Anti-metallothionein antibodies and uses thereof

Abstract

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

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 874,340, filed on July 15, 2019, the entire contents of which are incorporated by reference into this application.

[0003] Federal Funding Statement

[0004] This application was made with government support under Grant No. ES07408 from the National Institutes of Health. The government has certain rights in this application.

[0005] References to sequence listings

[0006] This application contains a sequence listing submitted in the form of an electronic text file named "19-1213-PCT_Sequence-Listing_ST25.txt", which is 21 kb in size and was created on July 13, 2020. The information contained in this electronic file is hereby incorporated by reference into this application in its entirety pursuant to 37 CFR §1.52(e)(5). Background Art

[0007] Diabetes is most commonly treated with insulin replacement. Research is ongoing to replace the islets that are damaged in diabetes, but unless the inflammatory process that destroys the insulin-producing islets is arrested, islet transplantation or repopulation of islet stem cells in the pancreas will be a temporary treatment. Similarly, current agents and methods for treating hepatitis and / or inflammatory liver disease are inadequate. Summary of the invention

[0008] In one aspect, the invention provides an isolated anti-metallothionein antibody or fragment thereof comprising one, two or all three complementarity determining regions (CDRs) selected from the group consisting of:

[0009] a light chain CDR1 (L-CDR1) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence QSIVHSNGNTYLE (SEQ ID NO: 1);

[0010] 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 of KVS (SEQ ID NO: 2); and

[0011] A light chain CDR3 (L-CDR3) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence FQGSHVPF(A / T) (SEQ ID NO:3); and / or comprising 1, 2 or all 3 complementarity determining regions (CDRs) selected from the following groups:

[0012] a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence GFSLSRYG (SEQ ID NO:4);

[0013] a heavy chain CDR2 (H-CDR2) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of IWSGGST (SEQ ID NO: 5); and

[0014] A heavy chain CDR3 (H-CDR3) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence WLLPGY (SEQ ID NO: 6).

[0015] In various embodiments, the antibody comprises a monoclonal antibody or a fragment thereof, and / or a humanized antibody or a fragment thereof.

[0016] In other embodiments, the isolated anti-metallothionein antibody or fragment thereof comprises a light chain comprising 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 the amino acid sequence of SEQ ID NO:7-9, wherein the light chain comprises SEQ ID NO:7-9 NO:1-3, respectively, and wherein the light chain includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 of the following residues: D1, V3, T5, S7, T14, Q17, P18, F41, Q42, R44, R50, V88, V90, T102, Q105 and K112; and / or includes an amino acid sequence that is 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 SEQ ID NO:11-15, wherein the heavy chain includes SEQ ID NO: NO:4-6 respective complementarity determining regions (CDRs), and wherein the heavy chain comprises at least 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 or all 43 of the following residues: E1, V5, G9, 88, V92, and Q109.

[0017] In one embodiment, the isolated anti-metallothionein antibody or fragment thereof comprises a Fab' fragment.

[0018] In other aspects, the present invention provides nucleic acids encoding isolated antibodies or fragments thereof, genetic engineering vectors comprising the nucleic acids operably linked to appropriate control sequences, host cells comprising the nucleic acids and / or the genetic engineering vectors, and pharmaceutical compositions, comprising:

[0019] (a) an isolated antibody or fragment thereof, nucleic acid, genetic engineering vector or host cell disclosed in the present application; and

[0020] (b) a pharmaceutically acceptable carrier.

[0021] On the other hand, the present application provides a method for treating or limiting the development of a condition selected from intestinal inflammation, diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease, comprising administering to a subject an effective amount of an isolated antibody or fragment thereof, or a pharmaceutically acceptable salt thereof, nucleic acid, vector, host cell, or a pharmaceutical composition described in any embodiment or combination of embodiments disclosed in the present application, sufficient to treat the condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A-1B :( Figure 1A ) Comparison of humanized kappa light chain with the original mouse UC1MT anti-MT L sequence using ClustalOmega software. Figure 1B ) Phylogram of the relative differences of the variable regions of UC1MT L-Hum1 (VK1), UC1MT L-Hum2 (VK2), and UC1MT L-Hum3 (VK3) when compared to the original hybridoma light chain sequence (UC1MT).

[0023] Figure 2A-2B :( Figure 2A ) Comparison of the humanized heavy chain V region with the original mouse UC1MT anti-MTH sequence using Clustal Omega software. Figure 2B )Systematic repertoire of relative differences of 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 when compared to the sequence of the original hybridoma heavy chain V sequence (UC1MT) (VH5).

[0024] Figure 3: ELISA measurements of humanized antibodies tested against immobilized metallothionein. Immulon 2HB ELISA plates were coated with 10ug / mL MT overnight at 4°C, wells washed with PBST and blocked with 2% BSA for 1.5 hours at room temperature. The plates were washed, coated with a dilution of each recombinant Fab fragment, and incubated for 1.5 hours at room temperature. The plates were washed and incubated with 1:1000 mouse anti-human Ig light chain kappa-biotin for 1 hour at room temperature, then washed and incubated with streptavidin-HRP (wells containing Fab) for 20 minutes at room temperature. After the last wash, substrate was added and incubated for 15 minutes at room temperature, followed by the addition of stop solution (2N H2SO4) and the OD measured in a Spectramax ELISA reader. 450 . This graph shows OD 405 The reactivity of each clone was characterized as low (L), medium (M), or high (H).

[0025] Figure 4A-4B :( Figure 4A ) Fab' fragments from computer modeling binding to metallothionein. ( Figure 4B ) The individual CDRs of the H and L chains of a representative humanized Fab fragment are shown simultaneously. DETAILED DESCRIPTION

[0026] As used in this application, unless otherwise specified, the terms "a" and "an" mean "one", "at least one" or "one or more". Unless the context requires otherwise, singular terms used in this application shall include the plural and plural terms shall include the singular. All embodiments of any aspect of the application may be used in combination unless the context clearly dictates otherwise.

[0027] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (A sp; 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).

[0028] On the one hand, the present application provides anti-metallothionein (MT) antibodies or fragments thereof. Anti-metallothionein antibodies or fragments thereof are useful for treating conditions (e.g., selected from intestinal inflammation, diabetes, prediabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease). The use of MT inhibitors for treating such diseases is described in WO2019 / 143767, which is incorporated herein by reference in its entirety.

[0029] As disclosed in the present application, "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that include an antigen binding site that immunospecifically binds to a metallothionein epitope. Therefore, the term antibody includes not only complete antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments. Such antibodies or their 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, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. In a specific embodiment, the antibody includes a monoclonal antibody or a fragment thereof. In a further specific embodiment, the antibody includes a humanized antibody or a fragment thereof. In one embodiment, the antibody includes a Fab' fragment.

[0030] In one embodiment, antibody is humanized.As used in the present application, the term "humanized antibody" refers to a chimeric antibody comprising a minimal sequence from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor CDR are replaced by residues from the CDR (donor antibody) of a non-human species (e.g., mice, rats, rabbits or non-human primates with required specificity, affinity and / or ability). In some cases, the framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in receptor antibodies or donor antibodies. These modifications may be performed to further improve antibody performance, such as binding affinity, expression and reduction of the immunogenicity of heterologous antibodies such as mouse antibodies, to be introduced into the human body, while maintaining complete antigen binding affinity and specificity of the antibody.

[0031] As used herein, "isolated" refers to the presence of the indicated molecule, in the substantial absence of other biological macromolecules of the same type.

[0032] In one embodiment, a humanized antibody or fragment thereof is provided, comprising 1, 2 or all 3 complementarity determining regions (CDRs) selected from the following group:

[0033] a light chain CDR1 (L-CDR1) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence QSIVHSNGNTYLE (SEQ ID NO: 1);

[0034] 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 of KVS (SEQ ID NO: 2); and

[0035] A light chain CDR3 (L-CDR3) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence FQGSHVPF(A / T) (SEQ ID NO: 3).

[0036] In another embodiment, a humanized anti-metallothionein antibody or fragment thereof is provided, comprising 1, 2 or all 3 complementarity determining regions (CDRs) selected from the following groups:

[0037] a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence GFSLSRYG (SEQ ID NO:4);

[0038] a heavy chain CDR2 (H-CDR2) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of IWSGGST (SEQ ID NO: 5); and

[0039] A heavy chain CDR3 (H-CDR3) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence WLLPGY (SEQ ID NO: 6).

[0040] In a further embodiment, the humanized anti-metallothionein antibody or fragment thereof of claim 1 comprises 4, 5 or all 6 complementarity determining regions (CDRs) selected from the group consisting of:

[0041] a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence GFSLSRYG (SEQ ID NO:4);

[0042] a heavy chain CDR2 (H-CDR2) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of IWSGGST (SEQ ID NO: 5); and

[0043] a heavy chain CDR3 (H-CDR3) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence WLLPGY (SEQ ID NO: 6);

[0044] a light chain CDR1 (L-CDR1) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence QSIVHSNGNTYLE (SEQ ID NO: 1);

[0045] 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 of KVS (SEQ ID NO: 2); and

[0046] A light chain CDR3 (L-CDR3) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence FQGSHVPF(A / T) (SEQ ID NO: 3).

[0047] In another aspect, an isolated metal chemical antibody or fragment thereof is provided, comprising a light chain, wherein the light chain comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 9%, 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: 7-9: as shown in Table 1 (see also FIG. 1 ), wherein the light chain comprises SEQ ID NOs: NO:1-3 each of the complementarity determining regions (CDRs), and wherein the light chain includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 of the following residues (indicated 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.

[0048] Table 1

[0049]

[0050] Bold letters represent changes relative to the parental mouse hybridoma; CDRs are underlined.

[0051] In a specific embodiment, the isolated anti-metallothionein antibody or fragment thereof comprises a light chain comprising 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 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of the following residues (noted in bold and enlarged font in Table 1): D1, V3, T5, S7, T14, Q17, P18, R44, R50, V88, V90, Q105 and K112.

[0052] 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 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following amino acid sequence: RTVAAPSVFIFPPSDEQLKSSGTASVVCLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

[0053] In another aspect, an isolated anti-metallothionein antibody or fragment thereof is provided, comprising a heavy chain comprising 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 SEQ ID NOs: 11-15, wherein the heavy chain comprises SEQ ID NOs: NO:4-6 in each of the complementary determining regions (CDR), and wherein the heavy chain includes at least 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 or all 43 of the following residues (in bold and enlarged font in Table 2; see also 2 ): E1 , V5, G9, K13, G15, E16 or G16, T17, R19, L20, S21 , A23, S35, 137, P40 or A40, G42, 148 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.

[0054] In the sequences that follow, bold letters indicate changes relative to the parental UC1MT mouse hybridoma sequence; CDRs are underlined.

[0055] Table 2

[0056]

[0057] In a specific embodiment, the isolated anti-metallothionein antibody or fragment thereof comprises a heavy chain comprising 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 the amino acid sequence shown in SEQ ID NO: 14, wherein the heavy chain comprises SEQ ID NO: NO:4-6 respective complementarity determining regions (CDRs), and wherein the heavy chain includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all of the following 13 residues (noted in bold and enlarged font in Table 2; see also Figure 2): K13, E16, V37, G42, K64, S70, N76, V78, S83, T86, A87, A88 and Q109.

[0058] In another specific embodiment, the isolated anti-metallothionein antibody or fragment thereof comprises a heavy chain comprising 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 the amino acid sequence shown in SEQ ID NO:15, wherein the heavy chain comprises SEQ ID NO:15 NO:4-6 respective complementarity determining regions (CDRs), wherein the heavy chain comprises at least 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 or all 34 of the following residues. Genes (noted 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, L80, Q81, N83, R86, A87, E88, V92, and Q109.

[0059] In another embodiment, the heavy chain also includes a human IgG1 sequence comprising a mutation that prevents glycosylation, including but not limited to an amino acid sequence that is 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 shown in SEQ ID NO:27.

[0060] astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqssglyslssvvt vpssslgtqtyicnvnhkps ntkvdkkvep kscdkthtcp pcpapellgg psvflfppkpkdtlmisrtp evtcvvvdvs hedpevkfnwyvdgvevhna ktkpreeqyQ styrvvsvlt vlhqdwlngkeykckvsnka lpapiektis kakgqprepq vytlppsrdeltknqvsltc lvkgfypsdi avewesngqpennykttppv ldsdgsffly skltvdksrw qqgnvfscsv mhealhnhytqkslslspgk (SEQ ID NO: 16).

[0061] In a further embodiment, the isolated anti-metallothionein antibody or fragment thereof comprises:

[0062] (a) a light chain comprising an amino acid sequence that is 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 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 of the following residues: D1, V3, T5, S7, T14, Q17, P18, F41, Q42, R44, R50, V88, V90, T102, Q105, K112; and

[0063] (b) comprises a heavy chain comprising an amino acid sequence that is 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 SEQ ID NOs: 16-26 or selected from SEQ ID NOs: 11-15, wherein the heavy chain comprises SEQ ID NOs: NO:4-6 respective complementarity determining regions (CDRs), and wherein the heavy chain comprises at least 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 or all 43 of the following residues (indicated in bold and enlarged font in Table 2; see also FIG2 ), E1, V5, G9, K13, G15, E16, G16, T17, R19, L20, S21, A23, S35, I37, P40, A40, G42, I48, V48, S49, N58, A60, P61, D61, S62, L63, V63, K64, G65, V67, F67, T68, S70, V71, T73, A74, N76, K76, S77, F78, S79, Y79, L80, Q81, L82, S83, N83, V85, T86, R86, A87, A88, E88, V92, and Q109.

[0064] In another specific embodiment, the isolated anti-metallothionein antibody or fragment thereof comprises:

[0065] (a) a light chain comprising an amino acid sequence that is 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of the following residues (indicated in bold and enlarged font in Table 1): D1, V3, T5, S7, T14, Q17, P18, R44, R50, V88, V90, Q105, K112; and

[0066] (b) comprises a heavy chain comprising an amino acid sequence that is 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 SEQ ID NOs: 14-15, wherein the heavy chain comprises a complementarity determining region (CDR) of each of SEQ ID NOs: 4-6, wherein the heavy chain comprises:

[0067] (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of the following residues: K13, E16, V37, G42, K64, S70, N76, V78, S83, T86, A87, A88 and Q109; or

[0068] (ii) 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 or all 34 of the following residues (in bold and enlarged in Table 2) Font size noted; 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, L80, Q81, N83, R86, A87, E88, V92, and Q109.

[0069] The anti-MT antibody of the present application can be connected to any other part that is considered suitable for the intended purpose. In one embodiment, the separated antibody or its fragment also includes a detectable label. For example, such an embodiment can be used to monitor the treatment process. Any suitable detectable label can be covalently, hereditarily or by any other means combined with the antibody, including but not limited to radioisotopes, fluorescent molecules, magnetic particles (including nanoparticles), metal particles (including nanoparticles), phosphorescent molecules and enzymes.

[0070] In another embodiment, the isolated antibody or fragment thereof further comprises an additional therapeutic agent. Any therapeutic agent suitable for the intended purpose can be covalently, genetically or by any other means linked to the antibody, including but not limited to insulin, metformin, pramlintide, sulfonylureas (including but not limited to glibenclamide, 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, cytokines, chemokines, interferons or ribavirin. Such conjugates can be used, for example, in the methods disclosed herein.

[0071] In another embodiment, the isolated antibody or fragment thereof further comprises a cell targeting moiety. Any cell targeting moiety may be considered suitable for the intended cell target, including but not limited to pancreatic or hepatocyte targeting moieties (e.g., pancreatic cell targeting moieties, which include 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 incretin analogs (exendin-4); or hepatocyte targeting moieties, wherein hepatocyte targeting moieties include but are not limited to circumsporozoite protein (CSP), CSP region I, CSPI plus, lactosamined human serum albumin, glycosylated lipoproteins and arabinogalactan). Such conjugates may be used, for example, in the methods disclosed herein.

[0072] On the other hand, an isolated nucleic acid is also disclosed, the nucleic acid encoding the antibody described in any embodiment or combination of embodiments disclosed in the present application. The isolated nucleic acid sequence may include RNA or DNA. Such an isolated nucleic acid sequence may include additional sequences for promoting expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals and secretion signals, nuclear localization signals, and plasma membrane localization signals.

[0073] On the other hand, an expression vector comprising the isolated nucleic acid of the application is provided." expression vector (Expression vector) " comprises a genetic engineering vector to which a nucleic acid coding region or a gene is operably connected to any promoter that can affect the expression of a gene product. The promoter sequence used to drive the disclosed nucleotide sequence to be expressed in a mammalian system can be a constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, EF) or an inducible (driven by any of many inducible promoters, including but not limited to tetracycline, ecdysone, steroid responsiveness). The expression vector can be replicated in a suitable host organism as an episome or by being integrated into the host chromosome DNA. In various embodiments, the expression vector comprises a plasmid or a viral vector.

[0074] In another aspect, a host cell is provided, which includes the antibody, nucleic acid and / or vector of the present application. The host cell can be a prokaryotic cell or a eukaryotic cell. 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 application. Therefore, the recombinant cell can be used, for example, in a method for producing an antibody, comprising:

[0075] (a) culturing the recombinant host cell under conditions suitable for expression of the antibody encoded by the nucleic acid; and

[0076] (b) isolating the antibody from the cultured cells.

[0077] Conditional vectors and conditions suitable for expressing nucleic acid-encoded antibodies can be determined by those skilled in the art based on the teachings of this application, the specific host cells and genetic engineering vectors used, and the general knowledge of those skilled in the art.

[0078] In another aspect, a pharmaceutical composition is provided, comprising:

[0079] (a) the antibody, isolated nucleic acid, expression vector or host cell according to any embodiment or combination of embodiments disclosed in the present application; and

[0080] (b) a pharmaceutically acceptable carrier.

[0081] For example, the antibody of the present application 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, etc.; and 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, p-aminobenzenesulfonic acid, etc. Suitable bases capable of forming such salts include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc.; and organic bases such as monoalkylamines, dialkylamines and trialkylamines and monoarylamines, diarylamines and triarylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, etc.)).

[0082] The pharmaceutical composition may include (a) a lyoprotectant in addition to the composition of the present invention; (b) a surfactant; (c) a filler; (d) a tension regulator; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the pharmaceutical composition in the buffer 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, benzyl thiophene, chlorine, phenol, m-cresol, benzyl alcohol, methyl paraben, propyl paraben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a filler, such as glycine. In some other embodiments, the pharmaceutical composition includes a surfactant such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monostearate, sorbitol monooleate, sorbitol trilaurate, sorbitol tristearate, sorbitol trioleate, or a combination thereof. The pharmaceutical composition may also include a tension modifier, for example, a compound that makes the formulation substantially isotonic or isotonic with human blood. Exemplary tension modifiers include sucrose, sorbitol, glycine, methionine, mannitol, glucose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, for example, a molecule that substantially prevents or reduces the chemical and / or physical instability of the protein of interest in lyophilized or liquid form when combined with the composition of the protein of interest. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0083] The pharmaceutical composition of the present application can be prepared into any suitable preparation, preferably into a preparation suitable for administration by injection. Such a pharmaceutical composition can be used, for example, in the treatment method disclosed in the present application.

[0084] The pharmaceutical composition may contain any other ingredients deemed suitable for a given use, such as additional therapeutic agents. In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent selected from insulin, metformin, pramlintide, sulfonylureas (including but not limited to glibenclamide, glipizide, 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, cytokines, chemokines, interferons or ribavirin.

[0085] On the other hand, the present application provides a method for treating or limiting the development of a disease selected from intestinal inflammation, diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease, comprising administering to a subject in need thereof an effective amount of an isolated antibody or fragment thereof, nucleic acid, vector, host cell and / or pharmaceutical composition disclosed in any embodiment or combination of embodiments of the present application.

[0086] As used herein, "therapeutically effective amount" refers to an amount of a composition that is effective in treating and / or limiting the relevant disorder.

[0087] As used herein, "treat" or "treating" means to accomplish one or more of the following in an individual suffering from one or more of the diseases: (a) reducing the severity of the disease; (b) limiting or preventing the development of symptoms characteristic of the treated disease; (c) inhibiting the worsening of symptoms characteristic of the treated disease; (d) limiting or preventing the recurrence of the disease in a patient who previously suffered from the disease; and / or (e) limiting or preventing the recurrence of symptoms in a patient who previously had symptoms of the disease. Any amount of such "treatment" is very beneficial to a subject suffering from one of the diseases.

[0088] As used herein, "limiting" or "limiting development" refers to accomplishing one or more of the following in an individual at risk for one or more of the diseases: (a) delaying disease progression and / or (b) limiting or preventing the development of symptoms characteristic of disease progression. Any amount of such "limiting development" is highly beneficial to a subject at risk for one of the diseases.

[0089] Such treatment or development of limitations may include the use of the extracellular MT inhibitor as the sole therapeutic agent, or may include its use to supplement or augment other therapeutic interventions, as deemed appropriate by the attending medical practitioner.

[0090] In one embodiment, the subject has or is at risk for developing diabetes and the method is used to treat or limit the development of diabetes.

[0091] In one such embodiment, the subject is at risk of type 1 diabetes, and the method is used to limit the development of type 1 diabetes in the subject. As described in WO2019 / 143767, human MT1 inhibitors prevent the development of type 1 diabetes in the NOD mouse model. Therefore, the method of this embodiment can be used to limit the development of type 1 diabetes (T1D) in subjects at risk of T1D. Limiting the development of T1D may include, but is not limited to, delaying the progression of T1D and / or delaying the development of T1D symptom characteristics. In this embodiment, a subject at risk of T1D has one or more T1D risk factors that the attending medical staff considers suitable for treatment. Such T1D risk factors include, but are not limited to, 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), and / or a variant of the IDDM1 gene selected from DRB10401, 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 has not yet been 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 development of T1D or its symptoms provides a great benefit to subjects at risk.

[0092] The insulin-dependent (I) type 1 diabetes (IDDM1) gene is located in the MHC class II region of chromosome 6. Certain variants of this gene increase the risk of reduced tissue compatibility in type 1 diabetes. Such variants include DRB1 0401, DRB10402, DRB1 0405, DQA 0301, DQB1 0302, and DQB1 0201. Similarly, the appearance of diabetes-related autoantibodies such as islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), insulinoma-related (IA-2) autoantibodies, zinc transporter autoantibodies (ZnT8) is usually earlier than type 1 hyperglycemic diabetes before any hyperglycemia occurs. The risk of T1D increases with the increase of antibody types, and the time interval from the appearance of autoantibodies to the clinical diagnosis of T1D may be several months in infants and young children, but may take several years in some people. Such autoantibodies can be detected by, for example, immunofluorescence or binding assays.

[0093] In another embodiment, the subject suffers from type 1 diabetes, and the method is used to treat type 1 diabetes in the subject. In this embodiment, the subject has been diagnosed with T1D, and the method can be used to treat T1D. T1D involves autoimmune destruction of beta cells in the pancreas, little or no insulin production, and hyperglycemia. Therefore, treating T1D involves 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, decreased wound healing ability, urinary tract infection, 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 need a pancreas or islet transplant. Thus, in various embodiments, treatment may include one or more of: reducing the frequency of insulin injection requirements; delaying the development or progression of complications of type 1 diabetes in a subject, including, but not limited to, destruction of pancreatic beta cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, decreased wound healing, urinary tract infection, 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 pancreatic or islet cell transplantation. In one embodiment, the treatment may include, for example, a 10%, 15%, 20% or more reduction in blood glucose levels (mg / dL) within 20-120 minutes after administration of the inhibitor.

[0094] In another embodiment, the subject suffers from the risk of suffering from type 2 diabetes, and the method is used to limit the subject's development into type 2 diabetes (T2D). T2D is a metabolic disease characterized by hyperglycemia, insulin resistance and relative lack of insulin. Symptoms and / or complications include but are not limited to hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular clearance, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss and stroke. In one embodiment, the limited development of type 2 diabetes may include, for example, within 20-120 minutes after the inhibitor is given, blood sugar levels reduce by 10%, 15%, 20% or more (mg / dL).

[0095] Risk factors for T2D include, but are not limited to, obesity, smoking, sedentary lifestyle, parent or sibling with type 2 diabetes, prediabetes, parent or sibling with prediabetes, poor diet (e.g., too much fat, not enough fiber, too much simple carbohydrates, etc.), age 50 or older, high blood pressure, high cholesterol, testosterone deficiency, metallothionein 1A (MT1A) rs8052394 locus (G change) single nucleotide polymorphism, and gestational diabetes. Therefore, in various embodiments, the subject has one or more of these risk factors, and the method is used to delay the development of T2D and / or (b) limit or prevent the development of symptoms characteristic of T2D.

[0096] In another embodiment, the subject suffers from T2D, and the method is used to treat the T2D of the subject. In this embodiment, treatment can include limiting one or more type 2 diabetes complications, including but not limited to hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular clearance, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, stroke, and reducing the frequency of demand for insulin or other treatments. Any amount of restriction on these symptoms / complications is very beneficial to subjects suffering from T2D. In one embodiment, the treatment may include, for example, within 20-120 minutes after the inhibitor is administered, blood sugar levels are reduced by 10%, 15%, 20% or more (mg / dL).

[0097] In another embodiment, the subject has prediabetes, and the method is used to treat prediabetes in the subject. In this embodiment, the subject is a person with prediabetes. As used in this application, "prediabetes" refers to a state that meets some but not all diagnostic criteria for diabetes. Therefore, a prediabetic subject may: (a) have impaired fasting glucose tolerance, which is a condition whereby the response of β cells to oral glucose tolerance challenge (OGT) is defective or (b) may have elevated fasting blood glucose (IFG), which is a condition where fasting blood glucose is higher than what is considered normal, but not enough to be classified as diabetes. The prediabetic state may be associated with increased risk of insulin resistance and cardiovascular disease. Individuals in prediabetes have a relatively high risk of T2D. The method of the present application can be used to treat a subject with prediabetes by, for example, 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, the treatment may include, for example, a 10%, 15%, 20% or more (mg / dL) reduction in blood glucose levels within 20-120 minutes after administration of the inhibitor.

[0098] In another embodiment, the subject suffers from impaired glucose tolerance, and the method is used to treat the impaired glucose tolerance of the subject. As used in the present application, impaired glucose tolerance is defined as a glucose level of 140 to 199 mg / dL (7.8 to 11.0 mmol / L) at two hours in a 75 g oral glucose tolerance test. When the patient's blood sugar level rises moderately after 2 hours, but is lower than the level that meets the criteria for type 2 diabetes, the patient is said to be in an IGT state. Fasting blood sugar may be normal or slightly elevated. Impaired glucose tolerance is a hyperglycemic state in the prediabetes stage, which is associated with increased risk of insulin resistance and cardiovascular disease. IGT may be many years earlier than type 2 diabetes. In this embodiment, treatment may include limiting or delaying the progress of one or more impaired glucose tolerance complications (including but not limited to type 2 diabetes, hyperglycemia, insulin resistance and / or cardiovascular disease). In one embodiment, the treatment may include, for example, within 20-120 minutes after the inhibitor is administered, blood sugar levels are reduced by 10%, 15%, 20% or more (mg / dL).

[0099] In another embodiment, the subject suffers from hepatitis, and the method is used to treat hepatitis in the subject. As described in WO2019 / 143767, MT inhibitors can effectively limit tissue inflammation, reduce proinflammatory cytokines MCP-1 and TNF-α, and increase anti-inflammatory IL-10 signals in liver tissue. Hepatitis is inflammation of liver tissue. Symptoms include but are not limited to yellowing of the skin and whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure or liver cancer. The most common causes of hepatitis are viral infections (types A, B, C, D and E), heavy drinking, certain drugs, toxins, other infections, autoimmune diseases and non-alcoholic fatty liver disease (NASH). Therefore, in various embodiments, treatment may include limiting or delaying the progression of one or more complications of hepatitis, including but not limited to yellow discoloration of the skin and / or whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure and liver cancer.

[0100] In a further embodiment, the subject suffers from inflammatory liver disease, and the method is used to treat the inflammatory liver disease of the subject. As used in the present application, "inflammatory liver disease" is a disease related to the accumulation of triglyceride fat vacuoles in the cytoplasm by steatosis (i.e., abnormal retention of intracellular lipids) in hepatocytes. The liver plays an important role in systemic metabolism, and energy imbalance is particularly relevant to liver lipid metabolism defects. Specifically, obesity and insulin resistance are generally associated with increased lipid deposition in the liver characteristic of non-alcoholic fatty liver disease (NAFLD). Although lipid metabolism is highly dynamic, chronic lipid overload can cause liver tissue damage, leading to the recruitment of liver resident immune cells and non-resident immune cells, which may lead to the fibrotic characteristics of non-alcoholic fatty liver disease (NASH). Liver fibrosis may lead to cirrhosis, cancer and significantly increase the risk of cardiovascular disease. This improves the potential for preventing immune cells from being recruited to the liver to improve the risk of non-alcoholic fatty liver disease (NAFLD). As shown in the examples below, MT inhibitor treatment resulted in increased wet tissue weight of epididymal white adipose tissue, decreased total triglyceride levels, and concomitant decreases in the pro-inflammatory cytokines MCP-1 and TNF-a and increased anti-inflammatory IL-10 signaling. The inflammatory liver disease may be steatosis (non-alcoholic fatty liver disease (NAFL). In another embodiment, the fatty liver disease may be non-alcoholic fatty liver disease (NAFLD), including but not limited to the most extreme form of NAFLD, non-alcoholic steatohepatitis (NASH). NAFLD is an inflammatory liver disease that occurs when fat is deposited in the liver (steatosis) due to reasons other than excessive drinking. Symptoms of NASH and NAFLD may include but are not limited to fatigue, discomfort, dull pain in the right upper abdomen, mild jaundice, and abnormal liver function tests on routine blood tests; complications of NASH and NAFLD may include but are not limited to liver fibrosis, liver cancer and / or cirrhosis. Therefore, in one embodiment, the inflammatory liver disease is selected from non-alcoholic steatohepatitis (NASH) and / or non-alcoholic fatty liver disease (NAFLD). In a further embodiment, treatment includes limiting or delaying the progression of one or more complications of inflammatory liver disease, including but not limited to fatigue, unexplained discomfort, liver fibrosis, liver cancer and / or cirrhosis.

[0101] In another embodiment, the subject suffers from intestinal inflammation. Such intestinal inflammation may include, but is not limited to, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, and indeterminate colitis, as well as Behcet's disease.

[0102] In one embodiment, MT inhibitors (including but not limited to anti-MT antibodies) can be connected to pancreatic cell targeting moieties to specifically target pancreatic cells that produce MT, such as pancreatic β cells. This embodiment will be particularly suitable for treating or limiting the development of T1D, T2D, prediabetes and / or impaired glucose tolerance. In one embodiment, pancreatic cell-specific targeting moieties include 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 incretin analogs (exendin-4). Exemplary amino acid sequences of such targeting moieties are provided in Table 3.

[0103] Table 3

[0104]

[0105] Attaching the pancreatic cell targeting moiety to the MT inhibitor (including but not limited to antibody MT or its fragment) can be achieved by any chemical reaction that combines the two molecules, as long as the pancreatic cell targeting moiety and the MT antibody or its fragment retain their respective activities. In one embodiment, the extracellular MT inhibitor includes an antibody or its fragment, and the composition includes a recombinant fusion protein. In other embodiments, the contact between the pancreatic cell targeting moiety and the MT antibody or its fragment can include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linkers can be used to couple protein molecules (such as antibodies) with other molecules. For example, representative non-limiting examples of coupling agents can be organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine.

[0106] In all embodiments, the subject can be any subject that can benefit from the treatment methods disclosed herein, including mammals, humans, cows, dogs, cats, horses, chickens, etc. In one embodiment, the subject is a human.

[0107] Compositions for administration are usually prepared into pharmaceutical compositions including pharmaceutically acceptable carriers. Suitable acids capable of forming pharmaceutically acceptable salts include inorganic acids (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, etc.) and 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, p-aminobenzenesulfonic acid, etc.). Suitable alkalis capable of forming such salts include inorganic alkalis such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, etc.; and organic alkalis such as monoalkylamines, dialkylamines and trialkylamines and monoarylamines, diarylamines and triarylamines (for example, triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (for example, ethanolamine, diethanolamine, etc.).

[0108] In addition to the composition and the carrier, the pharmaceutical composition may include (a) a lyoprotectant; (b) a surfactant; (c) a filler; (d) a tension regulator; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the pharmaceutical composition in the buffer 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, benzyl thiophene, chlorine, phenol, m-cresol, benzyl alcohol, methyl paraben, propyl paraben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a filler, such as glycine. In some other embodiments, the pharmaceutical composition includes a surfactant such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monostearate, sorbitol monooleate, sorbitol trilaurate, sorbitol tristearate, sorbitol trioleate, or a combination thereof. The pharmaceutical composition may also include a tension modifier, for example, a compound that makes the formulation substantially isotonic or isotonic with human blood. Exemplary tension modifiers include sucrose, sorbitol, glycine, methionine, mannitol, glucose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition also includes a stabilizer, for example, a molecule that substantially prevents or reduces the chemical and / or physical instability of the protein in lyophilized or liquid form when combined with a protein-based composition. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0109] The composition can be administered in the form of a dosage unit formulation containing a conventional pharmaceutically acceptable carrier, adjuvant and solvent by any suitable route, including oral, parenteral, inhalation aerosol, rectal or local routes. The term "parenteral" as used in the present application includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion technology or intraperitoneal. The dosage regimen can be adjusted to provide the best required response (e.g., treatment or prevention response). For example, a suitable dosage range can be 0.1ug / kg-100mg / kg body weight; or, it can be 0.5ug / kg to 50mg / kg, 1ug / kg to 25mg / kg, or 5ug / kg to 10mg / kg body weight. The composition can be delivered by a single bolus, or can be administered more than once (e.g., 2 times, 3 times, 4 times, 5 times or more) as determined by the attending medical staff. The composition can be the only therapeutic agent administered, or can be administered (alone or as a combination) with one or more other therapeutic agents deemed suitable by the attending medical staff. In one non-limiting embodiment, the subject suffers from or is at risk of T1D, and the inhibitor can be used with one or more of insulin, metformin, or pramlintide. In another embodiment, the subject suffers from or is at risk of T2D, prediabetes, and / or impaired glucose tolerance, and the inhibitor can be used with one or more of metformin, sulfonylureas (including but not limited to glibenclamide, 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 of developing hepatitis and the inhibitor can be used in combination with one or more of entecavir, tenofovir, lamivudine, adefovir, telbivudine, simeprevir, sofosbuvir, interferon, or ribavirin.

[0110] In another embodiment, a composition is provided comprising:

[0111] (a) the anti-metallothionein antibody disclosed in the present application; and

[0112] (b) insulin, metformin, pramlintide, sulfonylureas (including but not limited to glibenclamide, glipizide and glimepiride), glinides (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. One or more of these compositions can be used, for example, in the methods of the present application. All embodiments and combinations of the embodiments of the antibodies disclosed above are suitable for being included in the compositions of this aspect. In one embodiment, the anti-MT antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof. In another embodiment, the anti-MT antibody comprises a humanized anti-MT antibody or an antigen-binding fragment thereof.

[0113] In another aspect, the present application provides a composition comprising:

[0114] (a) the anti-metallothionein antibody disclosed in the present application; and

[0115] (b) Pancreatic cell or hepatocyte targeting moiety linked to extracellular human MT inhibitor.

[0116] These compositions can be used, for example, in the methods of the present application. All embodiments and combinations of embodiments of the antibodies disclosed above are suitable for being included in the compositions of this aspect. In one embodiment, the anti-MT antibody or its antigen-binding fragment comprises a monoclonal antibody or its antigen-binding fragment. In another embodiment, the anti-MT antibody comprises a humanized anti-MT antibody or its antigen-binding fragment.

[0117] In one embodiment, the cell targeting moiety is a hepatocyte targeting moiety. In an exemplary such embodiment, the hepatocyte targeting moiety includes but is not limited to circumsporozoite protein (CSP), CSP region I, CSP region I-plus, lactosamined human serum albumin, glycosylated lipoprotein and / or arabinogalactan. In one embodiment, the hepatocyte binding moiety is peptide and selected from CSP, CSP region I, CSP region I plus or its hepatocyte binding fragment. CSP attributes the targeting of Plasmodium sporozoites to the liver 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 about 400 amino acids long and consists of three domains: an N-terminal domain containing a conserved motif named "region I", a highly repetitive central domain, and a C-terminal domain containing another conserved sequence named "region II" (Singh et al. containing another conserved sequence named "region II" Singh et al. Cell. 2007; 131(3): 492–504). In addition to the conserved KLKQP sequence of region I, the N-terminal region also includes two consensus heparin sulfate binding sequences upstream of region I. The peptide containing the conserved region I amino acids and the two consensus heparin binding sequences upstream of region I was named "region I-plus" (Prudêncio et al., Nature Reviews Microbiology. 2006; 4(11): 849–856).

[0118] 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 incretin analogs (exendin-4). Exemplary amino acid sequences of such peptides are described above.

[0119] Attaching the cell targeting moiety to the anti-MT antibody or its fragment can be achieved by any chemical reaction combining the two molecules, as long as the cell targeting moiety and the anti-MT antibody or its fragment retain their respective activities. In one embodiment, the composition includes a recombinant fusion protein. In other embodiments, the contact between the cell targeting moiety and the anti-MT antibody or its fragment can include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules (such as antibodies) with other molecules. For example, representative non-limiting examples of coupling agents can be organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine.

[0120] In all embodiments, the subject can be any subject that can benefit from the treatment methods disclosed herein, including mammals, humans, cows, dogs, cats, horses, chickens, etc. In one embodiment, the subject is a human.

[0121] Example

[0122] Humanized UC1MT monoclonal anti-metallothionein antibody

[0123] The mouse 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 roughly in a nested manner, varying from more to less. The heavy and light chain designs were tested in all possible pairwise combinations with the parental V regions, resulting in 24 combinations. The constant regions used are common human heavy chains and are shared with all heavy and light chains used in these arrangements.

[0124] Parental sequences of the original UC1MT mouse hybridoma (V regions of heavy and light chains)

[0125] A. Primary UC1MT hybridoma derived sequence of heavy chain (ProMab):

[0126] GSEVKLQESGPGLVQPSQSLSITCTVSS GFSLSRYG VHWVRQSPVKGLEWLGV IWSGG ST DYNAAFISRLSINKDNSKSQVFFKMHSLQTYDTARYYCAR W LLPG YFDVWGGAGTTVT VSS(SEQ ID NO:23)

[0127] (Lake MHC2822HC copies the sequence to the original ProMab sequence):

[0128] QVQLTQSGPGLVQPSQSLSITCTVS GFSLSRYG VHWVRQSPVKGLEWLGV IWSGGST D YNAAFISRLSINKDNSKSQVFFKMHSLQTYDTARYYCAR WLJ YFDVWGAGTTVTVSS (SEQ ID NO: 24)

[0129] Light chain derived sequence of the original UC1MT hybridoma for CDR (underlined)

[0130] AVLMSQTPLSLPVSLGDQASISCRSS QSIVHSNGNTY LEWYLQKPGQSPKLLIY KVS NR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYC FQGSHVP FAFGSGTKLEIE (SEQ ID NO: 25)

[0131] Considerations for sequence design

[0132] The starting sequence was first compared to the mouse germline V region to identify potential mutations in the framework region that could cause poor folding. The heavy chain was most similar to the mouse IGHV2-2 germline sequence and the J1 J chain. Four mutations were noted in the framework region. The light chain was most similar to the mouse Igκ light chains IGKV6-13 and IGKV6-15. Six mutations were noted. In addition, a cross-over event appeared to have occurred between IGKV6-13 and IGKV6-15 near the 5' end of the coding sequence to produce the final light chain sequence. It appears that the J5 light chain mouse J region was used.

[0133] The sequences were mutated for codons used in human immunoglobulins, sequences with small, medium and large modifications were also selected, and the sequences were mutated to orient the antigen binding region (ABR) / complementarity determining region (CDR) towards the antigen binding pocket of the antibody.

[0134] Each of the three humanized light chains and five humanized heavy chains generated were co-expressed in H, L pairs in HEK 294 cells, and immunoglobulins were affinity purified from these cells. These antibodies were tested against MT in a standard ELISA, and the results were as follows Figure 3As shown. The combination of humanized heavy and light chains binds to metallothionein, and there are some differences in the binding potential between the different arrangements of the H and L chains. When the parental sequences of the mouse H and LV regions are combined with the human immunoglobulin H and L constant regions, the performance of the antibodies is similar, so the changes in the constant regions do not contribute significantly to antigen recognition.

[0135] We used software to identify the antigen binding region complementary determining region (CDR), described in this application as SEQ ID NO: 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 (WebServer issue): W521-4.)

[0136] We evaluated humanized antibodies that showed the best reactivity to bind to MT and other metallothioneins (alcohol dehydrogenase (ADH), carbonic anhydrase (CA), superoxide dismutase (SOD), cytochrome C oxide (CC), metallothionein (MT), and human insulin). All antibodies tested retained a high degree of specificity for MT (data not shown).

[0137] Finally, Fab' fragments were prepared from the best binding forms of the heavy and light chains combined and tested for binding to metallothionein. The data are shown in Figure 4 and demonstrate that they bind to metallothionein with improved binding relative to the Fab' fragment of the parental UC1MT.

Claims

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

2. An isolated anti-metallothionein antibody or fragment thereof comprising one, two or all three complementary determining regions (CDRs) selected from the group consisting of: a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence that is 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 that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of IWSGGST (SEQ ID NO: 5); and A heavy chain CDR3 (H-CDR3) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence WLLPGY (SEQ ID NO: 6).

3. The isolated anti-metallothionein antibody or fragment thereof according to claim 1 or 2, comprising 4, 5 or all 6 complementarity determining regions (CDRs) selected from SEQ ID NOs: 1-6.

4. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 3, wherein The antibody includes a monoclonal antibody or a fragment thereof.

5. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 4, wherein The antibody is a humanized antibody or a fragment thereof.

6. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1-5, comprising a light chain comprising 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 SEQ ID NOs: 7-9, wherein the light chain comprises SEQ ID NOs: NO:1-3 each of the amino acids, and wherein the light chain includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 of the following residues: D1, V3, T5, S7, T14, Q17, P18, F41, Q42, R44, R50, V88, V90, T102, Q105 and K112.

7. The isolated anti-metallothionein antibody or fragment thereof of claim 6, comprising a light chain comprising 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 the amino acid sequence shown in SEQ ID NO:8, wherein the light chain comprises the complementarity determining regions of SEQ ID NOs: 1-3, and wherein the light chain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of the following residues: D1, V3, T5, S7, T14, Q17, P18, R44, R50, V88, V90, Q105 and K112.

8. The isolated anti-metallothionein antibody or fragment thereof according to claim 6 or 7, further comprising a kappa constant region from a human light chain, including but not limited to 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 with the amino acid sequence shown in SEQ ID NO:

10.

9. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1-8, comprising a heavy chain comprising 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 the amino acid sequence shown in SEQ ID NO: 11-15, wherein the heavy chain comprises SEQ ID NO: NO:4-6 each of the complementary determining regions (CDRs), and wherein the heavy chain includes at least 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 or all 43 of the following residues: E1, V5, or G79, L80, Q81, L82, S83 or N83, V85, T86 or R86, A87, A88 or E88, V92, and Q109.

10. The isolated anti-metallothionein antibody of claim 9, comprising a heavy chain comprising 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 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, wherein the heavy chain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13, 14 of the following residues: K13, E16, V37, G42, K64, S70, N76, V78, S83, T86, A87, A88 and Q109.

11. The isolated anti-metallothionein antibody of claim 9 or 10, comprising a heavy chain comprising 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 the amino acid sequence of SEQ ID NO: 15, wherein the heavy chain comprises SEQ ID NO: NO:4-6 respective complementarity determining regions (CDRs), wherein the heavy chain comprises at least 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 or all 43 of the following residues: E1, V5, G9, K13 , G15, E16 or G16, T17, R19, L20, S21, A23, S35, 137, P40 or A40, G42, 148 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.

12. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 9 to 11, wherein The heavy chain also includes human IgG1 characteristics, which include mutations that prevent glycosylation, including but not limited to amino acid sequences that are 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 shown in SEQ ID NO:

16.

13. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 12, comprising: (a) a light chain comprising an amino acid sequence that is 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 SEQ ID NOs: 7-9, and wherein the light chain comprises amino acids of each of SEQ ID NOs: 1-3, and wherein 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: D1, V3, T5, S7, T14, Q17, P18, F41, Q42, R44, R50, V88, V90, T102, Q105, K112; and (b) a heavy chain comprising 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: 16-26 or SEQ ID NOs: 11-15, wherein the heavy chain comprises ... NO:4-6 respective complementarity determining regions (CDRs), wherein the heavy chain comprises at least 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 or all 43 of the following residues: E1, V5, G9, 88, V92, and Q109.

14. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 13, comprising: (a) a light chain comprising an amino acid sequence that is 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 shown in SEQ ID NO:8, wherein the light chain comprises the CDRs of sequence SEQ ID NO:1-3, and wherein the light chain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of the following residues: D1, V3, T5, S7, T14, Q17, P18, R44, R50, V88, V90, Q105, K112; and (b) comprises a heavy chain comprising an amino acid sequence that is 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 SEQ ID NOs: 14-15, wherein the heavy chain comprises a complementarity determining region (CDR) of each of SEQ ID NOs: 4-6, wherein the heavy chain comprises: (i) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13 of the following residues: K13, E16, V37, G42, K64, S70, N76, V78, S83, T86, A87, A88 and Q109; or (ii) at least 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, or all 43 of the following residues: El, V5, G9, K13, G15, E16, or 88, V92, and Q109.

15. The isolated anti-metallothionein antibody or fragment thereof according to claim 13 or 14, wherein The antibodies include monoclonal antibodies or fragments thereof, and / or humanized antibodies or fragments thereof.

16. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 14, wherein The antibodies include Fab' fragments.

17. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1-16, further comprising a detectable label.

18. The isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 17, further comprising: (a) a therapeutic agent conjugated to the antibody or a fragment thereof, the therapeutic agent including but not limited to insulin, metformin, pramlintide, sulfonylureas (including but not limited to glibenclamide, 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, cytokines, chemokines, interferons or ribavirin, and / or (b) a cell targeting moiety conjugated to the antibody portion or fragment thereof, the cell targeting moiety including but not limited to a pancreatic or hepatocyte targeting moiety (including but not limited to a pancreatic cell targeting moiety comprising one or more peptides or o-pancreatic cell targeting moiety therapeutic moiety preferentially binds 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 incretin analogs; or a hepatocyte targeting moiety, wherein the hepatocyte targeting moiety includes but is not limited to circumsporozoite protein (CSP), CSP region I, CSPIplus, lactosaminylated human serum albumin, glycosylated lipoproteins and arabinogalactan).

19. A nucleic acid encoding the isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 18.

20. A genetic engineering vector comprising the nucleic acid of claim 19 operably linked to a suitable control sequence.

21. A host cell comprising the nucleic acid of claim 19 and / or the genetic engineering vector of claim 20.

22. A pharmaceutical composition comprising: (a) the isolated anti-metallothionein antibody or fragment thereof according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, the nucleic acid according to claim 19, the genetic engineering vector according to claim 20, or the host cell according to claim 21; and (b) a pharmaceutically acceptable carrier.

23. A method for treating or limiting the progression of a disease selected from intestinal inflammation, diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease, comprising administering to a subject in need thereof an effective therapeutic amount of the isolated anti-metallothionein antibody or fragment thereof or a pharmaceutically acceptable salt thereof according to any one of claims 1-18, the nucleic acid according to claim 19, the vector according to claim 20, the host cell according to claim 21, or the pharmaceutical composition according to claim 22.

24. The method according to claim 23, wherein: The subject has diabetes or is at risk of developing diabetes, and the method is for treating or limiting the development of diabetes.

25. The method according to claim 24, wherein: The subject is at risk for developing type 1 diabetes, and the method is for limiting the development of type 1 diabetes in the subject.

26. The method according to claim 25, wherein: The subject has one or more risk factors for type 1 diabetes, including but not limited to a parent or sibling with type 1 diabetes, pancreatic tumors, pancreatitis, islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), insulinoma-associated (IA-2) autoantibodies, zinc transporter autoantibodies (ZnT8), an IDDM1 gene variant selected from DRB1 0401, DRB1 0402, DRB1 0405, DQA 0301, DQB1 0302 and DQB1 0201; polyuria, polydipsia, dry mouth, polyphagia, fatigue or weight loss.

27. The method according to claim 23, wherein: The subject has type 1 diabetes, and the method is for treating type 1 diabetes in the subject.

28. The method according to claim 27, wherein: The treatment includes one or more of the following: reducing the frequency of insulin injection requirements; delaying the development or progression of complications of type 1 diabetes in the subject; and delaying the need for pancreas or islet cell transplantation; the complications of type 1 diabetes include but are not limited to destruction of pancreatic beta cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, decreased wound healing ability, urinary tract infection, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure and foot ulcers.

29. The method of claim 23, wherein: The subject is at risk for developing type 2 diabetes, and the method is for limiting the development of type 2 diabetes in the subject.

30. The method of claim 29, wherein: The subject has one or more risk factors for type 2 diabetes, including but not limited to obesity, smoking, sedentary lifestyle, parents or siblings with type 2 diabetes, prediabetes, parents or siblings with prediabetes, poor eating habits (e.g., too much fat, not enough fiber, too much simple carbohydrates, etc.), age 50 years or older, high blood pressure, high cholesterol, testosterone deficiency, metallothionein 1A (MT1A) rs8052394 locus (G change) single nucleotide polymorphism and history of gestational diabetes.

31. The method of claim 23, wherein: The subject suffers from type 2 diabetes, and the method is for treating type 2 diabetes in the subject.

32. The method according to claim 31, wherein: The treatment includes limiting one or more complications of type 2 diabetes and reducing the frequency of the need for insulin or other treatments; the complications of type 2 diabetes include but are not limited to hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular cleaning, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, and stroke.

33. The method of claim 32, wherein: The subject has pre-diabetes, and the method is for treating pre-diabetes in the subject.

34. The method of claim 33, wherein: Such treatment includes limiting or delaying the progression of one or more complications of pre-diabetes, including but not limited to type 2 diabetes, hyperglycemia, insulin resistance, and / or cardiovascular disease.

35. The method of claim 23, wherein: The subject suffers from impaired glucose tolerance, and the method is for treating impaired glucose tolerance in the subject.

36. The method of claim 35, wherein: Such treatment includes limiting or slowing 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.

37. The method of claim 23, wherein: The subject has hepatitis, and the method is for treating hepatitis in the subject.

38. The method of claim 37, wherein: The treatment includes limiting or slowing the progression of one or more complications of hepatitis including, but not limited to, yellow discoloration of the skin and / or whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure, and liver cancer.

39. The method of claim 23, wherein: The subject has an inflammatory liver disease, and the method is for treating the inflammatory liver disease in the subject.

40. The method of claim 39, wherein: The inflammatory liver disease is selected from non-alcoholic steatohepatitis (NASH) and / or non-alcoholic fatty liver disease (NAFLD).

41. The method of claim 39 or 40, wherein The treatment includes 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.

42. The method of claim 23, wherein: The subject suffers from intestinal inflammation.

43. The method of claim 42, wherein: The intestinal inflammation is selected from Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, metastatic colitis and indeterminate colitis, and Behcet's disease.

44. The method according to any one of claims 23 to 43, wherein: The subject is a mammal.

45. The method according to any one of claims 23 to 44, wherein: The subject is a human.

Images