Cell surface antibodies against specific biomarkers of pancreatic beta cells

Abstract

The present invention relates to the field of immunology. More specifically, the present invention provides methods and compositions that are directed to the use of antibodies against ZnT8, pancreatic zinc transporter. In certain embodiments, anti-ZnT8 antibodies specifically bind to the transmembrane domain of ZnT8. In more specific embodiments, anti-ZnT8 antibodies are specific to the extracellular surface of the transmembrane domain.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 235,237, filed August 20, 2021, and U.S. Provisional Patent Application No. 63 / 388,005, filed July 11, 2022, which are incorporated by reference in their entireties.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically as an XML file named 448070440WO1.xml. The size of the XML file, created on August 17, 2022, is 54,472 bytes. The contents of the XML file are incorporated herein by reference in their entirety.

[0003] (Technical field) Provided herein are materials and methods related to the fields of immunology and diabetes. More specifically, provided herein are methods and compositions directed to the use of antibodies against the pancreatic zinc transporter, ZnT8.

[0004] (Federally sponsored research or development) This invention was made with Government support under Grants DK125746 and DK123435 awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0005] (Background technology) Pancreatic β-cells, as professional secretory cells, are the sole source of insulin to control blood glucose levels in the human body. Although β-cells have evolved a large dynamic capacity for insulin production in response to glucose fluctuations, they are poorly equipped to cope with islet inflammation and metabolic stress [1], which underlie β-cell autoimmune vulnerability in type 1 diabetes, and β-cell failure and loss in type 2 diabetes [2]. Thus, primary β-cell defects are at the heart of susceptibility to both forms of diabetes. To better understand the pathogenesis of diabetes and evaluate therapeutic interventions, accurate monitoring of β-cell fate under disease and treatment conditions is required. However, routine tests such as measuring insulin / C-peptide levels, fasting glucose levels, and oral glucose tolerance do not provide adequate information on the quantity and function of insulin-producing β-cells in the preclinical stages of diabetes and after receiving interventional therapy. Cell surface biomarkers directly related to insulin secretion biology at high cell surface density are valuable targets for cell surface monoclonal antibody (mAb) development that are applicable for noninvasive monitoring of β-cell function and drug delivery. Summary of the Invention

[0006] Dysfunction and loss of insulin-producing beta cells in pancreatic islets is a major cause of diabetes, yet methods for in vivo monitoring of beta cell mass and function and targeted drug delivery have yet to be developed. Insulin production and storage in beta cells is functionally coupled to cellular zinc enrichment, which is controlled by overexpression of the islet-specific zinc transporter-8 (ZnT8). Herein, we describe an autoreactive antibody (mAb43) with subnanomolar binding affinity and conformational specificity for an extracellular epitope of ZnT8. Glucose stimulation increased binding of ZnT8-mAb43 on the extracellular cell surface, enabling us to use mAb43 to isolate beta cells from single-cell suspensions of whole pancreas and induce islet homing of the fluorescent tag in mice following systemic administration. In some embodiments, the autoreactive antibody may target beta cell surface ZnT8 for in vivo delivery of imaging payloads and antibody-drug conjugates.

[0007] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to the three extracellular loops of the transmembrane domain of zinc transporter-8 (ZnT8). In some embodiments, the three extracellular loops of ZnT8 comprise amino acids 95-99, 169-175, and 242-245 of SEQ ID NO:31.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) heavy chain complementarity determining regions (CDRs) 1, 2, and 3 comprising SEQ ID NOs:3-5, respectively, and (b) light chain CDRs 1, 2, and 3 comprising SEQ ID NOs:8-10, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one conservative amino acid substitution within one or more of SEQ ID NOs:3-5, and / or at least one conservative amino acid substitution within one or more of SEQ ID NOs:8-10. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:19, and (b) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO:7. In some embodiments, the heavy chain variable region sequence has at least 95% sequence identity to SEQ ID NO:2 or SEQ ID NO:19, and the light chain variable region sequence has at least 95% sequence identity to SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment comprises (a) a heavy chain variable region sequence comprising SEQ ID NO:2 or SEQ ID NO:19, and (b) a light chain variable region sequence comprising SEQ ID NO:7.

[0009] In some embodiments, the fragments are Fab, Fab', F(ab')2, Fab 1-SH, Fv, diabody, linear antibody, or single chain variable fragment (scFv). In some embodiments, the heavy chain constant region is of the immunoglobulin G1 (IgG1) isotype. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an imaging agent.

[0010] Also provided herein is a pharmaceutical composition comprising a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof described herein.

[0011] Also provided herein is a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments described herein. Also provided herein is a vector comprising any of the nucleic acids described herein. Also provided herein is a host cell comprising any of the vectors described herein.

[0012] Also provided herein is a method for making an antibody-drug conjugate that specifically binds to the three extracellular loops of the transmembrane domain of ZnT8, the method comprising (a) culturing any of the host cells described herein under conditions suitable for production of the antibody, and (b) conjugating the antibody to a therapeutic agent.

[0013] Also provided herein is a method for producing an antibody-imaging agent conjugate that specifically binds to three extracellular loops of the transmembrane domain of ZnT8, the method comprising (a) culturing any of the host cells described herein under conditions suitable for production of the antibody, and (b) conjugating the antibody to an imaging agent.

[0014] Also provided herein is a method for treating a disease or condition associated with ZnT8 in a subject, comprising administering to the subject any of the antibodies or antigen-binding fragments described herein. In some embodiments, the disease or condition comprises type 1 or type 2 diabetes.

[0015] Also provided herein are methods for detecting pancreatic beta cells in vivo, comprising administering to a subject any of the antibodies or antigen-binding fragments described herein and detecting an imaging agent conjugated to the antibody or antigen-binding fragment thereof, in some embodiments, 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.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises a single chain variable fragment (scFv) comprising (a) a heavy chain variable region sequence comprising SEQ ID NO:2 or SEQ ID NO:19, and (b) a light chain variable region sequence comprising SEQ ID NO:7. In some embodiments, the heavy chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO:2 or SEQ ID NO:19, and (b) the light chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO:7.

[0017] In some embodiments, the imaging agent is a radioactive metal. In some embodiments, the imaging agent is a radioactive metal and the detecting step comprises PET. In some embodiments, the radioactive metal is 64 Cu, 67 Cu, 68 Ga, 60 Ga, 89 Zr, 86 Y, and 94m In some embodiments, the imaging agent is a radioactive metal and the detecting step comprises SPECT. 111 In, 67 Ga, 99 mTc, and 177 Lu.

[0018] Also provided herein is a single chain variable fragment comprising an (scFv) or antigen-binding fragment thereof that binds to the three extracellular loops of the transmembrane domain of ZnT8, comprising (a) a heavy chain variable region sequence of SEQ ID NO:2 or SEQ ID NO:19, and (b) a light chain variable region sequence of SEQ ID NO:7. In some embodiments, the heavy chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO:2 or SEQ ID NO:19, and (b) the light chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO:7.

[0019] In some embodiments, the scFv is conjugated to an imaging agent. In some embodiments, the imaging agent is a radioactive metal. In some embodiments, the radioactive metal is 64 Cu, 67 Cu, 68 Ga, 60 Ga, 89 Zr, 86 Y, 94 mTc, 111 In, 67 Ga, 99m Tc, and 177 Lu.

[0020] Also provided herein is an antibody, or antigen-binding fragment thereof, comprising (a) a light chain comprising SEQ ID NO:20, and (b) a heavy chain comprising one of SEQ ID NOs:21-24. Also provided herein is an antibody, or antigen-binding fragment thereof, comprising (a) a light chain comprising SEQ ID NO:20, and (b) a heavy chain comprising one of SEQ ID NOs:27-30.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and materials to those described herein can be used to practice the present invention, and 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 merely illustrative and are not intended to be limiting.

[0022] 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 become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0023] [Figure 1]Figure 1A shows the induction and biochemical characterization of anti-TMD antibodies. It shows the membrane-flush extracellular surface of ZnT8 (space-filling representation, left) formed by three short loops on the ZnT8 homodimer with bound zinc ions (ball-and-stick, right). The TMD is embedded in the lipid bilayer, while the CTD extends into the cytoplasm. Figure 1B shows the induction and biochemical characterization of anti-TMD antibodies. It shows the sequence alignment of the three extracellular loops (ECLs). Figure 1C shows the induction and biochemical characterization of anti-TMD antibodies. It shows the ELISA titration of mouse sera from ZnT8-KO mice injected with proteoliposomes or liposomes as indicated, against either flZnT8 or CTD. Figure 1D shows the induction and biochemical characterization of anti-TMD antibodies. It shows the same as in Figure 1C, except that the use of NOD female mice was assumed. Error bars are standard error, *p<0.01 (n=4) from 4 ZnT8-KO or 4 NOD mice. Figure 1E shows the induction and biochemical characterization of anti-TMD antibodies. Titration of mAb43 and mAb20 against detergent-solubilized flZnT8 as indicated. Figure 1F shows the induction and biochemical characterization of anti-TMD antibodies. Titration of mAb43 and mAb20 against the CTD is shown. Figure 1G shows the induction and biochemical characterization of anti-TMD antibodies. Titration of mAb43 and mAb20 against ZnT8 proteoliposomes. Note that ZnT8 within the proteoliposomes adopts a mixed transmembrane orientation exposing either the TMD or CTD to antibody binding as indicated. The solid lines are least-squares fits of the binding curves to hyperbolic functions with r2>0.98. [Diagram 2]Figure 2A shows antibody binding to ZnT8 and cell surface markers. IF labeling of live Endoc-βH1 cells with mAb43 or mAb20 as indicated. Cells were counterstained with CD71 antibody and DAPI. Figure 2B shows antibody binding to ZnT8 and cell surface markers. Parallel IF labeling of Endoc-βH1 cells after PFA fixation and detergent permeabilization. Figure 2C shows antibody binding to ZnT8 and cell surface markers. IF staining of live wild-type or ZnT8-KO INS-1E cells with mAb43, Na+ / K+ATPase antibody and DAPI as indicated. Figure 2D shows antibody binding to ZnT8 and cell surface markers. Parallel IF labeling of wild-type or ZnT8-KO INS-1E cells after PFA fixation and detergent permeabilization. Figure 2E shows antibody binding to ZnT8 and cell surface markers. IF labeling of live Endoc-bH1 cells with ZnT8ecA-positive human serum, mouse mAb43, or serum-mAb43 combination as indicated. Figure 2F shows antibody binding to ZnT8 and cell surface markers. Quantification of cell surface (S) and intracellular (I) fluorescence intensity by mAb43 or mAb20 immunolabeling of live Endoc-bH1 cells in Figure 2A-B, or mAb43 immunolabeling of either WT or ZnT8-KO INS-1E cells in Figure 2C-D. Fluorescence intensity was normalized to the intensity of mAb43 in each pair of control groups as indicated. Open circles are data points of individual cells. Error bars are standard error. Figure 2G shows antibody binding to ZnT8 and cell surface markers. Quantification of cell surface IF labeling of live Endoc-βH1 cells with ZnT8ecA-positive human serum, mouse mAb43, or serum / mAb43 combination as described in Figure 2 E. Fractional intensities are serum or mAb43 signals normalized to the sum of serum intensity and mAb43 intensity for each pair of control groups, as indicated. [Diagram 3]FIG. 3A shows mapping of the mAb43 epitope to the ECL. Sizing-HPLC chromatograms of stable protein-binding complexes of ZnT8-GFP with mAb43, mAb20, or FLAG antibody, as indicated. Dashed lines indicate alignment of peak positions of free or bound ZnT8-GFP, as indicated. FIG. 3B shows mapping of the mAb43 epitope to the ECL. Sizing-HPLC chromatograms of stable protein-binding complexes of ZnT8FLAG-GFP with mAb43, mAb20, or FLAG antibody, as indicated. FIG. 3C shows mapping of the mAb43 epitope to the ECL. Immunoblot analysis of mAb43, mAb20, and anti-peptide ZnT8 antibody with SDS-denatured total lysates of human Endoc-βH1 cells. Arrows indicate two splice variants of endogenous ZnT8. FIG. 3D shows mapping of the mAb43 epitope to the ECL. Side view of the negatively stained electron density map of the ZnT8-Fab43 complex showing a Fab43 molecule bound to one of the two ZnT8 protomers. The oval density consists of a ZnT8 homodimer and associated detergent / lipid molecules. The illustrations show docked human ZnT8 and Fab molecules, respectively. The dashed arrow indicates the twofold axis of the ZnT8 homodimer, which coincides with the minor axis of the oval. [Figure 4]Figure 4A shows the specificity of mAb43 for mouse β cells. mAb43 immunolabeling and diaminobenzidine detection of endogenous ZnT8 in paraffin-embedded mouse pancreatic sections with mAb20 and PBS as negative controls. Figure 4B shows the specificity of mAb43 for mouse β cells. IF labeling of enzymatically dispersed islet cells from isolated mouse islets using mouse mAb43 or mouse IgG2b isotype control followed by anti-mouse IgG-PE, anti-insulin-APC, anti-glucagon-488 and DCV. All islet cells were PFA fixed and detergent permeabilized prior to immunolabeling. Figure 4C shows the specificity of mAb43 for mouse β cells. mAb43 and anti-insulin co-immunolabeling of frozen sections of autopsy human pancreas with DAPI counterstain. Figure 4D shows the specificity of mAb43 for mouse β cells. Immunolabeling and fluorescence-activated cell sorting of single cell suspensions derived from enzymatically dispersed whole pancreas. Dispersed whole pancreatic cells were labeled with DCV, chimeric mAb43 or mAb20 and detected with PE-conjugated anti-human IgG as indicated. Intact cells (DCV positive) were gated and sorted into mAb43-PE positive (R1) and negative (R0) populations. Dashed lines indicate the thresholds for the DCV and mAb43-PE gates. The percentage of total intact cells within the R1 and R0 gates is shown. Data are representative of four independent experiments. Figure 4E shows the specificity of mAb43 for mouse β cells. Confocal microscopy analysis of insulin and glucagon expression in the different populations of mAb43-labeled cells as indicated. Sorted pancreatic cells were grown on matrigel-coated glass surfaces, fixed with PFA, permeabilized, and then immunolabeled with mAb43, followed by anti-insulin APC, anti-glucagon 488, and anti-human IgG-PE as indicated. The inset is a zoomed-in view of a normal β-cell in the R1 gate, showing colocalization of insulin with ZnT8 in the cytoplasm. Figure 4F shows the specificity of mAb43 for mouse β-cells. Quantification of mAb43 and anti-insulin IF intensity of enriched pancreatic cells in Figure 4E is shown. mAb43 or anti-insulin IF intensity is normalized to the intensity of the R1 cell population. Open circles are data points for individual cells.Error bars are standard error. [Diagram 5] Figure 5A shows glucose-stimulated uptake of ZnT8-mAb43. Figure 5B shows mAb43-A647 uptake in Endoc-βH1 cells at 37 °C. Live cells were exposed to mAb20-A647 or mAb43-A647 in the presence of either high glucose (20 mM) or basal glucose (2 mM) as indicated. For each image, the left panel shows A647-IF, and the right panel shows merged A647, CellMask green, and DAPI signals. All scale bars are 10 μm. Figure 5B shows glucose-stimulated uptake of ZnT8-mAb43. Figure 5C shows glucose-stimulated uptake of ZnT8-mAb43. Figure 5C shows image quantification of total A647-IF intensity in arbitrary units (au) with or without glucose stimulation (20 / 2 mM) at 8 °C or 37 °C as indicated. Open circles are data points for individual cells. Error bars are standard error. [Figure 6]Figure 6A shows the biodistribution of systemically administered antibodies in mice. Western blot analysis of SDS-solubilized tissues of different organs excised from C57BL / 6 mice 1 day after injection of the indicated chimeric mAb43 or mAb20. Tissue protein was loaded at 0.5 mg / lane and detected by horseradish peroxidase (HRP) chemiluminescence. Figure 6B shows the biodistribution of systemically administered antibodies in mice. Relative tissue abundance of mAb43 (black bars) or mAb20 (gray bars) in different organs is shown. Western blot intensities were normalized to pancreatic mAb43 signal at the same time point after injection and averaged from four independent measurements from tissues harvested 1, 3, 5, and 6 days after injection. Open circles are individual data points. Error bars are standard error. Figure 6C shows the biodistribution of systemically administered antibodies in mice. Time-dependent decrease of pancreatic mAb43 or kidney mAb20 is shown as indicated. Serial dilutions of human IgG standards were loaded on the same gel to calibrate the intensities of mAb43 and mAb20. Figure 6D shows the biodistribution of systemically administered antibodies in mice. Quantification of pancreatic mAb43 and kidney mAb20 at various time points after injection as indicated. Error bars are standard error from four independent Western blot measurements. Figure 6E shows the biodistribution of systemically administered antibodies in mice. Relative tissue uptake of mAb43 in NOD (black bars) or db / db mice (white bars) as indicated. Western blot intensities were normalized to pancreatic mAb43 signal and averaged over four NOD or four db / db mice from tissues harvested 2 days after injection. Open circles are individual data points. Error bars are standard error. Figure 6F shows the biodistribution of systemically administered antibodies in mice. Comparison of pancreatic mAb43 uptake in three different mouse strains as indicated. Pancreatic mAb43 uptake levels correlate with FBG levels in individual mice of different strains. Figure 6G shows the biodistribution of systemically administered antibodies in mice. Quantification of mean pancreatic mAb43 uptake in different mouse stains is shown, as indicated.Open circles are western blot data points for individual mice and corresponding FBG levels are shown in the right panel. Error bars are standard error from four mice in each mouse group as indicated. [Figure 7] Figure 7A shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Whole mount images of excised pancreas from MIP-GPF mice receiving 15 mg / kg mAb43-mScarlet injections are shown. GFP, mScarlet, and bright field images are merged and regions of interest (ROIs) used for close-up viewing are numbered. Figure 7B shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Zoomed views of different ROIs (1, 2, 3) are shown showing branching arterioles and colocalization of GFP and mScarlet in islet clusters. Figure 7C shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Zoom-in views of individual islets with overlapping GFP and mScarlet fluorescence in different ROIs (4, 5, 6) are shown. Figure 7D shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Zoom-in views of individual islets with additional scattered mScarlet fluorescence in different ROIs (7, 8, 9) are shown. Figure 7E shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Zoom-in views of individual islets with additional scattered mScarlet fluorescence in different ROIs (7, 8, 9) are shown. Figure 7F shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Zoom-in views of individual islets with additional scattered mScarlet fluorescence in different ROIs (7, 8, 9) are shown. Figure 7G shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Zoom-in views of individual islets with additional scattered mScarlet fluorescence in different ROIs (7, 8, 9) are shown. Figure 7H shows the distribution of mAb43-mScarlet in flat pancreas, demonstrating in vivo islet homing of mScarlet. Zoom-in views of individual islets with additional scattered mScarlet fluorescence in different ROIs (7, 8, 9) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] It is to be understood that the present invention is not limited to the specific methods and components described herein, which may be modified. It is also to be understood that the terms used herein are used merely to describe certain embodiments, and are not intended to limit the scope of the present invention. As used herein and in the appended claims, it should be noted that the singular forms "a", "an", and "the" include the plural, 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.

[0025] 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 belongs. Although specific methods, devices, and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0026] All publications cited herein, including all journal articles, books, manuals, published patent applications, and issued patents, are incorporated herein by reference.In addition, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided.These definitions are not intended to be limiting in nature, but are intended to help to more clearly understand certain aspects of the present invention.

[0027] ZnT8 is the major zinc transporter in beta cells, with protein expression levels comparable to those of housekeeping α-tubulin [3]. This extraordinary cellular ability to generate active zinc transporters results in one of the highest intracellular zinc contents of beta cells in the human body. The tissue distribution of ZnT8 is almost exclusively restricted to pancreatic islets [4,5]. ZnT8 mRNA in pancreatic islets was detected in all endocrine cell types, including α, β, γ, δ, and ε cells, although the mRNA levels may only loosely correlate with the corresponding protein levels of ZnT8 in different cell types [6,7]. Cell sorting based on cellular zinc content resulted in a clear separation of beta cells from other islet cells [8], suggesting that cellular zinc content and the associated ZnT8 protein levels are specific biomarkers for beta cells. The intracellular distribution of ZnT8 is in dynamic equilibrium between the cell surface membrane, insulin secretory granules, and the endoplasmic reticulum, where ZnT8 functions as a zinc sequestering transporter [3,9,10]. Concentrated zinc ions are required for proinsulin processing and crystal packaging of zinc-insulin hexamers [9–13]. As a result, the intracellular distribution of ZnT8 is tightly coupled to insulin processing, storage, and secretion

[14] . Glucose-stimulated insulin secretion promotes the transport of ZnT8 to the cell surface

[15] , making ZnT8 a major cell surface antigen target of autoantibodies in type 1 diabetes patients

[16] . Similarly, surfaced ZnT8 may potentially serve as a functional biomarker for mAb-based immune detection.

[0028] Initial ZnT8 mAbs against linear peptides derived from the extracellular loops of ZnT8 yielded moderate binding affinity (108 nM) and low specificity

[17] . In vivo β-cell imaging and targeting require the development of high-affinity mAbs with exquisite structural specificity for multiple extracellular loops arranged in a spatial configuration. ZnT8 is a bimodular protein consisting of a compact transmembrane domain (TMD) and a cytosolic C-terminal domain (CTD). While the TMD lacks an extracellular domain, its extracellular surface is flush with the membrane and is formed by three short extracellular loops (ECL1-3) (Figure 1A). In addition to limited epitope availability, these loops are quasi-invariant between mouse and human ZnT8, except for a highly conserved E-to-D substitution in ECL3, and therefore poorly antigenic (Figure 1B). As described herein, a mouse immunization strategy was established to enhance the immunogenicity of extracellular epitopes of natively folded ZnT8, identified mAb43 against the extracellular loops with conformational specificity, and demonstrated the utility of mAb43 in beta cell purification and targeted delivery of imaging probes.

[0029] (I. Definition) The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein (e.g., ZNT8, its subunits, or receptor complex), a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination thereof. The above reaction occurs through at least one antigen recognition site in the variable region of the immunoglobulin molecule. A typical antibody comprises at least two heavy (HC) chains and two light (LC) chains interconnected by disulfide bonds. Each heavy chain is composed 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 composed of three domains: CHI, CH2, and CH3. Each light chain is composed of a "light chain variable region" or "light chain variable domain" (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CI. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs, which are arranged from the amino terminus to the 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. 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 Fvs (scFvs), minibodies (e.g., sc(Fv)2, diabodies), multispecific antibodies such as bispecific antibodies generated from at least two complete antibodies, multispecific antibodies such as chimeric antibodies, humanized antibodies, and human antibodies, fusion proteins containing an antigenic determining portion of an antibody, and any other modified immunoglobulin molecule that contains an antigen recognition site, so long as the antibody exhibits the desired biological activity. Thus, the term "antibody" includes whole antibodies and any antigen-binding fragments or single chains thereof. Antibodies can be naked or conjugated to other molecules, such as toxins, radioisotopes, small molecule drugs, or polypeptides.

[0030] The term "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of the natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is (1) purified to greater than 95% by weight (including 99% by weight) of the antibody as determined, for example, by the Lowry method, (2) purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue or silver staining. An isolated antibody includes the antibody in situ in a recombinant cell, since at least one component of the antibody's natural environment is absent. Ordinarily, however, an isolated antibody is prepared by at least one purification step.

[0031] The term "humanized" immunoglobulin refers to an immunoglobulin that contains a human framework region and one or more CDRs derived from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly for the CDRs, are substantially identical to the corresponding parts of a native human immunoglobulin sequence. A "humanized antibody" is an antibody that contains a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a conventional chimeric antibody as defined above, since, for example, the entire variable region of the chimeric antibody is non-human.

[0032] The term "antigen-binding fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. It is known in the art that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Facb, Fd, and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments. In some cases, antibody fragments can be prepared by proteolytic digestion of intact or complete antibodies. For example, antibody fragments can be obtained by treating whole antibodies with enzymes such as papain, pepsin, and plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments. Pepsin digestion of whole antibodies produces F(ab')2 or Fab'. Plasmin digestion of whole antibodies produces Facb fragments.

[0033] The term "Fab" refers to an antibody fragment substantially equivalent to that obtained by digestion of an immunoglobulin (usually IgG) with the enzyme papain. The heavy chain segment of the Fab fragment is the Fd portion. Such fragments can be produced enzymatically or chemically by fragmentation of an intact antibody, or recombinantly from genes encoding partial antibody sequences, or wholly or partially synthetically produced. The term "F(ab')2" refers to an antibody fragment substantially equivalent to that obtained by digestion of an immunoglobulin (usually IgG) with the enzyme pepsin at pH 4.0-4.5. Such fragments can be produced enzymatically or chemically by fragmentation of an intact antibody, or recombinantly from genes encoding partial antibody sequences, or wholly or partially synthetically produced. The term "Fv" refers to an antibody fragment consisting of one NH domain and one N domain linked by non-covalent interactions.

[0034] The terms "ZNT8 antibody", "anti-ZNT8 antibody", "anti-ZNT8", "antibody that binds to ZNT8" and grammatical variations thereof refer to an antibody that can specifically bind to ZNT8 with sufficient affinity. The antibody is useful as a therapeutic agent or diagnostic reagent that targets ZNT8. The extent of binding of the anti-ZNT8 antibody disclosed herein to unrelated non-ZNT8 proteins is less than about 10% of the binding of the anti-ZNT8 antibody measured, for example, by radioimmunoassay (RIA), BIACORE™ (using recombinant ZNT8 as analyte and antibody as ligand, or vice versa), or other binding assays known in the art. In certain embodiments, the antibody that binds to ZNT8 has a dissociation constant (KD) of less than 1 μM, less than 100 nM, less than 50 nM, less than 10 nM, or less than 1 nM.

[0035] The term "% identity" between two polypeptide (or polynucleotide) sequences refers to the number of identical matching positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced to align the two sequences for optimal alignment. A matching position is any position where an identical nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted, since gaps are not nucleotides or amino acids. Similarly, gaps present in the reference sequence are not counted, since nucleotides or amino acids of the target sequence are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated by determining the number of positions where identical amino acid residues or nucleic acid bases are present in both sequences, finding the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity. Comparison of sequences and determination of the percent sequence identity between two sequences can be performed using software that is readily available for both online use and download. Software programs suitable for alignment of both protein and nucleotide sequences are available from a variety of sources. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST website. Bl2seq performs a comparison between two sequences using 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, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are available from the European Bioinformatics Institute (EBI) (www.ebi.ac.uk / Tools / psa).In certain embodiments, the percentage identity "X" of a first amino acid sequence to an amino acid of a second sequence 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 (aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the 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. Those skilled in the art will understand that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence comparisons driven solely by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2 (ClustalX is a version of the ClustalW2 program ported to a Windows environment). Another suitable program is MUSCLE. ClustalW2 and MUSCLE are available, for example, from the European Bioinformatics Institute (EBI).

[0036] The term "therapeutic agent" refers to any biological or chemical agent used to treat a disease or disorder. Therapeutic agents include any suitable biologically active 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 agent.

[0037] As used herein, the terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The term is also used in the context of administration of a "therapeutically effective amount" of an agent, such as an anti-ZnT8 antibody. The effect may be preventative in that it completely or partially prevents a particular outcome, disease or its symptoms, and / or it may be therapeutic in that it partially or completely cures the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a subject, particularly a human, and includes (a) preventing the onset of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., arresting its development, and (c) relieving the disease, e.g., causing regression of the disease, e.g., completely or partially eliminating the symptoms of the disease. In certain embodiments, the term is used in the context of the prevention or treatment of any ZnT8-mediated disease, including diabetes.

[0038] (II. Anti-ZnT8 antibody) The antibodies or antigen-binding fragments of the present disclosure specifically bind to ZNT8. In certain embodiments, these antibodies or antigen-binding fragments specifically bind to human ZNT8. In certain embodiments, these antibodies or antigen-binding fragments specifically bind to the transmembrane domain of ZNT8. As used herein, "specifically bind" means that the antibody or antigen-binding fragment preferentially binds to ZNT8 (e.g., human ZNT8, mouse ZNT8) over other proteins. In certain examples, the anti-ZNT8 antibodies of the present disclosure have a higher affinity for ZNT8 than other proteins. The anti-ZNT8 antibodies that specifically bind to ZNT8 have a higher affinity for human ZNT8 than 1x10 -7 M or less, 2×10 -7 M or less, 3×10 -7 M or less, 4×10 -7 M or less, 5×10 -7 M or less, 6×10 -7 M or less, 7×10 -7 M or less, 8×10 -7 M or less, 9×10 -7M or less, 1x10 -8 Below M, 2x10 -8 M or less, 3×10 -8 M or less, 4×10 -8 M or less, 5×10 -8 M or less, 6×10 -8 M or less, 7×10 -8 M or less, 8×10 -8 M or less, 9×10 -8 M or less, 1×10 -9 M or less, 2×10 -9 M, 3×10 -9 M or less, 4×10 -9 M or less, 5×10 -9 M or less, 6×10 -9 Below M, 7x10 -9 Below M, 8x10 -9 Below M, 9x10 -9 M or less, 1x10 -10 M or less, 2×10 -10 M or less, 3×10 -10 M or less, 4×10 -10 M or less, 5×10 -10 M or less, 6×10 -10 M or less, 7×10 -10 M or less, 8×10 -10 M or less, 9×10 -10 M or less, 1×10 -11 M or less, 2×10 -11 M or less, 3×10 -11 M or less, 4×10 -11 Below M, または5×10 -11 M or less, 6×10 -11 M or less, 7×10 -11 M or less, 8×10 -11 M or less, 9×10 -11 M or less, 1×10 -12 M or less, 2×10 -12 M or less, 3×10 -12 M or less, 4×10 -12 M or less, 5×10 -12 M or less, 6×10 -12 M or less, 7×10 -12 M or less, 8×10 -12 Below M, または9x10 -12It may have a binding affinity of less than or equal to M. Methods for measuring the binding affinity of antibodies 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), biolayer interferometry (Abdiche, "Determining Kinetics and Affinity of Protein Interactions using a Parallel Real-time Label-free Biosensor, the OCTET," Analytical Biochemistry (2008) 377, 209-217), kinetic exclusion assay (KinExA) (Darling and Braault, "Kinetic exclusion assay technology: characterization of tongue interactions," Assay and Drug Dev Tech (2004) 2, 647-657), isothermal calorimetry (Pierce et al., "Isothermal Titration Calorimetry of Protein-Protein Interactions," Methods in Enzymology (1998) 295, 268-294), and the like. These include analytical ultracentrifugation (Lebowitz et al., "Modem analytical ultracentrifugation in protein science: A tutorial review," Protein Science (2002), 11:2067-2079).

[0039] In one aspect, provided herein is an anti-ZnT8 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:5, and / or the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10, wherein the CDRs of the anti-ZnT8 antibody are defined according to the Kabat numbering scheme.

[0040] In one aspect, provided herein is an anti-ZnT8 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:7.

[0041] In some embodiments, provided herein is an anti-ZnT8 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:19. In certain embodiments, the heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:19 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence and retains the ability to bind to ZnT8 (e.g., human ZnT8). In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO:2 or SEQ ID NO:19. In certain embodiments, the substitutions, insertions or deletions (e.g., 1, 2, 3, 4 or 5 amino acids) occur in regions outside the CDRs (i.e., within the FRs). In some embodiments, the anti-ZnT8 antibody comprises a heavy chain variable domain sequence of SEQ ID NO:2 or SEQ ID NO:19 (including post-translational modifications of that sequence). In certain embodiments, the heavy chain variable domain sequence comprises one point mutation relative to SEQ ID NO:2 or SEQ ID NO:19. In further embodiments, the one point mutation is located in the CDR region.

[0042] In some embodiments, provided herein is an anti-ZnT8 antibody or antigen-binding fragment thereof comprising a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:7. In certain embodiments, the light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of SEQ ID NO:7 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence and retains the ability to bind to ZnT8 (e.g., human ZnT8). In certain embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO:7. In certain embodiments, the substitutions, insertions or deletions (e.g., 1, 2, 3, 4 or 5 amino acids) occur in regions outside the CDRs (i.e., within the FRs). In some embodiments, the anti-ZnT8 antibody comprises a light chain variable domain sequence of SEQ ID NO:7 (including post-translational modifications of that sequence). In certain embodiments, the light chain variable domain sequence comprises at least one point mutation relative to SEQ ID NO:7. In further embodiments, one point mutation is located in the CDR region.

[0043] Thus, in certain embodiments, a sequence may contain at least one conservative substitution. It is understood that the phrase "at least one" is synonymous with "one or more" and includes values ​​such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least "N", where N is equal to the total number of amino acids in a particular order (i.e., 1 or more, 2 or more, 3 or more, etc.).

[0044] The sequences may also be comprised of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, etc. up to "N", where "N" is equal to the total number of amino acids in the particular sequence. Alternatively, the particular sequence may include substitutions at 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, etc. amino acid positions.

[0045] In certain embodiments, the invention provides an isolated antibody or antibody-binding fragment thereof that specifically binds to ZnT8, the antibody or antibody-binding fragment comprising heavy chain complementarity determining regions (CDRs) 1, 2 and 3, wherein heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3 or the amino acid sequence set forth in SEQ ID NO:3 with no more than three amino acid positions substituted, heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4 or the amino acid sequence set forth in SEQ ID NO:4 with no more than seven amino acid positions substituted, and heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5 or the amino acid sequence set forth in SEQ ID NO:5 with no more than four amino acid positions substituted.

[0046] In a further embodiment, the isolated antibody or antigen-binding fragment further comprises light chain CDR1, 2 and 3, wherein light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8 or the amino acid sequence set forth in SEQ ID NO:8 with no more than six amino acid positions substituted, light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9 or the amino acid sequence set forth in SEQ ID NO:9 with no more than four amino acid positions substituted, and light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10 or the amino acid sequence set forth in SEQ ID NO:10 with no more than five amino acid positions substituted.

[0047] In some embodiments, the anti-ZnT8 antibody, or the anti-ZnT8 antibody of the anti-ZnT8 antibody-drug conjugate, is a monoclonal antibody.

[0048] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and μ classes are further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009.mAbs Vol 1 Issue41-7), any of which are suitable for use in some embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody can include a heavy chain Fc region that includes a human IgGFc region. In a further embodiment, the human IgGFc region includes human IgG4.

[0049] Antibodies may also include derivatives that are modified, i.e., modified by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from binding to ZnT8 or exerting a cytostatic or cytotoxic effect on cells. For example, but not limited to, antibody derivatives include, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands, or other proteins, etc. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, derivatives may include one or more non-classical amino acids.

[0050] (Antibody fragment) The present disclosure encompasses antibody fragments or domains described herein that retain the ability to specifically bind to ZNT8 (e.g., human ZNT8 (including but not limited to the transmembrane domain of ZNT8)). Antibody fragments include, for example, Fab, Fab', F(ab')2, Fab, and Fv. These fragments may be humanized and may be fully human. Antibody fragments may be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments may be obtained by treating whole antibodies with enzymes such as papain, pepsin, and plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments, pepsin digestion of whole antibodies produces F(ab')2 or Fab', and plasmin digestion of whole antibodies produces Facb fragments.

[0051] Alternatively, the antibody fragment can be produced recombinantly. For example, a nucleic acid encoding the antibody fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host cell. See, e.g., Co, MS et al., J Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, AH, 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 are expressed in and secreted from E. coli, so that these fragments can be easily produced in large quantities. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives that contain salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046.

[0052] (Mini Body) Also included herein are minibodies of the antibodies described herein. Minibodies of anti-ZNT8 antibodies include diabodies, single chain (scFv), and single chain (Fv)2 (sc(Fv)2).

[0053] A "diabody" is a bivalent minibody constructed by gene fusion (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); European Patent Application Publication No. 404,097; International Publication No. WO 93 / 11161). A diabody is a dimer composed of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are connected by a linker. The number of amino acid residues constituting the linker can be 2 to 12 residues (e.g., 3 to 10 residues, or 5 or about 5 residues). The linker of the polypeptides in the diabody is usually too short for the VL and VH to bind to each other. Therefore, the VL and VH encoded by the same polypeptide chain cannot form a single-chain variable region fragment, but form a dimer with a different single-chain variable region fragment. As a result, there are two antigen-binding sites in the diabody.

[0054] scFv is a single chain polypeptide antibody obtained by linking VH and VL with a linker (see, e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Pluckthun, "The Pharmacology of Monoclonal Antibodies," Vol. 113, Eds. Lisenberg and Moore, Springer-Verlag, New York, pp. 269-315, (1994)). Each variable domain (or part thereof) is derived from the same or different antibody. The single chain Fv molecule preferably comprises an scFv linker interposed between the VH and VL domains. Exemplary scFv molecules are known in the art and described, e.g., in U.S. Pat. No. 5,892,019; U.S. Pat. No. 5,892,019; and U.S. Pat. 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).

[0055] The term "scFv linker" as used herein refers to the moiety interposed between the VL and VH domains of an scFv. The scFv linker preferably maintains the scFv molecule in an antigen-binding conformation. In some embodiments, the scFv linker comprises or consists of an scFv linker peptide. In certain embodiments, the scFv linker peptide comprises or consists of a Gly-Ser peptide linker. In some embodiments, the scFv linker comprises a disulfide bond.

[0056] The order of VH and VL is not particularly limited and 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 the scFv may be derived from any anti-ZNT8 antibody or antigen-binding fragment thereof described herein.

[0057] sc(Fv)2 is a minibody in which two VHs and two VLs are linked by a linker to form a single chain (Hudson et al., J Immunol. Methods, (1999) 231:177-189 (1999)). sc(Fv)2 can be produced, for example, by linking scFvs with a linker. The sc(Fv)2 of the present invention preferably includes antibodies in which two VHs and two VLs are arranged in the order of VH, VL, VH, VL ([VH] linker [VL] linker [VH] linker [VL]), starting 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 can be arranged in any order. An example of the arrangement is shown below. [VL] linker [VH] linker [VH] linker [VL] [VH] linker [VL] linker [VL] linker [VH] [VH] linker [VH] linker [VL] linker [VL] [VL] linker [VL] linker [VH] linker [VH] [VL] linker [VH] linker [VL] linker [VH]

[0058] Typically, three linkers are required to link four antibody variable regions. The linkers used may be the same or different. There is no particular limitation on the linker that links the VH region and the VL region of the minibody. In some embodiments, the linker is a peptide linker. As the linker, any single-chain peptide of about 3 to 25 residues (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) may be used.

[0059] In some embodiments, the linker is a synthetic compound linker (chemical crosslinker). Examples of commercially available crosslinkers include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl ipropionate) (DSP), dithiobis(sulfosuccinimidyl ipropionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSocOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSocOES).

[0060] The amino acid sequence of VH or VL in an antibody fragment or minibody may include modifications such as substitutions, deletions, additions and / or insertions. For example, modifications may be in one or more of the CDRs of an anti-ZNT8 antibody described herein. In certain embodiments, modifications include 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 an anti-ZNT8 minibody. Such substitutions are made to improve the binding and / or functional activity of an anti-ZNT8 minibody. In some embodiments, one, two or three amino acids in one or more of the six CDRs of an anti-ZNT8 antibody or antigen-binding fragment thereof may be deleted or added, as long as there is ZNT8 binding and / or functional activity when the VH and VL are associated.

[0061] (VHH) VHHs, also known as nanobodies, are derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas that lack light chains. The present disclosure encompasses VHHs that specifically bind to ZNT8.

[0062] (Variable domains of novel antigen receptors (VNARs)) VNAR is a variable domain of a novel antigen receptor (IgNAR). IgNAR exists as a covalently linked heavy chain homodimer in shark serum. It exists as a soluble and receptor-bound form consisting of variable domains (VNAR) with different numbers of constant domains. VNAR is composed of CDR1 and CDR3, and has HV2 and HV4 domains instead of CDR2 (see, e.g., Barelle and Porter, Antibodies, 4:240-258 (2015)). The present disclosure encompasses VNARs that specifically bind to ZNT8.

[0063] (Constant region) The antibody of the present disclosure may be a whole antibody or a single chain Fc (scFc), and may include any constant region known in the art. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, for example, a human kappa or human lambda light chain constant region. The heavy chain constant region may be, for example, an alpha type, a delta type, an epsilon type, a gamma type, or a mu type heavy chain constant region, for example, a human alpha type, a human delta type, a human epsilon type, a human gamma type, or a human mu type heavy chain constant region. In a specific example, the anti-ZNT8 antibody is an IgA antibody, an IgD antibody, an IgE antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or an IgM antibody.

[0064] 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 anti-ZNT8 antibody described herein is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain is linked to a heavy chain constant region comprising a CH2 domain. In some embodiments, the variable heavy chain is linked to a heavy 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 a heavy chain constant region comprising a hinge region, a CH2 and a CH3 domain. The CH1, hinge region, CH2, and / or CH3 may be derived from an IgG antibody (e.g., IgG1, IgG4). In certain embodiments, the variable heavy chain of the anti-ZNT8 antibody described herein is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and a CH2 domain from IgG4, and a CH3 domain from IgG1. In certain embodiments, such chimeric antibodies may contain one or more additional mutations in the heavy chain constant region that enhance the stability of the chimeric antibody, hi certain embodiments, the heavy chain constant region contains substitutions that modify the properties of the antibody.

[0065] In certain embodiments, the anti-ZNT8 antibody of the present disclosure is an IgG isotype antibody. In some embodiments, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In yet another embodiment, the antibody is an IgG4. In some cases, the IgG4 antibody has one or more mutations that reduce or prevent it from adopting a functional monovalent format. For example, the hinge region of IgG4 can be mutated to have the same amino acid sequence as the hinge region of human IgG1 (serine to proline mutation in human IgG4 hinge). In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., has the CH1, hinge, and CH2 regions of IgG4 and the CH3 region of IgG1).

[0066] (bispecific antibody) In certain embodiments, the anti-ZNT8 antibody of the present disclosure is a bispecific antibody.A bispecific antibody is an antibody that has binding specificity for at least two different epitopes.An exemplary bispecific antibody can bind to two different epitopes of ZNT8 protein. Other such antibodies may combine the ZNT8 binding site with that of another protein. Bispecific antibodies can be prepared as full-length antibodies or lower molecular weight forms thereof (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).

[0067] Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, with the two chains having different specificities (Millstein et al., Nature, 305:537-539 (1983)). In another approach, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. DNA encoding the immunoglobulin heavy chain fusions, and optionally the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host cell. This allows for greater flexibility in adjusting the ratio of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector, provided that at least two polypeptide chains are expressed in equal ratios to obtain high yields.

[0068] According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (such as tyrosine or tryptophan). A compensatory "cavity" of the large side chain and identical or similar size is created at the interface of the second antibody molecule by replacing the large amino acid side chain with a small one (such as alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other undesirable end products such as homodimers.

[0069] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies of the heteroconjugate can be bound to avidin and the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method.

[0070] "Diabody" technology provides an alternative mechanism for creating bispecific antibody fragments. These fragments contain a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Thus, 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.

[0071] (conjugated antibody) The antibodies or antigen-binding fragments disclosed herein can be coupled to polymeric substances such as polymers (e.g., polyethylene glycol (PEG), PEG-modified polyethyleneimine (PEI) (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers), human serum albumin or fragments thereof, radioactive substances ( 90 Y, 131 I), fluorescers, luminescers, haptens, enzymes, metal chelates, and drugs.

[0072] In certain embodiments, the anti-ZNT8 antibody or antigen-binding fragment thereof is modified with a moiety that improves its stabilization and / or retention in circulation, e.g., in blood, serum, or other tissues, e.g., at least 1.5, 2, 5, 10, 15, 20, 25, 30, 40, or 50-fold. For example, the anti-ZNT8 antibody or antigen-binding fragment thereof can be conjugated (e.g., conjugated) with a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or polyethylene oxide. Suitable polymers vary substantially by weight. Polymers having number-average molecular 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, the anti-ZNT8 antibody or antigen-binding fragment thereof can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinyl alcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof, and block copolymers thereof, so long as the water solubility of the block copolymer is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylates, carbomers, and branched or unbranched polysaccharides.

[0073] The conjugated antibodies or fragments described above can be prepared by chemically modifying the antibodies or lower molecular weight forms thereof described herein. Methods for modifying antibodies are well known in the art.

[0074] III. Antibody Characterization The ZNT8 binding properties of the antibodies described herein can be measured by any standard method, such as one or more of the following methods: OCTET (registered trademark), surface plasmon resonance (SPR), BIACORE (registered trademark) analysis, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), and fluorescence resonance energy transfer (FRET).

[0075] The binding interaction of a protein of interest (anti-ZNT8 antibody or functional fragment thereof) with a target (such as ZNT8) can be analyzed using the OCTET® system. In this method, one of a variety of instruments from ForteBio (such as the OCTET® QKe and QK) is used to determine protein interactions, binding specificity, and epitope mapping. The OCTET® system provides a simple method of monitoring real-time binding by measuring the change in polarized light that passes through a custom chip and returns to the sensor.

[0076] The binding interaction of a protein of interest (anti-ZNT8 antibody or functional fragment thereof) with a target (such as ZNT8) can be analyzed using surface plasmon resonance (SPR). SPR or biomolecular interaction analysis (BIA) detects biospecific interactions in real time without labeling the interactants.

[0077] Mass changes on the binding surface of the BIA chip (indicating a binding event) cause changes in the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The change in refractive index generates a detectable signal that is measured as an indication of real-time reactions between biomolecules. Methods using SPR are described, for example, in U.S. Pat. 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 online resources provided by BIAcore International AB (Uppsala, Sweden). Information from SPR can be used to provide accurate and quantitative measurements of equilibrium dissociation constants (Kd) and kinetic parameters (including Kon and Koff) for the binding of biomolecules to targets.

[0078] Epitopes can also be directly mapped by assessing the ability of different anti-ZNT8 antibodies, or functional fragments thereof, to compete with each other for binding to human ZNT8 using BIACORE chromatography techniques (Pharmacia BIAtechnology Handbook, "Pitope Mapping", section 6.3.2, (May 1994); see also Johne et al., (1993) J. Immunol. Methods, 160:191-198).

[0079] When using enzyme immunoassay, a sample containing an antibody, such as 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 and incubated, and after washing, an enzyme substrate such as p-nitrophenyl phosphate is added and the antigen-binding activity is evaluated by measuring the absorbance.

[0080] Additional general guidance for evaluating antibodies, such as Western blot and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, eds. Harlow and Lane, Cold Spring Harbor Press (1988).

[0081] IV. Affinity Maturation In some embodiments, the anti-ZNT8 antibody or antigen-binding fragment thereof is modified, for example, by mutagenesis, to provide a pool of modified antibodies. The modified antibodies are then evaluated to identify one or more antibodies with altered functional properties (e.g., improved binding, improved stability, reduced antigenicity, or increased in vivo stability). In one embodiment, display library technology is used to select or screen the pool of modified antibodies. Higher affinity antibodies are then identified from the second library, for example, by using higher stringency or more competitive binding and washing conditions. Other screening techniques can also be used. Methods that effect 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 silica approaches (e.g., Lippow et al., Nat. Biotechnol, 25:1171-6 (2005)).

[0082] In some embodiments, mutagenesis targets regions that are known to be in the binding interface or likely to be in the binding interface.For example, if the identified binding protein is an antibody, mutagenesis can be directed to the CDR regions of heavy or light chains, as described herein.Furthermore, mutagenesis can be directed to framework regions near or adjacent to CDRs, for example, framework regions that are within 10, 5, or 3 amino acids of CDR junctions in particular.In the case of antibodies, mutagenesis can be limited to one or several CDRs, for example, for stepwise improvement.

[0083] In some embodiments, mutagenesis is used to make the antibody more similar to one or more germline sequences. One exemplary germline method may 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. Mutations (at the amino acid level) may then be added to the isolated antibody in a stepwise, combinatorial, or both manner. For example, a nucleic acid library is created that includes sequences that code for some or all of the possible germline mutations. The mutated antibodies are then evaluated to identify antibodies that, for example, have one or more additional germline residues compared to the isolated antibody and are still useful (e.g., have functional activity). In some embodiments, as many germline residues as possible are introduced into the isolated antibody.

[0084] In some embodiments, mutagenesis is used to replace or insert one or more germline residues into the CDR region. For example, the germline CDR residues can be derived from a similar (e.g., most similar) germline sequence to the variable region to be modified. After mutagenesis, the activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine whether the germline residues are tolerated. Similar mutagenesis can also be performed on framework regions.

[0085] Selection of germline sequences can be performed in various ways. For example, germline sequences can be selected if they meet a predefined criterion for selectivity or similarity, such as at least a certain percent identity, such as at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity compared to the donor non-human antibody. 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 of such sequences. In the case of CDR3, identifying a similar germline sequence can include selecting one of such sequences, but can include using two germline sequences that contribute separately to the amino-terminal and carboxy-terminal portions. In other embodiments, more than one or two germline sequences are used, for example to form a consensus sequence.

[0086] Calculation of "sequence identity" between two sequences is performed as follows: The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Optimal alignment is determined as the best score using the GAP program of the GCG software package, using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences.

[0087] In some embodiments, an antibody 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 that one or more carbohydrate moieties have been deleted and / or one or more glycosylation sites have been added to the original antibody. Addition of glycosylation sites to the antibodies disclosed herein may be accomplished by modifying the amino acid sequence to contain a glycosylation site consensus sequence. Such techniques are well known in the art. Another means of increasing the number of carbohydrate moieties on an antibody is by chemical or enzymatic coupling of glycosides to amino acid residues of the antibody. These methods are described, for example, in WO 87 / 05330, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., 22:259-306. Removal of carbohydrate moieties present on the antibody can 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, 118:131; and Thotakura et al., (1987) Meth. Enzymol., 138:350). See, e.g., U.S. Patent No. 5,869,046 for modifications that increase in vivo half-life by providing salvage receptor binding epitopes.

[0088] In some embodiments, the anti-ZNT8 antibody has one or more CDR sequences that differ from those described herein (e.g., Chothia, enhanced Chothia, or Kabat CDRs). In some embodiments, the anti-ZNT8 antibody has one or more CDR sequences that contain amino acid changes, such as substitutions of 1, 2, 3, or 4 amino acids if the CDR is 5-7 amino acids long, or substitutions of 1, 2, 3, 4, or 5 amino acids within the sequence of the CDR if the CDR is 8 amino acids long or longer. The substituted amino acids may have similar charge, hydrophobic, or stereochemical characteristics. In some embodiments, the amino acid substitution is a conservative substitution. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues with side chains with similar charges are defined in the art. 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), aromatic side chains (e.g., tyrosine), phenylalanine, tryptophan, histidine). In some embodiments, the amino acid substitutions are non-conservative substitutions. Antibodies or antibody fragments thereof containing substituted CDRs may be screened to identify an antibody of interest.

[0089] Unlike CDRs, more substantial changes can be made to the structural framework regions (FRs) without adversely affecting the binding properties of the antibody. Changes to FRs include, but are not limited to, humanizing non-human frameworks, or manipulating certain framework residues important for antigen contact or stabilizing the binding site, such as changing the class or subclass of the constant region, changing certain amino groups that may alter effector functions 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 originates.

[0090] V. Humanized Antibodies A humanized antibody is a genetically engineered antibody in which the CDRs of a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences. See, for example, 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. The acceptor antibody sequence 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 some embodiments, for the heavy chain, the acceptor sequence is a germline V H Exon V H1-2 (also referred to in the literature as HV1-2) (Shin et al., 1991, EMBO J. 10:3641-3645), and for the hinge region (JH), exon JH-6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the acceptor sequence may include exons VK2-30 (also referred to in the literature as KV2-30), and for the hinge region, exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Thus, a humanized antibody is an antibody having some or all of the CDRs derived completely or substantially from a donor antibody, as well as variable region framework sequences and, if present, constant regions derived completely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region, if present, substantially derived from a human heavy chain variable region framework and constant region sequence. Similarly, a humanized light chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody light chain, and a light chain variable region framework sequence and a light chain constant region, if present, substantially derived from a human light chain variable region framework and constant region sequence. Besides nanobodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. The CDRs of a humanized antibody are substantially derived from the corresponding CDRs in a non-human antibody when at least 60%, 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequences of the antibody chains or the constant regions of the antibody chains are substantially derived from human variable region framework sequences or human constant regions, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical. In some embodiments, the ZnT8 antibodies of the invention are humanized antibodies.

[0091] Humanized antibodies often incorporate all six CDRs of a murine antibody (preferably as defined by Kabat), but can be made using fewer than all CDRs of a murine antibody (e.g., at least three, four, or five). 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.

[0092] The heavy and light chain variable regions of the humanized antibody may be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotopes IgG1 and IgG3 have strong complement-dependent cytotoxicity, while human isotype IgG2 has weak complement-dependent cytotoxicity. IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region may be lambda or kappa. Antibodies may be expressed as tetramers containing two light and two heavy chains, as separate heavy and light chains, Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which the heavy and light chains are variable. The domains may be linked via spacers.

[0093] Human constant regions exhibit allotypic and isoallotypic variation between different individuals.That is, constant regions can vary between different individuals at one or more polymorphic positions.Isoallotypes differ from allotypes in that serum that recognizes isoallotypes binds to the non-polymorphic regions of one or more other isotypes.

[0094] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in some or all of the molecules. Substitutions may be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, for example, Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; Tso et al., U.S. Pat. No. 5,834,597, and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005,2006) or to increase half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213,2004).

[0095] Exemplary substitutions include amino acid substitutions of natural amino acids to cysteine ​​residues introduced at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332, preferably the S239C mutation in the human IgG1 isotype (US Patent Publication No. 20100158909). The presence of the additional cysteine ​​residue allows the formation of interchain disulfide bonds. Such interchain disulfide bond formation can cause steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. The cysteine ​​residues introduced in or near the Fc region of the IgG constant region also serve as sites for conjugation to therapeutic agents (i.e., conjugation of cytotoxic drugs using thiol-specific reagents such as maleimide derivatives of the drugs). The therapeutic agents cause steric hindrance, further reducing the affinity of the Fc region-FcγR binding interaction.

[0096] The in vivo half-life of an antibody can also affect its effector function. The half-life of an antibody can be extended or shortened to modify its therapeutic activity. FcRn is a receptor structurally similar to MHC class I antigens that bind non-covalently to β2-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). IgG-FcRn interaction occurs at pH 6.0 (pH of intracellular vesicles) but not at pH 7.4 (pH of blood). This interaction allows IgG to be recycled back into the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). Regions on human IgG1 involved in FcRn binding have 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 IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules with these substitutions have longer serum half-lives. As a result, these modified IgG1 molecules may be able to perform effector functions and thus exert their therapeutic effects for a longer period of time compared to unmodified IgG1. Other exemplary substitutions for increasing binding to FcRn include Gin at position 250 and / or Leu at position 428. EU numbering is used for all positions in the constant region.

[0097] Reference to a human constant region includes constant regions of any naturally occurring allotype, or of any permutation of residues occupying polymorphic positions of naturally occurring allotypes, and may have up to 1, 2, 5, or 10 mutations compared to a naturally occurring human constant region, as set forth above, to decrease Fc gamma receptor binding or increase binding to Fc RN.

[0098] VI. ANTIBODY DRUG CONJUGATES Anti-ZnT8 antibody can be conjugated to a therapeutic agent to form an antibody-drug conjugate (ADC). In certain embodiments, the therapeutic agent can include a cytotoxic agent, a prodrug-converting enzyme, a radioisotope or compound, or a toxin. For example, anti-ZnT8 antibody can be conjugated to a cytotoxic agent such as a toxin (e.g., a cytostatic or cytocidal agent such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin).

[0099] The anti-ZnT8 antibody can be conjugated to a prodrug-converting enzyme. The prodrug-converting enzyme can be recombinantly fused to the antibody using known methods or chemically conjugated to the antibody. Exemplary prodrug-converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, beta-glucosidase, nitroreductase and carboxypeptidase A.

[0100] Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known. See, for example, Arnon et al. in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., eds., Alan R. Liss, Inc., 1985); Hellstrom et al. in "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Controlled Drug Delivery, (Robinson et al., eds., Marcel Dekker, Inc., 2nd ed., 1987); Thorpe in "Antibodies For Drug Delivery", Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al., eds., 1985); and "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., eds., Academic Press, 1985) in "Analysis, Results, and Future Prospect of the Therapeutic Use of Radiolabeled Antibody In Cancer See, e.g., Immunol. Rev. 62:119-58, "Immunol. Therapy", Thorpe et al., 1982. See also, e.g., WO 89 / 12624.

[0101] The therapeutic agent may be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, antibody degradation, or a cleaving agent). Such a therapeutic agent is attached to the antibody using a cleavable linker that is susceptible to cleavage in the intracellular environment of a ZnT8-expressing cancer cell, but is substantially insensitive in the extracellular environment, such that the conjugate is cleaved from the antibody when it is taken up by the ZnT8-expressing cell (e.g., in an endosome, or, e.g., by pH sensitivity or protease sensitivity, in a lysosomal or caveolar environment).

[0102] Typically, ADCs include a linker region between the therapeutic agent and the anti-ZnT8 antibody. As described above, typically, the linker is cleavable under intracellular conditions, and cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., in lysosomes, endosomes, or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by intracellular peptidases or protease enzymes, including lysosomal or endosomal proteases. Typically, the peptidyl linker is at least 2 amino acids long, or at least 3 amino acids long. Most typically, it is a peptidyl linker that is cleavable by enzymes present in ZnT8-expressing cells. Other such linkers are described, for example, in U.S. Pat. No. 6,214,345. In certain embodiments, the peptidyl linker cleavable by an intracellular protease comprises a Val-Cit linker or a Phe-Lys dipeptide (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker).One advantage of using intracellular proteolytic release of a therapeutic agent is that the therapeutic agent is usually attenuated upon conjugation, and the serum stability of the conjugate is usually high.

[0103] Cleavable linkers can be pH sensitive, i.e., sensitive to hydrolysis at a certain pH value. Usually, pH sensitive linkers are hydrolyzable under acidic conditions. For example, acid labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. For example, see U.S. Pat. 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 in blood, but become unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929).

[0104] Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). 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-oxycarbonyl-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., "mmunoConjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer" (ed. C.W. Vogel, Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935.

[0105] The linker can also be a malonic acid 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). The linker can also be a malonic acid 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).

[0106] The linker may also be a non-cleavable linker, such as a maleimide-alkylene-linker or a maleimide-aryl linker that directly attaches to a therapeutic agent (e.g., a drug). The active drug linker is released by degradation of the antibody.

[0107] Typically, the linker is substantially insensitive 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%, no more than about 1% of the linkers in a sample of the ADC are cleaved when the ADC is present in an extracellular environment (e.g., in plasma).

[0108] Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by independently incubating both (a) an ADC ("ADC sample") and (b) an equimolar amount of unconjugated antibody or therapeutic agent ("control sample") with plasma for a predetermined period of time (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 to the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

[0109] The linker may also promote cellular internalization. The linker may promote cellular internalization when conjugated to a therapeutic agent (i.e., in the context of a linker-therapeutic agent portion of an ADC or ADC derivative described herein). Alternatively, the linker may promote cellular internalization when conjugated to both a therapeutic agent and an anti-ZnT8 antibody (i.e., in the context of an ADC described herein).

[0110] The anti-ZnT8 antibody can be attached to the linker through the heteroatom of the antibody. These heteroatoms can be naturally present on the antibody or can be introduced into the antibody. In some embodiments, the anti-ZnT8 antibody is attached to the linker through the nitrogen atom of a lysine residue. In other embodiments, the anti-ZnT8 antibody is attached to the linker through the sulfur atom of a cysteine ​​residue. The cysteine ​​residue can be naturally present or can be incorporated into the antibody. Methods for attaching linkers and drug linkers to antibodies through lysine and cysteine ​​residues are known in the art.

[0111] (VII. Imaging) In another embodiment, the antibody is conjugated to a labeling agent. By "labeling agent" (or "detectable label"), it is meant that the agent detectably labels the antibody so that the antibody can be detected in the intended application (e.g., in vitro and / or in vivo research and / or clinical application). Detectable labels of interest include radioisotopes, enzymes that generate detectable products (e.g., horseradish peroxidase, alkaline phosphatase, etc.), fluorescent proteins, paramagnetic atoms, etc. In certain embodiments, the antibody is conjugated to a specific binding partner of the detectable label (e.g., conjugated to biotin so that detection can occur via a detectable label that includes avidin / streptavidin).

[0112] In certain embodiments, the agent is a labeling agent used for 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), and nuclear magnetic resonance (NMR) spectroscopy. Labeling agents used for such applications include, but are not limited to, fluorescent labels, radioisotopes, and the like. In certain embodiments, the labeling agent is a multimodal in vivo imaging agent that allows in vivo imaging using two or more imaging approaches. See Thorp-Greenwood and Coogan (2011) Dalton Trans. 40:6129-6143. In other embodiments, the labeling agent is an in vivo contrast agent used in near infrared (NIR) imaging applications, the agent being selected from Kodak X-SIGHT dyes, Pz 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 fluorophores, Alexa Fluor 680 and 750 dyes, IRDye 680 and 800CW fluorophores. In some embodiments, the labeling agent is an in vivo contrast agent used in SPECT imaging applications, the agent being selected from: 99m Tc, In-111, 123-In, 201 T1, and 133 In certain embodiments, the labeling agent is an in vivo imaging agent used in positron emission tomography (PET) imaging applications, including, but not limited to, Xe. U C. 13 N, 15 O. 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, 68 These may include, but are not limited to, Ga.

[0113] (VIII. Method for producing anti-ZNT8 antibodies) The anti-ZNT8 antibody (or the antigen-binding domain of the antibody or its functional fragment) of the present disclosure can be produced in bacterial cells or eukaryotic cells. To produce a polypeptide of interest, a polynucleotide encoding the polypeptide is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies.

[0114] When the antibody is expressed in bacterial cells (e.g., E. coli), the expression vector should have properties that allow the vector to be amplified in the bacterial cell. In addition, when E. coli such as JM109, DH5a, HB101, or XL I-Blue is used as the host, the vector must have a promoter, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter that allows efficient expression in E. coli. Such vectors include, for example, M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably a T7 The expression vector may include 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 a signal sequence for antibody secretion. For bacterial expression, the calcium chloride method or electroporation method may be used to introduce the expression vector into bacterial cells.

[0115] When the antibody is expressed in animal cells such as CHO, COS, 293, 293T, and NIH3T3 cells, the expression vector contains a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), the MMLV-LTR promoter, the EF1a promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter. In addition to the nucleic acid sequence encoding an immunoglobulin or a domain thereof, the recombinant expression vector may carry additional sequences such as sequences that regulate the replication of the vector in host cells (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, U.S. Pat. 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 a host cell into which the vector has been introduced. Examples of vectors having selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0116] In some embodiments, the antibody is produced in mammalian cells.Exemplary mammalian host cells for expressing polypeptides 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 DHFR selection marker, e.g., 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 cells from transgenic animals, e.g., transgenic mammals.For example, the cell is a breast epithelial cell.

[0117] The antibody of the present disclosure may be isolated from the inside or outside of a host cell (such as the medium) and purified as a substantially pure and homogeneous antibody. Methods for isolation and purification commonly used for polypeptides may be used for the isolation and purification of the antibodies described herein, and are not limited to any particular method. The antibody may be isolated and purified by, for example, appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and the like. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed 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 may be performed using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Examples of columns using Protein A columns include Hyper D, POROS, Sepharose FF (GE Healthcare Biosciences), etc. The present disclosure also includes antibodies highly purified using these purification methods.

[0118] The present disclosure also provides a nucleic acid molecule or a set of nucleic acid molecules that encode the anti-ZNT8 antibody or its antigen-binding molecule disclosed herein.In some embodiments, the present invention comprises a nucleic acid molecule that encodes a polypeptide chain comprising the light chain of the anti-ZNT8 antibody or its antigen-binding molecule described herein.In some embodiments, the present invention comprises a nucleic acid molecule that encodes a polypeptide chain comprising the heavy chain of the anti-ZNT8 antibody or its antigen-binding molecule described herein.

[0119] Also provided are vectors or sets of vectors comprising such a nucleic acid molecule or set of nucleic acid molecules or their complements, as well as host cells comprising the vectors.

[0120] The present disclosure also provides a method for producing ZNT8 or its antigen-binding molecule, or chimeric molecule disclosed herein, comprising culturing a host cell disclosed herein and recovering the antibody, its antigen-binding molecule, or chimeric molecule from the culture medium.

[0121] Various methods are available for recombinantly producing the ZNT8 antibody or its antigen-binding molecule disclosed herein, or the chimeric molecule disclosed herein.It will be understood that due to the degeneracy of the code, various nucleic acid sequences code for the amino acid sequence of a polypeptide.Desired polynucleotides can be produced by denovo solid-phase DNA synthesis or by PCR mutagenesis of previously prepared polynucleotides.

[0122] For recombinant production, a polynucleotide sequence encoding a polypeptide (e.g., a ZNT8 antibody or antigen-binding molecule thereof disclosed herein, or any of the chimeric molecules disclosed herein) is inserted into an appropriate expression vehicle, i.e., a vector containing the necessary elements for transcription and translation of the inserted coding sequence, or, in the case of an RNA viral vector, a vector containing the necessary elements for replication and translation.

[0123] The nucleic acid encoding the polypeptide (e.g., ZNT8 antibody or its antigen-binding molecule disclosed herein, or any of the chimeric molecules disclosed herein) is inserted into the vector in the appropriate reading frame. The expression vector is then transfected into an appropriate target cell to 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., ZNT8 antibody or its antigen-binding molecule disclosed herein, or any of the chimeric molecules disclosed herein) in eukaryotic cells. In some embodiments, the eukaryotic cells are animal cells, including mammalian cells (e.g., 293 cells, PerC6, CHO, BHK, Cos, HeLa cells). When a polypeptide is expressed in a eukaryotic cell, the DNA encoding the polypeptide (e.g., the ZNT8 antibody or its antigen-binding molecule disclosed herein, or any of the chimeric molecules disclosed herein) can also encode a signal sequence that allows the polypeptide to be secreted. Those skilled in the art will understand that the signal sequence is cleaved by the cell during translation of the polypeptide to form a mature chimeric molecule. Various signal sequences are known in the art and are familiar to those skilled in the art. Alternatively, when no signal sequence is included, the polypeptide (e.g., the ZNT8 antibody or its antigen-binding molecule disclosed herein, or any of the chimeric molecules disclosed herein) can be recovered by lysing the cell.

[0124] IX. PHARMACEUTICAL COMPOSITIONS The present disclosure also provides a pharmaceutical composition comprising one or more of: (i) a ZNT8 antibody or antigen-binding molecule thereof disclosed herein, (ii) a nucleic acid molecule or set of nucleic acid molecules encoding a ZNT8 antibody or antigen-binding molecule disclosed herein, or (iii) a vector or set of vectors disclosed herein, and a pharma- ceutical acceptable carrier.

[0125] The anti-ZNT8 antibody or fragment thereof described herein can be formulated as a pharmaceutical composition for administration to a subject, for example, to treat a disorder described herein. Typically, a pharmaceutical composition can include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The composition can include a pharmaceutically acceptable salt, for example, an acid addition salt or a base addition salt (see, for example, Berge, SM et al., (1977) J.Pharm.Sci.66:1-19).

[0126] Pharmaceutical formulation is a well-established art and is further described, for example, 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).

[0127] Pharmaceutical compositions can be in various forms.These include, for example, liquid, semi-solid and solid dosage forms, such as solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.Preferred forms can depend on intended mode of administration and therapeutic application.Usually, the pharmaceutical compositions described herein are in the form of injectable or infusible solutions.

[0128] In some embodiments, the antibodies described herein are formulated with excipient materials such as sodium citrate, dibasic sodium phosphate heptahydrate, monobasic sodium phosphate, Tween®-80, and stabilizers. For example, they may be provided in a buffer solution of appropriate concentration and stored at 2-8° C. In some embodiments, the pH of the composition may be about 5.5-7.5 (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).

[0129] The pharmaceutical composition may also include an agent that reduces 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 at a concentration of about 0.5 mM to about 145 mM (e.g., 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM). The pharmaceutical composition may also include a sugar (e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol) and / or an isotonicity adjuster (e.g., sodium chloride, mannitol, or sorbitol) and / or a surfactant (e.g., polysorbate-20 or polysorbate-80).

[0130] Composition can be formulated as solution, microemulsion, dispersion, liposome or other ordered structure suitable for stable storage at high concentration.Sterile injection solution can be prepared by incorporating the agent described herein in the required amount in suitable solvent with one or combination of above-listed components as required, followed by filtration sterilization.

[0131] Generally, dispersion is prepared by incorporating the agent described herein into a sterile medium containing a basic dispersion medium and other necessary ingredients from those listed above.For the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying the powder of the agent described herein and any additional desired ingredients from its solution that has been previously sterile filtered.The appropriate fluidity of the solution can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size in the case of dispersion, and using surfactants.The sustained absorption of injectable composition can be achieved by including an agent that delays absorption, for example, monostearate salt and gelatin in the composition.

[0132] In certain embodiments, the antibody can be prepared with a carrier that protects the compound from rapid release, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known. For example, see Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York (1978).

[0133] In some embodiments, the pharmaceutical formulation comprises a concentration of from 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, In some embodiments, the antibody is formulated with a pharma- ceutically acceptable carrier at a concentration of 100 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). In some embodiments, the antibody is formulated in sterile distilled water or phosphate buffered saline. The pH of the pharmaceutical formulation can be 5.5 to 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).

[0134] The pharmaceutical composition may comprise a "therapeutically effective amount" of the agent described herein. Such an effective amount may be determined based on the effect of the agent administered, or the combined effect of the agents when multiple agents are used. The therapeutically effective amount of an agent may also vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the compound to induce a desired response in an individual, such as improvement of at least one disorder parameter, or improvement of at least one symptom of the disorder. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects.

[0135] The antibody or antigen-binding fragment thereof of the present disclosure, or the nucleic acid encoding same, can be administered to a subject, e.g., a subject in need thereof, e.g., a human or animal subject, by a variety of methods. In many applications, the route of administration is either intravenous injection or parenteral, infusion (IV), subcutaneous injection (SC), intraperitoneal (IP), or intramuscular injection, intratumoral (IT). Other parenteral administration methods may also be used. Examples of such modes include intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injection.

[0136] In some embodiments, the route of administration of the antibody of the present invention is parenteral. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or intravaginal administration. Intravenous forms of parenteral administration are preferred. Although all these forms of administration are clearly considered to be within the scope of the present invention, the form of administration will be a solution for injection, particularly a solution for intravenous or intraarterial injection or infusion. Typically, pharmaceutical compositions suitable for injection may include a buffer (e.g., acetate buffer, phosphate buffer, or citrate buffer), a surfactant (e.g., polysorbate), a stabilizer (e.g., human albumin) if necessary, and the like. According to the teachings of the present specification, a polypeptide can be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0137] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.Examples of non-aqueous solvents include 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).

[0138] Pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M, 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 may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0139] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (if 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 must be fluid to the extent that easy injection is possible. It should be stable under the conditions of manufacture and storage, and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. 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.

[0140] Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include an isotonic agent in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0141] In any case, sterile injectable solutions can be prepared by incorporating the active compound (e.g., the polypeptide alone 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.

[0142] Generally, dispersions are prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and the necessary other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which results in a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution. Injectable preparations are processed according to methods known in the art, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions. In addition, the preparations can be packaged and sold in the form of a kit. Such products preferably have a label or package insert indicating that the relevant composition is useful for treating subjects suffering from or susceptible to coagulation disorders.

[0143] The effective dosage of the disclosed composition for treating a condition varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs administered, and the drug administered, and whether the treatment is preventive or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dosage can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0144] The route and / or mode of administration of the anti-ZNT8 antibody or fragment thereof can also be adjusted to suit individual cases, for example, by monitoring the subject.

[0145] The antibody or fragment thereof may be administered as a fixed dose or in mg / kg doses. The dose may also be selected to reduce or avoid the production of antibodies against the anti-ZNT8 antibody or fragment thereof. The dosing regimen is adjusted to provide the desired response, e.g., a therapeutic response or a combination therapeutic effect. In general, a dose of the antibody or fragment thereof (and optionally a second agent) may be used to provide a bioavailable amount of agent to the subject. For example, a dose 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 may be administered. Other doses may also be used. In certain embodiments, the antibody or fragment thereof is administered to a subject in need of treatment with the antibody or fragment thereof at a dose of about 1 mg / kg to about 30 mg / kg. In some embodiments, a subject in need of treatment with an anti-ZNT8 antibody or fragment thereof is administered the antibody or fragment thereof at a dosage 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 certain embodiments, the antibody or fragment thereof is administered subcutaneously at a dosage of 1 mg / kg to 3 mg / kg. In another embodiment, the antibody or fragment thereof is administered intravenously at a dosage of 4 mg / kg to 30 mg / kg.

[0146] The composition may contain about 1 mg / mL to 100 mg / 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 the antibody or fragment thereof.

[0147] As used herein, dosage unit form or "fixed dose" refers to a physically discrete unit suitable as a unitary dosage for a subject to be treated. Each unit contains a predetermined amount of an antibody or fragment thereof calculated to produce a desired therapeutic effect in association with the necessary pharmaceutical carrier, and optionally in association with other agents. Single or multiple doses may be given. Alternatively, or in addition, the antibody or fragment thereof may be administered by continuous infusion.

[0148] Doses of the antibody or fragment thereof may be administered over a sufficient period (treatment course), for example at least 2, 3, 5, 10 or more times, for example once or twice daily, or about 1-4 times per week, or preferably weekly, every other week (every 2 weeks), every 3 weeks, monthly, for example, at regular intervals including about 1-12 weeks, preferably 2-8 weeks, more preferably about 3-7 weeks, and even more preferably about 4, 5, or 6 weeks. Factors that may affect the dosage and timing required to effectively treat a subject include, for example, the stage or severity of the disease or disorder, the formulation, the route of delivery, previous treatments, the general health and / or age of the subject, and other illnesses. Furthermore, treatment of a subject with a therapeutically effective amount of a compound may include a single treatment, or preferably, a series of treatments.

[0149] If a subject is at risk of developing a disorder described herein, the antibody or fragment thereof can be administered, for example, as a preventive measure, before the disorder fully develops.The duration of such preventive treatment can be a single administration of the antibody or fragment thereof, or the treatment can be continuous (e.g., multiple administrations).For example, a subject at risk of a disorder or a subject with a predisposition to a disorder can be treated with the antibody or fragment thereof for days, weeks, months, or even years to prevent the onset or progression of the disorder.

[0150] In certain embodiments, the antibody or fragment thereof is 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, The antibody or fragment thereof is administered subcutaneously at a concentration of about 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, the anti-ZNT8 antibody or fragment thereof is administered subcutaneously at a concentration of 50 mg / mL. In another embodiment, the antibody or fragment thereof is administered intravenously at a concentration of about 1 mg / mL to about 500 mg / mL. In some embodiments, the antibody or fragment thereof is administered intravenously at a concentration of 50 mg / mL.

[0151] Doses intermediate to the above ranges are also contemplated to be within the scope of the present invention.Subjects can be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis.Exemplary treatments require multiple administrations over an extended period of time, for example, at least 6 months.In some methods, two or more polypeptides can be administered simultaneously, in which case the dosage of each polypeptide falls within the indicated range.

[0152] The polypeptide of the present invention can be administered multiple times. The interval between single doses can be daily, weekly, monthly, or yearly. The interval can be irregular, as indicated by measuring the blood concentration of the modified polypeptide or antigen in the patient. Alternatively, the polypeptide can be administered as a sustained release formulation, which requires less dosage and less frequency. The dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0153] The dosage and frequency vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the polypeptides of the present invention or a cocktail thereof are administered to patients not yet in a disease state to enhance the patient's resistance or minimize the effects of the disease. Such an amount is defined as a "prophylactically effective dose." Relatively low dosages are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives.

[0154] (X. Therapeutic Equipment and Kits) Anti-ZNT8 antibody or fragment thereof can be provided as a kit. In some embodiments, the kit includes (a) a container that includes a composition comprising anti-ZNT8 antibody or fragment thereof described herein, and optionally (b) informational material. Informational material can be instructional, instructional, marketing, or other material related to the methods described herein and / or the use of the agent for therapeutic effect.

[0155] 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 containing a composition comprising an anti-ZNT8 antibody or a fragment thereof and a second container containing a second agent.

[0156] In some embodiments, the kit also includes a second agent, such as an imaging agent. For example, the kit includes a first container containing a composition including an anti-ZNT8 antibody or a fragment thereof and a second container containing a second agent.

[0157] The format of the informational material of the kit is not limited. In some embodiments, the informational material may include information regarding the manufacture of the compound, the molecular weight of the compound, the concentration, expiration date, batch or manufacturing site information, etc. In some embodiments, the informational material is about administration methods, such as administering an anti-ZNT8 antibody or fragment thereof in an appropriate dosage, form, or mode of administration (e.g., a dosage, form, or mode of administration described herein) to treat a subject who has suffered from or is at risk for a disease described herein. The information may be provided in a variety of formats, such as printed text, computer-readable material, video recording, audio recording, or information providing links or addresses to physical materials such as the Internet.

[0158] In addition to the anti-ZNT8 antibody or fragment thereof, the composition in the kit may include other components such as a solvent or buffer, stabilizer, or preservative. The anti-ZNT8 antibody or fragment thereof may be provided in any form, e.g., liquid, dry, or lyophilized, and is preferably substantially pure and / or sterile. When the agent is provided in a solution, the solution is preferably an aqueous solution. In certain embodiments, the anti-ZNT8 antibody or fragment thereof in the solution may be 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 the anti-ZNT8 antibody or fragment thereof is provided as a lyophilized product, the anti-ZNT8 antibody or fragment thereof is 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 adding a suitable solvent. The solvent (e.g., sterile water or a buffer (e.g., PBS)) can be included in the kit as needed.

[0159] The kit may include one or more containers for the composition or compositions including the agent. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and the informational material. For example, the composition may be contained in a bottle, vial, or syringe, and the informational material may 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 to which the informational material in the form of a label is attached. In some embodiments, the kit includes a plurality (e.g., a pack) of separate containers, each of which includes one or more unit dosage forms of the agent (e.g., dosage forms described herein). The container may include a combination unit dosage, e.g., a unit including both an anti-ZNT8 antibody or fragment thereof and a second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampoules, foil packets, blister packs, or medical devices, e.g., each of which includes a single combination unit dosage. The containers of the kits can be air tight, waterproof (eg, impermeable to changes in moisture or evaporation), and / or light-tight.

[0160] The kit optionally includes a device suitable for administration of the composition, such as a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents, or it can be provided empty and suitable for loading.

[0161] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The examples are merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. EXAMPLES

[0162] The following examples are presented 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 have been made and evaluated, and are intended to be merely illustrative and not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy of numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for herein. Unless otherwise indicated, parts are parts by weight, temperatures are degrees Celsius or ambient, and pressures are 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 purity and yield of products obtained from the described processes. Only reasonable and routine experimentation is required to optimize such process conditions.

[0163] Example 1: Generation and characterization of mAb43 (material and method) Animals: NOD, C57BL / 6, and MIP-GFP mice were purchased from Jackson Laboratory, and ZnT8-KO mice were purchased from Taconic. Mice were housed on a 12-h light / 12-h dark cycle and maintained in group housing in sterile containers in a pathogen-free barrier facility with free access to water and standard rodent chow. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University School of Medicine and the Barbara Davis Diabetes Center at the University of Colorado.

[0164] Generation of human ZnT8 antigen and reconstitution of proteoliposomes Human ZnT8 isoform-2 cDNA (NM_001172814.1) was subcloned into a mammalian pCMV6-based expression vector with a C-terminal His tag

[16] . The expression plasmid was introduced into FreeStyle 293-F cells and transiently expressed in suspension culture in serum-free medium according to the manufacturer's instructions. Human CTD-His was constructed by N-terminal deletion to remove the entire TMD sequence from the ZnT8-His construct and transiently expressed in 293-F cells as described above. Cells expressing ZnT8-His or CTD-His were harvested 18 h after transfection and homogenized using a microfluidizer. Cell membranes were separated from the cytosolic fraction by ultracentrifugation. Membrane-bound ZnT8-His was detergent extracted and purified as previously described

[16] . Purified ZnT8-His was reconstituted into proteoliposomes composed of DOPC, DOPE, and DOPG in a 2:1:1 ratio at a ZnT8 / lipid ratio of 1 / 20 (wt / wt). Lipid A auxiliary was added to the reconstituted lipid mixture to a concentration of 10% of the total lipid content. ZnT8-His reconstituted in proteoliposomes remains functionally active and can be resolubilized by detergent to form monodisperse species by sizing HPLC

[26] . Liposomes were prepared in parallel with the proteoliposomes without the addition of ZnT8-His to the lipid reconstitution mixture.

[0165] (Mouse immunization and generation of mAb43) Four pairs of 7-week-old male / female homozygous ZnT8-KO mice were used for proteoliposome immunization and one pair of male / female littermates was used for liposome immunization. Five 10-week-old NOD females were used for proteoliposome immunization and three NOD female littermates were used for liposome immunization. Each mouse was intraperitoneally injected weekly with 50-60 μg of purified ZnT8 in proteoliposome emulsion or an equal amount of liposome emulsion (100 μl). Submental bleeds were collected 3 weeks after injection and used for serum antibody titers by comparative ZnT8 and CTD ELISA. All mice were euthanized 5 weeks after injection. Draining lymph nodes and spleens were collected and hybridoma fusions were generated by electrofusion. Fused cells were selected in HAT, cloned in semi-solid ClonaCell™-HY medium D, and grown in medium E in 96-well plates for mAb screening by comparative ELISA (see below). Twelve positive clones were grown in AOF medium for large-scale mAb production. Cell culture grade mAbs were generated by size-exclusion HPLC purification in PBS and used for live cell screening based on ZnT8 binding on the surface of INS-1E cells stably expressing human ZnT8-GFP

[11] . The variable regions of mAb43 transcripts in hybridoma cells were sequenced and subcloned into mammalian bicistronic IRES expression vectors (Takara Bio, pIRES Vector, Addgene, pVITRO1-dV-IgG1 / κ; pVITRO1-trastuzumab-IgG2 / κ, pVITRO1-trastuzumab-IgG3 / κ, pVITRO1-trastuzumab-IgG4 / κ) carrying the human signal peptide, kappa and gamma constant regions. Recombinant mAb43 constructs of different IgG isotypes were transiently expressed in 293-F cells, then purified and verified for ZnT8 binding based on the formation of stable mAb43-ZnT8-GFP complexes by fluorescent size-exclusion HPLC.

[0166] (Comparison of ELISAs) For proteoliposome-based ELISAs, 4 μg of proteoliposomes (containing 5% human ZnT8-His by weight) diluted in 100 μl of PBS were added to each well of a high-binding 96-well plate and incubated overnight at 4°C. Passively immobilized proteoliposomes were blocked with 5% BSA and tested with hybridoma culture supernatants. For solution-based ELISAs, 293-F cells expressing human ZnT8-His or CTD-His were mechanically lysed using a microfluidizer and cell debris was removed by ultracentrifugation. Then, 0.2 μg of human ZnT8-His from detergent-solubilized cell membranes or 0.1 μg of CTD-His from cell lysates in 100 μl of PBS were immobilized via the C-terminal His tag in each well of a nickel-coated 96-well plate. The immobilized proteins were blocked with 5% BSA and then tested with threefold serial dilutions of mouse serum or hybridoma culture supernatants. Bound serum antibodies were detected by HRP-conjugated goat anti-mouse IgG secondary antibody (1:3000) on a Flexstation-3 microplate reader.

[0167] Immunofluorescence labeling and image analysis. Endoc-βH1 cells were seeded on glass-bottom microwell dishes precoated with β-coat and grown in OPTI cell culture medium at 37°C in a 5% CO2 humidified atmosphere for 2 days. For IF labeling of the cell surface, live cells were washed with high glucose (20 mM) Krebs buffer and chilled at 8°C for 30 min before being exposed to mAb43 (1:100), mAb20 (1:100), anti-CD71 (1:50) or anti-Na+ / K+ATPase (1:50) antibodies. After 1 h incubation at 8°C, unbound antibodies were removed by washing twice using high glucose Krebs buffer. Next, cells were exposed to fluorescent anti-IgG secondary antibody (1:400) for 0.5 h, and after washing to remove unbound secondary antibody, DAPI / DCV was added to the medium for fluorescence imaging on a Zeiss LSM 700 inverted confocal microscope with a 63x oil objective. For intracellular IF labeling, live cells were washed with high glucose (20 mM) Krebs buffer, fixed for 20 min at RT using flow cytometry fixation buffer, washed again using PBS, permeabilized for 20 min at RT using flow cytometry permeabilization buffer, exposed to PBS+5% BSA for 30 min, and then exposed to mAb43 (1:1000), mAb20 (1:1000), anti-CD71 (1:200) or anti-Na+ / K+ATPase (1:200) antibodies for 2 h at room temperature. Secondary antibody immunolabeling, DAPI counterstaining and immunofluorescence imaging were performed using the same procedures as described above. For experiments with wild-type INS-1E cells or ZnT8-KO INS-1E cells

[13] , cells were grown in RMPI 1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS), 100 units / ml penicillin, 100 μg / ml streptomycin, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate and 50 μM β-ME. Immunofluorescence labeling and imaging followed the same procedures as above. For antibody internalization in Endoc-βH1 cells, mAb43 (1:100) and NTPDase3 (1:100) were first co-incubated with Alexa fluor-647 (1:200) and Alexa fluor-488 secondary antibodies (1:200), respectively.To form the fluorescent antibody complex, it was added to live Endoc-βH1 cells for 1 h at 37 °C before IF imaging.

[0168] (Immunohistochemistry) Excised mouse pancreases were fixed in 4% PFA for 4 h at 4 °C, processed and then embedded in paraffin. Tissue sections (4 μm) were delipidated and rehydrated, blocked for 1 h, and then incubated with chimeric mAb43 or chimeric mAb20 at 1:50 in universal antibody dilution buffer for 16 h at 4 °C, followed by treatment with secondary biotinylated anti-human IgG antibody (1:400) for 30 min at 37 °C, followed by avidin-biotinylated peroxidase complex for 30 min at 37 °C. Diaminobenzidine substrate was then applied to develop optimal staining intensity. The colorimetric reaction was stopped by washing with dH2O. Pancreatic sections were then counterstained with eosin, dehydrated and mounted in xylene-compatible mounting medium for imaging.

[0169] (Fluorescence size-exclusion HPLC analysis) Approximately 3 × 106 stably transfected INS-1E cells expressing ZnT8-GFP or ZnT8FLAG-GFP were solubilized using 200 μl of assay buffer (20 mM HEPES, 100 mM NaCl, pH 7.0) + 0.5% DDM. Detergent crude extracts containing ZnT8-GFP or ZnT8FLAG-GFP were injected into a size-exclusion TSK HPLC column, and GFP fluorescence was monitored using a fluorescence detector (488 / 510 nm). ZnT8-GFP was collected as monodisperse peak fractions. HPLC-isolated ZnT8-GFP or ZnT8FLAG-GFP were then incubated with mAb43, mAb20, or anti-FLAG antibody for 1 h on ice and then re-injected into the HPLC column. ZnT8-antibody complexes were collected for immunoblotting analysis to verify the presence of both ZnT8 and antibody in the bound complex.

[0170] (Purification and EM single part analysis of ZnT8-Fab43) mAb43 was produced by hybridoma cells grown in serum-free AOF medium for 3 weeks, captured by protein A / G beads, eluted by IgG elution buffer, and concentrated to approximately 20mg / ml for Fab production using Piercers Fab preparation kit according to the manufacturer's protocol. Purified Fab43 was mixed with purified ZnT8 in reconstituted proteoliposomes at a 5:1 molar ratio and 1% DDM to solubilize Fab43-ZnT8 in lipid-rich detergent solution. Fab43-ZnT8 complex was purified through a TSK size-exclusion HPLC column equilibrated with 0.05% DDM in 20mM HEPES and 100mM NaCl, pH=7.0. After three HPLC delipidation runs, ZnT8-Fab complex was collected as a monodisperse elution peak. Purified protein samples were diluted to 20 μg / ml, and aliquots of 3 μl of the diluted samples were applied onto glow-discharged EM grids covered with a continuous thin carbon film and stained with 2% aqueous uranyl formate for 0.5 min. The grids were mounted on a Tecnai Spirit electron microscope operating at a high voltage of 120 kV. Electron micrographs were recorded in low-dose mode (10e- / angstroms) at 30,000x magnification using a Gatan Orius CCD camera, with underfocus values ​​ranging from 1 to 2.5 μm, corresponding to a specimen level of 2.3 angstroms / pixel. A total of 92 micrographs were collected, and contrast transfer function parameters for each image were determined by CTFFIND 4.1.10. 12,778 particles were extracted from the micrographs. After 2D classification with RELION 3.0 and 3D classification with cryoSPARC 3.1, 9,216 particle images were saved for 3D reconstruction. 3D refinement was performed using cryoSPARC3.1, resulting in a 3D EM map with an estimated resolution of 1.5 nm. We used the Fab structure (PDB 1M71), the cryo-EM structure of human ZnT8 (PDB 6XPD), and rigid-body docking to fit the component structures into the EM map of the ZnT8-Fab43 complex.

[0171] Tissue Dispersion and Pancreatic Cell Labeling. Pancreata excised from C57BL / 6 mice were cut into small pieces, minced, and washed with HBSS on a 70 μm strainer to remove hematopoietic cells. The washed tissue pellet was resuspended in Accutase and incubated at 37°C for 30 min. DCV was added to stain DNA in live cells. Dispersed cells were filtered through the strainer by gentle spinning at 1200 rpm for 2 min. The remaining tissue pellet underwent additional cycles of Accutase digestion and cell filtration to achieve complete cell dispersion. Dispersed cells were pooled and washed with cold cell culture medium containing DNase and trypsin / chymotrypsin inhibitors. At this point, cell viability, as measured by trypan blue exclusion, was typically 80% or higher. Dispersed cells were adjusted to a cell density of 106 / 100μl in flow cytometry tubes and incubated with chimeric mAb43 (106 cells / 1μL mAb43 stock at 1mg / ml) for 1 hour on ice, followed by PE-conjugation with anti-human IgG secondary antibody (106 cells / 1μL antibody stock at 1mg / ml) for 1 hour on ice. Chimeric mAb20 was used as an isotype control.

[0172] Fluorescence-activated cell sorting and confocal microscopy analysis. Labeled pancreatic cells were immediately analyzed and sorted on a MoFlo XDP cell sorter (Beckman Coulter) equipped with 405 and 561 nm lasers. Data were collected in forward scatter, side scatter, and 440 nm and 578 nm fluorescence channels. Cells gated on forward and side scatter yielded more than one million single-cell counting events. R0- or R1-gated sorted cells were deposited on the glass bottom of microwell dishes by gentle centrifugation (1200 rpm, 1 min). After attachment of cells to a surface coated with Matrigel (1:100), cells were fixed with 4% paraformaldehyde for 20 min and then permeabilized. Intracellular labeling was performed in permeabilization buffer containing 2% BSA with chimeric mAb43, followed by labeling with anti-human IgG-PE, anti-insulin APC, and anti-glucagon Alexa Fluor 488. After washing and nuclear DAPI counterstaining, immunofluorescence images were acquired using a Zeiss LSM 700 as described above.

[0173] (Western blot analysis of mAb biodistribution in mice). Male C57BL / 6 mice, 10–11 weeks of age, were administered chimeric mAb43 or chimeric mAb20 intravenously or intraperitoneally at a dose of 5 mg / kg. One to six days after injection, mice were euthanized and tissues from various organs were excised, dried by brief spinning on strainers, weighed, and homogenized in PBS containing DNase and protease inhibitors. Tissue suspensions were dissolved in 4X SDS-PAGE sampling buffer at a concentration of 50 mg / mL. Chimeric mAb43 or chimeric mAb20 in each tissue was detected by anti-human IgG immunoblotting and quantified using serial dilutions of human IgG standards on the same blot. Tissue uptake was corrected for tissue weight and total dose. The amount of antibody retained was calculated as the percentage of injected mAb per gram of each tissue collected (% mAb injected / g).

[0174] (Flattened whole-mount pancreas preparation) 10–11 week-old male / female C57BL / 6 mice were intravenously administered mAb43-mScarlet, mAb20-mScarlet, or PBS at a dose of 5 mg / kg. One day after injection, mice were euthanized, and whole pancreases were excised, placed between a pair of microscope slides, flattened by placing a weight on the glass sandwich, and fixed in 4% PFA for 2 h. The partially fixed pancreas was then removed from the glass sandwich and fixed for an additional 4 h. The fixed pancreas was then transferred to saturated sucrose for approximately 48 h and then to 100% glycerol overnight. The entire procedure, from tissue flattening to optical clarity, was performed in a cold room (8 °C) to minimize tissue degradation. For β-cell immunolabeling, the flattened and PFA-fixed pancreas was transferred to 1% Triton X-100 PBS + 2% BSA overnight. Pancreata were then incubated with anti-insulin-APC (1:50) in 0.1% Triton X-100 containing 0.2% BSA for 12 h, washed, and subjected to optical clearing as above. Cleared whole-mount pancreata were placed between a microscope slide and a coverslip, then flattened again using a heavy weight while the coverslip was sealed with fluorogel. Pairs of 10-week-old male / female MIP-GFP mice were intraperitoneally administered mAb43-mScarlet at a triple dose (15 mg / kg). Three days after injection, pancreata were excised and subjected to PFA fixation and optical clearing as above.

[0175] Whole-mount pancreas imaging and data analysis. Images of whole-mount pancreas were acquired with an ImageXpress Micro high content analysis system equipped with a 4x / 0.2 PlanApo objective. The laser autofocus controlled by MetaXpress software was fixed on the glass surface (20 mm W.D.), and maximal projections from 3D reconstructions of 17x10 μm Z-stacks (tissue thickness approx. 0.2 mm) yielded 2D projection images of each position in three colors from a Lumencor SOLA solid-state fluorescent light source with 16-bit planar resolution, 3-log intensity range, using GFP (488 nm), Rhodamine (585 nm), and Cy5 (692 nm) filter sets for GFP, mAb43-mScarlet, and insulin-APC fluorescence, respectively. Transmitted light scans were recorded simultaneously to generate bright-field images. Exposure times (100–200 ms) for each fluorescent channel were chosen to be just enough exposure to show autofluorescence of pancreas from mice given PBS or mAb20-mScarlet injections. Tiled scanning of the whole-mount pancreas on a motorized stage generated a grid of images, which were then stitched together using the Fiji stitching plugin to generate a merged image. Flattened pancreatic preparations and optical clearing provided a uniform autofluorescent background. A single background fluorescence level was measured for each fluorescence channel, numerically subtracted across the entire image, and displayed by ImageJ without further modification. Mander's overlap coefficients were calculated across the entire pancreas using all pixels above the automatic thresholds for GFP and mScarlet fluorescence without background correction.

[0176] Preparation and imaging of mouse islets Mouse pancreases were perfused with 5 ml of pre-chilled collagenase P (1 mg / ml) by cannulating the bile duct attached to the duodenum at the papilla while suturing the bile duct bundle near the liver

[44] . Fully distended pancreases were excised, digested for 7 min at 37°C, washed with G solution (HBSS plus 0.35 g / L NaHCO3 and 1% BSA), filtered through a mesh, and pelleted at 1200 rpm for 2 min. The islets were separated from tissue debris by resuspending the pellet in 15 ml of Histopaque 1100

[45] and centrifuging at 1200 rpm for 20 min. The upper layer was collected and diluted with 25 ml of G solution, after which the islets were pelleted at 1500 rpm for 4 min with two washes. The pellet was resuspended in islet culture medium (RPMI 1640 plus 2 mM L-glutamine, 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin). Healthy islets were harvested in fresh medium supplemented with 20 mM glucose in glass-bottom microwell dishes. mAb43-mScalet or mAb20-mScalet was added to the islet culture medium to a final concentration of 0.01 mg / mL, incubated for 2 h in a CO2 incubator at 37 °C, washed once with HBSS buffer, and mounted in a glass sandwich (approximately 0.4 mm apart) used for pancreatic whole-mount imaging. Islet imagers were acquired at room temperature in an ImageXpress Micro high content analysis system using the same settings as for pancreatic whole-mount imaging, as described above.

[0177] Statistical analysis: All values ​​are expressed as the mean ± standard error of the mean. Two-tailed Student's t-tests were used to compare groups. Significance indicated in the figures is indicated with *, P < 0.01.

[0178] Example 2: Induction and biochemical characterization of anti-TMD antibodies Lymphocytes responsible for the production of antibodies against the highly conserved epitopes of ZnT8 can be eliminated during the development of self-tolerance that prevents lymphocytes from attacking self-antigens. To overcome this obstacle, we used two different immunization strategies to induce antibody responses against human ZnT8: (1) deleting the ZnT8 gene to avoid negative selection in immunologically naive mice, and (2) stimulating autoreactivity against ZnT8 in immunologically constructed mice with defective immune tolerance. Thus, we immunized ZnT8-KO mice and non-obese diabetic (NOD) female mice, which are prone to developing spontaneous autoimmune diabetes. To preserve the native folding of the ZnT8 antigen after injection into the blood circulation, we developed a liposomal reconstituted ZnT8 formulation [14, 16]. ZnT8 is a bimodular protein consisting of a transmembrane domain (TMD) and a cytosolic C-terminal domain (CTD) (Figure 1A). As the native folding of the TMD requires the presence of the CTD, mouse antibody responses against the TMD were examined by comparative ELISA against full-length ZnT8 (flZnT8) and its CTD. Both mouse strains showed strong anti-flZnT8 (TMD+CTD) and anti-CTD responses above background levels in mice that received empty liposome injections as a control. ZnT8-KO mice showed no difference in serum titration against flZnT8 and CTD, suggesting that all serum antibodies were directed against the CTD (Figure 1C). In comparison, NOD mice showed significantly higher serum reactivity against flZnT8 at lower serum dilutions (Figure 1D), suggesting that anti-TMD reactivity is present in addition to CTD reactivity in NOD mice injected with proteoliposomes. Next, we generated hybridoma cells from immunized ZnT8-KO and NOD mice. All mAbs derived from ZnT8-KO mice targeted the intracellular CTD portion of ZnT8. Similarly, mAbs from NOD mice recognized primarily the CTD. Nevertheless, we identified an anti-TMD mAb (mAb43) that had no detectable reactivity to the CTD but was only reactive to flZnT8 (TMD+CTD) (Figures 1E-1F).Reconstitution of detergent-solubilized human ZnT8 into proteoliposomes increased mAb43 reactivity by 6.29-fold, demonstrating preferential recognition of the natively folded TMD conformation in the membrane (Figure 1G). Validated anti-CTD mAb20 was used as a binding control (17). No differences were observed in mAb20 reactivity to the three different antigen formats: detergent-solubilized ZnT8, CTD, and liposome-reconstituted ZnT8 (Figures 1E-1G). This mAb20 binding profile is consistent with the CTD as an independent natively folded soluble domain (18). Titration of mAb43 and mAb20 against ZnT8 proteoliposomes yielded binding affinities of 0.42 ± 0.05 and 0.57 ± 0.07 nM, respectively.

[0179] Example 3: Cell surface binding and specificity To confirm whether the observed anti-TMD reactivity of mAb43 was directed towards the extracellular surface of the TMD, we compared immunofluorescence (IF) labeling of live human β-cells (Endoc-βH1) with mAb43, mAb20, and an antibody against the abundant cell surface marker CD71. All experiments were performed at 8 °C to block endocytosis of the antibody and in the presence of 20 mM glucose to stimulate ZnT8 surfacing (11). mAb43 and anti-CD71 produced strong IF punctation at the cell surface, whereas mAb20 did not produce a detectable signal (Figure 2A). On the other hand, both mAb20 and mAb43 strongly labeled permeabilized Endoc-βH1 cells, as they recognize cytosolic CTD and luminal TMD epitopes, respectively (Figure 2B). We further investigated the cross-reactivity of mAb43 against a rat β-cell line (INS-1E) in comparison with a rodent-reactive antibody against the abundant cell surface marker Na+ / K+ ATPase. mAb43 and anti-Na+ / K+ATPase produced strong IF punctation on the cell surface of live INS-1E cells (Figure 2C). In comparison, immunolabeling of permeabilized INS-1E cells revealed vesicular and nuclear labeling by mAb43 and Na+ / K+ATPase antibodies, respectively (Figure 2D). Na+ / K+ATPase has previously been reported to localize to the nuclear membrane in addition to the cell surface

[19] . Finally, CRISPR / Cas9-mediated ZnT8 knockout in INS-1E cells abolished IF labeling of mAb43 on the cell surface and in intracellular vesicles, validating ZnT8 specificity in rodent β cells (Figure 2C-2D). We further validated the specific mAb43 immunolabeling of cell surface ZnT8 by quantifying the difference in mAb43 or mAb20 IF labeling of Endoc-bH1 cells and mAb43 IF labeling of wild-type or ZnT8-KO INS-1E cells (Figure 2F). Finally, we investigated competitive binding of ZnT8 by mouse mAb43 and human serum that had previously tested positive for ZnT8ecA. Exposure of live Endoc-bH1 cells to either mouse mAb43 or human serum resulted in strong mouse or human IgG punctuation at the cell surface.In comparison, exposure of live Endoc-βH1 cells to both mouse mAb43 and human serum resulted in punctuation of primarily mouse IgG, regardless of serum or mAb43 preblocking (Figure 2E). Image quantification showed that mAb43 displaced >80% of the serum IgG puncta on the cell surface (Figure 2G). This finding indicates that polyclonal serum ZnT8ecA from diabetic patients is nominally directed against cell surface ZnT8 epitopes shared by mAb43.

[0180] Example 4: Epitope Mapping and Conformational Specificity To map the mAb43 epitope to the ZnT8 extracellular loops (ECLs), we inserted FLAG-octapeptides into individual ECLs to perturb their local structure, and then compared mAb43 binding to native ZnT8 and ZnT8FLAG. Of the nine insertion constructs, only the ECL-2 insertion caused ZnT8FLAG expression in INS-1E cells

[11] . To monitor the formation of binary mAb-ZnT8 complexes by fluorescent size-exclusion HPLC, enhanced green fluorescent protein (GFP) was added to the ZnT8 C-terminus. mAb43 binding shifted the ZnT8-GFP peak to the left, indicating the formation of a stable mAb43-ZnT8-GFP complex (Figure 3A). The FLAG tag abolished mAb43 binding to ZnT8FLAG-GFP, but adding anti-FLAG binding formed a stable anti-FLAG-ZnT8FLAG-GFP complex (Figure 3B). The FLAG tag did not alter the monodispersity profile of ZnT8FLAG-GFP, nor did it affect the formation of the mAb20-CTD complex (Figure 3B). Thus, mAb43 and the FLAG antibody directly competed for ECL-2 on the TMD surface of natively folded ZnT8. Moreover, mAb43 showed no reactivity to SDS-denatured ZnT8 on immunoblots, despite the presence of an intact ECL-2 loop (Figure 3C). This finding further demonstrated the conformational specificity of mAb43. In comparison, mAb20 detected two SDS-denatured ZnT8 splice variants in lysates of Endoc-βH1 cells [4, 17], whereas the anti-peptide ZnT8 antibody detected denatured ZnT8 with high nonspecific reactivity (Figure 3C). Finally, negative staining electron microscopy (EM) single particle analysis was used to visualize the binding complex between the antigen-binding fragment of mAb43 (Fab43) and detergent-solubilized ZnT8. Delipidated ZnT8 was not able to form stable Fab43-ZnT8 complexes to withstand preparation of EM grids, nevertheless, Fab43-ZnT8 complexes were captured using minimally delipidated ZnT8, and only one Fab43 molecule was found complexed with the ZnT8 homodimer (Figure 3D).The binding point of Fab43 to the ZnT8 homodimer density was approximately 18° away from the 2-fold homodimer axis, consistent with an extended TMD. This Fab43 binding mode is distinct from the previously reported docking of Fab20 to the CTD at the 2-fold axis in the Fab20-ZnT8 complex [12, 17]. Because the two ZnT8 protomers in the ZnT8 homodimer adopt different conformations

[20] , Fab43 is likely to recognize an outward- or inward-facing conformation. Taken together, the biochemical data indicate that mAb43 forms a stable complex with ZnT8 through conformation-specific binding to the ECL-2 loop.

[0181] Example 5: Specificity for mouse pancreatic islets and β cells The specificity of mAb43 was investigated ex vivo in paraffin-embedded mouse pancreatic sections. mAb43 labeling and diaminobenzidine immunohistochemistry revealed specific localization of mAb43 binding to islets of Langerhans. In comparison, mAb20 did not immunolabel islets due to lack of cross-reactivity to mouse ZnT8 (Figure 4A). Co-immunolabeling of enzymatically dispersed and detergent-permeabilized mouse islet cells with anti-insulin, anti-glucagon and mAb43 showed that mAb43 recognized both a- and b-cells, whereas the isotype (IgG2b) control did not produce a detectable IF signal (Figure 4B). Simultaneous immunolabeling of human pancreatic frozen sections from two different T2D patients revealed co-localization of anti-insulin and mAb43 IF signals, demonstrating the specificity of mAb43 for human islets (Figure 4C). In some islets, a brown ring of unstained cells was evident. These cells are thought to be α- and δ-cells that are typically localized at the periphery of normal mouse islets. We next used fluorescence-activated cell sorting to investigate mAb43 labeling of β-cells in mixed cell populations of enzymatically dispersed mouse pancreas. mAb43-labeled cells were detected using phycoerythrin (PE)-conjugated secondary antibodies, and intact islet cells were gated for large cell debris and granular vesicles based on positive staining with the cell-permeable DNA dye DyeCycle Violet (DCV). Forward and side scatter restrictions were applied to gate on single-cell events. Only a small fraction (1.7%) of pancreatic dispersions fell into the DCV(+) / mAb43-PE(+) quadrant (Figure 4D). This low percentage is consistent with the pancreatic β-cell population constituting less than 2% of the entire pancreatic mass. Sorted cells were grown on Matrigel-coated glass surfaces and then fixed, permeabilized, and subjected to triple IF staining for ZnT8, insulin, and glucagon. All sorted DCV(+) / mAb43-PE(+) cells were positive for both ZnT8 and insulin, but negative for glucagon (Figure 4E). In comparison, most sorted DCV(+) / mAb43-PE(-) cells were negative for ZnT8, insulin, and glucagon.Quantification of insulin and ZnT8 immunolabeling revealed a clear enrichment of b cells that correlated with elevated mAb43 IF intensity (Figure 4F). Thus, by flow sorting of mAb43-labeled cells, it was possible to separate β cells from the acinar and ductal tissue that constitute the majority (98.3%) of the pancreatic mass. The mouse reactivity of mAb43 indicates that mAb43 arose from NOD autoimmunity against the ZnT8 self-epitope. The specificity of mAb43 for primary mouse β cells is consistent with the highly selective nature of NOD autoimmunity against β cells, while the remainder of the islet cells are autoimmune tolerant.

[0182] (Example 6: Glucose-stimulated uptake of ZnT8-mAb43) To track cell surface capture of mAb43 and subsequent ZnT8-mediated mAb43 endocytosis, we used fluorescent A647 secondary antibodies, which label mAb20 and mAb43, and CellMask green staining to demarcate cell boundaries. We monitored antibody surface binding and internalization in live EndoC-βH1 cells. mAb43-A647 was rapidly internalized at 37 °C, whereas exposure of mAb20-A647 did not yield a detectable signal (Figure 5A). Chilling EndoC-βH1 cells at 8 °C stopped endocytosis of mAb43-A647, but cell surface binding of mAb43-A647 persisted (Figure 5B). Importantly, lowering the glucose concentration from 20 mM to 2 mM significantly reduced both mAb43 cell surface binding at 8 °C and mAb43-A647 uptake at 37 °C (Figure 5A-5B). Image quantification suggested that glucose stimulation (20 mM) increased total mAb43-A647 IF labeling by 22.1- and 15.0-fold at 37°C and 8°C, respectively (Figure 5C). The difference in mAb43-A647 IF signal between 37°C and 8°C approximated the net mAb43-A647 uptake. Glucose stimulation increased ZnT8-mediated mAb43 uptake by 30.9-fold (Figure 5C). Glucose-dependent mAb43 capture and internalization was observed using a fusion of mAb43 with the monomeric red fluorescent protein mScarlet.

[0183] Example 7: In vivo mAb43 biodistribution in mice To characterize in vivo mAb43 uptake in mice, we generated a mouse Fab / human Fc chimeric mAb43 and injected it into four male C57BL / 6 mice (C1-4) at a low dose of 5 mg / kg, then used anti-human IgG immunoblotting to detect the chimeric mAb43 in a panel of excised organs. C1-C3 administered mAb43 intravenously and C4 administered intraperitoneally. Circulating mAb43 in plasma was rapidly cleared within 1 day (Figure 6A), consistent with a mouse pharmacokinetic model of target-mediated antibody clearance at low doses (Figure 6B). From 1 to 6 days post-injection, mAb43 was detected primarily in the pancreas, and its biodistribution profile did not change regardless of the route of administration (Figure 6B). Pancreas-to-serum ratios of mAb43 ranged from 24.6 to 66.2. Control experiments using mouse / human chimeric mAb 20 yielded no detectable signal in the pancreas by 3 days post-injection (Figures 6A-6C). In comparison, the half-life of mAb 43 in the pancreas is approximately 1 week, with initial pancreatic concentrations 1 day post-injection of 21.1 ± 0.9% of mAb injected / g, gradually decreasing to 14.3 ± 1.5% and 11.1 ± 1.0% mAb on days 5 and 6 after injection, respectively. injected / g (Figure 6D). Pancreas-specific mAb43 biodistribution indicates that it is possible to target mAb43 to the pancreas through systemic administration. This finding, together with the specificity of ex vivo mAb43 for pancreatic islets (Figure 4A-4F), further suggests that mAb43 is specifically directed to pancreatic islets. We next investigated the biodistribution of mAb43 in mouse models of T1D and T2D. Four 18-week-old NOD females (N1-N4) and four db / db males (D1-D4) were given a single intraperitoneal injection of 5 mg / kg mAb43, and mAb43 uptake into tissues was measured 48 h after injection. At 18 weeks of age, lymphocytic infiltration in the pancreatic islets of NOD females is well established, whereas db / db males develop overt obesity. Both mouse strains showed a similar biodistribution profile to C57BL / 6, with mAb43 accumulating primarily in the pancreas (Figure 6E). Pancreatic mAb43 uptake levels were compared between differently stained individual mice with different fasting blood glucose (FBG) levels ranging from normoglycemia to hyperglycemia (Figure 6F). On average, C57BL / 6 mice had slightly higher mAb43 uptake than NOD and db / db mice, respectively (Figure 6G). One NOD mouse and two db / db mice became diabetic (FBG >250 mg / dL), and these mice showed a significant decrease in pancreatic mAb43 uptake.

[0184] Example 8: Targeted delivery of mScarlet to pancreatic islets To assess the feasibility of mAb43 for in vivo delivery of imaging payloads, we injected C57BL / 6 mice with mAb43-mScarlet, mAb20-mScarlet, or PBS control and then performed whole-mount pancreatic imaging to detect mScarlet uptake in resected pancreata. Only mAb43-mScarlet injections resulted in characteristic mScarlet puncta throughout the pancreas. Anti-insulin APC immunolabeling of detergent-permeabilized pancreatic β-cells yielded a similar APC puncta distribution, but the mScarlet puncta disappeared due to the disappearance of mScarlet trapped inside the cells by detergent treatment. To directly assess islet homing of mScarlet, we used GFP-tagged β-cells in transgenic MIP-GFP mice that received mAb43-mScarlet injections (25). Whole-mount pancreatic imaging revealed a high degree of overall colocalization between GFP and mScarlet, with Mander's overlap coefficients of 0.93 and 0.79 for the fraction of mScarlet overlapping with GFP and the fraction of GFP overlapping with mScarlet, respectively (Figure 7A). The 21% mismatched GFP signal was primarily attributable to erythrocyte GFP autofluorescence in the pancreatic artery and its branches, where mScarlet signal was completely absent (Figure 7B). In contrast, GFP-mScarlet co-occurrence was nearly absolute in islet clusters surrounding large blood vessels (Figure 7B). Similar vasculature-associated islet clusters have also been reported in the human pancreas

[26] . High-power magnification confirmed that individual GFP and mScarlet puncta colocalized (Figure 7C). In some areas, minor mScarlet signal was scattered without overlap with GFP signal (Figure 7D). These were likely small β-cell clusters, whose GFP signal was not visible if no detergent was used during tissue removal

[27] .Finally, we investigated mScarlet uptake in isolated mouse islets ex vivo.The size of individual islets matched the size of mScarlet clusters revealed by whole-mount pancreatic imaging.mAb43-mScarlet exposure of isolated mouse islets resulted in strong mScarlet fluorescence, whereas exposure with mAb20-mScarlet showed no detectable uptake (Figures 7E-7F). These findings further demonstrated the specificity of mAb43-mediated mScarlet uptake via ZnT8 binding on the β-cell surface.

[0185] The data indicate that the generation of mAb43 depends on the breakdown of self-tolerance in NOD mice, where CD4+ autoreactive T cells against ZnT8 spontaneously arise, but they are weakly pathogenic. Thus, ZnT8 proteoliposome immunization is required to enhance autoreactivity against ECL. Deletion of the ZnT8 gene in ZnT8-KO mice is insufficient to induce antibodies against the ECL of human ZnT8, presumably because the extracellular ZnT8 epitope is conserved across species in other homologs of the ZnT protein family (ZnT1-10). Notably, part of the ZnT signature sequence is located in ECL1. The resulting mAb43 is an autoantibody that recognizes a cell surface ZnT8 epitope with remarkable in vivo islet specificity for T1D. The subnanomolar binding affinity of mAb43 occurs rarely in the spontaneous autoantibody repertoire of NOD mice. mAb43-ZnT8 binding is apparently structure-specific. Because individual ECLs are too short to fold independently, the formation of a recognizable conformation of mAb43 requires multiple ECLs and their interaction. Given that there is a multi-loop mAb43 epitope on the limited extracellular surface area of ​​ZnT8, the mAb43 epitope should be shared in its entirety or at least in part by polyclonal serum ZnT8ecA. Thus, mAb43 binding may effectively protect ZnT8 extracellular epitopes against serum ZnT8ecA of T1D patients. The IgD and IgM forms of mAb43 are BCRs of ZnT8-specific autoreactive B cells. Using mAb43 as a BCR, we can investigate the molecular recognition and engagement of β cells by autoreactive B cells through the formation of a ZnT8-BCR(mAb43)-centered immunological synapse.

[0186] Pancreatic-specific biodistribution of mAb43 combined with islet-specific immunolabeling of pancreatic sections suggests that systemically administered mAb43 may be specifically delivered to islets in vivo. Whole-mount pancreatic imaging using mScarlet as a probe revealed localized mAb43-mScarlet enrichment in islet clusters at the pancreatic periphery. These highly vascularized islets enable rapid release of insulin into the circulation, while local GSIS activity is functionally coupled to ZnT8 surfacing and subsequent capture of circulating mAb43. Pancreatic mAb43 uptake is maintained in both T1D and T2D models of diabetic mice, whereas mAb43 uptake levels are reduced, reflecting loss of β-cell mass and / or function in diseased mice.

[0187] The in vivo islet specificity of mAb43 is consistent with the islet-specific expression of ZnT8. Within islets, ZnT8 appears to be an intracellular protein expressed in all types of terminal cells by and large. β-cells are the second most abundant cell type after β-cells. Although detergent-permeabilized α- and β-cells were immunolabeled with mAb43, only intact β-cells were FACS-enriched from the total pancreatic cell population, suggesting that ZnT8 surface expression may be a β-cell specific feature driven by GSIS. Antibodies that recognize specific markers on the cell surface may be used to target β-cells to deliver toxic imaging agents or drugs to non-islet tissues. In addition to ZnT8, which is targeted by mAb43 and another ZnT8 antibody (Ab31) directed against the peptide sequence of ECL2, sphingomyelin patches and NTPDase3 have also been targeted as β-cell surface markers. Thus far, only mAb43 has demonstrated a pancreas-specific biodistribution profile supporting its utility for islet homing of imaging payloads and antidiabetogenic drugs.

[0188] Example 9: Use of mAb43 for In Vivo Imaging and Targeted Delivery of Antibody Drug Conjugates To evaluate the feasibility of mAb43 for in vivo imaging and targeted delivery of antibody-drug conjugates, we generate recombinant mAb43 with site-specific biotinylation at the C-terminus of the mAb43 heavy chain. The biotin label is used to conjugate fluorescent streptavidin as an imaging probe. Mouse pancreatic islet cells are labeled with mAb43-streptavidin and sorted based on cell surface IF intensity and cellular zinc-sensitive fluorescence. Positively or negatively gated cells from FACS are subcultured, PFA fixed, and then IF labeled with insulin and glycogen antibodies. Secondary flow cytometry analysis is expected to show that all mAb43-positive cells are insulin or glucagon positive, whereas mAb43-negative cells are insulin or glucagon negative. Further analysis may reveal a positive correlation between mAb43 and insulin positivity, indicating that surface-gated ZnT8 can be used as a biomarker to purify insulin-producing b cells from a mixed population of islet cells. Although some glucagon-producing a-cells may also be mAb43 positive, the IF intensity of mAb43 is expected to be significantly lower than that of b-cells and will not show a correlation with cellular zinc content. Finally, we will inject biotinylated mAb43 into mice and investigate the tissue distribution of mAb43 by streptavidin-HPR. We expect that tissue histology will reveal the accumulation of mAb43 in the islets, demonstrating the in vivo targeted delivery of mAb43 to the islets.

[0189] Example 10: Purification of live adult stem cell-derived beta cells (sBC) mAb43 is used to purify live mature sBCs from heterogeneous cell mixtures. In previous experiments, mAb20 was used to sort C-peptide positive sBCs after PFA fixation and permeabilization. Compared with mAb20, mAb43 has similar ZnT8 affinity and specificity, but the ZnT8 density on the cell surface is probably less than 5% of the intracellular density. Therefore, bright dyes such as PE or APC can be used for signal amplification. Recombinant mAb20 / 43 with site-specific fluorescent labeling is generated.

[0190] A study comparing mAb43 with ENTPD3 (NTPDase3) and INS / Cpep to identify adult stem cell-derived beta cells (sBCs) is performed. hES, iPSC, and T1D-iPSC sBC clusters containing immature and mature sBCs are used.

[0191] Single cell suspensions of live sBC clusters are prepared and labeling efficiency using mAbs is quantified. mAb positive / negative populations are sorted and correlation with insulin / C-peptide expression is investigated. As these clusters / cells also contain the pInsulin-GFP reporter, mAb43-GFP correlation can be directly examined.

[0192] mAb43 is a mouse IgG2b and the control mAb20 is a mouse IgG2a. The recombinant antibodies are switched to human IgG-4. See SEQ ID NOs:20-30.

[0193] [Table 1] [Prior art documents] [Non-patent literature]

[0194] [Non-Patent Document 1] Roep, BO, Thomaidou, S., van Tienhoven, R., and Zaldumbide, A. (2021) Type 1 diabetes mellitus as a disease of the beta-cell(do not blame the immune system?). Nat Rev Endocrinol 17,150-161. [Non-Patent Document 2] Liston, A., Todd, JA, and Lagou, V. (2017) Beta-Cell Fragility As a Common Underlying Risk Factor in Type 1 and Type 2 Diabetes.Trends Mol Med 23,181-194. [Non-Patent Document 3] Chimienti, F. (2013) Zinc, pancreatic islet cell function and diabetes: new insights into an old story. Nutr Res Rev 26, 1-11. [Non-Patent Document 4] Merriman, C., and Fu, D. (2019) Down-regulation of the islet-specific zinc transporter-8 (ZnT8) protects human insulinoma cells against inflammatory stress. J Biol Chem 294,16992-17006. [Non-Patent Document 5] Dodson, G., and Steiner, D. (1998) The role of assembly in insulin's biosynthesis. Curr Opin Struct Biol. 8, 189-194. [Non-Patent Document 6] Lemaire, K., Chimienti, F., and Schuit, F. (2012) Zinc transporters and their role in the pancreatic beta-cell. J Diabetes Investig 3, 202-211. [Non-Patent Document 7] Segerstolpe,A.,Palasanza,A.,Eliasson,P.,Andersson,EM,Andreasson,AC,Sun,X.,Picelli,S.,Sabi rsh,A.,Clausen,M.,Bjursell,MK,Smith,DM,Kasper,M.,Ammala,C.,およびSandberg,R.(2016)Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes.Cell Metab 24,593-607.

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Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the three extracellular loops of the transmembrane domain of zinc transporter-8 (ZnT8).

2. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the three extracellular loops of ZnT8 comprise amino acids 95-99, 169-175, and 242-245 of SEQ ID NO:

31.

3. The antibody or antigen-binding fragment thereof (a) heavy chain complementarity determining regions (CDRs) 1, 2, and 3 comprising SEQ ID NOs: 3-5, respectively; (b) light chain CDRs 1, 2, and 3 comprising SEQ ID NOs: 8-10, respectively; The antibody or antigen-binding fragment thereof of claim 1, comprising:

4. The antibody or antigen-binding fragment thereof (a) heavy chain complementarity determining regions (CDRs) 1, 2, and 3 comprising SEQ ID NOs: 3-5, respectively; (b) light chain CDRs 1, 2, and 3 comprising SEQ ID NOs: 8-10, respectively; and Including, the heavy chain CDRs comprise at least one conservative amino acid substitution within one or more of SEQ ID NOs: 3-5, and / or the light chain CDRs comprise at least one conservative amino acid substitution within one or more of SEQ ID NOs: 8-10; The antibody or antigen-binding fragment thereof of claim 1.

5. The antibody or antigen-binding fragment thereof (a) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:19; (b) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 7; The antibody or antigen-binding fragment thereof of claim 1, comprising:

6. 6. The antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain variable region sequence has at least 95% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 19, and the light chain variable region sequence has at least 95% sequence identity to SEQ ID NO:

7.

7. The antibody or antigen-binding fragment thereof (a) a heavy chain variable region sequence comprising SEQ ID NO: 2 or SEQ ID NO: 19; (b) a light chain variable region sequence comprising SEQ ID NO: 7; and The antibody or antigen-binding fragment thereof of claim 5, comprising:

8. The antigen-binding fragments include Fab, Fab', F(ab') ２ , Fab １ 2. The antibody or antigen-binding fragment thereof of claim 1, comprising an -SH, Fv, diabody, linear antibody, or single-chain variable fragment (scFv).

9. 2. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain constant region is of the immunoglobulin G1 (IgG1) isotype.

10. The antibody or antigen-binding fragment of claim 1 , wherein the antibody or antigen-binding fragment is a humanized antibody or a chimeric antibody.

11. The antibody or antigen-binding fragment of claim 1 , wherein the antibody or antigen-binding fragment is conjugated to a therapeutic agent.

12. The antibody or antigen-binding fragment of claim 1 , wherein the antibody or antigen-binding fragment is conjugated to an imaging agent.

13. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of claim 1.

14. A nucleic acid molecule encoding the antibody or antigen-binding fragment of claim 1.

15. A vector comprising the nucleic acid of claim 14.

16. A host cell comprising the vector of claim 15.

17. 1. A method for making an antibody-drug conjugate that specifically binds to three extracellular loops of the transmembrane domain of ZnT8, comprising: (a) culturing the host cell of claim 16 under conditions suitable for the production of antibodies; (b) conjugating the antibody to a therapeutic agent; 1. A method for producing an antibody-drug conjugate that specifically binds to the three extracellular loops of the transmembrane domain of ZnT8, comprising:

18. 1. A method for producing an antibody-imaging agent conjugate that specifically binds to the three extracellular loops of the transmembrane domain of ZnT8, comprising: (a) culturing the host cell of claim 16 under conditions suitable for the production of antibodies; (b) conjugating the antibody to an imaging agent; 1. A method for producing an antibody-imaging agent conjugate that specifically binds to the three extracellular loops of the transmembrane domain of ZnT8, comprising:

19. A method for treating a disease or condition associated with ZnT8 in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof described in claims 1 to 11 or a pharmaceutical composition described in claim 13.

20. 20. The method of claim 19, wherein the disease or condition comprises type 1 or type 2 diabetes.

21. A method for detecting pancreatic beta cells in vivo, comprising administering to a subject the antibody or antigen-binding fragment thereof described in claim 12 and detecting an imaging agent bound to the antibody or antigen-binding fragment thereof.

22. 22. The method of claim 21, wherein 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.

23. 22. The method of claim 21, wherein the antibody or antigen-binding fragment comprises (a) a heavy chain variable region sequence comprising SEQ ID NO: 2 or SEQ ID NO: 19, and (b) a light chain variable region sequence comprising SEQ ID NO: 7, and is a single-chain variable fragment (scFv).

24. 24. The method of claim 23, wherein (a) the heavy chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO: 2 or SEQ ID NO: 19, and (b) the light chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO:

7.

25. 22. The method of claim 21, wherein the imaging agent is a radioactive metal.

26. 22. The method of claim 21, wherein the imaging agent is a radioactive metal and the detecting step comprises PET.

27. The radioactive metal is ６４ Cu, ６７ Cu, ６８ Ga, ６０ Ga, ８９ Zr, ８６ Y, and ９４ｍ 27. The method of claim 26, wherein the agonist is selected from the group consisting of Tc.

28. 22. The method of claim 21, wherein the imaging agent is a radioactive metal and the detecting step comprises SPECT.

29. The radioactive metal is １１１ In, ６７ Ga, ９９ｍ Tc, and １７７ 29. The method of claim 28, wherein the cation is selected from the group consisting of Lu.

30. A single-chain variable fragment (scFv) or an antigen-binding fragment thereof that binds to the three extracellular loops of the transmembrane domain of ZnT8, comprising (a) the heavy chain variable region sequence of SEQ ID NO:2 or SEQ ID NO:19, and (b) the light chain variable region sequence of SEQ ID NO:

7.

31. 31. The scFv of claim 30, wherein (a) the heavy chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO: 2 or SEQ ID NO: 19, and (b) the light chain variable region sequence comprises at least one conservative amino acid substitution within SEQ ID NO:

7.

32. 31. The scFv of claim 30, wherein the scFv is conjugated to an imaging agent.

33. 32. The scFv of claim 31 , wherein the imaging agent is a radioactive metal.

34. Radioactive metals are ６４ Cu, ６７ Cu, ６８ Ga, ６０ Ga, ８９ Zr, ８６ Y. ９４ｍ Tc, １１１ In, ６７ Ga, ９９ｍ Tc, and １７７ 33. The scFv of claim 32, selected from the group consisting of:

35. An antibody or antigen-binding fragment thereof comprising: (a) a light chain comprising SEQ ID NO: 20; and (b) a heavy chain comprising one of SEQ ID NOs: 21-24.

36. An antibody or antigen-binding fragment thereof comprising: (a) a light chain comprising SEQ ID NO: 20; and (b) a heavy chain comprising one of SEQ ID NOs: 27-30.

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