Humanized cell-surface antibody to human zinc transporter-8, a biomarker of human pancreatic beta-cells

Abstract

Provided herein are materials and methods relating to the fields of immunology and diabetes. More specifically, provided herein are methods and compositions directed to humanized antibodies to human zinc transporter-8 (ZnT8), a biomarker of human pancreatic beta-cells.

Description

[0001] Attorney Docket No.: JHU-43512.601

[0002] HUMANIZED CELL-SURFACE ANTIBODY TO HUMAN ZINC TRANSPORTER S,

[0003] A BIOMARKER OF HUMAN PANCREATIC BETA-CELLS

[0004] CROSS REFERENCE TO RELATED APPLICATION

[0005] The present application claims priority to U.S. Provisional Application No. 63 / 684.987, filed August 20. 2024, which is incorporated herein by reference in its entirety.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named “JHU_43512_601_SequenceListing.xmF’. The XML file, created on August 20. 2025, is 40,885 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] Provided herein are materials and methods relating to the fields of immunology and diabetes. More specifically, provided herein are methods and compositions directed to humanized antibodies to human zinc transporter-8 (ZnT8), a biomarker of human pancreatic beta-cells.

[0010] BACKGROUND

[0011] Pancreatic 3-cell s as professional secretory cells provide the sole source of insulin in the human body to control blood glucose levels. While P-cells have evolved large dynamic capacities of insulin production in response to glucose fluctuations, they are poorly equipped to cope with islet inflammation and metabolic stress underlying 3-cell autoimmune vulnerability in type-1 diabetes (1), and 3-cell failure and loss in type-2 diabetes (2). Hence, primary 3-cel 1 defects he at the heart of susceptibility to both forms of diabetes. A better understanding of diabetes pathogenesis and evaluation of therapeutic interventions require exact monitoring of the fate of P-cells under disease and therapy conditions. However, routine tests such as measurements of the insulin / C-peptide level, fasting blood glucose level and oral glucose tolerance do not provide adequate information about the mass and function of insulin-producing P-cells in the pre-clinical phase of diabetes and after receiving intervention therapy. Cell surface biomarkers directly linked to the insulin secretory biology with a high cell-surface density are valuable targets for the development of humanized cellsurface monoclonal antibodies (mAbs) applicable as a therapeutic to prevent or reverse type- Attorney Docket No.: JHU-43512.601

[0012] 1 diabetes and other forms insulin-dependent diabetes, and for noninvasive monitoring of P- cell functions and drug delivery.

[0013] SUMMARY

[0014] The dysfunction and loss of insulin-producing -cells in pancreatic islets are primary causes of diabetes mellitus, but neither in vivo monitoring of 3-cell mass and function nor methods for targeted drug delivery have yet been developed. Insulin production and storage in -cells are functionally coupled with cellular zinc enrichment, which is controlled by the hyperexpression of an islet-specific zinc transporter-8 (ZnT8). Described herein are humanized antibodies (collectively called “Isle43” or “Isle43 variants”) with a subnanomolar binding affinity and conformation specificity for an extracellular epitope of human ZnT8 and improved properties over parental murine monoclonal antibody mAb43.

[0015] In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment that binds to human ZnT8, wherein the humanized antibody or antibody fragment comprises: (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (b) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 16, and CDR3 of SEQ ID NO: 19; (c) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5. FR3 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (d) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5. and CDR3 of SEQ ID NO: 9, and a light chain variable region compnsing CDR1 of SEQ ID NO: 13, FR2 of SEQ NO: 14. CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (e) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (I) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5, Attorney Docket No.: JHU-43512.601 and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17. and CDR3 of SEQ ID NO: 19; (g) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (h) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 9. and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; or (i) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 7, CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 15, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19.

[0016] In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19. In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5. FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 16, and CDR3 of SEQ ID NO: 19. In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain FR1 of SEQ ID NO: 1 ; a heavy chain FR4 of SEQ ID NO: 11 ; a light chain FR1 of SEQ ID NO: 12; or a light chain FR4 of SEQ ID NO: 20.

[0017] In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment that binds to human ZnT8, wherein the humanized antibody or antibody fragment comprises: (a) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 29; (b) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 30; (c) a heavy chain variable region comprising SEQ ID NO: 22 and a light chain variable region comprising SEQ ID NO: 29; (d) a heavy chain variable region comprising SEQ ID NO: 23 and a light chain variable region comprising SEQ ID NO: 29; (e) a heavy chain variable region comprising SEQ ID NO: 24 and a light chain variable region comprising SEQ ID NO: Attorney Docket No.: JHU-43512.601

[0018] 29; (f) a heavy chain variable region comprising SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 29; (g) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 29; (h) a heavy chain variable region comprising SEQ ID NO: 28 and a light chain variable region comprising SEQ ID NO: 29; or (i) a heavy chain variable region comprising SEQ ID NO: 22 and a light chain variable region comprising SEQ ID NO: 30.

[0019] In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 29. In further aspects, the humanized antibody or fragment comprises a human kappa light chain constant region (e.g., SEQ ID NO: 33) and / or a human IgGl constant region (e.g.. SEQ ID NO: 34 or 35). In still further aspects, the humanized antibody comprises a heavy chain and a light chain with amino acid sequences set forth in SEQ ID NO: 36 and 37, respectively.

[0020] In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 30. In further aspects, the humanized antibody or fragment comprises a human kappa light chain constant region (e.g., SEQ ID NO: 33) and / or a human IgGl constant region (e.g., SEQ ID NO: 34 or 35). In still further aspects, the humanized antibody comprises a heavy chain and a light chain with amino acid sequences set forth in SEQ ID NO: 36 and 38, respectively.

[0021] In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment, wherein the heavy chain variable region sequence comprises: SEQ ID NO: 22 or SEQ ID NO: 22 comprising 1-3 conservative amino acid substitution, SEQ ID NO: 27 or SEQ ID NO: 27 comprising 1-3 conservative amino acid substitution. In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment, wherein the heavy chain variable region sequence comprises: SEQ ID NO: 22 or SEQ ID NO: 22 comprising 1-3 conservative amino acid substitution, SEQ ID NO: 27 or SEQ ID NO: 27 comprising 1-3 conservative amino acid substitution, and the light chain variable region sequence comprises: SEQ ID NO: 29 or SEQ ID NO: 29 comprising 1-3 conservative amino acid substitution, or SEQ ID NO: 30 or SEQ ID NO: 30 comprising 1-3 conservative amino acid substitution.

[0022] In some aspects, a humanized antibody or antibody fragment described herein has an immunogenicity risk score (DRB1) of less than 1000. In some aspect, a humanized antibody or antibody fragment described herein has stronger cell surface labeling as compared to Attorney Docket No.: JHU-43512.601 mAb43. In some aspect, a humanized antibody or antibody fragment described herein has higher intracellular uptake levels as compared to mAb43. In some aspect, a humanized antibody or antibody fragment described herein has increased binding affinity as compared to tnAb43.

[0023] In some aspects, a humanized antibody or antibody fragment described herein comprises a heavy chain constant region from a human antibody sequence (e g., a human IgGI constant region, optionally with the amino acid sequence set forth in SEQ ID NO: 34 with or without the C-terminal lysine, or set forth in SEQ ID NO: 35 with or without the C- terminal lysine). In some aspect, a humanized antibody or antibody fragment described herein comprises a light chain constant region from a human antibody sequence (e.g., a human kappa light chain constant region, optionally with the amino acid sequence set forth in SEQ ID NO: 33). In some aspect, a humanized antibody or antibody fragment described herein specifically binds to three extracellular loops of a transmembrane domain of human Zinc Transporter-8 (ZnT8). In some aspects, the three extracellular loops of human ZnT8 comprise amino acids 95-99, 169-175 and 242-245 of SEQ ID NO: 21. In some aspect, the humanized antibody fragment comprises a Fab, Fab’, F(ab’)2, Fab’-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv). In some aspect, the heavy chain variable region is of the immunoglobulin (IgGI) isotype. In some aspects, a humanized antibody or antibody fragment described herein is conjugated to a therapeutic agent. In some aspect, a humanized antibody or antibody fragment described herein is conjugated to an imaging agent.

[0024] In some aspects, the presently disclosed subject matter provides a pharmaceutical composition comprising a therapeutically effective amount of a humanized antibody or antibody fragment described herein. In some aspects, the presently disclosed subject matter provides one or more nucleic acid molecules that encode a humanized antibody or antibody fragment described herein. In some aspects, the presently disclosed subject matter provides one or more expression vectors comprising the nucleic acid molecule(s). In some aspects, the presently disclosed subject matter provides a host cell comprising the expression vector(s) and a method of making the antibody or antibody fragment herein by culturing the host cell under conditions that allow expression of the antibody or antibody fragment and then isolating the antibody or antibody fragment from the cell culture.

[0025] In some aspects, the presently disclosed subject matter provides a method for treating a disease or condition associated with ZnT8 in a subject, the method comprising administering to the subject a humanized antibody or antibody fragment described herein or a Attorney Docket No.: JHU-43512.601 pharmaceutical composition described herein. In some aspects, the disease or condition comprises type 1 or type 2 diabetes.

[0026] In some aspects, the presently disclosed subject matter provides a method for detecting pancreatic beta cells in vivo, the method comprising (i) administering a humanized antibody or antibody fragment described herein to a subject; and (ii) detecting the imaging agent conjugated to the humanized antibody or antibody fragment. In some aspects, the method comprises (i) administering a humanized antibody or antibody fragment described herein, wherein the humanized antibody or antibody fragment comprises a single chain variable fragment (scFv) and is conjugated to an imaging agent; and (ii) detecting the imaging agent conjugated to the humanized antibody or antibody fragment. In some aspects, the detecting step comprises positron emission tomography (PET), single-photon emission computed tomography (SPECT)ZCT imaging, nuclear magnetic resonance (NMR) spectroscopy or near-infrared (NIR) optical imaging. In some aspects, the imaging agent is a radiometal. In some aspects, the imaging agent is a radiometal and the detecting step comprises PET. In some aspects, the radiometal is selected from the group consisting of64Cu,67Cu,68Ga,60Ga,89Zr,86Y, and94mTc. In some aspects, the imaging agent is a radiometal and the detecting step comprises SPECT. In some aspects, the radiometal is selected from the group consisting ofinIn,67Ga, "mTc, and177Lu.

[0027] In certain embodiments, the present invention provides methods for treating a disease or condition associated with ZnT8 in a human subject in need thereof, the method comprising administering to the subject the humanized antibody or antibody fragment or pharmaceutical composition as described herein. In further embodiments, the disease or condition associated with ZnT8 is type 1 diabetes (T1D). The T1D may be characterized by the existence in the subject two or more islet autoantibodies selected from the group consisting of insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GAD65), insulinoma- associated antigen-2 autoantibodies (IA-2), zinc transporter-8 autoantibodies (ZnT8), and islet cell cytoplasmic autoantibodies (ICA).

[0028] In some embodiments, the subject is classified as having Stage 1 T1D, which may be characterized by the presence of two or more islet autoantibodies, normoglycemia. and absence of clinical symptoms.

[0029] In some embodiments, the subject is classified as having Stage 2 T1D, which may be characterized by the presence of two or more islet autoantibodies, dysglycemia not meeting diagnostic thresholds for diabetes, and absence of clinical symptoms. Attorney Docket No.: JHU-43512.601

[0030] In some embodiments, the subject is classified as having Stage 3 (clinical) T1D, which may be characterized by hyperglycemia meeting diagnostic thresholds for diabetes and the presence of clinical symptoms of diabetes.

[0031] In some embodiments, the administration of the antibody, antibody fragment, or pharmaceutical composition delays or prevents progression of T1D in the subject. Accordingly, the present disclosure provides a method of delaying or preventing progression of T1D in a subject in need thereof, where the method comprises administration of the antibody, antibody fragment, or pharmaceutical composition herein to the subject. In some embodiments, the administration delays the progression of Stage 1 T1D to Stage 2 T1D, or the progression of Stage 2 T1D to Stage 3 T1D. In some embodiments, the administration delays the onset of Stage 3 T1D in a subject with Stage 2 T1D.

[0032] In some embodiments, the administering of the antibody, antibody fragment, or pharmaceutical composition alleviates or reverses T1D (e.g., alleviates or reverses one or more symptoms of T1D) in the subject having clinical T1D. Accordingly, the present disclosure provides a method of alleviating or reversing T1D (e.g., alleviating or reversing one or more symptoms of T1D) in a subject in need thereof, where the method comprises administration of the antibody, antibody fragment, or pharmaceutical composition herein to the subject. In some embodiments, the administration leads to regeneration of islet cells in the subj ect.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0034] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0035] BRIEF DESCRIPTION OF DRAWINGS Attorney Docket No.: JHU-43512.601

[0036] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0037] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying figures, which are not necessarily drawn to scale, and wherein:

[0038] FIG. 1A and FIG. IB show previously described induction of anti-TMD antibodies and biochemical characterization. (Diabetes 2023;72: 184-195) FIG. 1A shows a membraneflush extracellular surface of ZnT8 (space-filling representation, left) formed by three short loops (ball-and-sticks, right) on top of a ZnT8 homodimer with bound zinc ions. The TMD is imbedded in the lipid bilayer while the CTD is extended into the cytoplasm. FIG. IB shows sequence alignments of three extracellular loops (ECLs).

[0039] FIG. 2 shows representative SDS-PAGE results validating an Isle43 variant.

[0040] FIG. 3 shows a representative SEC-HPLC chromatogram validating an Isle43 variant.

[0041] FIG. 4 shows immunofluorescence labeling results comparing Isle43 variants and mAb43.

[0042] FIG. 5 shows the distribution of immunogenicity (DRB1) scores for marketed antibody drug products.

[0043] FIG. 6 An alignment of the Parental mAb43 VL domain sequences to the Acceptor framework.

[0044] FIG. 7 An alignment of the Parental mAb43 VH domain sequences to the Acceptor framework.

[0045] FIG. 8 shows production and validation of Isle43-12 (NA13) is shown in FIG. 8.

[0046] FIG. 9 shows specific immunolabeling of mouse islets with mAb43, NA13, and NA6.

[0047] FIG. 10 shows Isle43-12 biodistribution and dose-dependent uptake.

[0048] FIG. 11 shows Isle43-12 in vivo stability in blood and pancreas.

[0049] FIG. 12 shows T1D reversal with Isle43-12 in NOD.scid mice with adoptive transfer of TID.

[0050] FIG. 13 shows a head-to-head comparison of lsle43-12 and Teplizumab.

[0051] DETAILED DESCRIPTION

[0052] It is understood that the present invention is not limited to the particular methods and components, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only. Attorney Docket No.: JHU-43512.601 and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms “a,” '’an.” and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a “protein” is a reference to one or more proteins, and includes equivalents thereof known to those skilled in the art and so forth.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0054] All publications cited herein are hereby incorporated by reference including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present invention.

[0055] ZnT8 is a dominant zinc transporter in P-cells with a protein expression level comparable to that of the house-keeping a-tubulin (3). This extraordinary cellular capacity of producing an active zinc transporter brings about one of the highest cellular zinc contents of P-cells in the human body. The tissue distribution of ZnT8 is almost exclusively limited to pancreatic islets (4,5). Although ZnT8 mRNA in islets was detected in all endocrine cell types including a, , y, 5 and s cells, the mRNA level may be only loosely related to corresponding protein levels of ZnT8 in different cell ty pes (6,7). Cell sorting based on the cellular zinc content resulted in a clear separation of P-cells from other islet cells (8), suggesting that the cellular zinc content and its associated ZnT8 protein level are specific biomarkers for P-cells. The subcellular distribution of ZnT8 is in a dynamic equilibrium among the cell surface membrane, insulin secretory granule and endoplasmic reticulum where ZnT8 functions as a zinc-sequestering transporter (3,9,10). The enriched zinc ions are required for proinsulin processing and crystalline packaging of zinc-insulin hexamers (9-13). As a result. ZnT8 subcellular distribution is tightly coupled with insulin processing, storage and secretion (14). Glucose stimulated insulin secretion promotes ZnT8 trafficking to the cell surface (15), making it a major cell-surface antigenic target for autoantibodies in patients with type-1 diabetes (16). Likewise, the surfaced ZnT8 could potentially act as a functional biomarker for mAb-based immunodetection. Attorney Docket No.: JHU-43512.601

[0056] An earlier ZnT8 mAb to a linear peptide derived from an extracellular loop of ZnT8 yielded modest binding affinity (108 nM) and low specificity (17). In vivo P-cell imaging and targeting require the development of high-affinity mAbs with exquisite conformation specificity to multiple extracellular loops arranged in spatial configurations. ZnT8 is a two- modular protein consisting of a compact transmembrane domain (TMD) and a cytosolic C- terminal domain (CTD). The TMD lacks an ectodomain while its extracellular surface is membrane-flush and formed by three short extracellular loops (ECL 1-3) (FIG. 1A). In addition to limited epitope availability, these loops are poorly antigenic because they are quasi-invariant between mouse and human ZnT8 with the exception of a highly conserved E- to-D substitution in ECL3 (FIG. IB).

[0057] The present inventors have now identified a group of humanized antibodies (“Isle43” or ”Isle43 variants”) to extracellular loops with conformation specificity. These antibodies may be used as a delivery vehicle, e.g. for P-cell purification and targeted delivery of imaging-probes, and as a therapeutic to prevent or reverse type-1 diabetes and other forms of insulin-dependent diabetes.

[0058] Indeed, experiments conducted during the course of developing embodiments for the present invention demonstrated that Isle43 variants, such as Isle43- 11 (also called “NA6” herein) and Isle43-12 (also called “NA9” herein), are first-in-class, humanized monoclonal antibodies specifically engineered to target the zinc transporter ZnT8 on pancreatic P-cells for the treatment of type 1 diabetes (T1D). Isle43 variants were shown to introduce a novel. P- cell-directed therapeutic mechanism distinct from existing immunotherapies: they binds to ZnT8 on the P-cell surface to mask immunogenic epitopes and simultaneously act as a chaperone within the endoplasmic reticulum to mitigate inflammation-induced ER stress. The variants demonstrated selective immunolabeling of islets in mouse and human tissues, pancreas-specific uptake in vivo, and exceptional stability in both serum and pancreatic tissue for over six weeks after a single dose. In an adoptive transfer model of autoimmune diabetes, Isle43-12 (i.e., NA13) provided durable protection against disease relapse following insulin withdrawal, comparable in efficacy to teplizumab but without requiring T cell modulation. These findings highlight the therapeutic significance and novelty of Isle43 variants as nonimmunosuppressive, P-cell protective antibodies for disease modification in T1D.

[0059] I. Definitions

[0060] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein (e.g., human ZNT8, a subunit thereof, or the Attorney Docket No.: JHU-43512.601 receptor complex), polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.

[0061] A typical antibody comprises at least two heavy (HC) chains and two light (LC) chains interconnected by disulfide bonds. Each heavy chain is comprised of a 'heavy chain variable region” or ‘heavy chain variable domain” (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains. CHI, CH2, and CH3. Each light chain is comprised of a Tight chain variable region” or Tight chain variable domain” (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CI. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDR), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FRI, CDRI, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0062] As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab’, F(ab’)2, Fd, Facb, and Fv fragments), single chain Fv (scFv), minibodies (e.g., sc(Fv)2, diabody), multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, mixed antibodies, humanized antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. Thus, the term “antibody” includes whole antibodies and any antigenbinding fragment or single chains thereof. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, small molecule drugs, polypeptides, etc.

[0063] The term ‘Isolated antibody” refers to an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight of antibody as detennined by, for example, the Lowiy method, and including more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by Attorney Docket No.: JHU-43512.601 use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0064] The tenn ‘humanized antibody or antibody fragment” refers to an antibody or antibody fragment comprising one or more framework region sequence(s) entirely or substantially from or identical to one or more human immunoglobulin sequence(s); and one or more CDRs entirely or substantially from or identical to one or more non-human (usually a mouse or rat) immunoglobulin sequence(s). The non-human immunoglobulin providing the CDRs is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” The acceptor / human antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. In one aspect, a humanized antibody is an antibody having at least three, four, five or all CDRs entirely or substantially from or identical to a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from or identical to human antibody sequences. In one aspect, a humanized antibody fragment comprises at least one, two or three CDRs entirely or substantially from or identical to a donor antibody heavy chain, and a heavy chain variable region framework sequence substantially from or identical to a human heavy chain variable region framework sequence. In one aspect, a humanized antibody fragment comprises a at least one, two or three CDRs entirely or substantially from or identical to a donor antibody light chain, and a light chain variable region framework sequence substantially from or identical to human light chain variable region framework. In one aspect, a CDR is an abbreviated CDR. An “abbreviated CDR” is a portion of a CDR sequence that comprises specificity determining residues (SDRs). SDRs are CDR residues that are most crucial in antibody -ligand interaction or required for binding. In one aspect, a CDR is substantially from or identical to a corresponding CDR in a non-human antibody when at least 85%. 90%. 95% or 100% of corresponding residues (as defined by Chothia CDR definition (Al-Lazikani et al. 1997)) are identical to the corresponding non-human antibody CDR. In one aspect, a variable region framework sequence is substantially from or identical to a human variable region framework sequence when at least 85%. 90%. 95% or 100% of corresponding residues (as defined by Chothia CDR definition (Al-Lazikani et al. 1997)) are identical. In one aspect, a constant Attorney Docket No.: JHU-43512.601 region is substantially from or identical to a human constant region when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Chothia CDR definition (Al-Lazikani el al. 1997)) are identical. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. For example, ahumanized antibody would not encompass a typical chimeric antibody with an entire variable region that is non-human.

[0065] Examples of “antibody fragments” include(a) a Fab fragment, a monovalent fragment consisting of the Vi, VH, CI, and CHI domains; (b) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (c) a Fd fragment consisting of the VH and CHI domains; (d) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (e) a dAb fragment (Ward, et al. (1989) Nature 341 : 544-546), which consists of a VH domain; and (1) an isolated complementarily determining region (CDR). Further examples of antibody fragments include linear antibodies, single chain antibodies, and multi-specific antibodies formed from antibody fragments.

[0066] The humanized antibody or antibody fragment disclosed herein is capable of specifically binding to human ZNT8. In one aspect, the antibody or antibody fragment is capable of specifically binding to human ZNT8 with sufficient affinity such that the antibody or antibody fragment is useful as a therapeutic agent. In one aspect, the antibody or antibody fragment is capable of specifically binding to human ZNT8 with sufficient affinity such that the antibody or antibody fragment is useful as a therapeutic agent as a diagnostic reagent in targeting human ZNT8. The extent of binding of a humanized antibody or antibody fragment disclosed herein to an unrelated, non-ZNT8 protein is less than about 10% of the binding of the antibody to human ZNT8 as measured, e.g., by a radioimmunoassay (RIA), BIACORE™ (using recombinant human ZNT8 as the analyte and antibody as the ligand, or vice versa), or other binding assays known in the art. In certain embodiments, an antibody or antibody fragment that binds to human ZNT8 has a dissociation constant (KD) of <1 pM, <100 nM, <50 nM, <10 nM, or <1 nM.

[0067] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. Attorney Docket No.: JHU-43512.601

[0068] These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0069] The term “% identical’7or “% sequence identity” between two polypeptide (or polynucleotide) sequences refers to the number of identical matched positions shared by the sequences over a comparison window, considering additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated by determining the number of positions at which the identical ammo acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using readily available software both for online use and for download. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government’s National Center for Biotechnology Information BLAST web site. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. In certain embodiments, the percentage identity' X " of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the Attorney Docket No.: JHU-43512.601 total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program to generate multiple sequence alignments is ClustalW2 (ClustalX is a version of the ClustalW2 program ported to the Windows environment). Another suitable program is MUSCLE. ClustalW2 and MUSCLE are alternatively available, e.g., from the European Bioinformatics Institute (EBI).

[0070] The term 'therapeutic agent’’ refers to any biological or chemical agent used in the treatment of a disease or disorder. Therapeutic agents include any suitable biologically active chemical compounds, biologically derived components such as cells, peptides, antibodies, and polynucleotides, and radiochemical therapeutic agents such as radioisotopes. In some embodiments, the therapeutic agent comprises a chemotherapeutic agent or an analgesic.

[0071] As used herein, the terms “treatment,” “treating,” “treat” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The terms are also used in the context of the administration of a “therapeutically effective amount” of an agent, e.g., a humanized antibody or antibody fragment disclosed herein. The effect may be prophylactic in terms of completely or partially preventing a particular outcome, disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. '‘Treatment,” as used herein, covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease. In particular embodiments, the term is used in the context of preventing or treating any ZnT8-mediated disease including diabetes.

[0072] IL Humanized antibodies or antibody fragments that bind to human ZnT8

[0073] The humanized antibodies or antibody fragments of this disclosure specifically bind to human ZNT8. In one aspect, the antibody or antibody fragment specifically binds to three Attorney Docket No.: JHU-43512.601 extracellular loops of a transmembrane domain of human Zinc Transporter-8 (ZnT8). In one aspect, the three extracellular loops of human ZnT8 comprise amino acids 95-99, 169-175 and 242-245 of SEQ ID NO: 21.

[0074] In one aspect, the humanized antibody or antibody fragments described herein specifically bind to the transmembrane domain of human ZNT8. “Specifically binds” as used herein means that the antibody or antibody fragment preferentially binds human ZNT8 over other proteins. In certain instances, the anti-ZNT8 antibodies of the disclosure have ahigher affinity for human ZNT8 than for other proteins. Anti-ZNT8 antibodies that specifically bind human ZNT8 may have a binding affinity7for human ZNT8 of less than or equal to 1 x 10'7M, less than or equal to 2 x 10'7M, less than or equal to 3 x 10’7M. less than or equal to 4 x 10’7M, less than or equal to 5 x 10’7M. less than or equal to 6 x 10‘7M, less than or equal to 7 x 10‘7M, less than or equal to 8 x 10‘7M, less than or equal to 9 x ICT7M, less than or equal to 1 x 10'8M, less than or equal to 2 x 10'8M, less than or equal to 3 x 10'8M, less than or equal to 4 x 10'8M, less than or equal to 5 x 10’8M. less than or equal to 6 x 10’8M, less than or equal to 7 x 10’8M, less than or equal to 8 x IO'8M, less than or equal to 9 x 10'8M, less than or equal to l x 10‘9M, less than or equal to 2 x 10'9M, less than or equal to 3 x 10'9M, less than or equal to 4 x 10'9M, less than or equal to 5 x 10'9M, less than or equal to 6 x IO"9M, less than or equal to 7 x ICT9M, less than or equal to 8 x 10'9M, less than or equal to 9 x 10'9M, less than or equal to l x 10'10M. less than or equal to 2 x 10‘10M, less than or equal to 3 x 10'10M, less than or equal to 4 x I CT10M, less than or equal to 5 x 10‘10M, less than or equal to 6 x 10'10M, less than or equal to 7 x 10'10M, less than or equal to 8 x 10'10M, less than or equal to 9 x 10'10M, less than or equal to l x 10'11M, less than or equal to 2 x 10’11M, less than or equal to 3 x 10'11M, less than or equal to 4 x 1011M. less than or equal to 5 x 1011M, less than or equal to 6 x 10’11M, less than or equal to 7 x 10‘11M, less than or equal to 8 x 10’11M, less than or equal to 9 x ICT11M, less than or equal to 1 x ICT12M, less than or equal to 2 x 10'12M, less than or equal to 3 x 10'12M, less than or equal to 4 x 10'12M, less than or equal to 5 x 10"12M, less than or equal to 6 x 10'12M. less than or equal to 7 x 10’12M, less than or equal to 8 x IO’12M, or less than or equal to 9 x 10'12M. Methods of measuring the binding affinity' of an antibody are well known in the art and include Surface Plasmon Resonance (SPR) (Morton and Myszka “Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors” Methods in Enzymology (1998) 295, 268-294), Bio-Layer Interferometry. (Abdiche et al “Determining Kinetics and Affinities of Protein Interactions Using a Parallel Real-time Label -free Biosensor, the Octet” Analytical Biochemistry (2008) Attorney Docket No.: JHU-43512.601

[0075] 377, 209-217), Kinetic Exclusion Assay (KinExA) (Darling and Brault “Kinetic exclusion assay technology: characterization of molecular interactions’7Assay and Drug Dev Tech (2004) 2, 647-657), isothermal calorimetry (Pierce et al “Isothermal Titration Calorimetry of Protein-Protein Interactions” Methods (1999) 19, 213-221) and analytical ultracentrifugation (Lebowitz et al “Modem analytical ultracentrifugation in protein science: A tutorial review” Protein Science (2002), 11:2067-2079).

[0076] A prior mAb43 that binds to human ZnT8 is disclosed in PCT / US2022075156 and is the parental murine antibody. However, PCTUS2022075156 does not disclose a humanized antibody or antibody fragment comprising the specific humanized Isle43 variants, heavy chain variable domain sequences and light chain variable domain sequences, or combinations of CDR (1. 2, 3) and framework (1, 2, 3, 4) sequences described herein. See. for example. Table 7. The humanized antibody or antibody fragments described herein were engineered via CDR-grafting. The humanized antibody or antibody fragments described herein have reduced immunogenicity and unexpected improvements over mAb43. For example, the humanized Isle43 variants listed in Table 7 herein unexpectedly have stronger cell surface labeling as compared to mAb43, unexpectedly have higher intracellular uptake levels as compared to mAb43, and unexpectedly have increased binding affinity as compared to mAb43.

[0077] The unexpected nature of these properties is supported in view of other humanized Isle43 variants (NA9, NA10, NA11. NA12, NA14) listed in Table 6 that have weaker (NA10 and NA12) or similar (NA9, NA1 1 , NAM) cell surface labeling as compared to mAb43, and Isle43 variants that have lower intracellular uptake levels (NA9, NA10, NA11, NA 12. NAM) than that of mAb43. The humanized Isle43 variants in Table 6 also were engineered by the same methods used to engineer the humanized Isle43 variants in Table 7. Therefore, there is no reasonable expectation a priori that a humanized antibody with improved properties related to binding and intracellular uptake would normally or necessarily be obtained when carrying out standard humanization protocols. There is no guidance known in the art on which of the known humanization protocols should be followed or which back mutations or which forw ard mutations are necessary mutations in CDR-grafting to obtain a humanized anti-ZNT8 antibody or antibody fragment exhibiting improvements (e g. stronger cell surface labeling, higher intracellular uptake levels, or stronger binding affinity) as compared to the parent murine antibody, mAb43. There is no particular recipe known for reducing the immunogenicity of a specific antibody while not affecting or improving the affinity and specificity of the antibody. Without clear guidance on any specific set of mutations that Attorney Docket No.: JHU-43512.601 could reduce immunogenicity while improving or not affecting the affinity and specificity of the antibody, the impact of each mutation in the antibody on the final properties of the antibody is unpredictable.

[0078] In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment that binds to human ZnT8, wherein the humanized antibody or antibody fragment comprises: (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (b) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8. and CDR3 of SEQ ID NO: 10. and a light chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 16, and CDR3 of SEQ ID NO: 19; (c) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5. FR3 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (d) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (e) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (f) a heavy’ chain variable region comprising CDR1 of SEQ ID NO: 2. CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (g) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 6. and CDR3 of SEQ ID NO: 10, and a light chain variable region compnsing CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; (h) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; or (i) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 3, Attorney Docket No.: JHU-43512.601

[0079] CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 7, CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13. FR2 of SEQ ID NO: 15. CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19.

[0080] In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13. FR2 of SEQ ID NO: 14. CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19. In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 16, and CDR3 of SEQ ID NO: 19. In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain FR1 of SEQ ID NO: 1 ; a heavy chain FR4 of SEQ ID NO: 11 ; a light chain FR1 of SEQ ID NO: 12; or a light chain FR4 of SEQ ID NO: 20.

[0081] In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment that binds to human ZnT8, wherein the humanized antibody or antibody fragment comprises: (a) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 29; (b) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 30; (c) a heavy chain variable region comprising SEQ ID NO: 22 and a light chain variable region comprising SEQ ID NO: 29; (d) a heavy chain variable region comprising SEQ ID NO: 23 and a light chain variable region comprising SEQ ID NO: 29; (e) a heavy chain variable region comprising SEQ ID NO: 24 and a light chain variable region comprising SEQ ID NO: 29; (f) a heavy chain variable region comprising SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 29; (g) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 29; (h) a heavy chain variable region comprising SEQ ID NO: 28 and a light chain variable region comprising SEQ ID NO: 29: or (i) a heavy chain variable region comprising SEQ ID NO: 22 and a light chain variable region comprising SEQ ID NO: 30.

[0082] In some aspects, the humanized antibody or antibody fragment comprises: a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 29. In some aspects, the humanized antibody or antibody fragment Attorney Docket No.: JHU-43512.601 comprises: a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 30.

[0083] In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment, wherein the heavy chain variable region sequence comprises: SEQ ID NO: 22 or SEQ ID NO: 22 comprising 1-3 conserv ative amino acid substitution, SEQ ID NO: 27 or SEQ ID NO: 27 comprising 1-3 conservative amino acid substitution. In some aspects, the presently disclosed subject matter provides a humanized antibody or antibody fragment, wherein the heavy chain variable region sequence comprises: SEQ ID NO: 22 or SEQ ID NO: 22 comprising 1-3 conservative amino acid substitution, SEQ ID NO: 27 or SEQ ID NO: 27 comprising 1-3 conservative amino acid substitution, and the light chain variable region sequence comprises: SEQ ID NO: 29 or SEQ ID NO: 29 comprising 1-3 conservative amino acid substitution, or SEQ ID NO: 30 or SEQ ID NO: 30 comprising 1-3 conservative amino acid substitution.

[0084] In some aspect, a humanized antibody or antibody fragment described herein has an immunogenicity risk score (DRB1) of less than 1000. In some aspect, a humanized antibody or antibody fragment described herein has stronger cell surface labeling as compared to mAb43. In some aspect, a humanized antibody or antibody fragment described herein has higher intracellular uptake levels as compared to mAb43. In some aspect, a humanized antibody or antibody fragment described herein has increased binding affinity as compared to mAb43.

[0085] CDRs described herein are defined by the Chothia CDR definition (Al-Lazikani et al. 1997) numbering scheme. Variable region framework positions are in accordance with the Chothia CDR definition (Al-Lazikani et al. 1997) numbering scheme.

[0086] In some aspect, a humanized antibody or antibody fragment described herein comprises a heavy chain constant region from a human antibody sequence. In some aspect, a humanized antibody or antibody fragment described herein comprises a light chain constant region from a human antibody sequence. In some aspect, a humanized antibody or antibody fragment described herein specifically binds to three extracellular loops of a transmembrane domain of human Zinc Transporter-8 (ZnT8). In some aspects, the three extracellular loops of human ZnT8 comprise amino acids 95-99, 169-175 and 242-245 of SEQ ID NO: 21.

[0087] In one aspect, the humanized antibody or antibody fragment comprises a heavy chain variable framework region from a human antibody sequence. In one aspect, the heavy chain variable framework region is from human immunoglobulin. In one aspect, the humanized antibody or antibody fragment comprises a light chain variable framework region from a Attorney Docket No.: JHU-43512.601 human antibody sequence. In one aspect, the light chain variable framework region is from human immunoglobulin. In one aspect, the human antibody sequence is an individual human sequence, a human consensus sequence, an individual human germline sequence, or a human consensus germline sequence.

[0088] In one aspect, the humanized antibody or antibody fragment comprises at least one heavy’ chain variable framework region sequence selected from SEQ ID NO: 1, SEQ ID: 3, SEQ ID NO: 7, and SEQ ID NO: 11. In one aspect, the humanized antibody or antibody fragment comprises: (A) a heavy chain FR1 that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 1; (B) a heavy chain FR2 that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3; (C) a heavy chain FR3 that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7; and (D) a heavy chain FR4 that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to SEQ ID NO: 11.

[0089] In one aspect, the humanized antibody or antibody fragment comprises at least one heavy chain variable framework region sequence selected from SEQ ID NO: 1, SEQ ID: 4, SEQ ID NO: 8, and SEQ ID NO: 11. In one aspect, the humanized antibody or antibody’ fragment comprises: (A) a heavy chain FR1 that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1; (B) a heavy chain FR2 that has at least 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 4; (C) a heavy chain FR3 that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity’ to SEQ ID NO: 8; and (D) a heavy chain FR4 that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 11.

[0090] In one aspect, the humanized antibody or antibody fragment comprises at least one light chain variable framework region sequence selected from SEQ ID NO: 12, SEQ ID. NO: 14, SEQ ID NO: 17, and SEQ ID NO: 20. In one aspect, the humanized antibody or antibody fragment comprises: (A) a light chain FR1 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100% sequence identity to SEQ ID NO: 12; (B) a light chain FR2 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14; (C) a light chain FR3 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17; and (D) a light chain FR4 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20. Attorney Docket No.: JHU-43512.601

[0091] In one aspect, the humanized antibody or antibody fragment comprises at least one light chain variable framework region sequence selected from SEQ ID NO: 12. SEQ ID. NO: 15, SEQ ID NO: 18, and SEQ ID NO: 20. In one aspect, the humanized antibody or antibody fragment comprises: (A) a light chain FR1 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12; (B) a light chain FR2 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15; (C) a light chain FR3 has at least 85%. 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18; and (D) a light chain FR4 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20.

[0092] In one aspect, provided herein is a humanized antibody or antibody fragment that binds to human Zinc Transporter-8 (ZnT8), wherein the antibody or antibody fragment comprises: a heavy chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to SEQ ID NO: 22, or SEQ ID NO: 27.

[0093] In one aspect, provided herein is a humanized antibody or antibody fragment that binds to human Zinc Transporter-8 (ZnT8), wherein the antibody or antibody fragment comprises: (a) a heavy chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 27; and (b) a light chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to SEQ ID NO: 29 or SEQ ID NO: 30. In one aspect, the heavy chain variable region sequence has at least 98%, 99%, or 100% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 27; and the light chain variable region sequence has at least 98%, 99%, or 100% sequence identity to SEQ ID NO: 29 or SEQ ID NO: 30.

[0094] In one aspect, provided herein is a humanized antibody or antibody fragment that binds to human Zinc Transporter-8 (ZnT8), wherein the antibody or antibody fragment comprises: (a) a heavy chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:

[0095] 25, SEQ ID NO: 26, or SEQ ID NO: 28; and (b) a light chain variable region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. or 97% sequence identity to SEQ ID NO: 29. In one aspect, the heavy chain variable region sequence has at least 98%, 99%, or 100% sequence identity to SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:

[0096] 26, or SEQ ID NO: 28; and the light chain variable region sequence has at least 98%, 99%, or 100% sequence identity to SEQ ID NO: 29. Attorney Docket No.: JHU-43512.601

[0097] In one aspect, the heavy chain variable region sequence contains conservative amino acid substitutions, insertions, or deletions relative to SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28 and retains the ability to bind to a human ZNT8. In one aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one aspect, 1, 2. 3, 4, 5. 6. 7, 8, 9. or 10 amino acids have been substituted, inserted and / or deleted in framework regions of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one aspect, the heavy chain variable region sequence comprises post-translational modifications of SEQ ID NO: 22, SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one aspect, the heavy chain variable region sequence comprises one point mutation relative to SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one aspect, the one point mutation is located in a CDR region of the heavy chain variable region sequence.

[0098] In one aspect, the light chain variable region sequence contains conservative amino acid substitutions, insertions, or deletions relative to SEQ ID NO: 29 or SEQ ID NO: 30 and retains the ability to bind to a human ZNT8. In one aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 29 or SEQ ID NO: 30. In one aspect, 1. 2, 3, 4, 5. 6, 7, 8. 9, or 10 amino acids have been substituted, inserted and / or deleted in framework regions of SEQ ID NO: 29 or SEQ ID NO: 30. In one aspect, the light chain variable region sequence comprises post-translational modifications of SEQ ID NO: 29 or SEQ ID NO: 30. In one aspect, the light chain variable region sequence comprises one point mutation relative to SEQ ID NO: 29 or SEQ ID NO: 30. In one aspect, the one point mutation is located in a CDR region of the light chain variable region sequence.

[0099] In one aspect, provided herein is a humanized antibody or antibody fragment wherein the heavy chain variable region sequence comprises: SEQ ID NO: 22 or SEQ ID NO: 22 comprising less than 10, 9. 8, 7, 6, 5, 4, 3, or 2 conservative amino acid substitution(s). or SEQ ID NO: 27 or SEQ ID NO: 27 comprising less than 10, 9, 8. 7, 6, 5. 4. 3, or 2 conservative amino acid substitution(s); and the light chain variable region sequence comprises: SEQ ID NO: 29 or SEQ ID NO: 29 comprising less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 conserv ative amino acid substitution(s), or SEQ ID NO: 30 or SEQ ID NO: 30 comprising less than 10, 9. 8, 7, 6, 5. 4, 3, or 2 conservative amino acid substitution(s). Attorney Docket No.: JHU-43512.601

[0100] In one aspect, provided herein is a humanized antibody or antibody fragment wherein the heavy chain variable region sequence comprises: SEQ ID NO: 23 or SEQ ID NO: 23 comprising less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 conservative amino acid substitution(s), SEQ ID NO: 24 or SEQ ID NO: 24 comprising less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 conservative amino acid substitution(s), SEQ ID NO: 25 or SEQ ID NO: 25 comprising less than 10, 9, 8, 7, 6, 5, 4. 3, or 2 conservative amino acid substitution(s), SEQ ID NO: 26 or SEQ ID NO: 26 comprising less than 10. 9. 8, 7, 6. 5, 4, 3. or 2 conservative amino acid substitution(s). or SEQ ID NO: 28 or SEQ ID NO: 28 comprising less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 conservative amino acid substitution(s); and the light chain variable region sequence comprises: SEQ ID NO: 29 or SEQ ID NO: 29 comprising less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 conservative amino acid substitution(s).

[0101] In one aspect, provided herein is a humanized antibody or antibody fragment wherein the heavy chain variable region sequence comprises: SEQ ID NO: 22 or SEQ ID NO: 22 comprising 1-3 conservative amino acid substitution(s) or SEQ ID NO: 27 or SEQ ID NO: 27 comprising 1-3 conservative amino acid substitutions(s); and the light chain variable region sequence comprises: SEQ ID NO: 29 or SEQ ID NO: 29 comprising 1-3 conservative amino acid substitution(s), or SEQ ID NO: 30 or SEQ ID NO: 30 comprising 1-3 conservative amino acid substitution(s).

[0102] In one aspect, provided herein is a humanized antibody or antibody fragment wherein the heavy chain variable region sequence comprises: SEQ ID NO: 23 or SEQ ID NO: 23 comprising 1 -3 conservative amino acid substitutions(s), SEQ ID NO: 24 or SEQ ID NO: 24 comprising 1-3 conservative amino acid substitutions(s), SEQ ID NO: 25 or SEQ ID NO: 25 comprising 1-3 conservative amino acid substitutions(s), SEQ ID NO: 26 or SEQ ID NO: 26 comprising 1-3 conservative amino acid substitutions(s), or SEQ ID NO: 28 or SEQ ID NO: 28 comprising 1-3 conservative amino acid substitution(s); and the light chain variable region sequence comprises: SEQ ID NO: 29 or SEQ ID NO: 29 comprising 1-3 conservative amino acid substitution(s).

[0103] In some embodiments, a humanized antibody disclosed herein is a monoclonal antibody or a humanized antibody fragment disclosed herein is a fragment of a binding portion of a monoclonal antibody.

[0104] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a. 5, e, y, and p, respectively. The y and p classes are further divided into subclasses e.g., humans express the following subclasses: IgGl, IgG2. IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed Attorney Docket No.: JHU-43512.601 alloty pes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 41-7) any of which are suitable for use in some of the embodiments herein. Common allotypic variants in human populations are those designated by the letters a, f, n, z or combinations thereof. In any of the embodiments herein, a humanized antibody or antibody fragment may comprise a heavy chain Fc region comprising a human IgG Fc region. In some embodiments, the human IgG Fc region is a human IgG4 Fc region. In some embodiments, the human IgG Fc region is a human IgGl Fc region.

[0105] The humanized antibodies or antibody fragments may also include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to ZnT8 or from exerting a cytostatic or cytotoxic effect on cells. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, PEGylation, phosphylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acety lation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non- classical amino acids.

[0106] In one aspect, humanized sequences are generated using a two-stage PCR protocol that allows introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, B. A. (1999) BioTechniques 26:680- 682)]. In on aspect, a humanized antibody can be genetically engineered in which CDRs from a non-human “donor” antibody are grafted into human “acceptor” antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5.585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No. 6,881,557). A humanized antibody can be made with less than all six CDRs (e.g., at least 3, 4, or 5 CDRs) from a mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol.. 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36: 1079-1091. 1999; Tamura et al. J. Immunol., 164: 1432-1441, 2000). If more than one human acceptor antibody sequence is selected for a chain (either light or heavy), a composite or hybrid of those acceptors can be used for that chain. Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and / or binding to antigen. Investigation of such possible influences is by Attorney Docket No.: JHU-43512.601 modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.

[0107] Although humanized antibodies often incorporate all six CDRs (preferably as defined by Chothia CDR definition (Al-Lazikani et al. 1997)) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5) CDRs from a mouse antibody. See, e.g., Pascalis et al.. J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al.. Mol. Immunol. 36: 1079-1091. 1999; and Tamura et al, Journal of Immunology, 164: 1432-1441, 2000.

[0108] The heavy and light chain variable regions of humanized antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, whether antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis and / or complement dependent cytotoxicity are desired. For example, human isotopes IgGl and IgG3 have strong complement-dependent cytotoxicity, human isotype IgG2 weak complement-dependent cytotoxicity and human. IgG4 lacks complementdependent cytotoxicity. Human IgGl and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4. Light chain constant regions can be lambda or kappa. Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab’)2, and Fv, or as single chain antibodies in which heavy and light chain variable domains are linked through a spacer.

[0109] Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype binds to a non-polymorphic region of a one or more other isotypes.

[0110] One or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006). or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem.279:6213, 2004).

[0111] Exemplary7substitutions include the amino acid substitution of the native amino acid to a cysteine residue is introduced at amino acid position 234. 235, 237, 239, 267, 298, 299, 326, 330. or 332. preferably an S239C mutation in a human IgGl isotype (US 20100158909). The presence of an additional cysteine residue allows interchain disulfide bond formation. Attorney Docket No.: JHU-43512.601

[0112] Such interchain disulfide bond formation can cause steric hindrance, thereby reducing the affinity of the Fc region-FcyR binding interaction. The cysteine residue(s) introduced in or in proximity to the Fc region of an IgG constant region can also serve as sites for conjugation to therapeutic agents (i.e., coupling cytotoxic drugs using thiol specific reagents such as maleimide derivatives of drugs. The presence of a therapeutic agent causes steric hindrance, thereby further reducing the affinity of the Fc region-FcyR binding interaction.

[0113] The in vivo half-life of an antibody can also impact on its effector functions. The half-life of an antibody can be increased or decreased to modify its therapeutic activities. FcRn is a receptor that is structurally similar to MHC Class I antigen that non-covalently associates with p2-microglobulin. FcRn regulates the catabolism of IgGs and their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol.18:739- 766; Ghetie and Ward, 2002, Immunol. Res.25:97-113). The IgG-FcRn interaction takes place at pH 6.0 (pH of intracellular vesicles) but not at pH 7.4 (pH of blood); this interaction enables IgGs to be recycled back to the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol.18:739-766; Ghetie and Ward, 2002, Immunol. Res.25:97-113). The region on human IgGl involved in FcRn binding has been mapped (Shields et al, 2001, J. Biol. Chem.276:6591-604). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgGl enhance FcRn binding (Shields et al, 2001, J. Biol. Chem.276:6591- 604). IgGl molecules harboring these substitutions have longer serum half-lives. Consequently, these modified IgGl molecules may be able to carry out their effector functions, and hence exert their therapeutic efficacies, over a longer period of time compared to unmodified IgGl. Other exemplary' substitutions for increasing binding to FcRn include a Gin at position 250 and / or a Leu at position 428. EU numbering is used for all position in the constant region.

[0114] Reference to a human constant region includes a constant region with any natural allofype or any permutation of residues occupying polymorphic positions in natural allotypes. Also, up to 1, 2, 5, or 10 mutations may be present relative to a natural human constant region, such as those indicated above to reduce Fcgamma receptor binding or increase binding to FcRN.

[0115] In some aspect, the humanized antibody fragment comprises a Fab, Fab’, F(ab’)2, Fab’-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv).

[0116] In some aspect, a humanized antibody or antibody fragment described herein is conjugated to a therapeutic agent. In some aspect, a humanized antibody or antibodyfragment described herein is conjugated to an imaging agent. Attorney Docket No.: JHU-43512.601

[0117] Antibody fragments may be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin. Papain digestion of whole antibodies produces Fab fragments; pepsin digestion of whole antibodies yields F(ab’)2 or Fab’; and plasmin digestion of whole antibodies yields Facb fragments.

[0118] A Fab fragment can be obtained by digestion of immunoglobulin (typically IgG) with the enzyme papain. The heavy chain segment of the Fab fragment is the Fd piece. Such fragments can be enzymatically or chemically produced by fragmentation of an intact antibody, recombinantly produced from a gene encoding the partial antibody sequence, or it can be wholly or partially synthetically produced.

[0119] A F(ab’)2 fragment can be obtained by digestion of an immunoglobulin (typically IgG) with the enzyme pepsin at pH 4.0-4.5. Such fragments can be enzy matically or chemically produced by fragmentation of an intact antibody, recombinantly produced from a gene encoding the partial antibody sequence, or it can be wholly or partially synthetically produced. The term “Fv” refers to an antibody fragment that consists of oneNH and one N domain held together by noncovalent interactions.

[0120] Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). See e.g., Bird, et al.

[0121] (1988) Science 242: 423-426; Huston, et al. (1988) Proc Natl. Acad. Sci. USA 85: 5879-5883; and Osbourn, et al. (1998) Nat. Biotechnol. 16: 778. Any Viiand VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG molecules or other isotypes. VH and VL can also be used in the generation of Fab, Fv, or other fragments of immunoglobulins using either protein chemistry' or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary^ domains of another chain and creating two antigen-binding sites. See e.g. Holliger, et al. (1993) Proc Natl. Acad. Sci. USA 90: 6444-6448; Poljak, et al. (1994) Structure 2: 1121-1123. Still further, an antibody or antibody fragment may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion Attorney Docket No.: JHU-43512.601 with one or more other proteins or peptides. Examples of immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, et al. (1995) Hum. Antibodies Hybridomas 6: 93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules. Kipriyanov, et al. (199A) Mol. Immunol. 31: 1047-1058. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0122] Antibody fragments can be produced recombinantly. For example, nucleic acids encoding the antibody fragments of interest can be constructed, introduced into an expression vector, and expressed in suitable host cells. See. e.g., Co. M.S. et al., J Immunol., 152:2968-2976 (1994); Better, M. and Horwitz. A.H., Methods in Enzymology, 178:476-496 (1989); Pluckthun, A and Skerra. A, Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology’, 121:652-663 (1989); Rousseaux, J. et al., Methods in Enzymology’, (1989) 121:663-669 (1989); and Bird, RE. et al., TIBTECH, 9: 132-137 (1991)). Antibody fragments can be expressed in and secreted from A coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries. Alternatively, Fab’-SH fragments can be directly recovered fromE. coli and chemically coupled to formF(ab)2 fragments (Carter et al., Bio / 'Technology, 10: 163- 167 (1992)). According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab’) 2 fragment with increased in vivo halflife comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046.

[0123] Minibodies

[0124] Also encompassed are minibodies of the humanized antibodies described herein. Minibodies include diabodies, single chain (scFv), and single-chain (Fv)2 (sc(Fv)2).

[0125] A “diabody” is a bivalent minibody constructed by gene fusion (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. U S. A.. 90:6444-6448 (1993); EP 404,097; WO 93 / 1 1161). Diabodies are dimers composed of tw o polypeptide chains. The VL and VH domain of each polypeptide chain of the diabody are bound by linkers. The number of amino acid residues that constitute a linker can be between 2 to 12 residues (e.g., 3-10 residues or five or about five residues). The linkers of the polypeptides in a diabody are t pically too short to allow the VL and VH to bind to each other. Thus, the VL and VH encoded in the same Attorney Docket No.: JHU-43512.601 polypeptide chain cannot form a single-chain variable region fragment, but instead form a dimer with a different single-chain variable region fragment. As a result, a diabody has two antigen-binding sites.

[0126] An scFv is a single-chain polypeptide antibody obtained by linking the VH and VL with a linker (see e.g., Huston et al., Proc. Natl. Acad. Sci. U S. A., 85:5879-5883 (1988); and Pluckthun. ‘The Pharmacology of Monoclonal Antibodies” Vol. 113, Ed Resenburg and Moore. Springer Verlag. New York, pp.269-315. (1994)). Each variable domain (or a portion thereof) is derived from the same or different antibodies. Single chain Fv molecules preferably comprise an scFv linker interposed between the VH domain and the VL domain. Exemplary scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5.892,019; Ho et al, Gene. 77:51 (1989); Bird et al., Science. 242:423 (1988); Pantoliano et al, Biochemistry, 30: 101 17 (1991); Milenic et al, Cancer Research, 51 :6363 (1991); Takkinen et al. Protein Engineering, 4:837 (1991).

[0127] The term “scFv linker” as used herein refers to a moiety' interposed between the VL and VH domains of the scFv. The scFv linkers preferably maintain the scFv molecule in an antigen-binding conformation. In some embodiments, an scFv linker comprises or consists of an scFv linker peptide. In certain embodiments, an scFv linker peptide comprises or consists of a Gly-Ser peptide linker. In some embodiments, an scFv linker comprises a disulfide bond.

[0128] The order of VHs and VLs to be linked is not particularly limited, and they may be arranged in any order. Examples of arrangements include: [VH] linker [VL]; or [VL] linker [VH], The H chain V region and L chain V region in an scFv may be derived from any humanized antibody or antibody fragment described herein.

[0129] An sc(Fv)2 is aminibody in which two VHs and two VLs are linked by alinkerto form a single chain (Hudson, et al., J Immunol. Methods, (1999) 231 : 177-189 (1999)). An sc(Fv)2 can be prepared, for example, by connecting scFvs with a linker. The sc(Fv)2 of the present invention include antibodies preferably in which two VHs and two VLs are arranged in the order of: VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]), beginning from the N terminus of a single-chain polypeptide; however, the order of the two VHs and two VLs is not limited to the above arrangement, and they may be arranged in any order. Examples of arrangements are listed below: [VL] linker [VH] linker [VH] linker [VL] [VH] linker [VL] linker [VL] linker [VH] [VH] linker [VH] linker [VL] linker [VL] Attorney Docket No.: JHU-43512.601

[0130] [VL] linker [VL] linker [VH] linker [VH]

[0131] [VL] linker [VH] linker [VL] linker [VH]

[0132] Normally, three linkers are required when four antibody variable regions are linked; the linkers used may be identical or different. There is no particular limitation on the linkers that link the VH and VL regions of the minibodies. In some embodiments, the linker is a peptide linker. Any arbitrary single-chain peptide comprising about 3 to 25 residues (e.g.. 5. 6, 7, 8. 9, 10, 11, 12, 13, 14, 15. 16. 17. 18) can be used as a linker.

[0133] In some embodiments, the linker is a synthetic compound linker (chemical crosslinking agent). Examples of cross-linking agents that are available on the market include N- hydroxysuccinimide (NHS). disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidy Ipropionate) (DSP), dithiobis(sulfosuccinimidy Ipropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis [2-(sulfosuccinimidooxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).

[0134] The amino acid sequence of the VH or VL in the antibody fragments or minibodies may include modifications such as substitutions, deletions, additions, and / or insertions. For example, the modification may be in one or more of the CDRs of a humanized antibody or antibody fragments described herein. In certain embodiments, the modification involves one, two. or three amino acid substitutions in one, two. or three CDRs of the VH and / or one, two, or three CDRs of the VL domain of a humanized antibody or antibody fragments. Such substitutions are made to improve the binding and / or functional activity of the minibody. In some embodiments, one, two, or three amino acids of one or more of the six CDRs of a humanized antibody or antibody fragment may be deleted or added as long as there is human ZNT8 binding and / or functional activity when VH and VL are associated.

[0135] VHH

[0136] VHH also known as nanobodies are derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, which lack light chains. The present disclosure encompasses VHHs that specifically bind human ZNT8.

[0137] Variable Domain of New Antigen Receptors (VNARs) Attorney Docket No.: JHU-43512.601

[0138] A VNAR is a variable domain of anew antigen receptor (IgNAR). IgNARs exist in the sera of sharks as a covalently linked heavy chain homodimer. It exists as a soluble and receptor bound form consisting of available domain (VNAR) with differing numbers of constant domains. The VNAR is composed of a CDR1 and CDR3 and in lieu of a CDR2 has HV2 and HV4 domains (see, e.g., Barelle and Porter, Antibodies, 4:240-258 (2015)). The present disclosure encompasses VNARs that specifically bind human ZNT8.

[0139] Antibodies of this disclosure can be whole antibodies or single chain Fc (scFc) and can comprise any constant region of a human antibody sequence known in the art. In one aspect, the human antibody sequence is an individual human sequence, a human consensus sequence, an individual human germline sequence, or a human consensus germline sequence.

[0140] The light chain constant region can be, for example, a kappa- or lambda-ty pe light chain constant region, e.g., a human kappa or human lambda light chain constant region. In some embodiments, the light chain constant region is a human kappa light chain constant region (e.g., SEQ ID NO:33). The heavy chain constant region can be, e.g., an alpha-, delta- , epsilon-, gamma-, or mu-type heavy chain constant region, e.g., a human alpha-, human delta-, human epsilon-, human gamma-, or human mu-type heavy chain constant region. In certain instances, a humanized antibody is an IgA antibody, an IgD antibody, an IgE antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or an IgM antibody. In certain instances, a humanized antibody fragment is a fragment of an IgA antibody, an IgD antibody, an IgE antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or an IgM antibody.

[0141] In some embodiments, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region. In some embodiments, the variable heavy chain of the humanized antibody or antibody fragment described herein is linked to aheavy chain constant region comprising a CHI domain and a hinge region. In some embodiments, the variable heavy chain is linked to aheavy chain constant region comprising a CH2 domain. In some embodiments, the variable heavy chain is linked to aheavy chain constant region comprising a CH3 domain. In some embodiments, the variable heavy chain is linked to a heavy chain constant region comprising a CH2 and CH3 domain. In some embodiments, the variable heavy chain is linked to aheavy chain constant region comprising a hinge region, a CH2 and a CH3 domain. The CHI, hinge region, CH2, and / or CH3 can be from an IgG antibody (e.g., Attorney Docket No.: JHU-43512.601

[0142] IgGl, IgG4). In certain embodiments, the variable heavy chain of a humanized antibody or antibody fragmentdescribed herein is linked to aheavy chain constant region comprising a CHI domain, hinge region, and CH2 domain from IgG4 and a CH3 domain from IgGl.

[0143] In certain embodiments, a humanized antibody or antibody fragment of this disclosure is an IgG isolype antibody. In some embodiments, the antibody is IgGl. In some embodiments, the antibody comprises a human IgGl constant region with the amino acid sequence of SEQ ID NO: 34.

[0144] In another embodiment, the antibody is IgG2. In yet another embodiment, the antibody is IgG4. In some instances, the IgG4 antibody has one or more mutations that reduce or prevent it from adopting a functionally monovalent format. For example, the hinge region of IgG4 can be mutated to make it identical in amino acid sequence to the hinge region of human IgGl. In some embodiments, the hinge region of IgG4 comprises mutation of a serine in human IgG4 hinge to a proline at position 228 (S228P; Eu numbering). In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having the CHI, hinge, and CH2 regions of IgG4 and CH3 region of IgGl).

[0145] In some embodiments, the Fc domain of the IgG antibody contains mutations that improve the antibody’s biological properties.

[0146] In some embodiments, the Fc domain of a humanized anti-ZnT8 IgGl antibody herein may contain mutations that reduce or eliminate its effector functions (e.g., antibodydependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions). Such mutations include, without limitation, L234A / L235A (LALA), L234A / L235A / P329G (LALA-PG), GRLR (G236R / L328R), N297A / G / Q, L235E, K322A, and E233P / L234V / L235A / AG236 ( ‘PVLA deletion”). In some embodiments, the Fc domain comprises a human IgGl constant region with LALA-PG mutations (e.g., SEQ ID NO: 35).

[0147] In some embodiments, the Fc domain of a humanized anti-ZnT8 IgG4 antibody herein may contain mutations that further reduce an IgG4’s already low effector functions. Such mutations include, without limitation, L235E, P331S, and L235E / P331S.

[0148] In some embodiments, the Fc domain of a humanized anti-ZnT8 IgG (e.g., IgGl or IgG4) antibody herein contain mutations that improve the antibody’s serum half-life. Such mutations include, without limitation, M252Y / S254T / T256E (YTE), M428L / N434S (LS), T250Q / M428L (QL), Q311I / M428L / N434S (QLS). and N434H.

[0149] Unless otherwise indicated, all Fc residue numbers here are in accordance with the Eu numbering system. Attorney Docket No.: JHU-43512.601

[0150] In some embodiments, the humanized antibody herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of SEQ ID NO: 30. In further embodiments, the heavy chain comprises is of human IgGl isotype subtype (SEQ ID NO: 34 or 35; with or without the C-terminal lysine) and the light chain comprises a human kappa constant region (e.g., SEQ ID NO: 33). In particular embodiments, the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 36 and the light chain comprises the amino acid sequence of SEQ ID NO: 38.

[0151] In some embodiments, the humanized antibody herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:27 and a light chain comprising the amino acid sequence of SEQ ID NO:29. In further embodiments, the heavy chain comprises is of human IgGl isotype subtype (e.g., SEQ ID NO: 34 or 35; with or without the C-terminal lysine;) and the light chain comprises a human kappa constant region (e.g., SEQ ID NO: 33). In particular embodiments, the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO: 36 and the light chain comprises the amino acid sequence of SEQ ID NO: 37.

[0152] Bispecific Antibodies

[0153] In certain embodiments, a humanized antibody or antibody fragment of this disclosure is a bispecific antibody. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the human ZNT8 protein. Other such antibodies may combine a humanZNT8 binding site with a binding site for another protein. Bispecific antibodies can be prepared as full-length antibodies or low molecular weight forms thereof (e.g.. F(ab’) 2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).

[0154] Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). In a different approach, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the proportions of the three polypeptide fragments. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single Attorney Docket No.: JHU-43512.601 expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields.

[0155] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted endproducts such as homodimers.

[0156] Bispecific antibodies include cross-linked or ‘heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods.

[0157] The “diabody” technology provides an alternative mechanism for making bispecific antibody fragments. The fragments comprise aVH connected to aVL by a linker which is too short to allow pairing bet een the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

[0158] Conjugated Antibodies

[0159] A humanized antibody or antibody fragment disclosed herein may be conjugated to various molecules including macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers), 90 131 human serum albumin or a fragment, radioactive materials (e g., Y, I), fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, and drugs.

[0160] In certain embodiments, a humanized antibody or antibody fragment is modified with a moiety that improves its stabilization and / or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, 15, 20, 25, 30, 40, or 50 fold. For example, the humanized antibody or antibody fragment can be associated with Attorney Docket No.: JHU-43512.601

[0161] (e.g., conjugated to) a polymer, e.g.. a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, a humanized antibody or antibody fragment can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.. polyvinylalcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, poly oxypropylene, and block copolymers of polyoxyethylene and poly oxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides.

[0162] The above-described conjugated antibodies or fragments can be prepared hyperforming chemical modifications on the antibodies or the lower molecular weight forms thereof described herein. Methods for modifying antibodies are well known in the art.

[0163] III. Characterization of Antibodies

[0164] The human ZNT8 binding properties of the antibodies described herein may be measured by any standard method, e.g., one or more of the following methods: OCTET®, Surface Plasmon Resonance (SPR), BIACORE™ analysis, Enzyme Linked Immunosorbent Assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and Fluorescence Resonance Energy Transfer (FRET).

[0165] The binding interaction of a protein of interest (a humanized antibody or antibody fragment described herein) and a target (e.g.. human ZNT8) can be analy zed using the OCTET® systems. In this method, one of several variations of instruments (e.g., OCTET® QKe and QK), made by the ForteBio company are used to detennine protein interactions, binding specificity, and epitope mapping. The OCTET® systems provide an easy way to monitor real-time binding by measuring the changes in polarized light that travels down a custom rip and then back to a sensor.

[0166] The binding interaction of a protein of interest (a humanized antibody or antibody fragment described herein) and atarget (e.g.. human ZNT8) can be analyzed using Surface Plasmon Resonance (SPR). SPR or Biomolecular Interaction Analysis Attorney Docket No.: JHU-43512.601

[0167] (BIA) detects biospecific interactions in real time, without labeling any of the interactants.

[0168] Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity’ generate a detectable signal, which is measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden). Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (Kd), and kinetic parameters, including Kon and Koff, for the binding of a biomolecule to a target.

[0169] Epitopes can also be directly mapped by assessing the ability’ of different humanized antibodies or antibody fragments described herein to compete with each other for binding to human ZNT8 using BIACORE chromatographic techniques (Pharmacia BIAtechnology Handbook, “Epitope Mapping”, Section 6.3.2, (May 1994); see also Johne et al. (1993) J. Immunol. Methods, 160: 191-198).

[0170] When employing an enzyme immunoassay, a sample containing an antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added to an antigen-coated plate. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, and after washing, an enzyme substrate such as p-nitrophenylphosphate is added, and the absorbance is measured to evaluate the antigen binding activity.

[0171] Additional general guidance for evaluating antibodies, e.g., Western blots and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, ed. by Harlow and Lane, Cold Spring Harbor press (1988)).

[0172] IV. Affinity Maturation

[0173] In some embodiments, a humanized antibody or antibody' fragment described herein is modified, e.g., by mutagenesis, to provide a pool of modified antibodies. The modified antibodies are then evaluated to identify one or more antibodies having altered functional properties (e.g. , improved binding, improved stability, reduced antigenicity, or increased stability in vivo). In one implementation, display library technology is used to Attorney Docket No.: JHU-43512.601 select or screen the pool of modified antibodies. Higher affinity antibodies are then identified from the second library, e.g., by using higher stringency or more competitive binding and washing conditions. Other screening techniques can also be used. Methods of effecting affinity maturation include random mutagenesis (e.g., Fukuda et al., Nucleic Acids Res., 34:el27 (2006); targeted mutagenesis (e.g., Rajpal et al., Proc. Natl. Acad. Sci. USA, 102:8466-71 (2005); shuffling approaches (e.g., Jermutus et al., Proc. Natl. Acad. Sci. USA, 98:75-80 (2001); and in silica approaches (e.g.. Lippow et al., Nat. Biotechnol., 25: 1171-6 (2005).

[0174] In some embodiments, the mutagenesis is targeted to regions know n or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particularly within 10, 5, or 3 amino acids of a CDR junction. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make step-wise improvements.

[0175] In some embodiments, mutagenesis is used to make an antibody more similar to one or more germline sequences. One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In some embodiments, as many germline residues are introduced into an isolated antibody as possible.

[0176] In some embodiments, mutagenesis is used to substitute or insert one or more germline residues into a CDR region. For example, the germline CDR residue can be from agermline sequence that is similar (e.g., most similar) to the variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated. Similar mutagenesis can be performed in the framework regions.

[0177] Selecting agermline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criterion for selectivity or similarity, e.g., at least a certain percentage identity', e.g., at least 75, 80, 85, 90, 91, 92, 93, Attorney Docket No.: JHU-43512.601

[0178] 94, 95, 96, 97, 98, 99, or 99.5% identity, relative to the donor non-human antibody. The selection can be performed using at least 2. 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may include using two germline sequences that separately contribute to the amino-terminal portion and the carboxy -terminal portion. In other implementations, more than one or two germline sequences are used, e.g.. to form a consensus sequence.

[0179] In some embodiments, a humanized antibody or antibody fragment may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used in this context, ‘"altered” means having one or more carbohydrate moieties deleted, and / or having one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences; such techniques are well known in the art. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. These methods are described in, e.g., WO 87 / 05330, and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., 22:259-306. Removal of any carbohy drate moieties present on the antibodies may be accomplished chemically or enzymatically as described in the art (Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52; Edge et al. (1981) Anal. Biochem., 1 18: 131 ; and Thotakura et al. (1987) Meth. Enzymol., 138:350). See, e.g., U.S. Patent No. 5,869,046 for a modification that increases in vivo half-life by providing a salvage receptor binding epitope.

[0180] In some embodiments, a humanized antibody or antibody fragment described herein has one or more CDR sequences (e.g., a Chothia, an enhanced Chothia, or Chothia CDR definition (Al-Lazikani et al. 1997) CDR) that differ from those described herein. In some embodiments, a humanized antibody or antibody fragment has one or more CDR sequences that include amino acid changes, such as substitutions of 1.2, 3, or 4 amino acids if a CDR is 5-7 amino acids in length, or substitutions of 1, 2, 3. 4, or 5. of amino acids in the sequence of a CDR if a CDR is 8 amino acids or greater in length. The amino acid that is substituted can have similar charge, hydrophobicity, or stereochemical characteristics. In some embodiments, the amino acid substitution(s) is a conservative substitution. In some embodiments, the amino acid substitution(s) is anon-conservative substitution. The Attorney Docket No.: JHU-43512.601 antibody or antibody fragments thereof that contain the substituted CDRs can be screened to identify’ antibodies of interest.

[0181] Unlike in CDRs, more substantial changes in structure framework regions (FRs) can be made without adversely affecting the binding properties of an antibody. Changes to FRs include, but are not limited to, humanizing a nonhuman-derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter an effector function such as Fc receptor binding (Lund et al., J Immun., 147:26S7-62 (1991); Morgan et al., Immunology’, 86:319-24 (199S)), or changing the species from which the constant region is derived.

[0182] V. Antibody-Drug Conjugates

[0183] A humanized antibody or antibody fragment described herein can be conjugated to a therapeutic agent to form an antibody drug conjugate (ADC). In certain embodiments, the therapeutic agent can comprise cytotoxic agents, prodrug converting enzymes, radioactive isotopes or compounds, or toxins. For example, a humanized antibody or antibody fragment described herein can be conjugated to a cytotoxic agent such as a toxin (e.g., a cytostatic or cytocidal agent such as, e.g., abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin).

[0184] A humanized antibody or antibody fragment described herein can be conjugated to a pro-drug converting enzyme. The pro-drug converting enzyme can be recombinantly fused to the antibody or chemically conjugated thereto using knoyvn methods. Exemplary pro-drug converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin- V-amidase, penicillin- G-amidase, p-lactamase, p-glucosidase, nitroreductase and carboxypeptidase A.

[0185] Techniques for conjugating therapeutic agents to proteins, and in particular to antibodies, are well-known. See, e.g., Amon et al, “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds.. Alan R. Liss, Inc.. 1985); Hellstrom et al. “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed.1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies ‘84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy Attorney Docket No.: JHU-43512.601

[0186] (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al, 1982, Immunol. Rev.62:119- 58. See also, e.g., PCT publication WO 89 / 12624.

[0187] The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved off the antibody (e.g., by hydrolysis, by antibody degradation or by a cleaving agent). Such a therapeutic agent is attached to the antibody with a cleavable linker that is sensitive to cleavage in the intracellular environment of the ZnT8-expressing cancer cell but is not substantially sensitive to the extracellular environment, such that the conjugate is cleaved from the antibody when it is internalized by the ZnT8-expressing cell (e.g., in the endosomal or, for example by virtue of pH sensitivity' or protease sensitivity, in the lysosomal environment or in the caveolar environment).

[0188] Typically the ADC comprises a linker region between the therapeutic agent and the antibody or antibody fragment. As noted supra, typically, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. Most typical are peptidyl linkers that are cleavable by enzymes that are present in ZnT8-expressing cells. Other such linkers are described, e.g.. in U.S. Patent No. 6.214,345. In specific embodiments, the peptidyl linker cleavable by an intracellular protease comprises a Val-Cit linker or a Phe-Lys dipeptide (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[0189] The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. See, e.g.. U.S. Patent Nos. 5.122,368; 5.824,805; and 5.622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al, 1989, Biol. Chem.264: 14653-14661. Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0. the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether Attorney Docket No.: JHU-43512.601 attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Patent No. 5,622,929)).

[0190] Other linkers are cleavable under reducing conditions (e.g., a disulfide linker). Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S- acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N- succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxy carbonyl- alpha- methyl-alpha-(2-pyridyl-dithio)toluene). SPDB and SMPT. See, e.g., Thorpe et al. 1987, Cancer Res.47:5924-5931; Wawrzynczak et al, In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935.

[0191] The linker can also be a mal onate linker (Johnson et al, 1995, Anti cancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al, 1995, Bioorg-Med-Chem. 3(10): 1299- 1304), or a 3’-N-amide analog (Lau et al, 1995, Bioorg-Med-Chem. 3(10): 1305-12). The linker can also be a malonate linker (Johnson et al, 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al, 1995. Bioorg-Med-Chem .3(10): 1299-1304), or a 3’-N- amide analog (Lau et al, 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0192] The tinker also can be a non-cleavable tinker, such as a maleimido-alkylene- or maleimide-aryl linker that is directly attached to the therapeutic agent (e.g., a drug). An active drug-linker is released by degradation of the antibody.

[0193] Typically, the linker is not substantially sensitive to the extracellular environment meaning that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the tinkers in a sample of the ADC is cleaved when the ADC present in an extracellular environment (e.g., in plasma).

[0194] Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating independently with plasma both (a) the ADC (the “ADC sample’') and (b) an equal molar amount of unconjugated antibody or therapeutic agent (the “control sample”) for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample with that present in control sample, as measured, for example, by high performance liquid chromatography.

[0195] The linker can also promote cellular internalization. The linker can promote cellular internalization when conjugated to the therapeutic agent (i.e., in the milieu of the linker- therapeutic agent moiety of the ADC or ADC derivative as described herein). Alternatively, Attorney Docket No.: JHU-43512.601 the linker can promote cellular internalization when conjugated to both the therapeutic agent and a humanized antibody or antibody fragment described herein (i.e.. in the milieu of the ADC as described herein).

[0196] A humanized antibody or antibody fragment described herein can be conjugated to the linker via a heteroatom of the antibody. These heteroatoms can be present on the antibody in its natural state or can be introduced into the antibody. In some aspects, a humanized antibody or antibody fragment described herein is conjugated to the linker via a nitrogen atom of a lysine residue. In other aspects, a humanized antibody or antibody fragment described herein is conjugated to the linker via a sulfur atom of a cysteine residue. The cysteine residue can be naturally occurring or one that is engineered into the antibody. Methods of conjugating linkers and drug-linkers to antibodies via lysine and cysteine residues are known in the art.

[0197] VI. Imaging

[0198] In some aspects, the presently disclosed subject matter provides a method for detecting pancreatic beta cells in vivo, the method comprising (i) administering a humanized antibody or antibody fragment described herein to a subject; and (ii) detecting the imaging agent conjugated to the humanized antibody or antibody fragment. In some aspects, the method comprises (i) administering a humanized antibody or antibody fragment described herein, wherein the humanized antibody or antibody fragment comprises a single chain variable fragment (scFv) and is conjugated to an imaging agent; and (ii) detecting the imaging agent conjugated to the humanized antibody or antibody fragment. In some aspects, the detecting step comprises positron emission tomography (PET), single-photon emission computed tomography (SPECT) / CT imaging, nuclear magnetic resonance (NMR) spectroscopy or near-infrared (NIR) optical imaging. In some aspects, the imaging agent is a radiometal. In some aspects, the imaging agent is a radiometal and the detecting step comprises PET. In some aspects, the radiometal is selected from the group consisting of64Cu,67Cu,68Ga,60Ga,89Zr,86Y. and94mTc. In some aspects, the imaging agent is a radiometal and the detecting step comprises SPECT. In some aspects, the radiometal is selected from the group consisting ofmIn,67Ga, "mTc, and177Lu.

[0199] In another aspect, the antibody is conjugated to a labeling agent. By “labeling agent” (or “detectable label”) is meant the agent detectably labels the antibody, such that the antibody may be detected in an application of interest (e.g., in vitro and / or in vivo research and / or clinical applications). Detectable labels of interest include radioisotopes, enzymes that Attorney Docket No.: JHU-43512.601 generate a detectable product (e.g., horseradish peroxidase, alkaline phosphatase, etc.), fluorescent proteins, paramagnetic atoms, and the like. In certain aspects, the antibody is conjugated to a specific binding partner of detectable label (e.g., conjugated to biotin such that detection may occur via a detectable label that includes avidin / streptavidin).

[0200] In certain embodiments, the agent is a labeling agent that finds use in in vivo imaging such as, but not limited to, near-infrared (NIR) optical imaging, single-photon emission computed tomography (SPECT) / CT imaging, positron emission tomography (PET), nuclear magnetic resonance (NMR) spectroscopy, and the like. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, and the like. In particular embodiments, the labeling agent is a multi-modal in vivo imaging agent that permits in vivo imaging using two or more imaging approaches. See Thorp-Greenwood and Coogan (201 1) Dalton Trans. 40:6129-6143. In other embodiment, the labeling agent is an in vivo imaging agent that finds use in near-infrared (NIR) imaging applications, which agent is selected from a Kodak X-SIGHT dye, Pz 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDye 680 and 800CW Fluors. In some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, which agent can include, but is not limited to,99mTc, In-111, 123-In,2O1T1, and133Xe. In specific embodiments, the labeling agent is an in vivo imaging agent that finds use in positron emission tomography (PET) imaging applications, which agent can include, but is not limited to,UC,13N.15O,18F,64Cu,62Cu,124I.76Br.82Rb and68Ga.

[0201] VII. Methods of Producing Anti-ZNT8 Antibodies

[0202] The anti-ZNT8 antibodies (or antigen binding domain(s) of an antibody or functional fragment) of this disclosure may be produced in bacterial or eukaryotic cells. To produce the polypeptide of interest, a polynucleotide encoding the polypeptide is constructed, introduced into an expression vector, and then expressed in suitable host cells. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody.

[0203] If the antibody is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have characteristics that permit amplification of the vector in the bacterial cells. Additionally, when E. coli such as JM109, DH5a, HB1O1, or XL I-Blue is used as a host, the vector must have a promoter, for example, a lacZ promoter (Ward et al., 341:544-546 (1989), araB promoter (Better et al.. Science. 240: 1041-1043 (1988)), or Attorney Docket No.: JHU-43512.601

[0204] T7 promoter that can allow efficient expression in E. coli. Examples of such vectors include, for example, M13-series vectors. pUC-series vectors. pBR322, pBluescript, pCR-Script, pGEX-5X-l (Pharmacia), “QIAexpress system” (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al.. J. Bacteriol.. 169:4379 (1987)) may be used as the signal sequence for antibody secretion. For bacterial expression, calcium chloride methods or electroporation methods may be used to introduce the expression vector into the bacterial cell.

[0205] If the antibody is to be expressed in animal cells such as CHO, COS, 293, 293T, andNIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, for example, an SV40 promoter (Mulligan et al., Nature, 277 : 108 ( 1979)), MMLV -LTR promoter, EF la promoter (Mizushima et al. , Nucleic Acids Res., 18:5322 (1990)), or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or domain thereof, the recombinant expression vectors may carry / additional sequences, such as sequences that regulate replication of the vector in host cells (e.g. , origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4.399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418. hygromycin, or methotrexate, on ahost cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK- CMV, pOPRSV, and pOP13.

[0206] In some embodiments, the antibodies are produced in mammalian cells. Exemplary mammalian host cells for expressing a polypeptide include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with aDHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601 621). human embryonic kidney 293 cells (e.g.. 293, 293E. 293T), COS cells, NIH3T3 cells, lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, and a cell from a transgenic animal, e.g., atransgenic mammal. For example, the cell is a mammary epithelial cell.

[0207] The antibodies of the present disclosure can be isolated from inside or outside (such as medium) of the host cell and purified as substantially pure and homogenous antibodies. Methods for isolation and purification commonly used for polypeptides may be used for the Attorney Docket No.: JHU-43512.601 isolation and purification of antibodies described herein, and are not limited to any particular method. Antibodies may be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS- polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be carried out using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A column and protein G column. Examples of columns using protein A column include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.

[0208] The present disclosure also provides a nucleic acid molecule or a set of nucleic acid molecules encoding a humanized antibody or antibody fragment described herein. In some embodiments, the invention includes a nucleic acid molecule encoding a polypeptide chain, which comprises alight chain of a humanized antibody or antibody fragment described herein. In some embodiments, the invention includes a nucleic acid molecule encoding a polypeptide chain, which comprises a heavy chain of a humanized antibody or antibody fragment described herein.

[0209] Also provided are a vector or a set of vectors comprising such nucleic acid molecule or the set of the nucleic acid molecules or a complement thereof, as well as a host cell comprising the vector.

[0210] A variety ofmethods are available for recombinantly producing ahumanized antibody or antibody fragment described herein. It will be understood that because of the degeneracy of the code, a variety of nucleic acid sequences will encode the amino acid sequence of the polypeptide. The desired polynucleotide can be produced by denovo solid-phase DNAsynthesis or by PCR mutagenesis of an earlier prepared polynucleotide.

[0211] For recombinant production, a polynucleotide sequence encoding a polypeptide (e.g.. ahumanized antibody or antibody fragment described herein) is inserted into an appropriate expression vehicle, i.e.. a vector which contains the necessary elements Attorney Docket No.: JHU-43512.601 for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.

[0212] The nucleic acid encoding the polypeptide (e.g.. a humanized antibody or antibody fragment described herein) is inserted into the vector in proper reading frame. The expression vector is then transfected into a suitable target cell which will express the polypeptide. Transfection techniques known in the art include, but are not limited to. calcium phosphate precipitation (Wigler et al. 1978. Cell 14:725) and electroporation (Neumann et al. 1982, EMBO J. 1:841). A variety of host- expression vector systems can be utilized to express the polypeptides described herein (e.g., a humanized antibody or antibody fragment described herein ) in eukary otic cells. In some embodiments, the eukaryotic cell is an animal cell, including mammalian cells (e.g., 293 cells, PerC6, CHO, BHK, Cos, HeLa cells). When the polypeptide is expressed in a eukaryotic cell, the DNA encoding the polypeptide (e.g., a humanized antibody or antibody fragment described herein ) can also code for a signal sequence that will permit the polypeptide to be secreted. One skilled in the art will understand that while the polypeptide is translated, the signal sequence is cleaved by the cell to form the mature chimeric molecule. Various signal sequences are known in the art and familiar to the skilled practitioner. Alternatively, where a signal sequence is not included, the polypeptide (e.g., a humanized antibody or antibody fragment described herein ) can be recovered by lysing the cells.

[0213] VIII. Pharmaceutical Compositions

[0214] In some aspects, the presently disclosed subject matter provides a pharmaceutical composition comprising a therapeutically effective amount of a humanized antibody or antibody fragment described herein. In some aspects, the presently disclosed subject matter provides a nucleic acid molecule encoding a humanized antibody or antibody fragment described herein. In some aspects, the presently disclosed subject matter provides a vector comprising a nucleic acid described herein. In some aspects, the presently disclosed subject matter provides a host cell comprising a vector described herein.

[0215] The present disclosure also provides pharmaceutical compositions comprising one or more of: (i) ahumanized antibody or antibody fragment described herein ; (ii) a nucleic acid molecule or the set of nucleic acid molecules encoding a humanized antibody or antibody fragment described herein; or (iii) a vector or set of vectors disclosed herein, and a pharmaceutically acceptable carrier. Attorney Docket No.: JHU-43512.601

[0216] A humanized antibody or antibody fragment described herein can be formulated as a pharmaceutical composition for administration to a subject, e.g., to treat a disorder described herein. Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19).

[0217] Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472): Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).

[0218] The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.

[0219] In some embodiments, an antibody described herein is formulated with excipient materials, such as sodium citrate, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate, Tween®-80, and a stabilizer. It can be provided, for example, in a buffered solution at a suitable concentration and can be stored at 2-8°C. In some embodiments, the pH of the composition is between about 5.5 and 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1. 6.2, 6.3, 6.4. 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5).

[0220] The pharmaceutical compositions can also include agents that reduce aggregation of the antibody when formulated. Examples of aggregation reducing agents include one or more amino acids selected from the group consisting of methionine, arginine, lysine, aspartic acid, glycine, and glutamic acid. These amino acids may be added to the formulation to a concentration of about 0.5 mM to about 145 mM (e.g., 0.5 mM, 1 mM. 2 mM, 5 mM, lO mM, 25 mM, 50 mM, 100 mM). The pharmaceutical compositions can also Attorney Docket No.: JHU-43512.601 include a sugar (e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol) and / or atonicity modifier (e.g., sodium chloride, mannitol, or sorbitol) and / or a surfactant (e.g., polysorbate- 20 or polysorbate-80).

[0221] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0222] Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze dry ing that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0223] In certain embodiments, the antibodies may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery' systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such fonnulations are patented or generally known. See, e.g.. Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York (1978).

[0224] In some embodiments, the pharmaceutical formulation comprises an antibody at a concentration of about 0.005 mg / mL to 500 mg / mL (e.g.. 0.005 mg / ml. 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 250 mg / mL. 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL, 500 mg / mL), formulated with a pharmaceutically acceptable carrier. In some embodiments, Attorney Docket No.: JHU-43512.601 the antibody is formulated in sterile distilled water or phosphate buffered saline. The pH of the pharmaceutical formulation may be between 5.5 and 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 6.3, 6.4 6.5, 6.6 6.7, 6.8, 6.9 7.0, 7.1, 7.3, 7.4, 7.5).

[0225] A pharmaceutical composition may include a “therapeutically effective amount” of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual, e.g., amelioration of at least one disorder parameter or amelioration of at least one symptom of the disorder. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0226] The antibodies or antibody fragment, or nucleic acids encoding same of the disclosure can be administered to a subject, e g., a subject in need thereof, for example, a human or animal subject, by a variety of methods. For many applications, the route of administration is one of: intravenous injection or parenteral, infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection, intratumor (IT). Other modes of parenteral administration can also be used. Examples of such modes include: intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and epidural and intrastemal injection.

[0227] In some embodiments, the route of administration of the antibodies of the invention is parenteral. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous form of parenteral administration is preferred. While all these forms of administration are clearly contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection can comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer agent (e g., human albumin), etc. However, in other methods compatible with the teachings herein, the polypeptides can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent. Attorney Docket No.: JHU-43512.601

[0228] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media.

[0229] Pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0230] More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g.. glycerol, propylene glycol, and liquid polyethylene glycol, andthelike), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0231] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the inj ectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0232] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., a polypeptide by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Attorney Docket No.: JHU-43512.601

[0233] Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations can be packaged and sold in the form of a kit. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to clotting disorders.

[0234] Effective doses of the compositions of the present disclosure, for the treatment of conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety' and efficacy.

[0235] The route and / or mode of administration of a humanized antibody or antibody fragment described herein can also be tailored for the individual case, e.g., by monitoring the subject.

[0236] The humanized antibody or antibody fragment described herein can be administered as a fixed dose, or in a mg / kg dose. The dose can also be chosen to reduce or avoid production of antibodies against a humanized antibody or antibody fragment described herein. Dosage regimens are adjusted to provide the desired response, e.g., a therapeutic response or a combinatorial therapeutic effect. Generally, doses of a humanized antibody or antibody fragment described herein (and optionally a second agent) can be used in order to provide a subject with the agent in bioavailable quantities. For example, doses in the range of 0.1-100 mg / kg, 0.5-100 mg / kg. 1 mg / kg- 100 mg / kg. 0.5- 20 mg / kg, 0.1-10 mg / kg, or 1-10 mg / kg can be administered. Other doses can also be used. In certain embodiments, a subject in need of treatment with a humanized antibody or antibody fragment described herein is administered the humanized antibody or antibody fragment described herein at a dose of between about 1 mg / kg to about 30 mg / kg. In some embodiments, a subject in need of treatment with a humanized antibody Attorney Docket No.: JHU-43512.601 or antibody fragment described herein is administered the humanized antibody or antibody fragment at a dose of 1 mg / kg. 2 mg / kg. 4 mg / kg. 5 mg / kg. 7 mg / kg 10 mg / kg, 12 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 28 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, or 50 mg / kg. In a specific embodiment, a humanized antibody or antibody fragment described herein is administered subcutaneously at a dose of 1 mg / kg to 3 mg / kg. In another embodiment, a humanized antibody or antibody fragment described herein is administered intravenously at a dose of between 4 mg / kg and 30 mg / kg.

[0237] A composition may comprise about 1 mg / mL to lOOmg / ml or about 10 mg / mL to 100 mg / ml or about 50 to 250 mg / mL or about 100 to 150 mg / ml or about 100 to 250 mg / ml of a humanized antibody or antibody fragment described herein.

[0238] Dosage unit form or “fixed dose” as used herein refers to physically discrete units suited as unitary' dosages for the subjects to be treated; each unit contains a predetermined quantity of a humanized antibody or antibody fragment described herein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent. Single or multiple dosages may be given. Alternatively, or in addition, a humanized antibody or antibody fragment described herein may be administered via continuous infusion.

[0239] A dose of a humanized antibody or antibody fragment described herein can be administered, e.g., at a periodic interval over a period of time (a course of treatment) sufficient to encompass at least 2 doses, 3 doses, 5 doses, 10 doses, or more, e.g., once or twice daily, or about one to four times per week, or preferably weekly, biweekly (every two weeks), every three weeks, monthly, e.g., for between about 1 to 12 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. Factors that may influence the dosage and timing required to effectively treat a subject, include, e.g., the stage or severity of the disease or disorder, formulation, route of delivery, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments.

[0240] If a subject is at risk for developing a disorder described herein, a humanized antibody or antibody fragment described herein can be administered before the full onset of the disorder, e.g., as a preventative measure. The duration of such preventative treatment can be a single dosage of a humanized antibody or antibody fragment Attorney Docket No.: JHU-43512.601 described herein or the treatment may continue (e.g., multiple dosages). For example, a subject at risk for the disorder or who has a predisposition for the disorder may be treated with a humanized antibody or antibody fragment described herein for days, weeks, months, or even years so as to prevent the disorder from occurring or fulminating.

[0241] In certain embodiments, a humanized antibody or antibody fragment described herein is administered subcutaneously at a concentration of about 1 mg / mL to about 500 mg / mL (e g., 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL , 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL. 90 mg / mL, 95 mg / mL. 100 mg / mL. 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, 250 mg / mL, 275 mg / mL, 300 mg / mL. 325 mg / mL. 350 mg / mL, 400 mg / mL, 450 mg / mL). In some embodiments, ahumanized antibody or antibody fragment described herein is administered subcutaneously at a concentration of 50 mg / mL. In another embodiment, a humanized antibody or antibody fragment described herein is administered intravenously at a concentration of about 1 mg / mL to about 500 mg / mL. In some embodiments, a humanized antibody or antibody fragment described herein is administered intravenously at a concentration of 50 mg / mL.

[0242] Doses intermediate in the above ranges are also intended to be within the scope of the invention. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. In some methods, two or more polypeptides can be administered simultaneously, in which case the dosage of each polypeptide administered falls within the ranges indicated.

[0243] Polypeptides of the invention can be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring blood levels of modified polypeptide or antigen in the patient. Alternatively, polypeptides can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0244] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions Attorney Docket No.: JHU-43512.601 containing the polypeptides of the invention or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient’s resistance or minimize effects of disease. Such an amount is defined to be a “prophylactic effective dose.” A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.

[0245] IX. Methods of treatment.

[0246] In some aspects, the presently disclosed subject matter provides a method for treating a disease or condition associated with ZnT8 in a subject, the method comprising administering to the subject a humanized antibody or antibody fragment described herein or a pharmaceutical composition described herein. In some aspects, the disease or condition comprises Type 1 Diabetes (T1D) or Type 2 Diabetes (T2D).

[0247] In one aspect, provided herein is a method of treating T1D or T2D comprising administering to a patient a humanized antibody or antibody fragment described herein.

[0248] T1D is an autoimmune disease where pathogenic lymphocytes target autoantigens expressed in the pancreatic islets, leading to the destruction of insulin-producing beta-cells. Zinc transporter 8 (ZnT8) is a major autoantigen abundantly present on the beta-cell surface (47).

[0249] T2D is an immunometabolic disorder characterized by obesity and chronic low-grade inflammation (41). As a complex multifactorial polygenic disease, it involves numerous genetic variants (48) and targeting individual T2D associated genes are often insufficient to modify the disease course and outcome. Rare loss-of-function (LOF) mutations in T2D- associated genes can significantly alter the disease risk (49) and while most LOF mutations have deleterious effects, some are protective (50-52). These LOF mutations may modulate key tipping points of T2D pathophysiology, shifting the balance towards healthy states. The absence of adverse effects in carriers of protective LOF variants demonstrates the safety of life-long target inhibition in humans (53). One such gene is SLC30A8. which is associated with T2D (54). and contains rare heterozygous LOF mutations that strongly protect against the disease (odds ratio of 0.34) (50). SLC30A8 encodes zinc transporter-8 (ZnT8), a tissuespecific protein found exclusively in human pancreatic islets (55, 7). ZnT8 mutations are directly linked to islet-residing endocrine cells and functional perturbations of ZnT8 at different hierarchical levels — molecular, cellular, mouse modes of diabetes and human populations — have consistently shown that reducing ZnT8 transport activity or cellular Attorney Docket No.: JHU-43512.601 abundance improves beta cell function and reduces T2D risk (39, 56, 16). Therefore, the protective effects of ZnT8 LOF mutations may be potentially translated into safe and diseasetipping therapeutics (57).

[0250] ZnT8 expression in beta cells is dynamically distributed among the insulin secretory granule (58), cell surface membrane (11) and endoplasmic reticulum (ER) (4). ER-residing ZnT8 is prone to misfolding under low-grade inflammation and can significantly contribute to the burden of ER protein folding (4). Stress-induced ZnT8 misfolding may intensify a basal unfolded protein response (UPR) to the physiological insulin folding burden, and exceeding the capacity of this adoptive response can induce cellular dedifferentiation (59) and beta cell failure (60). Reducing the ZnT8 folding burden by siRNA knockdown in a human beta cell line was shown to protect against cytokine cytotoxicity, whereas blocking the clearance of misfolded ZnT8 increased the susceptibility to proinfl ammatory insults (4). Moreover, reducing ZnT8 expression through engineered nonsense mutations in human stem cell-derived beta cells improved graft stability and insulin secretion when transplanted into mice with strep tozotocin-induced diabetes (61). These findings suggest reducing ZnT8 misfolding may promote beta-cell adaptation to islet inflammation, which is primarily driven by islet-resident macrophages in T2D (62). Obesity -induced expansion of islet-resident macrophages initially induces beta-cell proliferation and temporary' hyperinsulinemia but eventually leads to cellular decompensation as islet inflammation persists (63).

[0251] It is hypothesized that pharmacological inhibition of ZnT8 misfolding could shift the balance from beta-cell decompensation to functional adaptation to islet inflammation, thereby halting the progression to beta-cell failure in T2D.

[0252] Type 1 Diabetes (T1D) is currently classified into three stages as defined by the American Diabetes Association. Stage 1 is characterized by the presence of two or more islet autoantibodies, such as anti-insulin autoantibodies (IAA), anti-GAD65 (glutamic acid decarboxylase) autoantibodies, anti-IA-2 (insulinoma-associated protein 2) autoantibodies, anti-ZnT8 autoantibodies, and ICA (islet cell antibodies), while blood glucose levels remain within the normal range and the subject is asymptomatic. Stage 2 likewise requires two or more islet autoantibodies but is further defined by the presence of dysglycemia, including impaired fasting glucose, impaired glucose tolerance, or elevated HbAlc below the diagnostic threshold for diabetes; individuals remain presymptomatic at this stage. Stage 3, also referred to historically as ‘‘clinical’' T1D, is defined by symptomatic hyperglycemia in conjunction with meeting diagnostic thresholds for diabetes, including fasting plasma glucose >126 mg / dL, a 2-hour plasma glucose >200 mg / dL during an OGTT, an HbAic >6.5%, or a Attorney Docket No.: JHU-43512.601 random plasma glucose >200 mg / dL in a symptomatic individual. Physical symptoms include polyuria, polydipsia, polyphagia, rapid weight loss, and lethargy.

[0253] In some embodiments, the subject to be treated by the method herein is classified as having Stage 1 T1D. In some embodiments, the subject is classified as having Stage 2 T1D. In some embodiments, the subject is classified as having Stage 3 T1D.

[0254] In some embodiments, the administration of the antibody or antibody fragment herein delays or prevents progression of T1D in the subject, e.g.. from Stage 1 or Stage 2 T1D to Stage 3 T1D. In some embodiments, the administration of the antibody or antibody fragment herein alleviates or reverses T1D symptoms such as polyuria, polydipsia, polyphagia, rapid weight loss, and / or lethargy.

[0255] X. Devices and Kits for Therapy

[0256] A humanized antibody or antibody fragment described herein can be provided in a kit. In some embodiments, the kit includes (a) a container that contains a composition that includes a humanized antibody or antibody fragment described herein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or the use of the agents for therapeutic benefit.

[0257] In certain embodiments, the kit also includes a second agent for treating a disorder described herein, i.e., a disease or condition mediated by or associated with ZnT8 (e.g.. Type 1 or Type 2 diabetes). For example, the kit includes a first container that contains a composition that includes a humanized antibody or antibody fragment described herein, and a second container that includes the second agent.

[0258] In some embodiments, the kit also includes a second agent such as an imaging agent. For example, the kit includes a first container that contains a composition that includes a humanized antibody or antibody fragment described herein, and a second container that includes the second agent.

[0259] The informational material of the kits is not limited in its form. In some embodiments, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In some embodiments, the informational material relates to methods of administering a humanized antibody or antibody fragment described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g.. a dose, dosage form, or mode of administration described herein), to treat a subject who has Attorney Docket No.: JHU-43512.601 had or who is at risk for a disease as described herein. The information can be provided in a variety of formats, including printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material, e.g., on the internet.

[0260] In addition to a humanized antibody or antibody fragment described herein, the composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. A humanized antibody or antibody fragment described herein can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and / or sterile. When the agents are provided in a liquid solution, the liquid solution preferably is an aqueous solution. In certain embodiments, a humanized antibody or antibody fragment described herein in the liquid solution is at a concentration of about 25 mg / mL to about 250 mg / mL (e.g., 40 mg / mL, 50 mg / mL, 60 mg / mL, 75 mg / mL, 85 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, and 200 mg / mL). When a humanized antibody or antibody fragment described herein is provided as a lyophilized product, the humanized antibody or antibody fragment is at about 75 mg / vial to about 200 mg / vial (e.g., 100 mg / vial, 108.5 mg / vial, 125 mg / vial, 150 mg / vial). The lyophilized powder is generally reconstituted by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer (e.g., PBS), can optionally be provided in the kit.

[0261] The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In some embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., a unit that includes both a humanized antibody or antibody fragment described herein and the second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be airtight, waterproof (e.g.. impermeable to changes in moisture or evaporation), and / or light-tight. Attorney Docket No.: JHU-43512.601

[0262] The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.

[0263] Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

[0264] EXAMPLES

[0265] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods described and claimed herein are made and evaluated, and are intended to be purely illustrative and are not intended to limit the scope of w hat the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for herein. Unless indicated otherwise, parts are parts by w eight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0266] EXAMPLE 1: Humanization and Deimmunization of mAb43

[0267] Humanization and deimniumzation using Epibasez® and in siiico tools was outsourced to Lonza Biologies pic. Humanized and deimmunized Ts1e43 variants of mAb43 were engineering by identifying optimal human germlines, humanization, deimmunization and de-risking steps. Preferred acceptor frameworks for the grafting of the complementaritydetermining regions (CDRs) w ere selected from sets of human antibody germline sequences and a structural model for the Fv-region of the antibody was constructed using Lonza Biologies molecular modelling platform. The CDR-grafting was accomplished by substituting any mismatched residues between the parental and acceptor frameworks. An alignment of the parental mAb43 VL domain sequences to the acceptor framework in shown in FIG. 6. An alignment of the parental mAb43 VH domain sequences to the acceptor Attorney Docket No.: JHU-43512.601 framework is shown in FIG. 7. The light chain constant region in the humanized antibodies was from human kappa light chain (SEQ ID NO: 33) and the heavy chain constant region in these antibodies was from human IgGl with the LALA-PG mutations (SEQ ID NO: 35).

[0268] Substitutions at potentially critical positions such as those in the Vernier zone, the VH / VL inter-chain interface or at positions determining the CDR canonical class were analyzed for prospective back mutations. Epibase® v.4.5 immunoprofiling of mAb43 against 85 HLA class II allotypes in the Global set was performed on the sequences. Predicted epitopes were evaluated for deimmunizing substitutions that would be considered effective in reducing the potential immunogenicity'. Lonza’s in silico Manufacturability' Assessment in conjunction with sequence and structural inspection was applied to design de-risk antibodyvariants. Engineered variants were then validated by structural modelling and Epibase® v.4.5 immunoprofile comparison.

[0269] Potential humanized variant sequences were screened using Epibase®. Each epitope or cluster of epitopes was analyzed for substitutions that would either remove the epitope or further reduce the predicted immunogenicity-. The focus was on the 43 DRB1 allotypes available in Epibase®, as DRB1 allotypes are the most relevant for immunogenicity assessments. The assessment of each position was updated as work progressed to reflect the impact of each position on all relevant epitopes. Although the focus during deimmunization was on DRB 1 , the epitopes affecting Human Leukocy te Antigen gene DR, chain B, haplotype 3, 4 or 5 (DRB3 / 4 / 5), Human Leukocyte Antigen gene DP (DP) and Human Leukocyte Antigen gene DQ (DQ) were also analyzed and taken into account before the set of proposed substitutions was finalized. The substitutions and their effects are described in Tables 1 and 2 below. Two deimmunizing substitutions have been proposed for the light chain and three for the heavy- chain. Additional protein engineering considerations such as the presence of potential post-translation modifications (PTMs) and alterations in the sequence based isoelectric point calculation were also taken into account as such parameters can influence binding affinity-, stability- and manufacturability . One derisking substitution has been proposed for the heavy chain. Two (2) engineered light chains and seven (7) engineered heavy chains were proposed. Table 2 lists the name of the engineered chains along with a description of the modifications. Experimental variant combinations are listed in Table 3 along with corresponding descriptions.

[0270] Table 1: Deimmunizing and PTM removal substitutions Attorney Docket No.: JHU-43512.601

[0271] Deimmunizing substitutions have been designed based on the humanized sequences and may have a different effect if performed in some other sequence context.

[0272] Table 2: Humanized and Engineered Chains Attorney Docket No.: JHU-43512.601

[0273] Table of engineered chains comprising humanized and deimmunized chains.

[0274] Attorney Docket No.: JHU-43512.601

[0275] Table 3: Variant Combinations

[0276] Attorney Docket No.: JHU-43512.601

[0277] Each variant is composed of two chains (light and heavy). The same chain can appear in multiple variants.

[0278] Attorney Docket No.: JHU-43512.601

[0279] Immunogenicity risk scores were predicted for each variant combination. The immunogenicity risk score is an approximate score expressing a worst-case immunogenic risk based on the critical Th epitopes of DRB1 (Human Leukocyte Antigen gene DR, chain B, haplotype 1) and is given for the parental sequence and the humanized / deimmunized variants. The present study has focused on the 43 DRB1 allotypes available in Epibase®, as DRB1 allotypes are the most relevant for immunogenicity assessments. The workflow to determine the immunogenicity risk score is as follows: (i) identification of potential T-cell epitopes in Isle43 variants, (ii) ranking of T-epitope risk based on binding strength, HLA- binding promiscuity, and HLA frequency in the general human population, and (iii) application of a calculation filter to generate overall immunogenicity scores. The immunogenicity risk score can be calculated as score = L( Epitope Count x Allotype Frequency).

[0280] The results are summarized in Table 4 (last column) and the distribution of marketed antibody drug products is shown in FIG. 5. Lonza’s in silico Manufacturability' Assessment platform was used to validate the engineered sequences.

[0281] The corresponding antibody names among variant Isle43 variant combinations, corresponding humanized and deimmunized Isle43 variants chains, Wuxi expression products (Example 2 below), and immunogenicity' (DRB1) risk score are summarized in Table 4. Compare the immunogenicity risk score for mAb43: 1728.0.

[0282] Table 4: Wuxi expression products and immunogenicity (DRB1) risk score for variant

[0283] Isle43 variant combinations Attorney Docket No.: JHU-43512.601

[0284] EXAMPLE 2: Transient Production of Isle43 Variants in CHO-K1 Cells

[0285] Manufacturability^ was assessed based on Isle43 variant expression levels in Wuxi's CHO cells. The coding sequences of individual Isle43 variants were analyzed, codon- optimized, and inserted into Wuxi’s expression vectors. The expression plasmids were synthesized, validated, and transiently expressed in CH0-K1 cells. The resultant Isle43 variants were purified through one-step protein A / G affinity7binding followed by SEC-HPLC. The purified Isle43 variants were then validated by SDS-PAGE and SEC-HPLC. Representative results are shown in FIG. 2 and FIG. 3.

[0286] EXAMPLE 3: Protein Yields of Individual Isle43 Variants

[0287] The protein yields from 1 mL scale production of individual Isle43 variants using Wuxi’s Ultra 96 high-throughput platform are summarized in Table 5. Table 5: Wuxi Yield and Glycosylation Table Attorney Docket No.: JHU-43512.601

[0288] EXAMPLE 4: Comparison of Isle43 Variants and mAb43 by Immunofluorescence Labeling and Imaging Analysis

[0289] EndoC-PHl cells were seeded onto a glass-bottom microwell dish pre-coated with P- coat and grown in OPTI cell culture medium at 37°C in a 5% CO2 humidified atmosphere for two days. For cell surface immunofluorescence labeling, live cells were washed with high glucose (20 mM) Krebs buffer, chilled at 8°C for 30 minutes, and then exposed to one of the 14 Isle43 variants (1 :25), mAb43 (1 :25), or mAb20 (1 :25). After a 1-hour incubation at 8°C, unbound antibodies were removed by two washes with high glucose Krebs buffer. Next, cells were exposed to a fluorescent anti-IgG secondary' antibody (1 :400) for 30 minutes, washed to remove unbound secondary antibody, and then DAPI was added to the medium for fluorescence imaging using a Zeiss LSM 700 inverted confocal microscope. For Isle43 uptake and intracellular immunofluorescence labeling, live cells were incubated with one of the 14 Isle43 variants (1 :250), mAb43 (1 :250), or mAb20 (1 :250) in high glucose (20 mM) Krebs buffer for 30 minutes at 37°C. Cells were then washed to remove extracellular antibodies, fixed with flow cytometry fixation buffer for 20 minutes at room temperature, washed with PBS, permeabilized with flow cytometry' permeabilization buffer for 20 minutes at room temperature, and blocked with PBS containing 5% BSA for 30 minutes. Secondary' antibody immunolabeling, DAPI counterstaining, and immunofluorescence imaging were then performed as described above. Cell surface immunofluorescence labeling intensities and intracellular uptake levels for individual Tsle43 variants were compared head-to-head, using mouse-human chimeric mAb20 and mAb43 as negative and positive controls, respectively. mAb20 and mAb43 are both ZnT8-specific monoclonal antibodies recognizing distinct extracellular and intracellular epitopes. The mouse Fc, human Fab chimeric constructs allow ed for direct comparison of immunofluorescence labeling with Isle43 variants using the same anti-human Fc antibody. The immunofluorescence labeling results are shown in FIG. 4.

[0290] The NA9, NA10, NA11, NA12, and NA14 Isle43 variants have w eaker (NA10 and NA12) or similar (NA9, NA11, and NAM) cell surface labeling as compared to mAb43. The NA9. NAI0. NA11, NAI 2. and NAM Isle43 variants have lower intracellular uptake levels than that of mAb43. The corresponding sequences for these Isle 43 variants are listed in Table 6 below':

[0291] Table 6: Isle43 Variant Sequences for NA9, NA10, NA11, NA12, and NA14 Attorney Docket No.: JHU-43512.601 Attorney Docket No.: JHU-43512.601

[0292] NA6, NA13, NA1, NA2, NA3, NA4, NA5, NA7, and NA8 Isle43 variants unexpectedly have stronger cell surface labeling as compared to mAb43 and have higher intracellular uptake levels than mAb43. The corresponding sequences for these Isle43 variants are listed in Tables 7 and 8 below:

[0293] Table 7: Isle43 Variant Sequences for NA6, NA13, NA1, NA2, NA3, NA4, NA5, NA7, and NA8 Attorney Docket No.: JHU-43512.601

[0294] Table 8: Sequence Identifier Number Table Attorney Docket No.: JHU-43512.601

[0295] Lead identification:

[0296] NA6 and NA13 demonstrated stronger cell surface labeling and intracellular uptake. The immunogenicity risk scores for NA6 and NA13 are 820.2 and 784.5. as indicated.

[0297] EXAMPLE 5: Identification of hAb43 Candidates

[0298] All possible combinations of humanized light and heavy chains were expressed in CHO cells to generate a panel of humanized mAb43 variants, which were individually purified. All variants showed expression levels exceeding 100 mg / L and proper folding, as confirmed by analytical size-exclusion HPLC. These variants were then tested head-to-head for ZnT8 cell surface immunolabeling using the human beta cell line EndoC-PHl. Cell surface binding was glucose-inducible and quantified by immunofluorescent intensity, benchmarked against the parental mAb43 under identical stimulation and labeling conditions. Internalization of each variant following glucose stimulation was also evaluated. Across all tested variants, surface binding intensity correlated with intracellular uptake. Based on these Attorney Docket No.: JHU-43512.601 results, together with predicted immunogenicity risk scores, the top three humanized mAb43 (hAb43) candidates were selected — NA6, NA13, and NA8. Among them, NA 13 (Isle43-12) had the lowest immunogenicity score and was selected as the lead candidate for further analysis.

[0299] EXAMPLE 6: Production and / n Vitro Characterization of Isle43-12

[0300] Isle43-12 and the parental mAb43 (Z43), used as a benchmark, were produced by transient CHO cell expression at the 2 L scale. Following HPLC purification, the antibody yields were 376 mg / L for Isle43-12 and 805 mg / L for mAb43. Both antibodies exhibited high purity (>99.6%), as confirmed by SDS-PAGE and analytical size-exclusion HPLC. ZnT8 binding affinity was determined by ELISA using membrane-bound ZnT8 reconstituted in proteoliposomes. The apparent binding affinities were 0.29 nM for Isle43- 12 and 0. 10 nM for mAb43. These results confirm that humanization of mAb43 preserves high-affinity ZnT8 binding while maintaining excellent production yield and biochemical quality'. The production and validation of Isle43-12 (NA13) is shown in FIG. 8.

[0301] Isle43- 12 was evaluated for islet immunolabeling. In mouse pancreas sections, Isle43-12, NA6, and the human IgGl chimeric version of mAb43 (used as a benchmark) all demonstrated specific immunohistochemical staining of islets, with minimal background in surrounding exocrine tissue. Isle43-12 also labeled isolated human islets, and this labeling was blocked by the mouse mAb43-IgG but not by an IgGl isotype control, indicating that Isle43- 12 and mAb43 compete for the same binding sites in human islets. These results confirm that humanization preserves the islet specificity' and epitope targeting of the original mAb43 antibody. FIG. 9 shows specific immunolabeling of mouse islets with mAb43, Isle43-12 (NA13), and NA6.

[0302] Example 7: In Vivo Characterization of Isle43-12

[0303] Isle43-12 was evaluated in C57BL / 6 (B6) mice following a single intravenous injection at varying doses. Tissues were collected post-mortem seven days after injection, and tissue-resident Isle43-12 was quantified by anti-human IgG immunoblotting. Isle43-12 was specifically detected in the pancreas, with no signal observed in other tissues, and remained at high levels in the serum, indicating both pancreas-specific uptake and favorable serum stability. Dose-response analysis showed that Isle43-12 levels in both the pancreas and serum increased in a hyperbolic manner, with half-maximal uptake (K0.5) values of 1.60 mg / kg and 1.76 mg / kg, respectively. These findings demonstrate the in vivo islet- Attorney Docket No.: JHU-43512.601 targeting specificity' of Isle43-12 and support its pharmacological potential for sustained pancreatic delivery. FIG. 10 shows Isle43-12 biodistribution and dose-dependent uptake.

[0304] In one study, pancreatic and plasma levels of Isle43- 12 were measured over time using anti-human IgG immunoblotting following a single intravenous dose of 5 mg / kg. No significant decline in Isle43-12 levels was observed in either serum or pancreas for up to six weeks post-injection. These findings indicate that Isle43-12 exhibits exceptional in vivo stability and prolonged tissue retention, supporting its suitability for long-acting therapeutic applications. FIG. 11 shows Isle43-12 in vivo stability in blood and pancreas.

[0305] To further evaluate the efficacy of Isle43-12 in vivo, an adoptive transfer model in NOD.scid mice was used, where ty pe 1 diabetes (T1D) was induced by transferring splenocytes from newly diabetic NOD donors. Within 48 hours of diabetes onset (fasting blood glucose >250 mg / dL), the recipient mice received a slow-releasing insulin implant to restore normoglycemia. Mice were then treated weekly with either Isle43-12 or an isotype control at an intravenous dose of 5 mg / kg. Four weeks after T1D onset, the insulin implant began to deplete. All mice in the isotype control group rapidly relapsed into hyperglycemia within 2-3 weeks. In contrast, all Isle43-12-treated mice maintained normoglycemia for six weeks or longer. These results demonstrate that Isle43-12 protects against diabetogenic T cell attack and promotes sustained -cell functional recovery' following insulin-mediated glycemic stabilization. FIG. 12 shows T1D reversal with Isle43-12 in NOD.scid mice with adoptive transfer of T1D.

[0306] EXAMPLE 8: Comparison of Isle43 with Teplizumab

[0307] Teplizumab is currently the only FDA-approved disease-modifying therapy for T1D. Its mechanism of action involves targeting CD3 on T cells to induce partial T cell depletion, promote T cell exhaustion, and modulate autoreactive immune responses. In contrast, Isle43 acts directly on beta cells, binding to their cell surface to mask antigenic epitopes and functioning intracellularly as a ZnT8 chaperone to reduce inflammation-induced ER stress.

[0308] To compare therapeutic efficacy, Isle43-12 and teplizumab were evaluated in parallel using the adoptive transfer model of T1D described above. Both treatments were administered under identical experimental conditions, following insulin-mediated glycemic stabilization. The head-to-head comparison showed that Isle43-12 and teplizumab provided comparable efficacy in prolonging T1D remission after insulin withdrawal. This finding, benchmarked against a well-established T1D immunotherapy, highlights the potential of Isle43- 12 as a non-immunosuppressive, beta cell-directed alternative for modifying disease Attorney Docket No.: JHU-43512.601 progression in T1D. FIG. 13 shows a head-to-head comparison of Isle43-12 and teplizumab.

[0309] EQUIVALENTS

[0310] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0311] INCORPORATION BY REFERENCE

[0312] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. Each of the following references (numerically identified herein) is incorporated by reference in its entirety:

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[0350] 38. Flannick, J., Thorleifsson, G., Beer, N. L., Jacobs, S. B , Grarup, N., Burtt, N. P., Mahajan, A., Fuchsberger, C., Atzmon, G., Benediktsson, R., Blangero, J., Bowden, D. W.. Brandslund, L. Brosnan, J., Burslem, F., Chambers. J., Cho, Y. S., Christensen, C., Douglas, D. A., Duggirala, R.. Dymek, Z., Faijoun, Y., Fennell, T.. Fontanillas, P.. Forsen, T., Gabriel, S., Glaser, B., Gudbjartsson, D. F., Hanis, C., Hansen, T., Hreidarsson, A. B., Attorney Docket No.: JHU-43512.601

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[0354] B., Payne, A. J., Moralli, D., Thomsen, S. K., Kramer, P., Spiliotis, L, Ramracheya, R., Chabosseau, P., Theodoulou, A., Cheung, R., van de Bunt, M., Flannick, J., Trombetta, M., Bonora, E.. Wolheim, C. B., Sarelin, L., Bonadonna, R. C., Rorsman, P., Davies. B., Brosnan, J., McCarthy, M. L, Otonkoski, T., Lagerstedt, J. O., Rutter, G. A., Gromada, J., Gloyn, A. L., Tuomi, T., and Groop, L. (2019) Loss of ZnT8 function protects against diabetes by enhanced insulin secretion. Nat Genet 51, 1596-1606.

[0355] 40. Kambe. T., Taylor, K. M., and Fu, D. (2021) Zinc transporters and their functional integration in mammalian cells. J Biol Chemi. 100320.

[0356] 41 . Donath, M. Y., and Shoelson, S. E. (201 1) Type 2 diabetes as an inflammatory disease. Nat Rev Immunol 11, 98-107.

[0357] 42. Peterson, L. B., Bell, C. J. M., Howlett, S. K., Pekalski, M. L., Brady, K, Hinton, H.. Sauter. D., Todd, J. A., Umana, P., Ast, O., Waldhauer. L, Freimoser- Grundschober, A., Moessner, E., Klein, C., Hosse, R. J., and Wicker, L. S. (2018) A long- lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease. J Autoimmun 95, 1-14.

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[0362] 47. Z. Guo et al., Cell-Surface Autoantibody Targets Zinc Transporter-8 (ZnT8) for In Vivo beta-Cell Imaging and Islet-Specific Therapies. Diabetes 'll, 184-195 (2023).

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[0366] 51 V. Steinthorsdottir et al., Identification of low-frequency and rare sequence variants associated with elevated or reduced risk of type 2 diabetes. Nat Genet 46, 294-298 (2014).

[0367] 52 J. Gudmundsson et al. , Two variants on chromosome 17 confer prostate cancer risk, and the one in TCF2 protects against ty pe 2 diabetes. Nat Genet 39, 977-983 (2007).

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Claims

1. Attorney Docket No.: JHU-43512.601WHAT IS CLAIMED IS:

1. A humanized antibody or antibody fragment that binds to human zinc transporter-8 (ZnT8), wherein the humanized antibody or antibody fragment comprises:(a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 16, and CDR3 of SEQ ID NO: 19;(b) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19;(c) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19;(d) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ NO:

14. CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19;(e) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19;(f) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO:

17. and CDR3 of SEQ ID NO: 19;(g) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO:

17. and CDR3 of SEQ ID NO: 19;Attorney Docket No.: JHU-43512.601(h) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19; or(i) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO:

7. CDR3 of SEQ ID NO: 9, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 15, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19.

2. The humanized antibody or antibody fragment of claim 1, wherein the humanized antibody or antibody fragment comprises:(a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, and CDR3 of SEQ ID NO: 19.

3. The humanized antibody or antibody fragment of claim 1, wherein the humanized antibody or antibody fragment comprises:(b) a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, FR2 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 10, and a light chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 16, and CDR3 of SEQ ID NO: 19.

4. The humanized antibody or antibody fragment of any one of claims 1-3, wherein the humanized antibody or antibody fragment comprises:- a heavy chain FR1 of SEQ ID NO: 1;- a heavy chain FR4 of SEQ ID NO: 1 I;- a light chain FR1 of SEQ ID NO: 12; and / or- a light chain FR4 of SEQ ID NO: 20.

5. A humanized antibody or antibody fragment that binds to human ZnT8. wherein the humanized antibody or antibody fragment comprises:Attorney Docket No.: JHU-43512.601(a) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 29;(b) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 30;(c) a heavy chain variable region comprising SEQ ID NO: 22 and a light chain variable region comprising SEQ ID NO: 29;(d) a heavy chain variable region comprising SEQ ID NO: 23 and a light chain variable region comprising SEQ ID NO: 29;(e) a heavy chain variable region comprising SEQ ID NO: 24 and a light chain variable region comprising SEQ ID NO: 29;(I) a heavy chain variable region comprising SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 29;(g) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 29;(h) a heavy chain variable region comprising SEQ ID NO: 28 and a light chain variable region comprising SEQ ID NO: 29; or(i) a heavy chain variable region comprising SEQ ID NO: 22 and a light chain variable region comprising SEQ ID NO: 30.

6. The humanized antibody or antibody fragment of claim 5, wherein the humanized antibody or antibody fragment comprises:(a) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 29.

7. The humanized antibody or antibody fragment of claim 5, wherein the humanized antibody or antibody fragment comprises:(b) a heavy chain variable region comprising SEQ ID NO: 27 and a light chain variable region comprising SEQ ID NO: 30.

8. A humanized antibody or antibody fragment, wherein the heavy chain variable region sequence comprises:SEQ ID NO: 22 or SEQ ID NO: 22 comprising 1-3 conservative amino acid substitution,Attorney Docket No.: JHU-43512.601SEQ ID NO: 27 or SEQ ID NO: 27 comprising 1-3 conservative amino acid substitution, and the light chain variable region sequence comprises:SEQ ID NO: 29 or SEQ ID NO: 29 comprising 1-3 conservative amino acid substitution, orSEQ ID NO: 30 or SEQ ID NO: 30 comprising 1-3 conservative amino acid substitution.

9. The humanized antibody or antibody fragment of any one of claims 1-8, wherein the humanized antibody or antibody fragment has an immunogenicity risk score (DRB1) of less than 1000, stronger cell surface labeling as compared to mAb43, higher intracellular uptake levels as compared to mAb43, and / or increased binding affinity' as compared to mAb43.

10. The humanized antibody or antibody fragment of any one of claims 1-9, wherein the antibody or antibody' fragment specifically binds to three extracellular loops of a transmembrane domain of human Zinc Transporter-8 (ZnT8).

11. The humanized antibody or antibody fragment of claim 10. wherein the three extracellular loops of human ZnT8 comprise amino acids 95-99, 169-175 and 242-245, respectively, of SEQ ID NO: 21.

12. The humanized antibody or antibody fragment of any one of claims 1-11, comprising a human kappa light chain constant region, optionally wherein the human kappa light chain constant region comprises the amino acid sequence of SEQ ID NO: 33.

13. The humanized antibody of any one of claims 1-12, wherein the antibody is of human IgG isotype, optionally of human IgGl subtype, further optionally wherein the antibody comprises the amino acid sequence of SEQ ID NO: 34 or 35.

14. The antibody fragment of any one of claims 1-12, wherein the fragment comprises a Fab, Fab’. F(ab’)2, Fab’-SH, Fv, diabody. linear antibody or single-chain variable fragment (scFv).Attorney Docket No.: JHU-43512.60115. The humanized antibody or antibody fragment of any one of claims 1-14, wherein the antibody or antibody fragment is conjugated to a therapeutic agent.

16. The humanized antibody or antibody fragment of any one of claims 1-14, wherein the antibody or antibody fragment is conjugated to an imaging agent.

17. A pharmaceutical composition comprising the humanized antibody or antibody fragment of any one of claims 1-16 and a pharmaceutically acceptable carrier.

18. Nucleic acid molecule(s) encoding the humanized antibody or antibody fragment of any one of claims 1-16.

19. Expression vector(s) comprising the nucleic acid molecule(s) of claim 18.

20. A host cell comprising the expression vector(s) of claim 19.

21. A method of producing an antibody or antibody fragment, comprising: culturing the host cell of claim 20 under conditions that allow expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the culture.

22. A method for treating a disease or condition associated with ZnT8 in a human subject in need thereof, the method comprising administering to the subject the humanized antibody or antibody fragment of claims 1-16 or the pharmaceutical composition of claim 17.

23. The method of claim 22, wherein the disease or condition is type 1 diabetes (T1D), optionally wherein the subject exhibits two or more islet autoantibodies selected from the group consisting of insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GAD65), insulinoma-associated antigen-2 autoantibodies (IA-2), zinc transporter-8 autoantibodies (ZnT8), and islet cell cytoplasmic autoantibodies (ICA).

24. The method of claim 23, wherein the subject is classified as having Stage 1 T1D.Attorney Docket No.: JHU-43512.60125. The method of claim 23, wherein the subject is classified as having Stage 2 T1D.

26. The method of claim 23, wherein the subject is classified as having Stage 3 T1D.

27. The method of any one of claims 23-26, wherein administering the antibody delays or prevents progression of T1D in the subject.

28. The method of any one of claims 23-27, wherein administering the antibody alleviates or reverses T1D in the subject.

29. A method for detecting pancreatic beta cells in vivo, the method comprising(i) administering the humanized antibody or antibody fragment of claim 16 to a human subject in need thereof; and(ii) detecting the imaging agent conjugated to the humanized antibody or antibody fragment.

30. The method of claim 29, wherein the humanized antibody or antibody fragment comprises a single chain variable fragment (scFv).

31. The method of claim 29 or 30, wherein the detecting step comprises positron emission tomography (PET), single-photon emission computed tomography (SPECT)ZCT imaging, nuclear magnetic resonance (NMR) spectroscopy or near-infrared (NIR) optical imaging.

32. The antibody or antibody fragment of any one of claims 1-16 or the pharmaceutical composition of claim 17 for use in the method of any one of claims 22-31.

34. Use of the antibody or antibody fragment of any one of claims 1-16 in the manufacture of a medicament for use in the method of any one of claims 22-31.

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