Metallothionein Antibodies and Their Uses - Patent application

Abstract

Disclosed herein are isolated anti-metallothionein antibodies, or fragments thereof, and their uses in treating intestinal inflammation, diabetes, pre-diabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 62 / 874,340, filed July 15, 2019, which is incorporated by reference herein in its entirety.

[0002] Federal Funding Statement This invention was made with Government support under Grant No. ES07408 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Reference to sequence listing This application contains a Sequence Listing that has been submitted as an electronic text file entitled "19-1213-PCT_Sequence-Listing_ST25.txt", having a byte size of 21 kb, created on July 13, 2020. The information contained in this electronic file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5). [Background technology]

[0004] Diabetes is most often treated with insulin replacement therapy. Although there are ongoing studies to replace pancreatic islets damaged by diabetes, islet transplantation or islet stem cell repopulation in the pancreas will be a short-lived treatment unless the inflammatory process that damages the insulin-producing islets is prevented. Similarly, current reagents and methods for treating hepatitis and / or inflammatory liver disease are inadequate. Summary of the Invention

[0005] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a light chain CDR1 (L-CDR1) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence QSIVHSNGNTYLE (SEQ ID NO: 1); a light chain CDR2 (L-CDR2) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence KVS (SEQ ID NO:2); and a light chain CDR3 (L-CDR3) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence FQGSHVPF(A / T) (SEQ ID NO:3); and / or a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence GFSLSRYG (SEQ ID NO: 4); a heavy chain CDR2 (H-CDR2) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence IWSGGST (SEQ ID NO:5); and Provided is an isolated anti-metallothionein antibody, or a fragment thereof, comprising one, two, or all three complementarity determining regions (CDRs) selected from the group consisting of a heavy chain CDR3 (H-CDR3) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence WLLPGY (SEQ ID NO:6).

[0006] In various embodiments, the antibody comprises a monoclonal antibody, or fragment thereof, and / or a humanized antibody, or fragment thereof.

[0007] In other embodiments, the isolated anti-metallothionein antibody, or fragment thereof, comprises a light chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9, wherein the light chain comprises an amino acid sequence of each of SEQ ID NOs: 1-3, and wherein the light chain comprises the following residues: D1, V3, T5, S7, T14, Q17, P18, F and / or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15. and the heavy chain comprises the complementarity determining regions (CDRs) of each of SEQ ID NOs: 4 to 6, and the heavy chain comprises the following residues: E1, V5, G9, K13, G15, E16 or G16, T17, R19, L20, S21, A23, S35, I37, P40 or A40, G42, I48 or V48, S49, N58, A60, P61 or D61, S62, L63 or V63, K64, G65, V67 or F67, T68, S70, V71, T73, A74, N76 or K76, S77 , F78, S79 or Y79, L80, Q81, L82, S83 or N83, V85, T86 or R86, A87, A88 or E88, V92, and Q109.

[0008] In one embodiment, the isolated anti-metallothionein antibody or fragment thereof comprises a Fab' fragment.

[0009] In other aspects, the disclosure provides nucleic acids encoding the isolated antibodies or fragments thereof, vectors comprising the nucleic acids operably linked to suitable control sequences, host cells comprising the nucleic acids and / or vectors, and (a) an isolated antibody or fragment thereof, nucleic acid, vector, or host cell disclosed herein; and (b) providing a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0010] In another aspect, the disclosure provides a method of treating or limiting the occurrence of a disorder selected from the group consisting of intestinal inflammation, diabetes, prediabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease, comprising administering to a subject in need thereof an isolated antibody or fragment thereof or a pharma- ceutically acceptable salt thereof, nucleic acid, vector, host cell, or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein in an amount effective to treat the disorder. [Brief description of the drawings]

[0011] [Figure 1A-B] (A) Clustal Omega software comparison of the humanized kappa light chain with the original murine UC1MT anti-MT L sequence. (B) Schematic representation of the relative differences between the variable regions of UC1MT L-Hum1 (VK1), UC1MT L-Hum2 (VK2), and UC1MT L-Hum3 (VK3) compared to the sequence of the original hybridoma light chain sequence (UC1MT). [Figure 2A-B] (A) Clustal Omega software comparison of the humanized heavy chain V regions with the original murine UC1MT anti-MT H sequence. (B) Graph of the relative differences between the humanized variable regions of UC1MT H-Hum1 (VH1), UC1MT H-Hum2 (VH2), UC1MT H-Hum3 (VH3), UC1MT H-Hum4 (VH4), and UC1MT L-Hum5 (VH5) compared to the sequence of the original hybridoma heavy chain V sequence (UC1MT). [Diagram 3]ELISA measurements of humanized antibodies tested against immobilized metallothionein. Immulon 2HB plates were coated with 10ug / mL MT overnight at 4°C, wells were washed with PBST and blocked with 2% BSA for 1.5 hours at room temperature. Plates were washed, coated with dilutions of each recombinant Fab fragment and incubated for 1.5 hours at room temperature. Plates were washed and incubated with 1:1000 mouse anti-human Ig light chain kappa-biotin for 1 hour at room temperature, then with streptavidin-HRP (Fab-containing wells) for 20 minutes at room temperature. After the final wash, substrate was added and incubated for 15 minutes at room temperature before the addition of stop solution (2N H2SO4) and OD450 was measured on a Spectramax plate reader. Chart represents OD405 values. Reactivity of each clone was characterized as low (L), medium (M), or high (H). [Figure 4A-B] (A) Graph of Fab' fragment binding to metallothionein from in silico modeling. (B) Individual CDRs are shown for both the heavy and light chains of a representative humanized Fab fragment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] As used herein, unless otherwise indicated, the terms "a" and "an" are to be construed to mean "one," "at least one," or "one or more." As used herein, the singular includes the plural and the plural includes the singular, unless otherwise required by context. All embodiments of any aspect of the present disclosure can be used in combination, unless the context clearly indicates otherwise.

[0013] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0014] In one aspect, the present disclosure provides an anti-metallothionein (MT) antibody, or fragment thereof.Anti-metallothionein antibody or fragment thereof is useful for treating disorders selected from the group consisting of, for example, intestinal inflammation, diabetes, pre-diabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease.The use of MT inhibitors for treating such disorders is described in WO2019 / 143767, the entirety of which is incorporated herein by reference.

[0015] As disclosed herein, "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an epitope of metallothionein. As such, the term antibody encompasses whole antibody molecules as well as antibody fragments and variants (including derivatives) of antibodies and antibody fragments. Such antibodies or antibody fragments may include, but are not limited to, monoclonal antibodies, humanized antibodies, chimeric antibodies, Fab', F(ab')2, Fab, Fv, rIgG, recombinant single chain Fv fragments (scFv), bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. In one particular embodiment, the antibody comprises a monoclonal antibody, or a fragment thereof. In a further particular embodiment, the antibody comprises a humanized antibody, or a fragment thereof. In one embodiment, the antibody comprises a Fab' fragment.

[0016] In one embodiment, the antibody is humanized. As used herein, the term "humanized antibody" refers to a chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDRs are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that has the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity, expression, and reduced immunogenicity of xenogeneic antibodies, such as murine antibodies, for introduction into humans, while maintaining the full antigen-binding affinity and specificity of the antibody.

[0017] As used herein, "isolated" means that the referenced molecule is present in the substantial absence of other biological macromolecules of the same type.

[0018] In one embodiment, a humanized antibody or fragment thereof is provided that comprises one, two, or all three complementarity determining regions (CDRs) selected from the group consisting of: a light chain CDR1 (L-CDR1) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence QSIVHSNGNTYLE (SEQ ID NO:1); a light chain CDR2 (L-CDR2) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence KVS (SEQ ID NO:2); and A light chain CDR3 (L-CDR3) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence FQGSHVPF(A / T) (SEQ ID NO:3).

[0019] In another embodiment, a humanized anti-metallothionein antibody or fragment thereof is provided that comprises one, two, or all three complementarity determining regions (CDRs) selected from the group consisting of: a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence GFSLSRYG (SEQ ID NO: 4); a heavy chain CDR2 (H-CDR2) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence IWSGGST (SEQ ID NO:5); and A heavy chain CDR3 (H-CDR3) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence WLLPGY (SEQ ID NO:6).

[0020] In a further embodiment, the humanized anti-metallothionein antibody, or fragment thereof, of claim 1 comprises four, five, or all six complementarity determining regions (CDRs) selected from the group consisting of: a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence GFSLSRYG (SEQ ID NO: 4); a heavy chain CDR2 (H-CDR2) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence IWSGGST (SEQ ID NO:5); and A heavy chain CDR3 (H-CDR3) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence WLLPGY (SEQ ID NO:6); a light chain CDR1 (L-CDR1) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence QSIVHSNGNTYLE (SEQ ID NO:1); a light chain CDR2 (L-CDR2) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence KVS (SEQ ID NO:2); and A light chain CDR3 (L-CDR3) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence FQGSHVPF(A / T) (SEQ ID NO:3).

[0021] In another aspect, an isolated anti-metallothionein antibody, or fragment thereof, is provided, comprising a light chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9, as set forth in Table 1 (see also FIG. 1 ), The chains comprise the complementarity determining regions (CDRs) of each of SEQ ID NOs: 1-3, and the light chain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following residues (listed in bold and enlarged font in Table 1): D1, V3, T5, S7, T14, Q17, P18, F41, Q42, R44, R50, V88, V90, T102, Q105, and K112. [Table 1]

[0022] Bold indicates changes relative to the parental mouse hybridoma, and the CDRs are underlined.

[0023] In one particular embodiment, the isolated anti-metallothionein antibody, or fragment thereof, comprises a light chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8, wherein the light chain comprises the CDRs of each of SEQ ID NOs:1-3, and wherein the light chain comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen of the following residues (listed in bold and enlarged font in Table 1): D1, V3, T5, S7, T14, Q17, P18, R44, R50, V88, V90, Q105, and K112.

[0024] In another embodiment, the light chain of the isolated anti-metallothionein antibody, or fragment thereof, further comprises a kappa constant region from a human light chain, including, but not limited to, an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0025] In another aspect, an isolated anti-metallothionein antibody, or fragment thereof, is provided, comprising a heavy chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15, wherein the heavy chain comprises a complementarity determining region (CDR) of each of SEQ ID NOs: 4-6, and wherein the heavy chain is comprised of the following residues (set out in bold and enlarged font in Table 2, see also FIG. 2): E1, V5, G9, K13, G15, E16 or G16, T17, R19, L20, S21, A23, S35, I37, P40 or A40, G42, I4 8 or V48, S49, N58, A60, P61 or D61, S62, L63 or V63, K64, G65, V67 or F67, T68, S70, V71, T73, A74, N76 or K76, S77, F78, S79 or Y79, L80, Q81, L82, S83 or N83, V85, T86 or R86, A87, A88 or contains at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty-one, thirty-two, thirty-three, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, twenty-nine, twenty-one, twenty-one, twenty-one, twenty-one, twenty-one, twenty-one, twenty-one, twenty-one, twenty-one, twenty-one,

[0026] In the sequences below, bold indicates changes relative to the parental UC1MT mouse hybridoma sequence, and the CDRs are underlined. [Table 2]

[0027] In one particular embodiment, the isolated anti-metallothionein antibody, or fragment thereof, comprises a heavy chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14, wherein the heavy chain comprises the complementarity determining regions (CDRs) of each of SEQ ID NOs: 4-6, and wherein the heavy chain comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen of the following residues (listed in bold and enlarged font in Table 2, see also FIG. 2): K13, E16, V37, G42, K64, S70, N76, V78, S83, T86, A87, A88, and Q109.

[0028] In another specific embodiment, the isolated anti-metallothionein antibody, or fragment thereof, comprises a heavy chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain comprises a complementarity determining region (CDR) of each of SEQ ID NOs: 4-6, and wherein the heavy chain comprises the following residues (listed in bold and expanded font in Table 2, see also FIG. 2): E1, V5, G9, G15, G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13, G14, G15, G16, G17, G18, G19, G20, G21, G22, G23, G24, G25, G26, G27, G28, G29, G30, G31, G32, G33, G34, G35, G36, G37, G38, G39, G40, G41, G42, G43, G44, G45, G46, G47, G48, G49, G50, G51, G52, G53, G54, G55, G56, G57, G58, G59, G60, G61, G62, G63, G64, G65, G66, G67, G68, G69, G70, G71, G72, G73, G74, G75, G75, G76, G77, G78, G79, G82, G83, G84, G85 , G16, R19, L20, S21, A23, A40, G42, V48, S49, A60, D61, S62, V63, K64, G65, F67, T68, A74, K76, S77, V78, Y79, L80, Q81, N83, R86, A87, E88, V92, and Q109.

[0029] In another embodiment, the heavy chain further comprises a human IgG1 constant sequence containing a mutation that prevents glycosylation, including, but not limited to, an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:27. [Table 3]

[0030] In a further embodiment, the isolated anti-metallothionein antibody, or fragment thereof, (a) a light chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9, wherein the light chain comprises a complementarity determining region (CDR) of each of SEQ ID NOs: 1-3, and wherein the light chain comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or all sixteen of the following residues: D1, V3, T5, S7, T14, Q17, P18, F41, Q42, R44, R50, V88, V90, T102, Q105, and K112; (b) a heavy chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-26 or selected from the group consisting of SEQ ID NOs: 11-15, wherein the heavy chain comprises a complementarity determining region (CDR) of each of SEQ ID NOs: 4-6, and wherein the heavy chain comprises the following residues (listed in bold and enlarged font in Table 2, see also FIG. 2): E1, V5, G9, K13, G15, E16 or G16, T17, R19, L20, S21, A23, S35, I37, P40 or A40, G42, I48 or V48. , S49, N58, A60, P61 or D61, S62, L63 or V63, K64, G65, V67 or F67, T68, S70, V71, T73, A74, N76 or K76, S77, F78, S79 or Y79, L80, Q81, L82, S83 or N83, V85, T86 or R86, A87, A88 or E8 8, V92, and Q109.

[0031] In another specific embodiment, the isolated anti-metallothionein antibody, or fragment thereof, (a) a light chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8, wherein the light chain comprises the CDRs of each of SEQ ID NOs:1-3, wherein the light chain comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or all thirteen of the following residues (set out in bold and enlarged font in Table 1): D1, V3, T5, S7, T14, Q17, P18, R44, R50, V88, V90, Q105, and K112; (b) a heavy chain comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 15, wherein the heavy chain comprises a complementarity determining region (CDR) of each of SEQ ID NOs: 4 to 6, (i) one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen of the following residues: K13, E16, V37, G42, K64, S70, N76, V78, S83, T86, A87, A88, and Q109, or (ii) the following residues (listed in bold and enlarged font in Table 2, see also Figure 2): E1, V5, G9, G15, G16, R19, L20, S21, A23, A40, G42, V48, S49, A60, D61, S62, V63, K64, G65, F67, T68, A74, K76, S77, V78, Y79, Contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine ...

[0032] The anti-MT antibodies of the present disclosure may be linked to any other moiety deemed suitable for the intended purpose. In one embodiment, the isolated antibody or fragment thereof further comprises a detectable label. Such an embodiment may be useful, for example, for monitoring the course of treatment. Any suitable detectable label, including but not limited to radioisotopes, fluorescent molecules, magnetic particles (including nanoparticles), metal particles (including nanoparticles), phosphorescent molecules, and enzymes, may be covalently, genetically, or by any other means attached to the antibody.

[0033] In another embodiment, the isolated antibody or fragment thereof further comprises an additional therapeutic agent. Therapeutic agents suitable for the intended purpose include insulin, metformin, pramlintide, sulfonylureas (including but not limited to glyburide, glipizide, and glimepiride), meglitinides (including but not limited to repaglinide and nateglinide), thiazolidinediones (including but not limited to rosiglitazone and pioglitazone), DPP-4 inhibitors (including but not limited to sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists, and the like. The conjugates may be linked, either covalently, genetically, or by any other means, to antibodies, including, but not limited to, stimulants (including, but not limited to, exenatide, liraglutide, and semaglutide), SGLT2 inhibitors (including, but not limited to, canagliflozin, dapagliflozin, and empagliflozin), entecavir, tenofovir, lamivudine, adefovir, telbivudine, simeprevir, sofosbuvir, cytokines, chemokines, interferons, or ribavirin. Such conjugates may be useful, for example, in the methods disclosed herein.

[0034] In another embodiment, the isolated antibody or fragment thereof further comprises a cell targeting moiety. Any cell targeting molecule can be used as appropriate for the intended cell target, including but not limited to pancreatic or hepatocyte targeting moiety (such as pancreatic cell targeting moiety comprising one or more peptides or other moieties that preferentially bind to pancreatic β cells, including but not limited to glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), and exendin-4, or hepatocyte targeting moiety, where the hepatocyte targeting moiety comprises but is not limited to circumsporozoite protein (CSP), CSP domain I, CSP I plus, lactosaminylated human serum albumin, glycosylated lipoprotein, and arabinogalactan). Such conjugates can be useful, for example, in the methods disclosed herein.

[0035] In another aspect, disclosed is an isolated nucleic acid encoding the antibody of any embodiment or combination of embodiments disclosed herein.The isolated nucleic acid sequence may comprise RNA or DNA.Such isolated nucleic acid sequence may comprise additional sequences useful for facilitating the expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, transport signals, and secretion signals, nuclear localization signals, and plasma membrane localization signals.

[0036] In a further aspect, an expression vector is provided that comprises the isolated nucleic acid of the present disclosure. An "expression vector" includes a vector that operably links a nucleic acid coding region or gene to any promoter that can cause expression of a gene product. The promoter sequence used to drive the expression of the disclosed nucleic acid sequence in a mammalian system can be constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters, including but not limited to tetracycline, ecdysone, steroid responsive). The expression vector can replicate in a suitable host organism as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector comprises a plasmid or a viral vector.

[0037] In a further aspect, a host cell is provided that comprises the antibody, nucleic acid, and / or vector of the present disclosure. The host cell can be either prokaryotic or eukaryotic. The cell can be transiently or stably transfected. In one embodiment, the cell is a hybridoma cell that expresses and secretes the antibody of the present disclosure. Thus, the recombinant host cell can be used in, for example, a method for producing an antibody, comprising: (a) culturing the recombinant host cell under conditions suitable for expression of the nucleic acid encoding the antibody; (b) isolating the antibody from the cultured cells.

[0038] The conditions appropriate for expression of a nucleic acid encoded antibody can be determined by one of skill in the art based on the teachings of the present specification, the particular host cells and vectors used, and the general knowledge of one of ordinary skill in the art.

[0039] In another aspect, (a) an antibody, isolated nucleic acid, expression vector, or host cell of any embodiment or combination of embodiments disclosed herein; and (b) A pharmaceutical composition is provided, comprising a pharma- ceutically acceptable carrier.

[0040] For example, the antibody of the present disclosure may be present in a pharmaceutical preparation. In this embodiment, the antibody is combined with a pharmaceutically acceptable carrier. Suitable acids capable of forming such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid, and the like. Suitable bases capable of forming such salts include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like; and organic bases such as mono-, di-, and trialkyl and arylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, and the like), and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, and the like).

[0041] In addition to the compositions of the present invention, the pharmaceutical composition may include (a) a lyoprotectant, (b) a surfactant, (c) a bulking agent, (d) a tonicity adjusting agent, (e) a stabilizer, (f) a preservative, and / or (g) a buffering agent. In some embodiments, the buffering agent in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, such as glycine. In yet other embodiments, the pharmaceutical composition comprises a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or combinations thereof. The pharmaceutical composition may also comprise a tonicity adjuster, such as a compound that renders the formulation substantially isotonic or isosmotic with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that, when combined with the protein of interest, substantially prevents or reduces the chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0042] The pharmaceutical compositions of the present invention may be comprised in any suitable formulation, preferably a formulation suitable for administration by injection. Such pharmaceutical compositions may be used, for example, in the therapeutic methods disclosed herein.

[0043] The pharmaceutical composition may contain any other components deemed appropriate for a given use, such as additional therapeutic agents. In one embodiment, the pharmaceutical composition is selected from the group consisting of insulin, metformin, pramlintide, sulfonylureas (including but not limited to glyburide, glipizide, and glimepiride), meglitinides (including but not limited to repaglinide and nateglinide), thiazolidinediones (including but not limited to rosiglitazone and pioglitazone), DPP-4 inhibitors (including but not limited to sitagliptin, saxagliptin, and linagliptin). and further comprising an additional therapeutic agent selected from the group consisting of: an anti-inflammatory drug (e.g., sirolimus), a sirolimus inhibitor (e.g., sirolimus), a sirolimus agonist ...

[0044] In another aspect, the disclosure provides a method of treating or limiting the occurrence of a disorder selected from the group consisting of intestinal inflammation, diabetes, prediabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease, comprising administering to a subject in need thereof an isolated antibody or fragment thereof, nucleic acid, vector, host cell, and / or pharmaceutical composition of any embodiment or combination of embodiments disclosed herein in an amount effective to treat the disorder.

[0045] As used herein, a "therapeutically effective amount" refers to an amount of a composition that is effective for treating and / or limiting the associated disorder.

[0046] As used herein, "treat" or "treating" means achieving one or more of the following in an individual having one or more of the listed disorders: (a) reducing the severity of the disorder, (b) limiting or preventing the onset of symptoms characteristic of the disorder(s) being treated, (c) inhibiting the worsening of symptoms characteristic of the disorder(s) being treated, (d) limiting or preventing the recurrence of the disorder(s) in patients who have previously had the disorder(s), and / or (e) limiting or preventing the recurrence of symptoms in patients who were previously symptomatic of the disorder(s). Any such degree of "treating" will be of great benefit to a subject with one of the listed disorders.

[0047] As used herein, "limit" or "limiting the onset" means achieving one or more of the following in an individual at risk for one or more of the enumerated disorders: (a) slowing the progression to the disorder, and / or (b) limiting or preventing the onset of symptoms characteristic of progression to the disorder. Any degree of such "limiting the onset" is of great benefit to a subject at risk for one of the enumerated disorders.

[0048] Such treatment or limitation of onset may involve the use of an extracellular MT inhibitor as the sole therapeutic agent or may involve its use to complement or augment other therapeutic interventions as deemed appropriate by the attending medical practitioner.

[0049] In one embodiment, the subject has or is at risk of developing diabetes, and the method serves to treat or limit the onset of diabetes.

[0050] In one such embodiment, the subject is at risk for type 1 diabetes, and the method helps to limit the onset of type I diabetes in the subject. As described in WO2019 / 143767, human MT1 inhibitors prevented the onset of type 1 diabetes in a NOD mouse model. Thus, the method of this embodiment can be used to limit the onset of type 1 diabetes (T1D) in a subject at risk for T1D. Limiting the onset of T1D can include, but is not limited to, delaying the progression to T1D and / or delaying the onset of symptoms characteristic of T1D. In this embodiment, a subject at risk for T1D has one or more risk factors for T1D from which a medical professional in charge considers treatment appropriate. Such risk T1D risk factors include, but are not limited to, a parent or sibling with type 1 diabetes, pancreatic tumor, pancreatitis, islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), insulinoma-associated (IA-2) autoantibodies, zinc transporter autoantibodies (ZnT8), and / or variants of the IDDM1 gene selected from the group consisting of DRB1 0401, DRB1 0402, DRB1 0405, DQA 0301, DQB1 0302, and DQB1 0201. Alternatively, or in combination, the subject may exhibit one or more symptoms of T1D (but not yet diagnosed with T1D), such symptoms may include, but are not limited to, polyuria (increased urination), polydipsia (increased thirst), dry mouth, polyphagia (increased hunger), fatigue, and weight loss. As will be appreciated by those skilled in the art, any limitation on the onset of T1D or its symptoms provides a great benefit for at-risk subjects.

[0051] The insulin-dependent (type I) diabetes mellitus 1 (IDDM1) gene is located in the MHC class II region of chromosome 6. Certain variants of this gene increase the risk of the reduced histocompatibility characteristic of type 1 diabetes. Such variants include DRB1 0401, DRB1 0402, DRB1 0405, DQA 0301, DQB1 0302, and DQB1 0201. Similarly, the appearance of diabetes-associated autoantibodies, such as islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), insulinoma-associated (IA-2) autoantibodies, and zinc transporter autoantibodies (ZnT8), often precedes hyperglycemic type 1 diabetes before any hyperglycemia occurs. The risk of T1D increases with the number of antibody types, and the time interval from the appearance of autoantibodies to clinically diagnosable T1D can be several months in infants and young children, but may take several years in some individuals. Such autoantibodies can be detected, for example, by immunofluorescence or binding assays.

[0052] In another embodiment, the subject has type 1 diabetes, and the method is useful for treating type I diabetes in the subject. In this embodiment, the subject has already been diagnosed with T1D, and the method can be used to treat T1D. T1D is characterized by autoimmune destruction of pancreatic beta cells, little or no insulin production, and hyperglycemia. Treatment of T1D includes administering insulin to the subject. Subjects with T1D may have symptoms or complications including, but not limited to, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, impaired wound healing ability, urinary tract infections, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure, and foot ulcers. In some cases, subjects with T1D may require pancreas or islet transplantation. Thus, in various embodiments, treatment may include one or more of reducing the frequency of need for insulin injections, delaying the onset or progression of type 1 diabetes complications in a subject, including, but not limited to, destruction of pancreatic beta cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, impaired wound healing ability, urinary tract infections, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure, and foot ulcers, as well as delaying the need for pancreas or islet cell transplantation. In one embodiment, treatment may include, for example, reducing blood glucose levels (mg / dL) by 10%, 15%, 20% or more within 20-120 minutes after administration of the inhibitor.

[0053] In another embodiment, the subject is at risk for type 2 diabetes, and the method is useful for limiting the onset of type 2 diabetes (T2D) in the subject. T2D is a metabolic disorder characterized by hyperglycemia, insulin resistance, and a relative lack of insulin. Symptoms and / or complications include, but are not limited to, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, glomerular clearance defects, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, and stroke. In one embodiment, limiting the onset of type 2 diabetes can include, for example, reducing blood glucose levels (mg / dL) by 10%, 15%, 20% or more within 20-120 minutes after administration of the inhibitor.

[0054] Risk factors for T2D include, but are not limited to, obesity, smoking, a sedentary lifestyle, a parent or sibling with type 2 diabetes, prediabetes, a parent or sibling with prediabetes, a poor diet (e.g., too much fat, not enough fiber, too many simple carbohydrates, etc.), age 50 years or older, high blood pressure, high cholesterol, testosterone deficiency, metallothionein 1 A (MT1A) rs8052394 locus (G change) single nucleotide polymorphism, and a history of gestational diabetes. Thus, in various embodiments, a subject has one or more of these risk factors, and the method helps to delay progression to T2D and / or (b) limit or prevent the onset of symptoms characteristic of T2D.

[0055] In another embodiment, the subject has T2D and the method is useful for treating T2D in the subject. In this embodiment, the treatment may include limiting one or more of type 2 diabetes complications, including but not limited to hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, glomerular clearance defects, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, stroke, and reducing the frequency of need for insulin or other therapy. Any degree of limiting these symptoms / complications is of great benefit to the subject with T2D. In one embodiment, the treatment may include reducing blood glucose levels (mg / dL) by 10%, 15%, 20% or more, for example, within 20-120 minutes after administration of the inhibitor.

[0056] In another embodiment, the subject has prediabetes and the method is useful for treating prediabetes in the subject. In this embodiment, the subject is a subject with prediabetes. As used herein, "prediabetes" is a condition in which some, but not all, diagnostic criteria for diabetes are met. Thus, a prediabetic subject may have (a) impaired fasting glucose tolerance, a condition due to a lack of beta cell response to oral glucose load (OGT), or (b) consistently high fasting blood glucose (IFG), a condition in which fasting blood glucose levels are elevated above what is considered normal levels, but not high enough to be classified as diabetes. The prediabetic state may be associated with increased risk of insulin resistance and cardiovascular pathology. Individuals with a prediabetic state are at relatively high risk of developing T2D. The methods of the present disclosure may be used to treat a subject with prediabetes, for example, by limiting or delaying the progression of one or more complications of prediabetes, including but not limited to T2D, hyperglycemia, insulin resistance, and / or cardiovascular disease. In one embodiment, treatment can include, for example, reducing blood glucose levels (mg / dL) by 10%, 15%, 20% or more within 20-120 minutes after administration of the inhibitor.

[0057] In another embodiment, the subject has impaired glucose tolerance and the method is useful for treating impaired glucose tolerance in the subject. As used herein, impaired glucose tolerance is defined as a 2-hour glucose level of 140-199 mg per deciliter (7.8-11.0 mmol / l) in a 75-g oral glucose tolerance test. A patient is below the condition of IGT if they have a moderately elevated glucose level after 2 hours, but below that level they have type 2 diabetes. Fasting blood glucose levels may be normal or mildly elevated. Impaired glucose tolerance is a pre-diabetic state of hyperglycemia associated with increased risk of insulin resistance and cardiovascular pathology. IGT may occur several years before type 2 diabetes. In this embodiment, the treatment may include limiting or delaying the progression of one or more complications of impaired glucose tolerance, including but not limited to type 2 diabetes, hyperglycemia, insulin resistance, and / or cardiovascular disease. In one embodiment, treatment can include, for example, reducing blood glucose levels (mg / dL) by 10%, 15%, 20% or more within 20-120 minutes after administration of the inhibitor.

[0058] In another embodiment, the subject has hepatitis, and the method is useful for treating hepatitis in the subject. As described in WO2019 / 143767, MT inhibitors are effective in limiting tissue inflammation and increasing anti-inflammatory IL-10 signaling in liver tissue while lowering pro-inflammatory cytokines MCP-1 and TNF-a. Hepatitis is inflammation of liver tissue. Symptoms include, but are not limited to, yellow discoloration of the skin and whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, scarring of the liver, liver failure, or liver cancer. The most common causes of hepatitis are viral infections (types A, B, C, D, and E), heavy alcohol use, certain drugs, toxins, other infections, autoimmune diseases, and nonalcoholic steatohepatitis (NASH). Thus, in various embodiments, treatment may include limiting or slowing the progression of one or more complications of hepatitis, including, but not limited to, yellowing of the skin and / or whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, scarring of the liver, liver failure, and liver cancer.

[0059] In further embodiments, the subject has an inflammatory liver disease, and the method is useful for treating the inflammatory liver disease in the subject. As used herein, "inflammatory liver disease" is a condition associated with the cytoplasmic accumulation of large vacuoles of triglyceride fat in hepatocytes via steatosis (i.e., abnormal retention of lipids within the cells). The liver plays a major role in whole body metabolism, and energy imbalance is associated with defects in lipid metabolism, particularly in the liver. Specifically, obesity and insulin resistance are often associated with increased lipid deposition in the liver, characteristic of non-alcoholic fatty liver disease (NAFLD). While lipid metabolism is highly dynamic, chronic lipid overload leads to recruitment of liver-resident and non-resident immune cells that can cause tissue damage in the liver and fibrosis characteristic of non-alcoholic steatohepatitis (NASH). Liver fibrosis can lead to cirrhosis, cancer, and significantly increase the risk of cardiovascular disease. This raises the possibility of blocking immune cell recruitment to the liver to improve the risk of non-alcoholic fatty liver disease (NAFLD). As shown in the following examples, treatment with MT inhibitors caused an increase in wet tissue weight of epididymal white adipose tissue, and a decrease in total triglyceride levels, as well as an increase in anti-inflammatory IL-10 signaling and a decrease in proinflammatory cytokines MCP-1 and TNF-a. Inflammatory liver disease can be steatosis (non-alcoholic fatty liver disease (NAFL)). In another embodiment, fatty liver disease can be non-alcoholic fatty liver disease (NAFLD), including but not limited to non-alcoholic steatohepatitis (NASH), which is the most extreme form of NAFLD. NAFLD is one of the types of inflammatory liver disease that occurs when fat accumulates in the liver (steatosis) due to causes other than excessive alcohol use. Symptoms of NASH and NAFLD may include, but are not limited to, fatigue, lethargy, dull right upper quadrant discomfort, mild jaundice, and abnormal liver function test results during routine blood work, and complications of NASH and NAFLD may include, but are not limited to, liver fibrosis, liver cancer, and / or cirrhosis.Thus, in one embodiment, the inflammatory liver disease is selected from the group consisting of nonalcoholic steatohepatitis (NASH) and / or nonalcoholic fatty liver disease (NAFLD).In a further embodiment, the treatment comprises limiting or slowing the progression of one or more complications of inflammatory liver disease, including but not limited to fatigue, malaise, liver fibrosis, liver cancer, and / or liver cirrhosis.

[0060] In another embodiment, the subject has intestinal inflammation, which may include, but is not limited to, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, and undifferentiated colitis, and Behcet's disease.

[0061] In one embodiment, MT inhibitors, including but not limited to anti-MT antibodies, can be linked to pancreatic cell targeting moieties to specifically target MT-producing pancreatic cells, such as pancreatic beta cells. This embodiment would be particularly useful for treating or limiting the onset of T1D, T2D, prediabetes, and / or impaired glucose tolerance. In one embodiment, the pancreatic beta cell-specific targeting moiety comprises one or more peptides or other moieties that preferentially bind to pancreatic beta cells, including but not limited to glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), and exendin-4. Exemplary amino acid sequences of such targeting moieties are provided in Table 3. [Table 4]

[0062] Attachment of the pancreatic cell targeting moiety to the MT inhibitor, including but not limited to the MT antibody or fragment thereof, can be accomplished by any chemical reaction that bonds the two molecules, so long as the pancreatic cell targeting moiety and the MT antibody or fragment thereof retain their respective activities. In one embodiment, where the extracellular MT inhibitor comprises an antibody or fragment thereof, the composition comprises a recombinant fusion protein. In other embodiments, the link between the pancreatic cell targeting moiety and the MT antibody or fragment thereof can include many chemical mechanisms, such as covalent bonds, affinity bonds, intercalation, coordinate bonds, and complex formation. Covalent bonds can be achieved by direct condensation of existing side chains or by incorporating external cross-linking molecules. Many bivalent or multivalent linking agents are useful for linking protein molecules, such as antibodies, to other molecules. For example, representative non-limiting examples of linking agents can be organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine.

[0063] In all embodiments, the subject can be any subject that can benefit from the therapeutic methods disclosed herein, including mammals, humans, cows, dogs, cats, horses, chickens, etc. In one embodiment, the subject is a human.

[0064] The composition for administration is typically formulated as a pharmaceutical composition to include a pharmaceutically acceptable carrier.Suitable acids that can form pharmaceutically acceptable salts include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, etc.; organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid, etc.Suitable bases that can form such salts include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc.; and organic bases such as mono-, di-, and trialkyl and arylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, etc.), and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, etc.).

[0065] In addition to the composition and carrier, the pharmaceutical composition may include (a) a lyoprotectant, (b) a surfactant, (c) a bulking agent, (d) a tonicity adjusting agent, (e) a stabilizer, (f) a preservative, and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, such as glycine. In yet other embodiments, the pharmaceutical composition comprises a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or combinations thereof. The pharmaceutical composition may also comprise a tonicity adjuster, such as a compound that renders the formulation substantially isotonic or isosmotic with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that, when combined with the protein-based composition, substantially prevents or reduces chemical and / or physical instability of the protein in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0066] The composition may be administered in a dosage unit formulation containing conventional pharma- ceutically acceptable carriers, adjuvants, and vehicles via any suitable route, including orally, parenterally, inhalation spray, rectally, or topically. The term parenteral as used herein includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneally. Dosage regimens may be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). A suitable dose range may be, for example, 0.1 ug / kg to 100 mg / kg body weight, or it may be 0.5 ug / kg to 50 mg / kg, 1 ug / kg to 25 mg / kg, or 5 ug / kg to 10 mg / kg body weight. The composition may be delivered in a single bolus, or may be administered two or more times (e.g., 2, 3, 4, 5 or more times) as determined by the attending medical professional. The composition may be the only therapeutic agent administered or may be administered together (separately or in combination) with one or more other therapeutic agents as deemed appropriate by the attending medical professional. In one non-limiting embodiment, the subject has or is at risk for T1D, and the inhibitor may be used together with one or more of insulin, metformin, or pramlintide. In another embodiment, the subject has or is at risk for T2D, prediabetes, and / or impaired glucose tolerance, and the inhibitor may be used in combination with one or more of metformin, sulfonylureas (including but not limited to glyburide, glipizide, and glimepiride), meglitinides (including but not limited to repaglinide and nateglinide), thiazolidinediones (including but not limited to rosiglitazone and pioglitazone), DPP-4 inhibitors (including but not limited to sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists (including but not limited to exenatide, liraglutide, and semaglutide), SGLT2 inhibitors (including but not limited to canagliflozin, dapagliflozin, and empagliflozin), or insulin.In one non-limiting embodiment, the subject has or is at risk for hepatitis and the inhibitor may be used in combination with one or more of entecavir, tenofovir, lamivudine, adefovir, telbivudine, simeprevir, sovosbuvir, interferon, or ribavirin.

[0067] In another embodiment, (a) an anti-metallothionein antibody disclosed herein, and (b) compositions are provided that comprise one or more of insulin, metformin, pramlintide, sulfonylureas (including but not limited to glyburide, glipizide, and glimepiride), meglitinides (including but not limited to repaglinide and nateglinide), thiazolidinediones (including but not limited to rosiglitazone and pioglitazone), DPP-4 inhibitors (including but not limited to sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists (including but not limited to exenatide, liraglutide, and semaglutide), SGLT2 inhibitors (including but not limited to canagliflozin, dapagliflozin, and empagliflozin), entecavir, tenofovir, lamivudine, adefovir, telbivudine, simeprevir, sofosbuvir, interferon, or ribavirin. The composition may be used, for example, in the methods of the present disclosure. All of the antibody embodiments and combinations of embodiments disclosed above are suitable for inclusion in the composition of this aspect. In one embodiment, the anti-MT antibody or antigen-binding fragment thereof comprises a monoclonal antibody or antigen-binding fragment thereof. In another embodiment, the anti-MT antibody comprises a humanized anti-MT antibody or antigen-binding fragment thereof.

[0068] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: (a) an anti-metallothionein antibody disclosed herein, and (b) A composition is provided, comprising a pancreatic or hepatocyte targeting moiety linked to an inhibitor of extracellular human MT.

[0069] The composition may be used, for example, in the methods of the present disclosure. All of the antibody embodiments and combinations of embodiments disclosed above are suitable for inclusion in the composition of this aspect. In one embodiment, the anti-MT antibody or antigen-binding fragment thereof comprises a monoclonal antibody or antigen-binding fragment thereof. In another embodiment, the anti-MT antibody comprises a humanized anti-MT antibody or antigen-binding fragment thereof.

[0070] In one embodiment, the cell targeting moiety is a hepatocyte targeting moiety.In an exemplary embodiment, the hepatocyte targeting moiety includes, but is not limited to, circumsporozoite protein (CSP), CSP domain I, CSP domain I plus, lactosaminylated human serum albumin, glycosylated lipoprotein, and / or arabinogalactan.In one embodiment, the hepatocyte binding moiety is peptidic and is selected from CSP, CSP domain I, CSP domain I plus, or hepatocyte binding fragments thereof. CSP targets Plasmodium sporozoites to the liver and is attributed to the circumsporozoite protein (CSP) present on the surface of Plasmodium sporozoites (Rathore D, et al. The Journal of Biological Chemistry. 2005; 280(21): 20524-20529). CSP is approximately 400 amino acids long organized into three domains: an N-terminal domain containing a conserved KLKQP motif named "region I", a highly repetitive central domain, and a C-terminal domain containing another conserved sequence named "region II" (Singh et al. Cell. 2007; 131(3): 492-504). In addition to the conserved region I KLKQP sequence, the N-terminal region also contains, upstream of region I, two consensus heparin sulfate binding sequences. Peptides that contain both the conserved domain I amino acids and two consensus heparin-binding sequences upstream from domain I are designated "domain I-plus" (Prudencio et al., Nature Reviews Microbiology. 2006;4(11):849-856).

[0071] In another embodiment, the cell targeting moiety is a pancreatic cell targeting moiety, including, but not limited to, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), and exendin-4, exemplary amino acid sequences of such peptides are described above.

[0072] Attaching the cell targeting moiety to the anti-MT antibody or fragment thereof can be accomplished by any chemical reaction that bonds the two molecules, so long as the cell targeting moiety and the anti-MT antibody or fragment thereof retain their respective activities. In one embodiment, the composition comprises a recombinant fusion protein. In other embodiments, the link between the cell targeting moiety and the anti-MT antibody or fragment thereof can include many chemical mechanisms, such as covalent bonds, affinity bonds, intercalation, coordinate bonds, and complex formation. Covalent bonds can be achieved by direct condensation of existing side chains or by incorporating external cross-linking molecules. Many bivalent or multivalent linking agents are useful for linking protein molecules, such as antibodies, to other molecules. For example, representative non-limiting examples of linking agents can be organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine.

[0073] In all embodiments, the subject can be any subject that can benefit from the therapeutic methods disclosed herein, including mammals, humans, cows, dogs, cats, horses, chickens, etc. In one embodiment, the subject is a human. EXAMPLES

[0074] Humanization of UC1MT monoclonal anti-metallothionein antibody The murine UC1MT monoclonal anti-metallothionein (MT) antibody was humanized using the sequences PromabKV6,10 (light chain) and MHC2822HC / Promab (heavy chain). Three human light chain sequences and five human heavy chain sequences were designed. The sequences were designed in a nested manner, loosely from more to less altered. The heavy and light chain designs were tested in all possible pairwise combinations with the parent V regions, resulting in 24 combinations. The constant region used was the consensus human heavy chain, shared by all heavy and light chains used in these permutations.

[0075] Parental sequences of the original UC1MT mouse hybridoma (heavy and light chain V regions) A. Primary UC1MT hybridoma source sequence of the heavy chain (ProMab) [Table 5] (Lake MHC2822HC replica sequence relative to the original ProMab sequence): [Table 6] Light chain source sequence from the original UC1MT hybridoma of the CDRs (underlined) [Table 7]

[0076] Sequence Design Considerations The starting sequence was first compared to mouse germline V regions to identify potential mutations in the framework regions that may cause poor folding. The heavy chain was most similar to the mouse IGHV2-2 germline sequence and the J1 J chain. Four mutations in the framework regions were noted. The light chain was most similar to the mouse Ig kappa light chains IGKV6-13 and IGKV6-15. Six mutations were noted. It also appeared that there had been a crossover event between IGKV6-13 and IGKV6-15 near the 5' end of the coding sequence to generate the final light chain sequence. It appears that the J5 light chain mouse J region was used.

[0077] The sequences were mutated for codon usage in human immunoglobulins, sequences with minor, moderate and major modifications were also selected, and sequences were mutated to orient the antigen binding region (ABR) / complementarity determining region (CDR) into the antigen binding pocket of the antibody.

[0078] Each of these three humanized light chains and five humanized heavy chains generated were co-expressed in a H,L pairwise fashion in HEK 294 cells and the immunoglobulins were affinity purified from these cells. These antibodies were tested against MT in a standard ELISA and the results are shown in Figure 3. The humanized heavy and light chain combinations bind metallothionein, with some differences in binding ability between the different permutations of H and L chains. When the parental sequences of the mouse H and LV regions are combined with human immunoglobulin H and L constant regions, the antibodies function similarly, so the variation in the constant regions does not contribute significantly to antigen recognition.

[0079] We used software to identify the antigen-binding region complementarity determining regions (CDRs), which are set forth herein as SEQ ID NOs: 1-6. (See Kunik V, et al., (2012). PLoS Comput Biol 8(2): e1002388. doi: 10.1371 / journal.pcbi.1002388; Kunik V, et al., Y(2012). Nucleic Acids Res. 2012 Jul; 40(Web Server issue): W521-4.)

[0080] We evaluated the humanized antibodies that showed the best reactivity with MT for their binding to other metalloproteins (alcohol dehydrogenase (ADH), carbonic anhydrase (CA), superoxide dismutase (SOD), cytochrome C oxide (CC), metallothionein (MT), and human insulin). All antibodies tested retain high specificity for MT (data not shown).

[0081] Finally, Fab' fragments were generated with the best binding versions of the heavy and light chain combinations and tested for binding to metallothionein. The data are presented in Figure 4 and show that they bind to metallothionein with improved binding compared to the Fab' fragment of the parent UC1MT.

Claims

1. An isolated anti-metallothionein antibody, or antigen-binding fragment thereof, comprising: (a) a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9; and (b) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15; 2. An isolated anti-metallothionein antibody, or an antigen-binding fragment thereof, comprising:

2. An isolated anti-metallothionein antibody, or an antigen-binding fragment thereof, described in claim 1, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

8.

3. The isolated anti-metallothionein antibody, or antigen-binding fragment thereof, of claim 1 or 2, wherein the light chain further comprises a kappa constant region from a human light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

10.

4. An isolated anti-metallothionein antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

14.

5. An isolated anti-metallothionein antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

15.

6. The isolated anti-metallothionein antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the heavy chain further comprises a human IgG1 constant region comprising a mutation that prevents glycosylation, and the IgG1 constant region comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

16.

7. (a) the light chain comprises the amino acid sequence of SEQ ID NO:8, and (b) the isolated anti-metallothionein antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-15.

8. The isolated anti-metallothionein antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody comprises a monoclonal antibody or antigen-binding fragment thereof.

9. The isolated anti-metallothionein antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody comprises a Fab' fragment.

10. The isolated anti-metallothionein antibody or antigen-binding fragment thereof of any one of claims 1 to 9, further comprising a detectable label.

11. (a) a therapeutic agent conjugated to said anti-metallothionein antibody or antigen-binding fragment thereof, and / or (b) a cell targeting moiety conjugated to the anti-metallothionein antibody or antigen-binding fragment thereof. The isolated anti-metallothionein antibody or antigen-binding fragment thereof of any one of claims 1 to 10, further comprising:

12. One or more nucleic acids encoding the light chain and the heavy chain of the isolated anti-metallothionein antibody or antigen-binding fragment thereof of any one of claims 1-11.

13. One or more vectors comprising one or more nucleic acids according to claim 12 operably linked to suitable control sequences.

14. A host cell comprising one or more nucleic acids according to claim 12 and / or one or more vectors according to claim 13.

15. 1. A pharmaceutical composition for use in treating or limiting the occurrence of a disorder selected from the group consisting of intestinal inflammation, diabetes, pre-diabetes, impaired glucose tolerance, hepatitis, and inflammatory liver disease, comprising administering to a subject in need thereof: (a) an isolated anti-metallothionein antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a pharma- ceutically acceptable salt thereof; (b) a pharma- ceutically acceptable carrier.

16. 16. The pharmaceutical composition of claim 15, wherein the subject has or is at risk of developing diabetes, and the use serves to treat or limit the onset of diabetes.

17. 17. The pharmaceutical composition of claim 16, wherein the subject is at risk for type 1 diabetes and the use serves to limit the onset of type 1 diabetes in the subject.

18. 18. The pharmaceutical composition of claim 17, wherein the subject has one or more of the following risk factors for type 1 diabetes selected from the group consisting of: a parent or sibling with type 1 diabetes, a pancreatic tumor, pancreatitis, islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), insulinoma-associated (IA-2) autoantibodies, zinc transporter autoantibodies (ZnT8), a variant of the IDDM1 gene selected from the group consisting of DRB1 0401, DRB1 0402, DRB1 0405, DQA 0301, DQB1 0302, and DQB1 0201; polyuria, polydipsia, dry mouth, polyphagia, fatigue, or weight loss.

19. 16. The pharmaceutical composition of claim 15, wherein the subject has type 1 diabetes and the use is useful for treating type I diabetes in the subject.

20. 20. The pharmaceutical composition of claim 19, wherein said treating comprises one or more of: reducing the frequency of need for insulin injections, delaying the onset or progression of type 1 diabetes complications in the subject selected from the group consisting of: destruction of pancreatic beta cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, impaired wound healing ability, urinary tract infections, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure, and foot ulcers, and delaying the need for pancreas or pancreatic islet cell transplantation.

21. 16. The pharmaceutical composition of claim 15, wherein the subject is at risk for type 2 diabetes and the use serves to limit the onset of type 2 diabetes in the subject.

22. 22. The pharmaceutical composition of claim 21, wherein the subject has one or more risk factors for type 2 diabetes selected from the group consisting of obesity, smoking, a sedentary lifestyle, a parent or sibling with type 2 diabetes, prediabetes, a parent or sibling with prediabetes, age 50 years or older, high blood pressure, high cholesterol, testosterone deficiency, metallothionein 1 A (MT1A) rs8052394 locus (G alteration) single nucleotide polymorphism, and a history of gestational diabetes.

23. 16. The pharmaceutical composition of claim 15, wherein the subject has type 2 diabetes and the use is useful for treating type 2 diabetes in the subject.

24. 24. The pharmaceutical composition of claim 23, wherein the treating comprises limiting one or more of the type 2 diabetes complications selected from the group consisting of hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, glomerular clearance abnormalities, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, and stroke; and reducing the frequency of the need for insulin or other therapy.

25. 16. The pharmaceutical composition of claim 15, wherein the subject has prediabetes and the use serves to treat prediabetes in the subject.

26. 26. The pharmaceutical composition of claim 25, wherein said treating comprises limiting or slowing the progression of one or more complications of prediabetes selected from the group consisting of type 2 diabetes, hyperglycemia, insulin resistance, and cardiovascular disease.

27. 16. The pharmaceutical composition of claim 15, wherein the subject has impaired glucose tolerance and the use is useful for treating impaired glucose tolerance in the subject.

28. 28. The pharmaceutical composition of claim 27, wherein said treating comprises limiting or slowing the progression of one or more complications of impaired glucose tolerance selected from the group consisting of type 2 diabetes, hyperglycemia, insulin resistance, and cardiovascular disease.

29. 16. The pharmaceutical composition of claim 15, wherein the subject has hepatitis and the use serves to treat hepatitis in the subject.

30. 30. The pharmaceutical composition of claim 29, wherein said treating comprises limiting or slowing the progression of one or more complications of hepatitis selected from the group consisting of yellowing of the skin and / or whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, scarring of the liver, liver failure, and liver cancer.

31. 16. The pharmaceutical composition of claim 15, wherein the subject has an inflammatory liver disease and the use is useful for treating the inflammatory liver disease in the subject.

32. 32. The pharmaceutical composition of claim 31, wherein the inflammatory liver disease is selected from the group consisting of nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD).

33. 33. The pharmaceutical composition of claim 31 or 32, wherein the treating comprises limiting or slowing the progression of one or more complications of inflammatory liver disease selected from the group consisting of fatigue, malaise, liver fibrosis, liver cancer, and liver cirrhosis.

34. The pharmaceutical composition of claim 15 , wherein the subject has intestinal inflammation.

35. 35. The pharmaceutical composition of claim 34, wherein the intestinal inflammation is selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, and unspecified colitis, and Behcet's disease.

36. The pharmaceutical composition according to any one of claims 15 to 35, wherein the subject is a mammal.

37. The pharmaceutical composition according to any one of claims 15 to 35, wherein the subject is a human.

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