has a slower developmental course. It is estimated that up to about 50% of adults diagnosed with non-obesity-related type 2 diabetes may have LADA. In some embodiments of the methods disclosed herein, the autoimmune disease comprises a decrease in the number of insulin-producing beta cells in the pancreas in the subject compared to a reference standard or normal control subject.

[0113] In some embodiments of the methods disclosed herein, the subject has been diagnosed with an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes). In certain embodiments, the subject has been diagnosed with an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) for less than 3 months, less than 6 months, less than 9 months, less than 1 year, or less than 1.5 years.

[0114] Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject has detectable levels of autoimmune antibodies but does not develop symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes), such as hyperglycemia. The autoimmune antibodies can be specific for islet autoantigens. For example, in some embodiments, the islet autoantigen comprises insulin. In other embodiments, the autoimmune antibodies are anti-insulin antibodies.

[0115] In certain aspects of the methods disclosed herein, the subject does not have detectable levels of insulin autoantibodies and does not develop symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes), e.g., does not develop hyperglycemia.

[0116] In some embodiments of the methods disclosed herein, the subject has detectable levels of pathogenic or disease-causing B cell populations (e.g., anti-insulin B cells, insulin-specific B cells, or insulin-specific B cells). + B cells) and do not develop symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes), e.g., do not develop hyperglycemia.

[0117] In certain aspects of the methods disclosed herein, the subject is diagnosed with a detectable level of a pathogenic or disease-causing B cell population (e.g., anti-insulin B cells, insulin-specific B cells, or insulin-specific B cells). + B cells) but are not developing symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes), e.g., are not developing hyperglycemia. In some embodiments, the B cell population comprises insulin-specific B cells. In some embodiments, the insulin-specific B cells display specific cell surface receptors, such as B220, CD19, CD20, CD22, or other similar cell surface receptors and isoforms thereof. In certain embodiments, the insulin-specific B cells express two or more cell surface receptors, such as B220, CD19, CD20, CD22, and other similar cell surface receptors and isoforms thereof.

[0118] Additionally or alternatively, in some embodiments of the methods disclosed herein, the level of endogenous C-peptide in a subject is evaluated to aid in the diagnosis and extent of autoimmune diabetes (e.g., type 1 diabetes or latent autoimmune diabetes). C-peptide is often utilized as a biomarker for residual beta cell function, since it is produced in amounts equivalent to insulin and therefore can represent total insulin secretion in a subject (Jones, AG and Hattersley, AT, Diabet Med (2013) 30, 803-817). Measurement of C-peptide in a subject can be useful in directing the determination of insulin levels, since any exogenous insulin present in the subject is also detected in a direct insulin assay. Healthy subjects (e.g., subjects without autoimmune diabetes) have endogenous C-peptide levels in the range of about 0.6 nmol / L to about 0.8 nmol / L (e.g., about 0.65 nmol / L). In contrast, subjects with autoimmune diabetes (eg, type 1 diabetes or LADA) have endogenous C-peptide levels ranging from undetectable to about 0.6 nmol / L (eg, about 0.05 nmol / L).

[0119] In some embodiments, the subject has been diagnosed with autoimmune diabetes (e.g., type 1 diabetes) and has, for example, a serum IL-1 level of about 0.01 nmol / L, about 0.02 nmol / L, about 0.03 nmol / L, about 0.04 nmol / L, about 0.05 nmol / L, about 0.06 nmol / L, about 0.07 nmol / L, about 0.08 nmol / L, about 0.09 nmol / L, about 0.1 nmol / L, about 0.125 nmol / L, about 0.15 nmol / L, about 0.175 nmol / L, about 0.2 nmol / L, about 0.3 nmol / L, about 0.4 nmol / L, or about 0.5 nmol / L, or about 0.6 nmol / L, or about 0.7 nmol / L, or about 0.8 nmol / L, or about 0.9 nmol / L, or about 0.1 nmol / L, or about 0.2 nmol / L, or about 0.3 nmol / L, or ... or has an endogenous C-peptide level of less than about 0.5 nmol / L, or about 0.01 nmol / L, about 0.02 nmol / L, about 0.03 nmol / L, about 0.04 nmol / L, about 0.05 nmol / L, about 0.06 nmol / L, about 0.07 nmol / L, about 0.08 nmol / L, about 0.09 nmol / L, about 0.1 nmol / L, about 0.125 nmol / L, about 0.15 nmol / L, about 0.175 nmol / L, about 0.2 nmol / L, about 0.3 nmol / L, about 0.4 nmol / L, or about 0.5 nmol / L, or less than 0.6 nmol / L.

[0120] In some embodiments, the subject has been diagnosed with autoimmune diabetes (e.g., type 1 diabetes) and has, for example, an autoimmune level of about 0.1 nmol / L, about 0.09 nmol / L, about 0.08 nmol / L, about 0.07 nmol / L, about 0.06 nmol / L, about 0.05 nmol / L, about 0.04 nmol / L, about 0.03 nmol / L, about 0.02 nmol / L, or about 0.2 nmol / L prior to treatment with a conjugate described herein. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 108, 109, 108, 109, 101, 108, 109, 101, 110, 111, 120, 121, 122, 123, 124 In some embodiments, the subject has been diagnosed with autoimmune diabetes (e.g., type 1 diabetes) and has, for example, an autoimmune inflammatory response (EGF) level of about 0.01 nmol / L, about 0.009 nmol / L, about 0.008 nmol / L, about 0.007 nmol / L, about 0.006 nmol / L, about 0.005 nmol / L, about 0.004 nmol / L, about 0.003 nmol / L, about 0.002 nmol / L, or about 0.01 nmol / L, or about 0.009 nmol / L, or about 0.008 nmol / L, or about 0.007 nmol / L, or about 0.006 nmol / L, or about 0.005 nmol / L, or about 0.004 nmol / L, or about 0.003 nmol / L, or ... , or less than about 0.001 nmol / L, or about 0.01 nmol / L, about 0.009 nmol / L, about 0.008 nmol / L, about 0.007 nmol / L, about 0.006 nmol / L, about 0.005 nmol / L, about 0.004 nmol / L, about 0.003 nmol / L, about 0.002 nmol / L, about 0.001 nmol / L, or less. In some embodiments, the subject has been diagnosed with autoimmune diabetes (e.g., type 1 diabetes) and has endogenous C-peptide levels of less than about 0.001 nmol / L, about 0.1 pmol / L, about 0.01 pmol / L, about 0.001 pmol / L, or less than about 0.001 nmol / L, about 0.1 pmol / L, about 0.01 pmol / L, about 0.001 pmol / L, or less, e.g., prior to treatment with a conjugate described herein.In certain embodiments, the subject has been diagnosed with autoimmune diabetes (e.g., type 1 diabetes), e.g., has undetectable levels of endogenous C-peptide prior to treatment with a conjugate described herein.

[0121] In some embodiments, the subject has been diagnosed with autoimmune diabetes (e.g., type 1 diabetes) and has an endogenous C-peptide level of about 95% or less compared to a reference standard, e.g., prior to treatment with a conjugate described herein. In some embodiments, the subject has been diagnosed with autoimmune diabetes (e.g., type 1 diabetes) and has an endogenous C-peptide level of about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, or about 1% or less compared to a reference standard.

[0122] In some embodiments, endogenous C-peptide levels can be measured in subjects in a fasting (e.g., glucose-deprived) or fed (e.g., glucose-stimulated) state.For example, a fasting subject can fast for about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 18 hours, about 20 hours, or about 24 hours before the analysis of endogenous C-peptide levels.In another example, a fed subject can consume a meal within 12 hours, 10 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, 30 minutes, 15 minutes, or simultaneously with the analysis of endogenous C-peptide levels.

[0123] In some embodiments, the subject is fasted (e.g., glucose deprived) and has undetectable levels of endogenous C-peptide, e.g., prior to treatment with a conjugate described herein. In some embodiments, the subject is fasted (e.g., glucose deprived) and has endogenous C-peptide levels of less than or about 0.06 nmol / L, e.g., prior to treatment with a conjugate described herein. In some embodiments, the subject is in a fasted (e.g., glucose deprived) state, e.g., has a glucose level of about 0.06 nmol / L, about 0.07 nmol / L, about 0.08 nmol / L, about 0.09 nmol / L, about 0.1 nmol / L, about 0.125 nmol / L, about 0.15 nmol / L, about 0.175 nmol / L, about 0.2 nmol / L, about 0.3 nmol / L, about 0.4 nmol / L, about 0.5 nmol / L, about 0.6 nmol / L, about 0.7 nmol / L, about 0.8 nmol / L, about 0.9 nmol / L, about have an endogenous C-peptide level of less than 1.0 nmol / L or more, or of about 0.06 nmol / L, about 0.07 nmol / L, about 0.08 nmol / L, about 0.09 nmol / L, about 0.1 nmol / L, about 0.125 nmol / L, about 0.15 nmol / L, about 0.175 nmol / L, about 0.2 nmol / L, about 0.3 nmol / L, about 0.4 nmol / L, about 0.5 nmol / L, about 0.6 nmol / L, about 0.7 nmol / L, about 0.8 nmol / L, about 0.9 nmol / L, about 1.0 nmol / L or more. In some embodiments, the subject is fasted and has an endogenous C-peptide level of less than or about 0.2 nmol / L, or less than or about 0.5 nmol / L, or less than or about 1.0 nmol / L, e.g., prior to treatment with a conjugate described herein.

[0124] In some embodiments, the subject is fed (e.g., glucose stimulated) and has undetectable levels of endogenous C-peptide, e.g., prior to treatment with a conjugate described herein. In some embodiments, the subject is fed (e.g., glucose stimulated) and has endogenous C-peptide levels of less than or about 0.2 nmol / L, e.g., prior to treatment with a conjugate described herein. In other embodiments, the subject is in a fed (e.g., glucose stimulated) state and has an endogenous C-peptide level of less than about 0.2 nmol / L, about 0.25 nmol / L, about 0.3 nmol / L, about 0.4 nmol / L, about 0.5 nmol / L, about 0.6 nmol / L, about 0.7 nmol / L, about 0.8 nmol / L, about 0.9 nmol / L, about 1.0 nmol / L, or more, e.g., prior to treatment with a conjugate described herein, of about 0.2 nmol / L, about 0.25 nmol / L, about 0.3 nmol / L, about 0.4 nmol / L, about 0.5 nmol / L, about 0.6 nmol / L, about 0.7 nmol / L, about 0.8 nmol / L, about 0.9 nmol / L, about 1.0 nmol / L, or more. In certain embodiments, the subject is fed and has an endogenous C-peptide level of less than or about 0.6 nmol / L, or less than or about 0.75 nmol / L, or less than or about 1.0 nmol / L, e.g., prior to treatment with a conjugate described herein.

[0125] In some embodiments, the conjugate is administered prophylactically. In some embodiments, the subject does not have detectable levels of autoimmune antibodies (e.g., insulin autoantibodies) and the conjugate is administered prophylactically. In some embodiments, the subject does not have detectable levels of pathogenic or disease-causing B cell populations (e.g., anti-insulin B cells, insulin-specific B cells, or insulin-specific B cells). +In some embodiments, the subject does not have detectable levels of autoimmune antibodies (e.g., insulin autoantibodies) and does not have detectable levels of pathogenic or disease-causing B cell populations (e.g., anti-insulin B cells, insulin-specific B cells, or insulin-specific B cells), and the conjugate is administered prophylactically. + In some embodiments, the subject does not have an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) and the conjugate is administered prophylactically. In some embodiments, the subject does not have an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) and the conjugate is administered prophylactically. In some embodiments, the subject is at risk of developing T1D, e.g., the subject has a first-degree relative diagnosed with T1D. The subject can be an adult or a child.

[0126] In some embodiments, the subject is at risk of developing T1D, e.g., the subject has one or more alleles at the DRB1, DQA1, and / or DQB1 loci (e.g., a DR-DQ haplotype) that are associated with a higher risk of developing T1D, e.g., as described in Erlich, et al., Diabetes. 2008 April; 57(4): 1084-1092, which is incorporated herein by reference. In some embodiments, the subject has one or more of the following human leukocyte antigen (HLA) haplotypes: (a) DRB1*0301-DQA1*0501-DQB1*0201; (b) DRB1*0405-DQA1*0301-DQB1*0302; (c) DRB1*0401-DQA1*0301-DQB*0302; (d) DRB1*0402-DQA1*0301-DQB1*0302; (e) DRB1*0404-DQA1*0301-DQB1*0302; or (f) DRB1*0801-DQB1*0401-DQB1*0402.

[0127] In some embodiments, upon administration of the conjugate, the subject does not develop symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes). In some embodiments, upon administration of the conjugate, the subject does not develop symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) for at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 10 years, at least about 15 years, at least about 20 years, at least about 25 years, at least about 30 years, at least about 40 years, at least about 50 years, or more.

[0128] In some embodiments, upon administration of the conjugate, the subject does not develop an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes). In some embodiments, upon administration of the conjugate, the subject does not develop an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) for at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 1.5 years, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 10 years, at least about 15 years, at least about 20 years, at least about 25 years, at least about 30 years, at least about 40 years, at least about 50 years, or more.

[0129] In some embodiments, upon administration of the conjugate, the subject has a delayed rate of onset of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) that is delayed compared to a reference standard or reference treatment. In some embodiments, upon administration of the conjugate, the rate of onset of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) is delayed by at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 40 years, at least 50 years, or longer, compared to a reference standard or reference treatment. In some embodiments, upon administration of the conjugate, the rate of onset of symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) is delayed by about 2%, about 3%, about 4%, about 5%, about 7%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more compared to a reference standard or reference treatment.

[0130] In some embodiments, upon administration of the conjugate, the subject has a delayed rate of onset of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) compared to a reference standard or reference treatment. In some embodiments, upon administration of the conjugate, the rate of onset of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) is delayed by at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 40 years, at least 50 years, or longer, compared to a reference standard or reference treatment. In some embodiments, upon administration of the conjugate, the rate of occurrence of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) is delayed by about 2%, about 3%, about 4%, about 5%, about 7%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more compared to a reference standard or reference treatment.

[0131] In some embodiments, the conjugate is administered therapeutically. In some embodiments, the subject has detectable levels of autoimmune antibodies (e.g., insulin autoantibodies) and the conjugate is administered therapeutically. In some embodiments, the subject has detectable levels of pathogenic or disease-causing B cell populations (e.g., anti-insulin B cells, insulin-specific B cells, or insulin-specific B cells) and the conjugate is administered therapeutically. + In some embodiments, the subject has detectable levels of autoimmune antibodies (e.g., insulin autoantibodies) and detectable levels of pathogenic or disease-causing B cell populations (e.g., anti-insulin B cells, insulin-specific B cells, or insulin-specific B cells), and the conjugate is administered therapeutically. +In some embodiments, the subject has an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) and the conjugate is administered therapeutically. In some embodiments, the subject has an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) and the conjugate is administered therapeutically.

[0132] In some embodiments, administration of the conjugate treats, reverses, or ameliorates symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) in the subject compared to those observed in the subject prior to administration. In some embodiments, administration of the conjugate treats, reverses, or ameliorates symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) in the subject for at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 40 years, at least 50 years, or more, compared to those observed in the subject prior to administration. In certain embodiments, upon administration of the conjugate, symptoms of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) in a subject are treated, regressed, or ameliorated by about 2%, about 3%, about 4%, about 5%, about 7%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to those observed in the subject prior to administration.

[0133] In some embodiments, administration of the conjugate treats, reverses, or ameliorates the autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) in the subject compared to that observed in the subject prior to administration. In some embodiments, administration of the conjugate treats, reverses, or ameliorates the autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) in the subject for at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, at least 30 years, at least 40 years, at least 50 years, or more, compared to that observed in the subject prior to administration. In certain embodiments, upon administration of the conjugate, an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) in a subject is treated, regressed, or ameliorated by about 2%, about 3%, about 4%, about 5%, about 7%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to that observed in the subject prior to administration.

[0134] In some embodiments, the subject receives one course of treatment with the conjugate described herein. A course of treatment as used herein refers to a specific dosage or regimen, as determined by a suitable medical practitioner, provided to the subject until the autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) is treated, cured, alleviated, or symptoms are reduced. In other embodiments, the subject receives more than one course of treatment with the conjugate. In other embodiments, the subject receives multiple courses of treatment with the conjugate. In still other embodiments, the subject receives multiple courses of treatment with the conjugate, each course of treatment being separated by a specific length of time (e.g., about 1 day, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year, about 1.5 years, about 2 years, about 3 years, about 4 years, about 5 years, about 7.5 years, about 10 years, about 12.5 years, about 15 years, about 20 years, or more).

[0135] In some embodiments, the subject is an adult (e.g., at least 18 years old, e.g., at least 19, 20, 21, 22, 23, 24, 25, 25-30, 30-35, 35-40, 40-50, 50-60, 60-70, 70-80, or 80-90 years old). In some embodiments, the subject is a child (e.g., under 18 years old, e.g., under 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year old, or younger). In some embodiments, the subject is male or female.

[0136] In some embodiments, the reference treatment used in any of the methods described herein includes, but is not limited to, insulin or an insulin analog, e.g., an insulin or insulin analog described herein; islet cell transplantation; pancreatic transplantation; or an antibody (e.g., a cytotoxic antibody) against a pan-B cell antigen (e.g., an anti-CD20 antibody, an anti-CD22 antibody, or an anti-CD19 antibody). In some embodiments, the reference treatment can include natural insulin (e.g., proinsulin or mature insulin) from a mammal, e.g., a human or mouse, or a composition described in US2013 / 0028918A1 or CN103509118A.

[0137] In some embodiments, the reference standard used in any method described herein includes the outcome of autoimmune disease therapy, for example type 1 diabetes therapy, for example the outcome described herein.In some embodiments, the reference standard is the level of marker (for example blood glucose or C-peptide level) in the subject before the start of therapy, for example conjugate therapy described herein, for example, when the subject has risk of developing T1D (for example, the subject is a first-degree relative of a T1D patient); when the subject is pre-diabetic (for example, the subject is autoantibody positive); when the subject has recently experienced the onset of T1D (for example, the time since onset is less than 12 months); when the subject has long-standing T1D (for example, the time since onset is more than 12 months or 12 months); or when the subject is a healthy subject (for example, a healthy, age- and / or sex-matched subject). In some embodiments, the reference standard is a measure of the presence / progression / severity of the disease, or the presence / severity of symptoms of the disease, prior to initiation of a therapy, e.g., a conjugate therapy described herein, e.g., if the subject is at risk of developing T1D (e.g., the subject is a first-degree relative of a T1D patient); if the subject is pre-diabetic (e.g., the subject is autoantibody positive); if the subject has recently experienced an onset of T1D (e.g., time since onset is less than 12 months); if the subject has long-standing T1D (e.g., time since onset is greater than or equal to 12 months); or if the subject is a healthy subject (e.g., a healthy, age- and / or gender-matched subject).

[0138] Pharmaceutical Compositions Provided herein are pharmaceutical compositions containing the conjugates described herein that can be used to induce anergy in insulin-binding B cells and / or treat or prevent an autoimmune disease, e.g., autoimmune diabetes, e.g., type 1 diabetes.

[0139] The amount and concentration of the conjugate in the pharmaceutical composition and the amount of the pharmaceutical composition administered to the subject can be selected based on clinically relevant factors, such as the medically relevant characteristics of the subject (e.g., age, weight, sex, other medical conditions, etc.), the solubility of the compound in the pharmaceutical composition, the potency and activity of the compound, and the mode of administration of the pharmaceutical composition.For further information on administration route and dosage regime, see Chapter 25.3, Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0140] Provided herein are pharmaceutical compositions containing the conjugates described herein that can be used to induce anergy in insulin-binding B cells and / or treat or prevent an autoimmune disease, e.g., autoimmune diabetes, e.g., type 1 diabetes.

[0141] Although the conjugates described herein may be administered alone, in some embodiments, the conjugates of the present technology may be administered as a pharmaceutical formulation (composition), in which the conjugates are combined with one or more pharma- ceutically acceptable diluents, excipients, or carriers. Pharmaceutical compositions having one or more conjugates disclosed herein may contain a carrier, which may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and others. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it is advantageous to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0142] The conjugate can be formulated for administration in any convenient way for use in human medicine.In certain embodiments, the conjugate contained in the pharmaceutical product can be active per se, or can be, for example, a prodrug, which can be converted into an active compound in a physiological setting.Regardless of the selected administration route, the conjugate of the present technology, which can be used in a suitable hydrated form, and / or the pharmaceutical composition of the present technology, can be formulated into a pharmaceutically acceptable dosage form as described herein, or by other conventional methods known to those skilled in the art.

[0143] In another aspect, the present technology provides a pharma- ceutically acceptable composition comprising a therapeutically effective amount or a prophylactically effective amount of the conjugate described herein, formulated together with one or more pharma- ceutically acceptable carriers (additives) and / or diluents.The pharmaceutical compositions described herein can be specially formulated for administration in solid or liquid form, for example, in sterile solution or suspension, including those adapted for parenteral administration, for example, by subcutaneous, intramuscular, or intravenous injection.In certain embodiments, the pharmaceutical composition can simply be dissolved or suspended in sterile water.In some embodiments, the pharmaceutical product is non-pyrogenic, i.e., does not elevate the patient's body temperature.

[0144] Some examples of substances which can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycosaminoglycans, such as glyceryl stearate, glyceryl stearate, and glyceryl stearate; (11) glyceryl stearate, ... Examples of suitable carriers include ethanol, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffered saline; (21) cyclodextrins, such as Captisol®; and (22) other non-toxic, compatible substances employed in pharmaceutical formulations. In some embodiments, the carrier includes phosphate buffered saline (PBS).

[0145] Wetting agents, emulsifying agents and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0146] As described herein, certain embodiments of the conjugates of the present technology may contain basic functional groups, such as amines, and thus may form pharma-ceutically acceptable salts with pharma-ceutically acceptable acids. In these cases, the term "pharma-ceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of the conjugates of the present technology. These salts may be prepared in situ during the final isolation and purification of the conjugates, or by separately reacting the purified conjugates described herein in their free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate, among others (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0147] In other cases, certain embodiments of the conjugates of the present technology may contain one or more acidic functional groups, and thus can form pharma-ceutically acceptable salts with pharma-ceutically acceptable bases.These salts can be prepared in situ during the final isolation and purification of the conjugate, or by separately reacting the purified conjugate in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharma-ceutically acceptable metal cation, with ammonia, or with a pharma-ceutically acceptable organic primary, secondary, or tertiary amine.Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, etc.Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (see, for example, Berge et al., supra).

[0148] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0149] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following 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 methylparabens; antioxidants, such as ascorbic acid, or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials. For the convenience of the patient or treating physician, the dosage formulations can be provided as a kit containing all the necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a course of treatment (e.g., a 7-day treatment).

[0150] The formulations of the present disclosure include those suitable for parenteral administration. The formulations may be conveniently presented in unit dosage form and may be prepared by any method known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of compound that produces a therapeutic effect. Generally, this amount will range from about 1 percent to about 99 percent of active ingredient, from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent, out of one hundred percent.

[0151] Pharmaceutical compositions of the present technology suitable for parenteral administration include the conjugates described herein in combination with one or more pharma- ceutically acceptable, sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately prior to use. These may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0152] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the present technology include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants, such as Tween-like surfactants. In some embodiments, the pharmaceutical compositions (e.g., those described herein) include a Tween-like surfactant, such as Tween-80. In some embodiments, the pharmaceutical compositions (e.g., those described herein) include a Tween-like surfactant, such as Tween-80, at a concentration of about 0.001% to about 2%, or about 0.005% to about 0.1%, or about 0.01% to about 0.5%.

[0153] In some embodiments, it may be desirable to slow down the absorption of the drug from the subcutaneous or intramuscular injection site in order to prolong the effect of the conjugate.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.The rate of absorption of the drug then depends on its dissolution rate, which in turn can depend on the crystal size and crystalline form.Alternatively, delayed absorption of the parenterally administered form of the conjugate can be achieved by dissolving or suspending the conjugate in an oil medium.Alternatively, delayed absorption of the drug can be achieved by using a concentrated form of the conjugate.

[0154] In some embodiments, the conjugate is administered as a bolus injection or intravenous infusion, hi some embodiments, the conjugate is administered by syringe injection, pump, pen, needle, or indwelling catheter.

[0155] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, compositions for parenteral administration must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0156] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of the components listed above as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into sterile medium that contains basic dispersion medium and other necessary components from the above-listed ones.For the preparation of sterile injectable solution, typical preparation methods include vacuum drying and freeze-drying, which can produce powder of active ingredient plus any additional desired components from the solution that has been previously sterile filtered.

[0157] Oral compositions generally contain an inert diluent or edible carrier. For oral therapeutic administration, active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using liquid carriers for use as mouthwash. Pharmaceutically compatible binders and / or auxiliary substances can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients: binders such as microcrystalline cellulose, tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; flow agents such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring, or compounds of a similar nature.

[0158] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0159] Systemic administration of the therapeutic compounds described herein can also be by transmucosal or transdermal / topical means. For transmucosal or transdermal / topical administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or inhalants. For transdermal / topical administration, the active compound is formulated into powders, solutions, ointments, lotions, gels, patches, pastes, salves, or creams generally known in the art. The active compound can be mixed under sterile conditions with a pharma- ceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required. In one embodiment, transdermal administration can be carried out by iontophoresis.

[0160] The method of introduction can be provided by rechargeable or biodegradable devices.Various slow-release polymeric devices have been developed and have been tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biologics.Various biocompatible polymers, including both biodegradable and non-biodegradable polymers (including hydrogels), can be used to form implants for the sustained release of compounds at specific target sites.

[0161] The present technology contemplates the formulation of the conjugate as the aforementioned pharmaceutical compositions and preparations.Furthermore, the present technology contemplates administration by any of the aforementioned administration routes.Those skilled in the art can select the appropriate formulation and administration route based on the condition being treated and the general health, age, and size of the patient being treated.

[0162] Effective Dosage The dosage, toxicity and therapeutic effectiveness of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, and can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indexes are advantageous. Although compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to affected tissue sites, in order to minimize potential damage to non-affected cells, thereby reducing side effects.

[0163] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compounds can fall within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the method, the therapeutically effective dose can be estimated initially from cell culture assays. Dosages can be determined based on the IC as determined in cell culture. 50 It is possible to establish in animal models to achieve a circulating plasma concentration range that includes. Such information can be used to accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0164] Actual dosage levels of the conjugates in the pharmaceutical compositions described herein can be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic or prophylactic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0165] The selected dosage level will depend upon a variety of factors, including the activity of the particular conjugate employed, or esters, salts, or amides thereof, the route of administration, the time of administration, the rate of excretion of the particular conjugate employed, the duration of treatment, other drugs, compounds and / or substances used in combination with the particular conjugate employed, the severity of the disease or disorder, previous treatments, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical art. Moreover, treatment of a subject with a therapeutically or prophylactically effective amount of a pharmaceutical composition described herein can include a single treatment or a series of treatments.

[0166] A physician of ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician can start the dose of the conjugate employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic or prophylactic effect, and gradually increase the dosage until the desired effect is achieved. When the conjugate is administered to a subject as a pharmaceutical product, it can be given by itself or in combination with a pharma- ceutically acceptable carrier, for example, as a pharmaceutical composition containing 0.1-99.5% (or 0.5-90%) of the active ingredient.

[0167] In general, a suitable daily dose of a conjugate is that amount of the conjugate that is the lowest dose useful to produce a therapeutic effect. Such an effective dose generally depends on the factors described above. Typically, an effective amount of one or more conjugates disclosed herein sufficient to achieve a therapeutic or prophylactic effect ranges from about 0.000001 mg / kilogram body weight / day to about 10,000 mg / kilogram body weight / day. Suitably, the dosage range is from about 0.0001 mg / kilogram body weight / day to about 100 mg / kilogram body weight / day. For example, dosage can be in the range of 1 mg / kg body weight or 10 mg / kg body weight every day, every second or third day, or 1 to 10 mg / kg every week, every two or three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001 to 10,000 micrograms / kg body weight. In one embodiment, the concentration of one or more conjugates in the carrier ranges from 0.2 to 2000 micrograms / milliliter delivered. Exemplary therapeutic forms involve administration once a day or once a week. In therapeutic applications, sometimes relatively high dosages are required at relatively short intervals until the progression of the disease is reduced or terminated, or until the subject shows partial or complete improvement of the symptoms of the disease. Thereafter, a prophylactic form can be administered to the patient. In some embodiments, a therapeutically effective amount of one or more conjugates is administered at a dose of 100 mg / kg or more in the target tissue. -32 ~10 -6 Molar concentration, e.g., about 10 -7 It may be defined as the molar conjugate concentration. This concentration may be delivered by a systemic dose of 0.001-100 mg / kg or the equivalent dose per body surface area. To maintain therapeutic concentrations in the target tissue, dosing regimens are optimized, for example, by daily or weekly administration, but also including continuous administration (e.g., parenteral injection or transdermal application).

[0168] Typically, intravenous and subcutaneous doses of the conjugate for a patient range from about 0.0001 to about 100 mg / kilogram of body weight / day, e.g., from about 0.0001 to about 0.001 mg / kg / day, from about 0.001 to about 0.01 mg / kg / day, from about 0.01 to about 0.1 mg / kg / day, from about 0.1 to about 1 mg / kg / day, from about 1 to about 10 mg / kg / day, or from about 10 to about 100 mg / kg / day. In some embodiments, the conjugate is administered at a dose of greater than or equal to 60 nmol / kg / day. In some embodiments, the conjugate is administered at a dose of greater than or equal to 75 nmol / kg / day, greater than or equal to 100 nmol / kg / day, greater than or equal to 150 nmol / kg / day, or greater than or equal to 200 nmol / kg / day. In certain embodiments, the conjugate is administered at a dose of greater than or equal to 1 mg / kg / day, e.g., 2 mg / kg / day, 4 mg / kg / day, 8 mg / kg / day, 16 mg / kg / day, 32 mg / kg / day, 64 mg / kg / day, 100 mg / kg / day, 200 mg / kg / day, or higher.

[0169] The conjugate may be present at a concentration of about 100 mg / mL or less (e.g., 100 mg / mL or less, e.g., 90 mg / mL, 80 mg / mL, 70 mg / mL, 60 mg / mL, 50 mg / mL, 40 mg / mL, 30 mg / mL, 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.01 mg / mL, or less). In some embodiments, the conjugate is present at a concentration of about 0.25 mg / mL to about 1 mg / mL, e.g., about 0.25 mg / mL, about 0.5 mg / mL (e.g., 0.5 mg / mL), about 0.75 mg / mL, or about 1 mg / mL.

[0170] If desired, the effective daily dose of the conjugate can be administered as 2, 3, 4, 5, 6 or more subdoses, which are administered separately over the course of a day at appropriate intervals, optionally in unit dosage form. In some embodiments, the conjugate is administered once a day. In some embodiments, the conjugate is administered at least twice a week. In some embodiments, the conjugate is administered at least once a week. In certain embodiments, the conjugate is administered twice a week.

[0171] The conjugates can be administered per se or in admixture with pharma- ceutically acceptable and / or sterile carriers, and can be administered with antibacterial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides. Thus, conjunctive therapy includes sequential, simultaneous, and separate administration of conjugates such that when the subsequent therapy is administered, the therapeutic or prophylactic effect of the first administered therapy is still detectable.

[0172] Combination therapy In some embodiments, one or more conjugates disclosed herein may be combined with one or more additional therapies for inducing anergy in insulin-binding B cells and / or for the prevention or treatment of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes).

[0173] In any case, the multiple therapeutic agents may be administered in any order, or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single unified form, or in multiple forms (for example only, as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given in multiple doses. If not simultaneously, the timing of multiple doses may vary from more than 0 weeks to less than 4 weeks. In addition, the combination method, composition and formulation are not limited to the use of only two active substances.

[0174] In some embodiments, the at least one additional therapy is administered in a formulation comprising a combination with a conjugate described herein for treating or preventing an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes). In certain embodiments, the at least one additional therapy is administered simultaneously with the conjugate described herein. In certain embodiments, the at least one additional therapy is administered sequentially (at different times) with the conjugate described herein. In examples, the at least one additional therapy is administered about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks away from the conjugate described herein. The at least one additional therapy may or may not be administered by the same route as the conjugate. In examples, the conjugate may be administered in one manner (eg, intravenously or subcutaneously), while at least one additional therapy may be administered separately in another manner (eg, orally).

[0175] In some embodiments, at least one additional therapy is an insulin sensitivity enhancer. Insulin sensitivity enhancers (e.g., biguanides (e.g., metformin) and glitazones (e.g., rosiglitazone and pioglitazone)) act by increasing the subject's response to a given amount of insulin (or insulin analogue). Thus, patients receiving insulin sensitivity enhancers may require lower doses of the conjugates described herein compared to patients not receiving insulin sensitivity enhancers. Thus, in certain embodiments, the conjugates are administered to subjects in combination with insulin sensitivity enhancers. In some embodiments, the conjugate may be administered at about 95% of the standard dose required in the absence of the insulin sensitizer, e.g., about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less of the standard dose required in the absence of the insulin sensitizer.

[0176] In some embodiments, the at least one additional therapy is administered to prevent the full development of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) or a symptom thereof, resulting in a maintenance of at least about 90% beta cell mass compared to a healthy subject. In some embodiments, the at least one additional therapy is administered to prevent the full development of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) resulting in a maintenance of at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, or at least about 5% beta cell mass compared to a healthy subject. In some embodiments, the conjugate is administered to the subject in combination with a therapy to prevent a symptom of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) resulting in a maintenance of at least about 90% beta cell mass compared to a healthy subject. In some embodiments, the conjugate is administered to a subject in combination with a therapy to prevent a symptom of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes), resulting in preservation of beta cell mass of at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, or at least about 5% compared to a healthy subject.

[0177] In some embodiments, at least one additional therapy is administered to prevent the full development of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) or a symptom thereof, resulting in a maintenance of endogenous C-peptide levels of at least about 90% compared to a healthy subject. In some embodiments, at least one additional therapy is administered to prevent the full development of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes) or a symptom thereof, resulting in a maintenance of endogenous C-peptide levels of at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, or at least about 5% compared to a healthy subject. In some embodiments, the conjugate is administered to a subject in combination with a therapy that prevents a symptom of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes), resulting in a maintenance of endogenous C-peptide levels of at least about 90% compared to a healthy subject. In some embodiments, the conjugate is administered to a subject in combination with a therapy to prevent a symptom of an autoimmune disease (e.g., autoimmune diabetes, e.g., type 1 diabetes), resulting in maintenance of endogenous C-peptide levels of at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, or at least about 5% compared to a healthy subject.

[0178] kit The present disclosure also provides a kit for inducing anergy in insulin-binding B cells and / or for preventing and / or treating autoimmune diseases (e.g., autoimmune diabetes, e.g., type 1 diabetes), the kit comprising one or more of the conjugates described herein, and optionally instructions for use. Optionally, the above components of the kit of the present technology are packaged in a suitable container and labeled for preventing and / or treating autoimmune diseases (e.g., autoimmune diabetes, e.g., type 1 diabetes).

[0179] The above-mentioned components may be stored in unit-dose or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous, preferably sterile solutions, or as lyophilized, preferably sterile formulations for reconstitution. The kit may further include a second container that contains a suitable diluent for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, the pharma- ceutically acceptable excipients of the pharmaceutical composition and saline solution. In addition, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, diluted or undiluted. The container may be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The kit may further include more containers that contain pharma-ceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, media for one or more suitable hosts. The kit may optionally include instructions customarily included in commercial packaging of therapeutic products, the instructions containing, for example, information regarding the indications, use, dosage, manufacture, administration, contraindications and / or warnings regarding the use of such therapeutic agent or product.

[0180] The kit may also include, for example, a buffer, a preservative, or a stabilizer. The kit may also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package with instructions for interpreting the results of the assay performed using the kit. The kit of the present technology may contain a written product on or in the container of the kit. The written product describes how to use the reagent contained in the kit. In certain aspects, the use of the reagent may be according to the method of the present technology. EXAMPLES

[0181] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

[0182] Example 1: Materials and Methods Protein and plasmid design. The selection of the vector (Pet9a), construction of the plasmid, and the modified signal peptide sequence (MGSSHHHHHHSSFLDPVLM (SEQ ID NO:5)) are disclosed in RB Mackin (Mackin, RB MethodsX 2014, 1, 108-117), who describes a method for expressing and purifying correctly folded native proinsulin with a yield of over 15 mg / L culture (99% purity). Expression of proinsulin in E. coli poses several challenges, including the formation of insoluble inclusion bodies and preventing N-terminal degradation. Other papers have addressed these challenges but have not exceeded yields of 5 mg / L culture (Cowley, DJ; Mackin, RB FEBS Letters 1997, 402, 124-130; Redwan, EM et al., Preparative Biochemistry and Biotechnology 2007, 38, 24-39), have required fusion of proinsulin with other large proteins (Trabucchi, A et al., Appl Microbiol Biotechnol 2012, 94, 1565-1576; Winter, J. et al. Journal of Biotechnology 2000, 84, 175-185), or have not provided sufficient details due to commercial interests (Tikhonov et al. Protein Expression and Purification 2001, 21, 176-182). The N-terminal tag of GGG-sortase is fused directly to the proinsulin sequence without a peptide linker.

[0183] Expression and purification of proinsulin Human proinsulin and variants, referred to herein as proinsulin, were expressed in BL21(DE3) E. coli as fusion proteins with a precursor peptide, isolated as inclusion bodies by centrifugation, and the fusion peptide was cleaved with cyanogen bromide. The details of the expression protocol for proinsulin are detailed below.

[0184] Day 1: Prepare medium in baffled shake flasks as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl per liter of ultrapure water. Mix 20 g / liter low salt LB-Lennox premixed medium with 5 g NaCl / liter. Mix 25 g / liter LB-Luria broth premixed medium with medium so that it does not exceed 50% of the flask volume. Prepare growth cultures as well as starter cultures (2 x 50 ml starter cultures are sufficient for up to 4 L of growth culture). Cover shake flasks with aluminum foil and autoclave at setting 2 (liquid, medium, etc.) for approximately 80 minutes. After cooling, add 1000x kanamycin antibiotic stock solution to starter cultures only and swirl briefly to mix. Add 1 μl of 1000x stock solution per ml of medium. Inoculate starter cultures with frozen stock solution that has been placed in a -80° freezer. Use a 200 ml pipette tip to scrape the small ice crystals from the vial and pipette the mixture into the medium. Place the shake flask in a shaking incubator. Run at 30 °C and 200 RPM overnight.

[0185] Day 2: Check growth of starter culture. Add 1000x kanamycin antibiotic stock solution to growth culture flask and swirl briefly to mix. Add 1 μl of 1000x stock solution per ml of medium. Inoculate growth culture by adding 1% of the growth culture volume of the starter culture using a stripette. A 1% inoculation volume results in an OD600nm>0.6 at 2.0 hours. Return to shaking incubator. Run at 37°C and 200 RPM. For 1L baffled flasks, 160 RPM. At 1.5 hours, check OD600nm (growth density) by removing 1ml of medium from the flask and checking with a UV-Vis spectrophotometer. Use water as blank. Induce when OD600nm is 0.6-0.9 by adding 1M IPTG stock solution to a final concentration of 0.5 mM and allow expression for 3-4 hours.

[0186] Harvesting Proinsulin: Harvest the E. coli by transferring the medium to 750 ml centrifuge bottles (blue screw cap). Spin down at 4500 g for 45 minutes at 4°C in a swinging bucket centrifuge. Carefully discard the supernatant without disturbing the pelleted cells. Suspend the cells from each centrifuge bottle in 20 ml of lysis buffer without Triton X100 (50 mM Tris, 50 mM NaCl (pH 8), 0.5 mM EDTA, 5% glycerol, 1% Triton X100). Transfer to a 200 ml glass homogenizing jar.

[0187] Inclusion body isolation: Homogenize on ice for 2 minutes at 85% intensity using a 10 sec on, 20 sec off method. Transfer contents evenly into four 50 ml falcon tubes. Centrifuge at 8000g for 15 minutes at 20°C. Discard the supernatant. Suspend contents of each falcon tube in 40 ml lysis buffer with Triton X100. Repeat centrifugation and washing steps two more times (total of 3). Suspend each pellet in 2 ml DI H2O. Pipette and sonicate the mixture to resuspend. Freeze at -80°C to aid dispersion.

[0188] Cleavage of signal peptide from proinsulin: Thaw and sonicate pellet to suspend. Transfer to a 250 ml round bottom flask with stir bar. Add 32 ml (for a 4 L batch) of 88% formic acid. Process 4 L of culture and collect 4 pellets, placing each in a 50 ml falcon tube. In a fume hood, weigh out 1.6 g cyanogen bromide and transfer to flask. Cap flask with a rubber stopper, cover with aluminum foil, and stir at 200 rpm overnight at room temperature (RT).

[0189] Load the cooling chamber of a rotary evaporator (Roto-Vap) with 3 lb of dry ice and approximately 200 ml of acetone. Attach the 50 ml round bottom flask containing the CNBr cleavage product. Turn on the vacuum pump and close the inlet port. Spin at 200 rpm or less and place in a water bath at RT. Watch for "bumping" and counteract by admitting air to the inlet port. After bumping subsides, turn on the water bath heater to 40°C. Run to dryness.

[0190] Reduction of disulfide bonds: Add 40 ml of 6 M guanidinium, 500 mM Tris (pH 8.1) to the flask containing the dried CNBr cleavage product and stir / sonicate until dissolved. Add a stopper and seal with parafilm. Inject 0.6 ml of β-mercaptoethanol, then stir at RT for 10 min.

[0191] Purification of reduced proinsulin: Set up the Prep-LC by standard protocol using solvent with 0.05% TFA. Equilibrate the column with 30% ACN. Acidify the sample to pH 3-4 by adding 0.5 ml of concentrated HCl (12 M). Monitor the addition with a pH meter / test paper to avoid dropping below pH 2. Syringe filter 40 ml of reduced proinsulin solution through a Millex AA 0.8 um MCE membrane filter (blue) or Millex GP 0.22 um PES membrane filter (green). Keep the filtered solution on ice. Inject a volume of 4.5 ml and collect the main peak (35 ml) eluting at 9-11.5 min. Combine fractions and place in a 500 ml flask and rotary evaporate to remove ACN.

[0192] Refolding reduced proinsulin: Add appropriate volume of 20x refold buffer to flask with stir bar. Add 2mM β-mercaptoethanol and 2mM L-cystine to flask. Add stopper and seal with parafilm. 1.5μl BME, 5mg L-cystine per 10ml volume. Stir overnight at 4°C in refrigerator.

[0193] Purification of folded proinsulin: Check folding by analytical HPLC by removing 0.2 ml using needle and syringe. Set up Prep-LC by standard protocol using solvent with 0.05% TFA. Equilibrate column with 25% ACN. Add 5% ACN (volume) and acidify to pH 3-4 using concentrated HCl. Monitor by pH meter. Syringe filter through a Millex 0.22 μm filter. Keep filtered solution on ice. Stop pump and attach sample inlet line (pre-loaded with sample) to pump A (top). Load 50-80 ml of sample at 10 ml / min. Stop pump, attach solvent A inlet line (loaded with water) and flush with 5% B at 14 ml / min for 8 min. Collect refolded proinsulin eluting at 9.5-12 min (35 ml) and improperly folded proinsulin eluting at 12-14.5 min (35 ml). Similar fractions are combined, placed in a 500 ml flask and rotary evaporated to remove ACN. Aqueous folded proinsulin is transferred to a 50 ml falcon tube. Freeze at -80°C and then placed in a lyophilizer.

[0194] Analysis of purified folded proinsulin: Weigh dry proinsulin on a balance. Dissolve a portion of the sample at 1mg / ml in 0.02% formic acid. Check absorbance using nanodrop and calculate protein concentration. Run analytical HPLC and SDS-PAGE (native and reduced) to check purity. Analyze samples by mass spectrometry to confirm identity.

[0195] Proinsulin was denatured in 6 M guanidine and reduced with β-mercaptoethanol (BME). Reduced proinsulin was purified by RP LC and refolded at 0.5 mg / ml in oxidation buffer. Refolded proinsulin was purified by RP LC and lyophilized.

[0196] Sortase ligation:Enhanced penta-mutant sortase A (eSrtA) was expressed in E. coli and purified by Ni-IMAC. Ligations were typically carried out with 100 μM Fc-LPETGGH6, proinsulin-LPETGGHG (SEQ ID NO:6), or azide-linker-LPETGGHG (SEQ ID NO:6); 100 μM GGG-proinsulin or GGG-linker-azide; and 10 μM enhanced sortase A (penta-mutant) in Tris-buffered saline, 20% DMSO, 6 mM CaCl2, and 200 μM NiSO4 at 37° C. for 4 hours or at RT for 16 hours. Reaction progress was monitored by HPLC using RP C4 and SEC columns. Ligation products were purified by RPLC or Protein A.

[0197] Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC): 1 equivalent (eq.) of proinsulin-azide, 4 eq. of alkyne-PEG n -alkyne, 1eq. CuSO4, 5eq. THPTA, and 20eq. sodium ascorbate to synthesize proinsulin-PEG. n -alkyne conjugate was synthesized. Proinsulin-PEG n -alkyne was purified by RPLC. 40 μM Fc-LPETGGG-azide or 100 μM CD22L-azide, 4 eq. alkyne-PEG n The final CuAAC reaction to recover the Fc / CD22L-conjugate was carried out using -proinsulin, 12 eq. CuSO4, 60 eq. THPTA, and 240 eq. sodium ascorbate. n -Proinsulin was purified by SEC. n - Proinsulin was purified by RPLC.

[0198] Example 2: Synthesis of the conjugate Proinsulin-LPETGGG-PEG3-N3. A solution of 0.2 mM Proinsulin-LPETGGHG, 0.8 mM GGG-PEG3-azide, 0.02 mM pentamutant sortase A, 10 mM CaCl2, and 0.4 mM NiSO4 in 50 mM Tris, 150 mM NaCl (pH 8) buffer is stirred at room temperature for 2 h. The reaction is quenched and acidified with 0.5% v / v of 88% formic acid, after which the desired product is isolated by preparative reversed-phase liquid chromatography (Prep-LC) using a C18 stationary phase column. Rotary evaporation under reduced pressure is used to remove acetonitrile, followed by freezing and lyophilization to recover a white powder. 80% isolated recovery with purity >95% is routinely achieved at the 10+ mg scale. Product identity is confirmed by MALDI mass spectrometry.

[0199] Insulin-CD22L. A solution of 0.4 mM insulin-B29-alkyne, 0.48 mM mCD22L-N3 or 0.48 mM hCD22L-N3 (structures of each are provided below this paragraph), 0.4 mM CuSO4, 2 mM tris-hydroxypropyltriazolylmethylamine (THPTA), and 8 mM sodium ascorbate in 50 mM sodium phosphate buffer (pH 7.6) with 20% by volume DMSO was stirred at room temperature for 2 hours. The reaction was quenched and acidified with an equal volume of 50 mM HCl, after which the desired product is isolated by Prep-LC using a C18 stationary phase column. Rotary evaporation under reduced pressure is used to remove acetonitrile, followed by freezing and lyophilization to recover a white powder. 70% isolated recovery with purity >95% is routinely achieved at the 5+mg scale. Product identity was confirmed by MALDI mass spectrometry. TIFF2024505002000017.tif181147

[0200] Conjugation of insulin-B29-azide or proinsulin-LPETG-N3 with PEG-alkyne. The synthesis was carried out essentially according to the procedures listed and cited in Johnson, SN et al., ACS Appl. Bio Mater. 2020, 3, 6319-6330. A solution of 0.4 mM PEG-alkyne (4-arm or bifunctional), 0.5 equivalents of insulin-B29-azide / alkyne or 0.5 equivalents of proinsulin-LPETG-N3 / alkyne, 0.8 mM CuSO4, 4.0 mM THPTA, and 16 mM sodium ascorbate in 50 mM sodium phosphate buffer (pH 7.6) with 20% DMSO by volume was stirred at room temperature for 1 hour. The reaction was quenched and acidified with an equal volume of 50 mM HCl, after which the desired product was isolated by Prep-LC using a C18 stationary phase column. Separate PEG-insulin conjugates are isolated from the mixture by collecting each peak separately. 4-arm PEG(20K)-alkyne reaction, for example, recovers 4-arm PEG-insulin (1), 4-arm PEG-insulin (2), 4-arm PEG-insulin (3), and 4-arm PEG-insulin (4). Bifunctional PEG(10K)-alkyne reaction, for example, recovers bifunctional PEG-insulin (1) and bifunctional PEG-insulin (2). Rotary evaporation under reduced pressure is used to remove acetonitrile, followed by freezing and lyophilization to recover a white powder. The isolated products are >95% pure, and their identity is confirmed by MALDI mass spectrometry. Data for exemplary synthetic products are presented in Table 1 below.

[0201] Conjugation of CD22L-N3 with PEG-alkyne and PEG-insulin. The synthesis was carried out substantially according to the procedures listed and cited in Johnson, SN et al., ACS Appl. Bio Mater. 2020, 3, 6319-6330. In particular, a solution of 0.1 mM PEG-alkyne or PEG-insulin (4-arm or bifunctional), 3 equivalents of m / hCD22L-N3 / alkyne, 0.2 mM CuSO4, 1.0 mM THPTA, and 4 mM sodium ascorbate in 50 mM sodium phosphate buffer (pH 7.6) with 20% by volume DMSO was stirred at 37° C. for 2 hours. The reaction is quenched and acidified with an equal volume of 50 mM HCl, after which the desired product is isolated by Prep-LC using a C18 stationary phase column. Rotary evaporation under reduced pressure is used to remove acetonitrile, followed by freezing and lyophilization to recover a white powder. Isolated recoveries of 60% with purity >95% are routinely achieved at the 5 mg scale. Product identity is confirmed by MALDI mass spectrometry. Data for exemplary synthetic products are presented in Table 1 below. TIFF2024505002000018.tif61131TIFF2024505002000019.tif173131

[0202] Conjugates with the general formula Fc-(proinsulin)2 and CD22L-proinsulin (Figure 2) were designed. The following five human proinsulins (Figure 3) were expressed at a titer of 40 mg / L culture and purified to >90% purity with a final yield of 5-10 mg / L (Figure 4): native proinsulin, G3-proinsulin, G3-proinsulin F25D, proinsulin-LPETG, and proinsulin-LPETG F25D. The F25D mutation was incorporated to reduce binding to insulin-receptor B to prevent hypoglycemia in future in vivo testing, and this mutation was selected to minimally disrupt T-cell and B-cell epitopes. Alkyne-PEG-alkyne with molecular weights of 10 and 20 kDa was purchased with G3-PEG3-azide linkers. The N3-PEG3-LPETGGHG (SEQ ID NO:6) linker was synthesized from N3-PEG3-COOH and LPETGGHG (SEQ ID NO:6) peptides. The G3-proinsulin variant was ligated with the N3-LPETGGHG (SEQ ID NO:6) linker by eSrtA to give N3-G3-proinsulin (Figure 5A) and with the alkyne-LPETGGHG (SEQ ID NO:6) linker to give alkyne-G3-proinsulin (Figure 5B).

[0203] A disubstituted sialic acid CD22 ligand bearing an azide handle, CD22L-N3 (Figure 6A), was synthesized.

[0204] Additionally, the proinsulin-LPETG variant is ligated to a G3-azide linker by eSrtA to give proinsulin-LPETG-N3 (Figure 5C). N3-G3-proinsulin and proinsulin-LPETG-N3 are then ligated to alkyne-PEG by CuAAC. n -alkyne conjugated to alkyne-PEG, respectively n -G3 Proinsulin and Alkyne-PEG n -(proinsulin LPETG) is given (Figures 5A, 5C). The proinsulin-CD22L conjugate is constructed according to Figures 6B-6C.

[0205] Example 3: Determination of insulin variants that bind to anti-insulin antibodies Binding kinetics were determined using biolayer interferometry (BLI) on an Octet RED96e (ForteBio) according to the manufacturer's instructions. Monoclonal anti-insulin antibody (125mAb) was loaded onto anti-mouse IgG Fc Capture Dip and Read Biosensors (ForteBio) at 40μg / ml for 300s, then equilibrated for 300s in 1x kinetic buffer. A baseline was collected for 180s in 1x kinetic buffer, association with each insulin variant at concentrations between 0.5 and 20x the estimated KD was performed for 300s in 1x kinetic buffer, and dissociation was performed for 600s in 1x kinetic buffer. Reference sensor data and reference sample data were subtracted for each sample, and binding kinetics were calculated using a 1:1 global fit.

[0206] As shown in Figure 7, most insulin variants had similar binding kinetics and affinity (<10 nM) to native human insulin, demonstrating that the insulin variants can bind to anti-insulin antibodies.

[0207] Example 4: Functional characterization of insulin variants Determination of hormonal activity of insulin variants. Insulin variants at concentrations between 1 nM and 100 μM were incubated with a human insulin receptor (hCD220) luciferase reporter cell line (iLite Insulin Assay Ready Cells, Svar Life Science BM3060) in 96-well plates according to the manufacturer's specifications. After incubation, cells were equilibrated to room temperature and hCD220 signaling was measured by luciferase luminescence using a luminescence plate reader (Biotek Synergy H4 Hybrid Reader H4MLFPTAD) using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's specifications. Firefly luciferase luminescence was normalized by subtracting background luminescence and then dividing by background-subtracted Renilla luciferase luminescence. EC50 values ​​were calculated using nonlinear regression analysis with variability slope and four parameters.

[0208] As shown in Figure 8, the EC50 concentrations of the insulin variants ranged from 240 to >40,000 fold greater than native human insulin, demonstrating a reduced ability of the insulin variants to antagonize the hormone insulin receptor.

[0209] Conjugation of insulin variants with functionalized oligoglycines. A solution of 0.2 mM insulin variant, 0.8 mM GGG-PEG3-azide, 0.02 mM pentamutant sortase A, 10 mM CaCl2, and 0.4 mM NiSO4 in Tris-buffered saline was stirred at room temperature for 2 h. The reaction was quenched and acidified with 0.5% v / v 88% formic acid, after which the desired product was isolated by preparative reversed-phase liquid chromatography (Prep-LC) using a C18 stationary phase column with acidified (0.05% trifluoroacetic acid) water and acetonitrile as the mobile phase. Rotary evaporation under reduced pressure was used to remove the acetonitrile, which was then frozen and lyophilized to recover a white powder. Isolated recoveries of 80% were routinely achieved at the 10+ mg scale with purities >95%. Product identity was confirmed by MALDI mass spectrometry.

[0210] FIG. 9 shows HPLC identification and quantification of sortase-mediated ligation recovery over time.

[0211] Determination of 4-arm PEG-insulin binding to anti-insulin antibodies. Binding kinetics were determined using biolayer interferometry (BLI) using an Octet RED96e (ForteBio) according to the manufacturer's instructions. Monoclonal anti-insulin antibodies (125mAb) were loaded onto anti-mouse IgG Fc Capture Dip and Read Biosensors (ForteBio) at 10μg / ml for 300 seconds and then equilibrated in 1x kinetic buffer for 300 seconds. A baseline was collected for 180 seconds in 1x kinetic buffer, association with 4-arm PEG-insulin (1-4) at a concentration of 40nM was performed in 1x kinetic buffer for 600 seconds, and dissociation was performed in 1x kinetic buffer for 1800 seconds. Reference sensor data and reference sample data were subtracted for each sample to calculate binding kinetics using a 1:1 local fit.

[0212] FIG. 10 demonstrates that 4-arm PEG-insulin with 2-4 insulins / scaffold can bind strongly to anti-insulin antibody 125 mAb, and that increasing the insulin binding valency increases the binding affinity and decreases the dissociation rate as predicted by avidity.

[0213] Determination of insulin self-association within 4-arm PEG-insulin. Near UV circular dichroism (near UV CD) using a Jasco 1500 circular dichroism spectrophotometer was used to determine the insulin self-association state within 4-arm PEG-insulin. Samples were dissolved at insulin concentrations of 0.6 and 0.06 mM in sodium phosphate buffer (pH 7.4) with cell path lengths of 1 and 10 mm, respectively. Samples with zinc contained 0.4 molar equivalents of zinc sulfate / insulin. CD spectra were recorded from 250 to 310 nm and results of triplicate measurements were averaged. Near UV CD temperature melting was performed at 274 nm, 20 to 90 °C, with 0.6 mM samples placed in a cuvette with a 1 mm path length.

[0214] FIG. 11 demonstrates that 4-arm PEG-insulin exhibits minimal intramolecular and no intermolecular self-association.

[0215] Determination of the hydrodynamic radius of insulin variants and 4-arm PEG-insulin variants. The hydrodynamic radius of insulin variants and 4-arm PEG-insulin variant conjugates was determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern). Insulin variants were dissolved in Tris-buffered saline at 0.6 mM and 4-arm PEG-insulin variant conjugates were dissolved at 25 μM and then filtered through a 0.22 μm PVDF filter. The results of triplicate measurements were averaged.

[0216] FIG. 12 demonstrates that insulin variants with lispro and DKP mutations exist as lower oligomeric species in solution, that the 4-arm PEG-insulin variant conjugates do not self-associate intermolecularly, and that the 4-arm PEG-insulin variant conjugates have favorable hydrodynamic radii.

[0217] Determination of secondary structure of insulin variants and 4-arm PEG-insulin variants. The secondary structures of insulin variants and 4-arm PEG-insulin variant conjugates were determined using far-UV circular dichroism (far-UV CD) on a Jasco 1500 circular dichroism spectrophotometer. Samples were dissolved at an insulin concentration of 0.2 mg / ml in sodium phosphate buffer (pH 7.4) with a cell path length of 1 mm. CD spectra were recorded from 190 to 260 nm and triplicate measurements were averaged. The α-helical characterization was calculated using the BeStSeL online server.

[0218] FIG. 13 demonstrates the expected reduction in the α-helical character of insulin variants that retain a predominantly disordered proinsulin C-peptide and the retention of insulin variant secondary structure when conjugated with 4-arm PEG.

[0219] Determination of size heterogeneity of insulin variants and 4-arm PEG-insulin variants. Size exclusion chromatography using a Superdex 200 Increase HiScale 16 / 40 (GE) column in Tris-buffered saline was used to determine the heterogeneity of insulin variants and 4-arm PEG-insulin variant conjugates. 50 μl of sample was injected at concentrations between 0.5 and 2 mg / ml. Data are representative of triplicate measurements.

[0220] FIG. 14 shows the size distribution of the 4-arm PEG-insulin variant species after HPLC purification, predicting that their hydrodynamic radii are similar to those of 200-300 kDa proteins.

[0221] Determination of insulin variant binding to anti-insulin B cells. VH125 non-obese diabetic mouse splenocytes containing 1-3% insulin-specific B cells were incubated with 2 μM FITC-labeled insulin variants and co-labeled with Alexa Fluor 647 anti-mouse CD19 antibody and PI / Cyanine 7 anti-mouse CD3 antibody for 30 min at 4 °C. Samples were then washed and analyzed by flow cytometry (BD FACSFusion). Splenocytes were gated on the basis of CD19 and CD3 expression and FITC signal. Insulin-specific B cells (FITC Hi ) and specific interactions with B and T cells (FITC Lo The binding of the insulin variants was compared to that of native human insulin for non-specific interactions with insulin.

[0222] FIG. 15 shows that insulin variants inhibit non-insulin-specific B cells (FITC Lo CD19+) or T cells (FITC + Insulin-specific B cells (FITC Hi These results demonstrate improved selectivity for binding to CD19+ T cells.

[0223] Example 5: Effect of 4-arm PEG-insulin variants of the present technology on anti-insulin B cells Determination of binding of 4-arm PEG-insulin variants to anti-insulin B cells. 125Tg non-obese diabetic mouse splenocytes, containing >90% insulin-specific B cells, are incubated with 2 μM FITC-labeled 4-arm PEG-insulin variants and co-labeled with Alexa Fluor 647 anti-mouse CD19 antibody and PI / Cyanine 7 anti-mouse CD3 antibody for 30 min at 4 °C. Samples are washed and then analyzed by flow cytometry. Splenocytes are gated based on CD19 and CD3 expression and FITC signal. Binding of 4-arm PEG-insulin variants is compared to that of native human insulin for specific interactions with insulin-specific B cells and non-specific interactions with T cells (Apley et. al. J. Vis. Exp. 2020, 164, e61827).

[0224] The four-arm PEG-insulin variants are predicted to exhibit strong binding kinetics and affinity with insulin-specific B cells and little or no nonspecific interactions with T cells.

[0225] Determination of the in vitro B cell activation prevention of 4-arm PEG-insulin variants. Splenocytes isolated from 125Tg non-obese diabetic (NOD) mice are incubated with treatment (4-arm PEG-insulin variant or 4-arm PEG-insulin variant / CD22L) and induced with anti-IgM Fab2', CpG, or anti-CD40 / IL2 (Acevedo-Suarez et. al. J Immunol 2005, 174, 827). Splenocytes are analyzed by flow cytometry and B cell expression of CD86 is quantified along with B cell proliferation by CellTrace Violet. Additionally, cytokine secretion is analyzed by ELISA for IFN-gamma, TNF-α, IL-2, IL-17A, IL-6, and IL-10 (Johnson et. al. ACS Appl. Bio. Mater. 2020, 3, 6319).

[0226] It is predicted that the 4-arm PEG-insulin variant or 4-arm PEG-insulin variant / CD22L conjugates will effectively prevent B cell activation in vitro compared to untreated controls.

[0227] Prevention of disease onset in non-obese diabetic mice. Non-obese diabetic (NOD) and VH125 NOD mice are treated with weekly subcutaneous injections of 0.2-1.0 mg of 4-arm PEG-insulin variant or 4-arm PEG-insulin variant / CD22L and monitored for the development of type 1 diabetes by two consecutive measurements of blood glucose >200 mg / dL (Henry et. al. Diabetes 2012, 61, 2037).

[0228] It is predicted that the 4-arm PEG-insulin variant or 4-arm PEG-insulin variant / CD22L conjugate will effectively prevent disease onset in non-obese diabetic mice compared to untreated controls.

[0229] Equivalent The present technology should not be limited by the specific embodiment described in this application, which is intended as a single illustration of each aspect of the present technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from the spirit and scope of the present technology. In addition to those listed herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the present technology. It should be understood that the present technology is not limited to specific methods, reagents, compounds, compositions or biological systems, which may of course vary. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0230] Additionally, when features or aspects of the disclosure are described in terms of Markush groups, those of skill in the art will recognize that the disclosure is also described in terms of any individual members or subgroups of members of the Markush group.

[0231] As will be understood by those skilled in the art, virtually all ranges disclosed herein, especially with respect to the provision of a written description, also encompass any and all possible subranges and combinations of subranges thereof. It can be readily recognized that any range described is fully descriptive and allows for the same range to be equally divided into at least one-half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range described herein can be readily divided into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all phrases such as "up to," "at least," "greater than," "less than," etc., include the recited numbers and refer to ranges that can be subsequently divided into the subranges described above. Finally, as will be understood by those skilled in the art, ranges include individual members. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0232] All publications, patent applications, issued patents, and other documents (e.g., journals, articles, and / or textbooks) referenced herein are incorporated by reference as if each individual publication, patent application, issued patent, and other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in textbooks incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

[0233] It is understood that the present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, and that the following paragraphs should not be construed as limiting the scope of the claims appended hereto or as mandating that all such features must necessarily be included in the scope of such claims. A. Formula I A conjugate of TIFF2024505002000020.tif69128, or a pharma- ceutically acceptable salt and / or solvate thereof, During the ceremony, R 1 , R 2 , R 3 , and R 4 are each independently TIFF2024505002000021.tif93146; R 5 is independently at each occurrence aryl or aralkyl; R 6 is independently at each occurrence halo, hydroxyl, aryl, or heteroaryl; X 1 is independently, at each occurrence, the epsilon nitrogen atom of a lysine of SEQ ID NO:2, or a substitution variant thereof in the first proinsulin polypeptide; X 2 is independently, in each occurrence, the carbonyl carbon of the nitrogen atom of the C-terminal glycine of a sortase moiety, wherein said sortase moiety is fused to a second proinsulin polypeptide at the C-terminus of said second proinsulin polypeptide; x is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; m is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; n is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and p is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; The conjugate, or a pharma- ceutically acceptable salt and / or solvate thereof. B. The conjugate of paragraph A, having a volume weighted mean diameter of about 2 nm to about 15 nm as measured by dynamic light scattering. C. The conjugate of paragraph A or paragraph B, having a volume weighted average hydrodynamic diameter of about 4 nm to about 8 nm as measured by dynamic light scattering. D. The conjugate of any of paragraphs A through C, having a number average molecular weight of about 10,000 to about 150,000. E. The conjugate of any of paragraphs A through D, having a number average molecular weight of about 40,000 to about 80,000. F.R. 6 The conjugate of any of paragraphs A through E, wherein, independently at each occurrence, is halo or hydroxyl. G.R. 5 but, independently in each case, A conjugate of any of paragraphs A-F, which is TIFF2024505002000022.tif19128. H. The conjugate of any of paragraphs A-G, wherein the first proinsulin polypeptide is fused to a sortase moiety, and the first proinsulin polypeptide comprises an insulin B chain of SEQ ID NO:2, an insulin C chain of SEQ ID NO:3, and an insulin A chain of SEQ ID NO:4. I. The conjugate of paragraph H, wherein said sortase moiety is located at the N-terminus or C-terminus of said first proinsulin polypeptide. J. The conjugate of any of paragraphs AI, wherein the sequence of the sortase moiety is SEQ ID NO:6. K. The conjugate of any of paragraphs A-J, wherein the first proinsulin polypeptide comprises an F25D substitution in the insulin B chain of SEQ ID NO:2, and / or a Y19A or Y19L substitution in the insulin A chain of SEQ ID NO:4. L. The conjugate of any of paragraphs A-K, wherein the first proinsulin polypeptide comprises an H10D substitution in the insulin B chain of SEQ ID NO:2. M. The conjugate of any of paragraphs A-L, wherein said first proinsulin polypeptide contains both a P28K and a K29P substitution in the insulin B chain of SEQ ID NO:2. N. The conjugate of any of paragraphs A-M, wherein said first proinsulin polypeptide comprises P28K, K29P, and H10D substitutions in the insulin B chain of SEQ ID NO:2. O. The conjugate of any of paragraphs A-N, wherein said first proinsulin polypeptide comprises a signal peptide sequence. P. The conjugate of paragraph O, wherein said signal peptide sequence is a native or an engineered signal peptide sequence. Q. The conjugate of paragraph O or paragraph P, wherein said signal peptide sequence is SEQ ID NO:5. R. The conjugate of any of paragraphs A-Q, wherein said second proinsulin polypeptide comprises an F25D substitution in the insulin B chain of SEQ ID NO:2, and / or a Y19A or Y19L substitution in the insulin A chain of SEQ ID NO:4. S. The conjugate of any of paragraphs A-R, wherein said second proinsulin polypeptide comprises an H10D substitution in the insulin B chain of SEQ ID NO:2. T. The conjugate of any of paragraphs A-S, wherein said second proinsulin polypeptide contains both a P28K and a K29P substitution in the insulin B chain of SEQ ID NO:2. U. The conjugate of any of paragraphs A-T, wherein said second proinsulin polypeptide comprises P28K, K29P, and H10D substitutions in the insulin B chain of SEQ ID NO:2. V. The conjugate of any of paragraphs A-U, wherein the second proinsulin polypeptide comprises a signal peptide sequence. W. The conjugate of paragraph V, wherein said signal peptide sequence is a native or an engineered signal peptide sequence. X. The conjugate of paragraph V or paragraph W, wherein said signal peptide sequence is SEQ ID NO:5. Y. The conjugate of any of paragraphs A-X, wherein the first proinsulin polypeptide and / or the second proinsulin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8-10. Z. The conjugate of any of paragraphs A-Y, wherein said first proinsulin polypeptide and said second proinsulin polypeptide exhibit reduced activation of the hormone insulin receptor (IR) compared to native human insulin. AA. A composition comprising the conjugate of any of paragraphs A-Z and a pharma- ceutically acceptable carrier. AB. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and an effective amount of any of the compounds of paragraphs A-Z. AC. The pharmaceutical composition of paragraph AB, wherein an effective amount of the compound is effective to treat one or more of type 1 diabetes, juvenile diabetes, insulin-dependent diabetes, or latent autoimmune diabetes in a subject. AD. The pharmaceutical composition of paragraph AC, wherein the subject is not hyperglycemic. AE. The pharmaceutical composition of paragraph AC or paragraph AD, wherein said subject has a detectable level of insulin autoantibodies (IAA). AF. The pharmaceutical composition of any of paragraphs AC-AE, wherein the subject has a detectable level of an insulin-specific B cell population. AG. The pharmaceutical composition of any of paragraphs AC-AF, wherein the subject possesses one or more human leukocyte antigen (HLA) haplotypes selected from the group consisting of: (a) DRB1*0301-DQA1*0501-DQB1*0201; (b) DRB1*0405-DQA1*0301-DQB1*0302; (c) DRB1*0401-DQA1*0301-DQB*0302; (d) DRB1*0402-DQA1*0301-DQB1*0302; (e) DRB1*0404-DQA1*0301-DQB1*0302; and (f) DRB1*0801-DQB1*0401-DQB1*0402. AH. The pharmaceutical composition of any of paragraphs AB-AG, formulated for parenteral, intravenous, or subcutaneous administration. AI. A kit comprising a conjugate of any of paragraphs A-Z and instructions for use. AJ. A method for treating or preventing autoimmune diabetes in a subject in need thereof comprising administering to the subject an effective amount of a conjugate of any of paragraphs A-Z. AK. The method of paragraph AJ, wherein the autoimmune diabetes comprises type 1 diabetes, juvenile onset diabetes, insulin-dependent diabetes, or latent autoimmune diabetes. AL. The method of paragraph AJ or paragraph AK, wherein the subject has been diagnosed with or is at risk for autoimmune diabetes. AM. The method of any of paragraphs AJ-AL, wherein the subject is not hyperglycemic. AN. The method of any of paragraphs AJ-AM, wherein the subject has a detectable level of insulin autoantibodies (IAA). AO. The method of any of paragraphs AJ-AN, wherein the subject has a detectable level of an insulin-specific B cell population. AP. The method of any of paragraphs AJ-AO, wherein the subject possesses one or more human leukocyte antigen (HLA) haplotypes selected from the group consisting of: (a) DRB1*0301-DQA1*0501-DQB1*0201; (b) DRB1*0405-DQA1*0301-DQB1*0302; (c) DRB1*0401-DQA1*0301-DQB*0302; (d) DRB1*0402-DQA1*0301-DQB1*0302; (e) DRB1*0404-DQA1*0301-DQB1*0302; and (f) DRB1*0801-DQB1*0401-DQB1*0402. AQ. The method of any of paragraphs AJ-AP, wherein the conjugate is administered parenterally, intravenously, or subcutaneously. AR. The method of any of paragraphs AJ-AQ, wherein administration of the conjugate induces anergy in insulin-binding B cells in the subject. AS. The method of any of paragraphs AJ-AR, wherein the subject's blood glucose level after at least one administration of the conjugate is equivalent to the blood glucose level observed in the subject prior to at least one administration. AT. The method of any of paragraphs AJ-AS, wherein the subject is a child or an adult.

[0234] Other aspects are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. Formula I or a pharma- ceutically acceptable salt and / or solvate thereof, During the ceremony, R 1 , R 2 , R 3 , and R 4 are each independently and R 5 is independently at each occurrence aryl or aralkyl; R 6 is independently at each occurrence halo, hydroxyl, aryl, or heteroaryl; X 1 is independently at each occurrence the epsilon nitrogen atom of a lysine of SEQ ID NO:2, or a substitution variant thereof in the first proinsulin polypeptide; X 2 is independently, at each occurrence, the carbonyl carbon of the nitrogen atom of the C-terminal glycine of a sortase moiety, wherein said sortase moiety is fused to a second proinsulin polypeptide at the C-terminus of said second proinsulin polypeptide; x is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; m is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; n is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; and p is independently at each occurrence 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; Optionally, the sequence of the sortase portion is SEQ ID NO:

6. The conjugate, or a pharma- ceutically acceptable salt and / or solvate thereof.

2. having a volume weighted mean diameter of about 2 nm to about 15 nm as measured by dynamic light scattering; have a volume weighted average hydrodynamic diameter of about 4 nm to about 8 nm as measured by dynamic light scattering; having a number average molecular weight of about 10,000 to about 150,000; or having a number average molecular weight of about 40,000 to about 80,000; The conjugate of claim 1.

3. R 6 is independently, at each occurrence, halo or hydroxyl; R 5 but, independently in each case, That is, The conjugate of claim 1.

4. the first proinsulin polypeptide is fused to a sortase moiety, the first proinsulin polypeptide comprising an insulin B chain of SEQ ID NO:2, an insulin C chain of SEQ ID NO:3, and an insulin A chain of SEQ ID NO:4; Optionally, the sortase moiety is located at the N-terminus or C-terminus of the first proinsulin polypeptide. The conjugate of claim 1.

5. the first proinsulin polypeptide comprises an F25D substitution in the insulin B chain of SEQ ID NO:2 and / or an Y19A or Y19L substitution in the insulin A chain of SEQ ID NO:4; the first proinsulin polypeptide comprises an H10D substitution in the insulin B chain of SEQ ID NO:2; the first proinsulin polypeptide comprises both a P28K and a K29P substitution in the insulin B chain of SEQ ID NO:2; or the first proinsulin polypeptide comprises P28K, K29P, and H10D substitutions in the insulin B chain of SEQ ID NO:2; The conjugate of claim 1.

6. the first proinsulin polypeptide comprises a signal peptide sequence; Optionally, the signal peptide sequence is a native or engineered signal peptide sequence, or the signal peptide sequence is SEQ ID NO:

5. The conjugate of claim 1.

7. the second proinsulin polypeptide comprises an F25D substitution in the insulin B chain of SEQ ID NO:2 and / or a Y19A or Y19L substitution in the insulin A chain of SEQ ID NO:4, or the second proinsulin polypeptide comprises an H10D substitution in the insulin B chain of SEQ ID NO:2; or the second proinsulin polypeptide comprises both a P28K and a K29P substitution in the insulin B chain of SEQ ID NO:2; or the second proinsulin polypeptide comprises P28K, K29P, and H10D substitutions in the insulin B chain of SEQ ID NO:2; The conjugate of claim 1.

8. the second proinsulin polypeptide comprises a signal peptide sequence; Optionally, the signal peptide sequence is a native or engineered signal peptide sequence, or the signal peptide sequence is SEQ ID NO:

5. The conjugate of claim 1.

9. the first proinsulin polypeptide and / or the second proinsulin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-10, and / or said first proinsulin polypeptide and said second proinsulin polypeptide exhibit reduced activation of the hormone insulin receptor (IR) compared to native human insulin; The conjugate of claim 1.

10. A composition comprising the conjugate of any one of claims 1 to 9 and a pharma- ceutically acceptable carrier.

11. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and an effective amount of the conjugate of any one of claims 1 to 9, Optionally, formulated for parenteral, intravenous, or subcutaneous administration; Pharmaceutical compositions.

12. 12. The pharmaceutical composition of claim 11, wherein an effective amount of the conjugate is effective to treat one or more of type 1 diabetes, juvenile diabetes, insulin-dependent diabetes, or latent autoimmune diabetes in a subject.

13. the subject is not hyperglycemic, or the subject has a detectable level of insulin autoantibodies (IAA); or the subject has a detectable level of an insulin-specific B cell population; or the subject possesses one or more human leukocyte antigen (HLA) haplotypes selected from the group consisting of: (a) DRB1*0301-DQA1*0501-DQB1*0201; (b) DRB1*0405-DQA1*0301-DQB1*0302; (c) DRB1*0401-DQA1*0301-DQB*0302; (d) DRB1*0402-DQA1*0301-DQB1*0302; (e) DRB1*0404-DQA1*0301-DQB1*0302; and (f) DRB1*0801-DQB1*0401-DQB1*0402.

13. The pharmaceutical composition of claim 12.

14. A kit comprising the conjugate of any one of claims 1 to 9 and instructions for use.

15. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 9 for use in treating or preventing autoimmune diabetes in a subject in need thereof.

16. 16. The pharmaceutical composition of claim 15, wherein the autoimmune diabetes comprises type 1 diabetes, juvenile diabetes, insulin-dependent diabetes, or latent autoimmune diabetes.

17. the subject has been diagnosed with or is at risk for autoimmune diabetes, or the subject is not hyperglycemic, or the subject has a detectable level of insulin autoantibodies (IAA); or the subject has a detectable level of an insulin-specific B cell population; or the subject possesses one or more human leukocyte antigen (HLA) haplotypes selected from the group consisting of: (a) DRB1*0301-DQA1*0501-DQB1*0201; (b) DRB1*0405-DQA1*0301-DQB1*0302; (c) DRB1*0401-DQA1*0301-DQB*0302; (d) DRB1*0402-DQA1*0301-DQB1*0302; (e) DRB1*0404-DQA1*0301-DQB1*0302; and (f) DRB1*0801-DQB1*0401-DQB1*0402; or The subject is a child or an adult.

16. The pharmaceutical composition of claim 15.

18. 16. The pharmaceutical composition of claim 15, formulated for parenteral, intravenous, or subcutaneous administration.

19. The pharmaceutical composition of claim 15, wherein administration of the conjugate induces anergy in insulin-binding B cells in the subject.

20. 16. The pharmaceutical composition of claim 15, wherein the blood glucose level of the subject after at least one administration of the conjugate is equivalent to the blood glucose level observed in the subject before at least one administration.

Images